Recombinant vector comprising a gene for a binding domain and a secretable peptide
阅读说明：本技术 包含用于结合结构域和可分泌肽的基因的重组载体 (Recombinant vector comprising a gene for a binding domain and a secretable peptide ) 是由 艾米·H·林 道格拉斯·J·乔利 于 2019-11-13 设计创作，主要内容包括：本公开提供了经修饰的重组逆转录病毒,其包含编码具有异源分泌信号的蛋白质、含有可操作地连接至异源多核苷酸的2A-肽或肽样编码序列的转基因。本公开还涉及表达或包含此类载体的细胞和载体,以及在疾病和病症治疗中使用此类经修饰的载体的方法。(The present disclosure provides modified recombinant retroviruses comprising a transgene encoding a protein having a heterologous secretion signal comprising a 2A-peptide or peptide-like coding sequence operably linked to a heterologous polynucleotide. The disclosure also relates to cells and vectors expressing or comprising such vectors, and methods of using such modified vectors in the treatment of diseases and disorders.)
1. A recombinant replication competent retrovirus,
comprises the following steps:
retroviral GAG proteins;
a retroviral POL protein;
a retroviral envelope;
a retroviral polynucleotide comprising a Long Terminal Repeat (LTR) sequence 3 'to the retroviral polynucleotide sequence, a promoter sequence 5' to the retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain, said promoter suitable for expression in a mammalian cell;
a cassette comprising a2A peptide or 2A peptide-like coding sequence followed by a secretion signal peptide coding sequence operably linked to a heterologous polynucleotide, wherein the cassette is located 5' to the 3' LTR and operably linked to the 3' end of the env nucleic acid domain encoding the retroviral envelope; and
the cis-acting sequences necessary for reverse transcription, packaging and integration in the target cell.
2. The recombinant replication competent retrovirus of claim 1, wherein the envelope is selected from one of an amphotropic envelope, an pantropic envelope, a heterotropic envelope, a 10a1 envelope, a GALV envelope, a baboon endogenous virus envelope, a RD114 envelope, a rhabdovirus envelope, an alphavirus envelope, a measles envelope, or an influenza virus envelope.
3. The retrovirus of claim 1, wherein the retroviral polynucleotide sequence is engineered by a virus selected from the group consisting of: murine Leukemia Virus (MLV), Moloney murine leukemia virus (Mo MLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PE RV), feline-derived retrovirus RD114, squirrel monkey retrovirus, xenotropic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon ape leukemia virus (GAL V).
4. The retrovirus of claim 1, wherein the retrovirus is a gammaretrovirus.
5. The retrovirus of claim 1, wherein the target cell is a mammalian cell.
6. The retrovirus of claim 1, wherein the 2A peptide or 2A peptide-like coding sequence encodes a polypeptide comprising the sequence of SEQ ID NO: 1.
7. The retrovirus of claim 1, wherein the 2A peptide or 2A peptide-like coding sequence encodes a polypeptide of SEQ ID No: 55-125.
8. The retrovirus of claim 1, wherein the 2A peptide or 2A peptide-like coding sequence comprises the amino acid sequence of SEQ ID No: 8-19.
9. The retrovirus according to any one of claims 1 to 8, wherein the heterologous polynucleotide is > 500 bp.
10. The retrovirus of claim 1, wherein the heterologous polynucleotide comprises at least 2 coding sequences.
11. The retrovirus of claim 1, further comprising a second cassette comprising a2A peptide or 2A peptide-like coding sequence downstream of the cassette.
12. The retrovirus of claim 1, wherein the secretion signal peptide coding sequence encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 289 and 302.
13. The retrovirus of any one of the preceding claims, wherein the heterologous polynucleotide encodes an antibody, antibody fragment, scFv, antigen-binding domain or peptide homologous to a biomolecule.
14. The retrovirus of any one of claims 1-12, wherein the heterologous polynucleotide comprises a sequence encoding a secretory signal peptide operably linked to a heterologous protein or polypeptide, wherein the heterologous protein or polypeptide is selected from the group consisting of a prodrug activating enzyme, a cytokine, a receptor ligand, an immunoglobulin-derived binding polypeptide, a non-immunoglobulin binding polypeptide, and any combination thereof, wherein the plurality of heterologous proteins or polypeptides are separated by a2A or 2A-like peptide.
15. The retrovirus of any one of claims 1-12, wherein the retrovirus further comprises a second cassette comprising an internal promoter or gene expression element operably linked to a different heterologous polynucleotide downstream of the cassette.
16. The retrovirus of claim 5, wherein the target cell is selected from the group consisting of a lung cancer cell, a colorectal cancer cell, a breast cancer cell, a prostate cancer cell, a urinary tract cancer cell, a uterine cancer cell, a brain cancer cell, a head and neck cancer cell, a pancreatic cancer cell, a melanoma cell, a gastric cancer cell, and an ovarian cancer cell.
17. The retrovirus of claim 1, wherein the gag nucleic acid domain is derived from a gammaretrovirus.
18. The retrovirus of claim 17, wherein the gag nucleic acid domain comprises SEQ ID NO:2 from about nucleotide number 1203 to about nucleotide 2819, or a sequence having at least 95%, 98%, 99%, or 99.8% identity thereto, wherein T may be U.
19. The retrovirus of claim 1, wherein the pol nucleic acid domain is derived from a gammaretrovirus.
20. The retrovirus of claim 19, wherein the pol nucleic acid domain comprises SEQ ID NO:2 from about nucleotide number 2820 to about nucleotide number 6358, or a sequence at least 95%, 98%, 99%, or 99.9% identical thereto, wherein T may be U.
21. The retrovirus of claim 1, wherein the env nucleic acid domain comprises SEQ ID NO:2 from about nucleotide number 6359 to about nucleotide 8323, or a sequence at least 95%, 98%, 99%, or 99.8% identical thereto, wherein T may be U.
22. The retrovirus of claim 1, wherein the LTR sequence at the 3' end is derived from a gammaretrovirus.
23. The retrovirus of claim 22, wherein the 3' LTR comprises a U3-R-U5 domain.
24. The retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immune enhancing cytokine.
25. The retrovirus of claim 24, wherein the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 38, interferons, Tumor Necrosis Factor (TNF), and granulocyte-macrophage colony stimulating factor (GM-CSF).
26. The retrovirus of claim 1, wherein the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug into a toxic drug.
27. The retrovirus of claim 26, wherein the polypeptide that converts a nontoxic prodrug to a toxic drug is thymidine kinase, Purine Nucleoside Phosphorylase (PNP), or cytosine deaminase.
28. The retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a receptor domain, an antibody fragment, or a non-immunoglobulin binding domain.
29. A recombinant polynucleotide for producing the retrovirus of claim 1.
30. The polynucleotide of claim 29 comprising an MLV 4070A envelope protein gene in frame with a2A peptide or peptide-like coding sequence with or without a GSG linker coding sequence, and a second gene in frame with the 2A peptide or 2A-like coding sequence.
31. The polynucleotide of claim 29 comprising an MLV 10a1 envelope protein gene in frame with a2A peptide or peptide-like coding sequence with or without a GSG linker coding sequence, and a second gene in frame with the 2A peptide or 2A-like coding sequence.
32. The polynucleotide of claim 29, comprising an XMRV envelope protein gene in frame with a2A peptide or peptide-like coding sequence with or without a GSG linker coding sequence, and a second gene in frame with the 2A peptide or 2A-like coding sequence.
33. The polynucleotide of claim 29, comprising a non-Friend MLV envelope protein gene in frame with a2A peptide or peptide-like coding sequence with or without a GSG linker coding sequence, and a second gene in frame with the 2A peptide or 2A-like coding sequence.
34. The polynucleotide of any one of claims 29-33, wherein the polynucleotide further comprises a secretion peptide sequence located between the 2A peptide or 2A peptide-like coding sequence with or without the GSG linker coding sequence and a gene encoding a polypeptide to be secreted or a heterologous polynucleotide.
35. The polynucleotide of claim 34, wherein the gene encoding the polypeptide to be secreted or the heterologous polynucleotide is a secretory, membrane, cytoplasmic, nuclear or cellular compartment specific protein.
36. The retrovirus of claim 1 or the polynucleotide of claim 29, wherein the retrovirus and/or the polynucleotide has been engineered to remove a tryptophan codon that is sensitive to a human APOB EC hypermutation.
37. The retrovirus or polynucleotide of claim 36, wherein the heterologous polynucleotide encodes a polypeptide having cytosine deaminase activity.
38. The retrovirus or polynucleotide of claim 37, wherein the polypeptide having cytosine deaminase activity encodes a polypeptide of SEQ ID NO: 29, wherein Xaa is any amino acid other than tryptophan.
Technical Field
The present disclosure relates to viral vectors. The disclosure also relates to the use of such viral vectors for the delivery and expression of heterologous nucleic acids in cells, and their expression and secretion.
Background
Efficient methods of delivering genes and heterologous nucleic acids to cells and subjects have been the target of scientific development by researchers and possible treatment of diseases and disorders.
Disclosure of Invention
The present disclosure provides viruses comprising a 2A-peptide cassette containing a secretory peptide coding sequence downstream of the 2A-peptide and upstream of a heterologous gene to be secreted. Further embodiments comprise heterologous genes encoding antibodies, single chain antibodies or other antibody-related structures, binding proteins derived from non-immunoglobulin scaffold proteins, and the like. In further embodiments, the antibody-related peptide or non-immunoglobulin binding protein comprises sequences that cause multimerization of the binding protein to provide higher binding affinity for the target entity. Still further embodiments include viruses comprising a heterologous gene having a heterologous secretion signal for the virus and gene upstream of the heterologous gene product to be secreted.
The present disclosure further describes polypeptide subunits of immunoglobulin (Ig) and non-immunoglobulin (non-Ig) scaffold proteins, each subunit comprising a fusion polypeptide of an antigen binding domain, a multimerization domain (e.g., dimerization, trimerization, and pentamultimerization domains), and optionally an IgG Fc domain, capable of forming stable homologous and dimeric proteins. Oligomeric complexes of non-Ig scaffold proteins may also be formed by single or multiple Gly-Ser linkers.
The present disclosure includes engineered Ig scaffold proteins including heavy chain variable domains derived from human, mouse, camel (Camelidae), shark and bovine (Curr Opin Struct biol.2017 Aug; 45:10-16.doi:10.1016/j. sbi.2016.2016.10.019, incorporated herein by reference), (Nat Biotechnol.2017Dec 8; 35(12):1115-1117.doi:10.1038/nbt1217-1115, incorporated herein by reference), and non-Ig scaffold proteins (see, e.g., Skrlex et al, Trends Biotechnol, 33. (408-7), Chem.18, Jul.2015; and Simeon & n Protein & 9:2-14,2018; both incorporated herein by reference) including Adnens, affinis (Affibodies), affiides, Affinisins, Affinis, Airlins, Abies & n Protein (Abies & n proteins), Abies & n proteins, Abies & n # III, Abies #5, Abies 3, Abies # Cys, Abies 3, Abies 3, Abies 3, Abies # Cys, Abies 3, Abies # Cys, Abies 3, Abies, Eps8L1, FISH #5, CMS #1, and OSTF1, all of which may be operably linked to the N-terminal portion of human IgG Fc, thereby allowing monomeric or oligomeric scaffold proteins to dimerize via disulfide bond formation to form highly complex oligomeric proteins.
Compositions and methods useful for cancer immunotherapy delivered by viral vectors, including retroviral replicating vectors and retroviral non-replicating vectors, other viral vectors, oncolytic viral vectors and non-viral expression vectors, are provided.
In one embodiment, the non-Ig scaffold is of human origin to minimize anti-scaffold protein immune responses.
In one embodiment, the antigen-specific binding subunit of a non-Ig scaffold protein is used as an agonist or antagonist that targets CTLA-4, PD-1, PDL1, GITR, ICOS, LAG-3, TIM-3, OX40, CD40L, CD137/4-1BB, CD27, TIGIT, VISTA, BTLA, IL-2 Ra, IL-2 Rbeta, IL-2 Rgamma, IL-15 Ra, IL-15 Rbeta, or IL-15 Rgamma, CD19, CD20, mesothelin, ganglioside GD2, fibroblast-associated protein FAP, BCMA, CD3, FOXP3, IL-12 Ralpha or beta, CD47, SIRPa, CD94/NKG2, CD 244/EGF 2B4, adenosine receptor A2A, EGFR, FR, PDGF, HGF/MET, VEGFR, IGF, IR-HER-1, HER-1 IGF, HER 3, HER-3, IGF, CEA, EB-D, TRAILR1/DR4, TRAILR2/DR5, Extra Domain B (ED-B), IL-10, and IL-35.
In another embodiment, the antigen-specific binding subunit of a non-Ig scaffold protein functions as an agonist or antagonist that targets at least one of interleukins 1 through 38, with interleukins 1 through 38 consisting of greater than 60 existing members; and their receptors, such as the IL-10 and IL-35 receptors of the IL-2 family, which consist of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 (these receptors contain a common cytokine receptor gamma chain (CD132, gammac)); IL-13R shares IL-4 Ra with IL-4, the receptors for IL-4 and IL-13 consist of 2 receptor chains-IL-4 and IL-13 binds IL-4R, IL-4R consists of IL-4 Ra (CD124) and IL-13 Ra 1 chains, and IL-13R consists of 2 subunits IL-13 Ra 1 and IL-13 Ra 2, and signaling occurs through a type II IL-4R complex consisting of IL-4 Ra and IL-13 Ra; TSLPR (CRFLR-2) shares IL-7R with IL-7; receptors for IL-3, IL-5, and GM-CSF, which is a heterodimer with distinct alpha chain and common beta chain (β c, CD131) subunits; IL-10 family members (IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28 and IL-29) and corresponding receptors sharing a common receptor subunit, as shown; TNF- α and its receptors TNFRI and TNFR 2; TGF-beta and its heterodimeric receptor consisting of TGF-beta R1 and TGF-beta R2; IL12 and its receptor IL-12R, receptor IL-12R is composed of 2 subunits: IL-12R beta 1 and IL-12R beta 3. IL-23 and/or its heterodimeric receptor subunits IL-12R β 1 and IL-23R; IFN- α and IFN- β and/or their heterodimeric receptors consisting of IFNAR1 and IFNAR 2; IFN-gamma and/or its heterodimeric receptor subunits IFN-gamma R1 and IFN-gamma R2.
In one embodiment, the antigen binding domains of the non-Ig scaffold proteins are fusion proteins, each comprising an antigen binding non-Ig scaffold protein, a glycine-serine linker, a functional multimerization domain, wherein the non-Ig scaffold proteins can self-assemble into homodimers, homotrimers, homopentameric protein complexes, homohexamers, or other types of protein complexes, including heteromeric complexes.
In one embodiment, the homo-hexamer non-Ig scaffold protein complex is a fusion protein comprising 6 antigen binding non-Ig scaffold proteins, each consisting of a non-Ig scaffold protein, a glycine-serine linker, a functional trimerization domain, and an IgG Fc domain.
In one embodiment, the homodecameric non-Ig scaffold protein complex is a fusion protein comprising 10 antigen-binding non-Ig scaffold proteins, each consisting of a non-Ig scaffold protein, a glycine-serine linker, a functional pentameric domain, and an IgG Fc domain.
In another embodiment, the antigen binding domain of the non-Ig scaffold protein is a multivalent fusion protein complex comprising different antigen binding non-Ig scaffold proteins, glycine-serine linkers, wherein the non-Ig scaffold protein can self-assemble into a heterodimeric, heterotrimeric, or heteromultimeric protein.
In one embodiment, a method of promoting antigen-experienced T cells and/or activated NK cells and for neoantigen-primed dendritic cell survival or proliferation is provided, wherein the oligomeric form of the non-Ig scaffold protein can specifically bind to an antigen on the surface of a tumor cell, T cell, NK cell, dendritic cell, myeloid cell, tumor-associated fibroblast, B cell.
In a further embodiment, the transgene encodes a prodrug activating protein that is formulated as a secretable peptide or protein. In a further embodiment, the prodrug-activating transgene is a yeast-derived cytosine deaminase.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
Drawings
FIG. 1 shows an alignment of the amino acid sequences of the 2A region of foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Spodoptera litura beta-virus (T2A) and porcine Tespidae virus-1 (P2A) (SEQ ID Nos: 55 to 58).
FIG. 2 shows an alignment of the 2A peptide sequences present in different virus species (SEQ ID Nos: 59 to 125).
FIG. 3 is a schematic representation of RRV-scFv-PDL1 plasmid DNA. (A) Two pairs of single-chain variable fragments (scFv) targeting PD-L1 are encoded in the pAC3 RRV backbone. A pair consisting of scFv with and without Fc from human IgG1 was named pAC3-scFv-PDL1 and pAC3-scFvFc-PDL1, respectively. The other pair consisted of scFv-PDL1 and scFvFc-PDL1, incorporating HA and Flag epitopes at the C-terminus, designated pAC3-scFv-HF-PDL1, pAC3-scFvFc-HF-PDL 1. Solid grey rectangles indicate 2A peptide, IRES or mini-promoter located downstream of the env gene; solid black rectangles (SP ═ signal peptide, table a) represent secretion/leader sequences, e.g. derived from human IL-2.
FIGS. 4A-B show expression of PDL1scFv and PDL1scFvFc protein and efficiency of separation of Env-scFv and Env-ScFvFc polyprotein in transiently transfected 293T cells. (A) Expression of scFv-tag (. about.30 kDa) and scFvFc-tag (. about.55 Kd) proteins by HEK293T cells transiently transfected with pAC3-GSG-T2A-PDL1scFv, pAC3-GSG-T2A-PDL1scFvFc, pAC3-GSG-T2A-PDL1 scFv-tag, pAC3-GSG-T2A-PDL1 scFvFc-tag. (B) anti-2A immunoblotting of cell lysates of transiently transfected 293T cells. The protein bands detected above-110 kDa represent the Env-scFv and Env-ScFvFc fusion polyproteins. The protein band detected at-85 kDa represents the Pr85 virus envelope protein isolated from the fusion polyprotein, and the protein band detected at-15 kDa represents the p15E-2A protein processed from the Pr85 virus envelope protein.
FIG. 5 shows Western blot analysis of viral envelope proteins produced in transiently transfected 293T cells. Twenty micrograms of total protein lysate was loaded per well. The membrane was incubated with (left panel) anti-HA detecting HA-and Flag-labeled scFv-PD-L1 and scF vFc-PD-L1 or (right panel) anti-2A peptide antibodies detecting Env-scFv polyprotein (Env-scFv), unprocessed viral precursor envelope protein isolated from Env-scFv polyprotein (Env-2A) and processed viral envelope protein labeled with 2A peptide at the C-terminus (p 15E-2A). anti-GAPDH antibodies (bottom left panel) served as loading controls, including housekeeping protein GAPDH.
FIGS. 6A-B show scFv PD-L1 detecting binding to PD-L1 by competitive ELISA. Wells in a 96-well microtiter plate were coated with (a) recombinant human or (B) mouse PD-L1-Fc, then co-incubated with His-tagged recombinant PD-1-Fc, competing with undefined scFv PD-L1(scFv) and scfvfcfpd-L1 (scfvffc) proteins of the supernatant, which were collected concentrated from CT26 cells maximally infected with RRV-scFv-PDL1 and RRV-scfvffc-PDL 1, respectively. An anti-PD-L1 antibody was included as a positive control. anti-6X His-tag antibody was used to detect bound His-tagged PD-1-Fc. The optical density was measured at 450 nm. Percent inhibition was calculated relative to the supernatant from CT26 maximally infected with RRV-GFP (non-scFv-PD-L1) used in the competition. Error bars indicate the standard deviation of the data set.
FIGS. 7A-B show scFv PD-L1 trans-binding activity to PD-L1 on the cell surface of bystander cells. IFN γ -treated EMT6 cells maximally infected with either RRV-scFv-HF PDL1 (HA-labeled scFv-PD-L1) or RRV-GFP were divided into 2 groups at the indicated ratios. (A) One group of cells was stained with Alexa Flu or 647-conjugated anti-HA antibody, and (B) a second group of cells was stained with PE-conjugated anti-mouse PD-L1 antibody. HA-positive, PD-L1-positive and GFP-positive cell populations were measured by flow cytometry analysis.
FIGS. 8A-D show pre-transduced tumor cells expressing scFv PD-L1 and scFv Fc PD-L1 that exhibited dose-dependent anti-tumor activity. (A) An in situ breast cancer model using a mixture of EMT6 tumor cells pre-transduced with RRV-scFv-PDL1 or RRV-scfvffc-PDL 1 and tumor cells pre-transduced with RRV-GFP at a predetermined ratio was implanted into the mammary fat pad of 8 week old BALB/c female mice (each group n-10). Survival was monitored for 90 days. anti-PD-1 antibody was included as a control and was administered intraperitoneally on days 10 (300. mu.g per mouse), 13, 16 and 19 (200. mu.g per mouse). For the0% scFv/scFv vFc relative to anti-PD-1, p ═ 0.2529; 0% versus 2%,. P — 0.2529; 0% versus 30%,. P ═ 0.0919; 0% vs 100%,. p. 0.1674. Crosses (Ticks) on the graph indicate mice that were examined and terminated due to tumor necrosis; these mice were not scored as dead and were not excluded from the figure. (B-D) use of 1X 10 on the flanks6An EMT6 tumor cell challenged mice with surviving initial tumor implants from the RRV-scFv-PDL1 and RRV-scfvffc-PDL 1 treated groups (n-5) and monitored tumor growth over time. Initial animals (n-5) were included as controls. Error bars represent SEM of the data set.
FIGS. 9A-B show data from an in situ glioma model, which was intracranially injected with RRV-scFv-PDL1, showing dose-dependent anti-tumor activity. (A) Female B6C3F1 mice (8 weeks old; n-10 per group) were implanted i.c. at 1 × 104Tu-2449 cells. Survival analysis was monitored for 90 days. On day 4 after tumor implantation, mice of the experimental group were injected with 1 × 105Or 1X 106Purified RRV-scFv-PDL1 of Transduction Units (TU). The control groups were mice carrying 100% pre-transduced scFv-PD-L1 expressing tumor cells and mice treated with anti-PD-1 antibody or isotype control. Tu-2449 cells pre-transduced with RRV-scFv-PDL 1100% expressing scFv PD-L1 and anti-PD-1 antibody (300. mu.g intraperitoneally induced per mouse on day 4; 200. mu.g maintenance dose per mouse on days 10, 14 and 17) were used as controls. Survival data were plotted using the Kaplan-Meier method. Statistical significance of survival between mice treated with isotype and mice pre-transduced with RRV-scFv-PD-L1100% or the injection-treated RRV-scFv-PDL1 group was determined by log rank (Mantel-Cox). (B) Mice surviving the initial tumor implantation from the RRV-scFv-PDL1 treated group were flanked on the right by 2X 106Each Tu-2449 cell was challenged. Tumor growth was monitored and measured over time. Error bars represent SEM of the data set.
FIG. 10 shows the detection of epitope-tagged Affimer-SQT protein from the supernatant of 293T cells transiently transfected with pAC3-gT2A-Affimer-SQT by direct immunoblotting and immunoprecipitation.
FIGS. 11A-B show detection of epitope-tagged Hck protein by direct immunoblotting and immunoprecipitation from supernatants of 293T cells transiently transfected with pAC3-gT2A-Hck shown in (A) and pAC3-IRES-Hck shown by arrows in (B).
FIG. 12 shows a schematic representation of RRV-scaffold plasmid DNA. The antigen binding domain [0066] derived from non-Ig scaffold is encoded in pAC3-2A, pAC3-IRES or pAC 3-mini promoter backbone. Solid grey rectangles represent 2A peptides, IRES or mini-promoters placed downstream of the env gene to direct transgene expression; the solid black rectangle represents the leader sequence (table a). The oligomerization domains (tables 4, 5 and 6) can be placed at the N-terminus or C-terminus of the non-Ig scaffold with a linker to form an oligomer. The last two lines also show configurations leading to the secretion of bispecific and trispecific binding molecules with properties for a ligation response against two or three targets, such as bispecific or trispecific antibodies (Labrijn et al, Nature Rev. drug Disc.18: 585-.
FIG. 13 shows a schematic representation of RRV-syCD2 plasmid DNA. The secreted form of yCD2 was encoded in pAC3-2A, pAC3-IRES, pAC 3-mini promoter backbone. Solid grey rectangles represent 2A peptides, IRES or mini-promoters placed downstream of the env gene to direct transgene expression; the solid black rectangle represents the Signal Peptide (SP) (Table A).
Detailed Description
As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the vector" includes reference to one or more vectors, and so forth.
Further, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "including" and "including" are interchangeable and not intended to be limiting.
It should also be understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some particular cases embodiments may alternatively be described using the language "consisting essentially of … …" or "consisting of … ….
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein.
General textbooks describing the molecular biology techniques useful herein (including the use of vectors, promoters and many other related subjects) include: berger and Kimmel, Guide to Molecular Cloning technologies, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); sambrook et al, Molecular Cloning- -A Laboratory Manual,2d ed., Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989 ("Sambrook"); current Protocols in Molecular Biology, f.m. Ausubel et al, eds., Current Protocols, a joint vector between green Publishing Associates, inc.and John Wiley & Sons, inc., (possessed by thread 1999) ("Ausubel"); and s.carson, h.b.miller & d.s.witherow and Molecular Biology Techniques: a classrom Laboratory Manual, Third Edition, Elsevier, San Diego (2012). Examples of protocols sufficient to instruct the skilled artisan to produce homologous nucleic acids of the present disclosure by in vitro amplification methods, for example, include Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), amplification by O β -replicase, and other RNA polymerase mediated techniques (e.g., NASBA), see Berger, Sambrook, and Ausubel, and Mullis et al (1987) U.S. patent nos. 4,683,202; innis et al, eds. (1990) PCR Protocols A guides to Methods and Applications (Academic Press Inc. san Diego, Calif.) ("Innis"); arnheim & Levinson (1990, 1/10) C & EN 36-47; the Journal Of NIH Research (1991)3: 81-94; kwoh et al (1989) Proc. Natl. Acad. Sci. USA 86: 1173; guatelli et al (1990) Proc.nat' l.Acad.Sci.USA 87: 1874; lomell et al (1989) j.clin.chem 35: 1826; landegren et al (1988) Science 241: 1077-1080; van Brunt (1990) Biotechnology 8: 291-294; wu and Wallace (1989) Gene 4: 560; barringer et al (1990) Gene 89: 117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564. An improved method for cloning in vitro amplified nucleic acids is described in U.S. Pat. No.5,426,039 to Wallace et al. Improved methods for amplifying nucleic acids by PCR are summarized in Cheng et al (1994) Nature 369:684-685 and references cited therein, in which PCR amplicons of up to 40kb are generated. One skilled in the art will appreciate that essentially any RNA can be converted to double stranded DNA suitable for restriction digestion, PCR amplification, and sequencing using reverse transcriptase and polymerase. See, e.g., Ausubel, Sambrook, and Berger, all supra.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
The terms "expression" and "expression" refer to allowing or causing information in a gene or DNA sequence to become apparent, for example by activating cellular functions involved in transcription and translation of the corresponding gene or DNA sequence to produce a protein, or in the case of the inhibitor Rna (RNAi), transcribing an RNAi molecule so that it is processed and is capable of inhibiting expression of a target gene.
The DNA sequence is expressed in or by a cell to form an "expression product," e.g., a polypeptide or protein. The expression product itself, e.g., the resulting polypeptide or protein, may also be referred to as being "expressed" by the cell. For example, a polynucleotide or polypeptide is recombinantly expressed or produced in an exogenous host cell under the control of an exogenous or native promoter, or expressed or produced in a native host cell under the control of an exogenous promoter.
As noted above, in some instances, the term "expression" includes the production of inhibitory RNA molecules (RNAi). Expression of such molecules does not involve the translation machinery of the cell, but rather utilizes mechanisms within the cell to modify gene expression of the host cell. In some embodiments, a recombinant viral vector of the present disclosure can be modified to deliver a coding sequence (e.g., a polypeptide or protein), an RNAi molecule, or both a coding sequence (e.g., expressing a polypeptide or protein) and an RNAi molecule to a host cell, which can then express the coding sequence and/or RNAi molecule.
"2A peptide or 2A peptide-like sequence" refers to a peptide having the sequence of SEQ ID NO:1, which sequence is 97% identical to any of the sequences in figures 1 and 2 and which comprises the sequence of SEQ ID NO: 1. A sequence "encoding" a2A peptide or 2A peptide-like sequence is a sequence encoding a polypeptide having, for example, SEQ ID NO:1, or a peptide-like sequence of 2A. In one embodiment, the coding sequence is operably linked to and placed between the ENV and the heterologous sequence such that once the sequence is transcribed, it is transcribed as a single transcript (e.g., multiple mrnas) and when the transcript is translated, two polypeptides (e.g., the ENV and the heterologous polypeptide) are produced.
An internal ribosome entry site ("IRES") refers to a nucleic acid fragment that facilitates ribosome entry or retention during translation of a coding sequence (typically 3' to the IRES). In some embodiments, the IRES may comprise a splice acceptor/donor site, however, it is preferred that the IRES lacks a splice acceptor/donor site. Typically, ribosomal entry into messenger RNA is via a cap located at the 5' end of all eukaryotic mrnas. However, there are exceptions to this general rule. The absence of a cap in some viral mrnas indicates that alternative structures exist that allow ribosomes to enter the internal site of these RNAs. To date, many of these structures have been identified in the 5' non-coding region of uncapped viral mrnas, designated IRES by their function, such as picornaviruses, in particular polioviruses (Pelletier et al, 1988, mol. cell. biol.,8, 1103-.
The term "promoter region" as used herein refers in a general sense to a nucleotide region comprising DNA regulatory sequences from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3' -direction) coding sequence. The regulatory sequences may be homologous or heterologous to the desired gene sequence. For example, a wide variety of promoters may be used, including viral or mammalian promoters.
The term "regulatory nucleic acid sequence" refers collectively to promoter sequences/regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present, so long as the selected coding sequence is capable of replication, transcription and translation in an appropriate host cell. Regulatory nucleic acid sequences can be readily identified from public databases and materials by those skilled in the art. Furthermore, one skilled in the art can identify regulatory sequences suitable for the intended use (e.g., in vivo, ex vivo, or in vitro).
As used herein, the term "RNA interference" (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering nucleic acids (siRNA or microrna (mirna)). The term "agent capable of mediating RNA interference" refers to siRNA as well as DNA and RNA vectors that encode siRNA when transcribed in a cell. The term siRNA or miRNA is intended to include any nucleic acid molecule capable of mediating sequence-specific RNA interference, such as short interfering RNA (siRNA), double stranded RNA (dsrna), microrna (miRNA), short hairpin RNA (shrna), short interfering oligonucleotides, short interfering nucleic acids, short interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsrna), and the like.
The term "secretory signal domain" or "secretory signal peptide" (SSP) or "signal peptide" refers to a short peptide usually located at the N-terminus as part of the precursor protein sequence. Translation mechanisms in eukaryotic cells utilize these short peptides to sort proteins to target destinations. The general features of SSPs consist of three domains: (1) n-region: positively charged domain, (2) H-region: hydrophobic core and (3) C-region: cleavage sites (Owji et al, Euro j.of Cell biol., 2018). The endoprotease SPase I cleaves SSP from its passenger proteins or polypeptides. The level of polypeptide or protein expression is not only related to translation efficiency, but also to translocation efficiency determined by the secretion machinery and SSP. The sequence of the SSP can affect translocation efficiency, and thus the combination of heterologous SSPs linked to passenger polypeptides or proteins can be engineered at the nucleic acid level to modulate the level of secreted proteins (Kober et al, 2013; Zamani et al, 2015; negahderipoiur et al, 2017; mouswavi et al, 2017). Furthermore, there are artificial SSPs designed to enhance protein secretion in prokaryotic and eukaryotic systems (Barash et al, Biochem and Biophy Res Comm, 2002; Cl rico et al, Biopolymers, 2008). Although the existence and general function of SSPs has been known for decades, it has not previously been described that SSPs have the ability to allow functional, non-naturally expressed gene products to be secreted from a host cell, particularly when combined with other membrane proteins such as the ENV protein and the 2A expression system of retroviral vectors.
The terms "vector", "vector construct" and "expression vector" refer to a vector through which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. Vectors usually comprise DNA or RNA into which foreign DNA encoding proteins, polypeptides, nucleic acids, etc., has been inserted by restriction enzyme techniques. A common type of vector is a "plasmid", which is generally a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and can be readily introduced into a suitable host cell. A number of vectors (including plasmids and fungal vectors) have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.) or pMAL plasmids (New England Biolabs, Beverly, mas.). A number of suitable host cells have been used for such transfection, using the methods disclosed or referenced herein or other methods known to those skilled in the relevant art. Recombinant cloning vectors typically include one or more replication systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes.
The present disclosure provides methods and compositions useful for delivering genes or proteins to cells or subjects. In one such embodiment, the methods and compositions allow the protein or polypeptide to be secreted from a cell that has taken up a gene encoding the protein or polypeptide. Such methods and compositions may be used to treat a variety of diseases and disorders in a subject, including cancer and other cell proliferative diseases and disorders. The present disclosure provides replication-competent viral vectors, and in one embodiment, the viral vectors are replication-competent retroviral vectors, for delivering genes to cells.
The present disclosure provides viral vectors containing heterologous polynucleotides encoding, for example, cytosine deaminase or a mutant thereof, miRNA or siRNA, a cytokine, an antigen binding domain (e.g., an antibody or antibody fragment; or a non-antibody binding domain), a non-immunoglobulin (Ig) scaffold protein, or a combination of coding sequences, etc., that can be delivered to a cell or a subject. The viral vector can be an adenoviral vector, a measles vector, a herpesvirus vector, a retroviral vector (including alpha-, beta-, gamma-, delta-retroviral vectors, foamy viral vectors such as Simian Foamy Virus (SFV) or Human Foamy Virus (HFV) or lentiviral vectors), a rhabdovirus vector such as vesicular stomatitis virus vector, a reovirus vector, a seneca valley virus vector, a poxvirus vector (including an zoopox or vaccinia derived vector), a parvovirus vector (including an AAV vector), an alphavirus vector, or other viral vectors known to those of skill in The art (see also, e.g., Concepts in Genet medical, ed. Borabulic and Carrier, Wiley,2008, Hoboken, NJ.; The Development of Human Therapy, ed. Nancy Smyth Templeton, Marcel Dekker inc, New York, 2000; and Gene&Cell Therapy:Therapeutic Mechan ism and Strategies,3rdEd., ed.nancy Smyth Templetone, CRC Press, Boc a Raton, FL, 2008; the disclosure of which is incorporated herein by reference).
As described below, the RRVs of the present disclosure may be derived from (i.e., the parent nucleotide sequence is obtained from) MLV, Mo MLV, GALV, FELV, etc., and engineered to contain a2A peptide or 2A-like peptide (sometimes referred to herein as a "2A-peptide cassette") operably linked to a heterologous nucleotide sequence. In some cases, the 2A peptide or 2A-like peptide is separated from the heterologous nucleotide sequence by an oligonucleotide encoding a secretion signal peptide.
Recombinant replication competent retroviral vector or Retroviral Replication Vector (RRV) refers to a vector based on a member of the family of retroviridae. The structure of the retrovirus is fully characterized as described more fully below. Retroviruses have been classified in various ways, but nomenclature has been standardized in The last decade (see The world Wide Web (www) ncbi. nlm. nih. gov/ICTTVdb/ICTTVb/ICTTB on ICTTB-The Universal Virus Database, v4, and The textbook "Retroviruses" eds. coffee, Hughs and Varmus, Cold Spring Harbor Press 1997, The disclosure of which is incorporated herein by reference). Such vectors may be engineered by recombinant genetic techniques to modify the parental virus into non-naturally occurring RRVs by insertion of heterologous genes or sequences. Such modifications may provide attributes to the vector that enable it to deliver the gene to be expressed to a host cell in vitro or in vivo.
Retroviruses are defined by the way they replicate their own genetic material. During replication, the viral RNA genome is converted to DNA (referred to as proviral DNA). After infection of a cell, a double-stranded DNA molecule is produced from two RNA molecules carried in a viral particle by a molecular process called reverse transcription. The DNA form is covalently integrated as a provirus in the host cell genome from which the viral RNA is expressed by means of cellular and/or viral factors. The expressed viral RNA is packaged into particles and released as infectious virions.
Retroviral particles consist of two identical RNA molecules. Each wild-type genome has a plus-sense single-stranded RNA molecule that is capped at the 5 'end and polyadenylated at the 3' tail end. Diploid viral particles contain two RNA strands complexed with gag protein, viral enzymes (pol gene product) and host tRNA molecules that are within the 'nuclear' structure of the gag protein. Surrounding and protecting the capsid is a lipid bilayer (lipid envelope) derived from the host cell membrane and containing viral envelope (env) proteins. The env protein binds to the cellular receptor of the virus and the particle enters the host cell, typically by receptor-mediated endocytosis and/or membrane fusion.
After release of the viral particles into the target cell, the outer envelope is sloughed off and the viral RNA is replicated into DNA by reverse transcription. This is catalyzed by the reverse transcriptase encoded by the pol region and uses the host cell tRNA packaged into the virion as a primer for DNA synthesis. In this way, the RNA genome is converted into a more complex DNA genome.
Double-stranded linear DNA produced by reverse transcription may or may not have to be circularized in the nucleus. The provirus now has two identical repeats at both ends, called Long Terminal Repeats (LTRs). The ends of the two LTR sequences create sites recognized by the pol product- -the integrase protein- -which catalyzes integration such that the provirus is always linked from the end of the LTR to the host DNA by two base pairs (bp). Replication of the cellular sequence is seen at the ends of both LTRs, suggesting a pattern of integration of transposable genetic elements. Retroviruses can integrate their DNA at many sites in the host DNA, but different retroviruses have different integration site preferences. HIV-1 and simian immunodeficiency virus DNA preferentially integrate into expressed genes, Murine Leukemia Virus (MLV) DNA preferentially integrate near the Transcription Start Site (TSS), and Avian Sarcoma Leukemia Virus (ASLV) and human T cell leukemia virus (HTLV) DNA integrate almost randomly, showing slight preference for genes (Derse D, et al. (2007), J Virol 81: 6731-6741; Lewinki MK, et al. (2006), PLoS Patholog 2: e 601).
Transcription, RNA splicing, and translation of the integrated viral DNA are mediated by host cell proteins. Various spliced transcripts are generated. In the case of human retroviruses, HIV-1/2 and HTLV-I/II viral proteins are also used to regulate gene expression. The interaction between cellular and viral factors is a factor in controlling the viral latency and the temporal sequence of viral gene expression.
Retroviruses can spread horizontally and vertically. Effective infectious transmission of retroviruses requires the expression of receptors on target cells that specifically recognize viral envelope proteins, although viruses can use receptor-independent, non-specific entry pathways with low efficiency. Typically, viral infection results in only a single or a few copies of the viral genome per cell because of receptor masking or down-regulation, which in turn results in resistance to superinfection (Ch 3p 104, JM coffee, SH Hughes, & HE Varmus,1997, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY; Fan et al.J.Virol 28:802,1978 in "retroviruses"). By manipulating the conditions in tissue culture, one can obtain a level of multiple infection, but this is typically less than 5 copies per diploid genome. In addition, the target cell type must be able to support all phases of the replication cycle after virus binding and penetration. Vertical transmission occurs when the viral genome is integrated into the germline of the host. The provirus will then pass from one generation to the other as if it were a cellular gene. Thus, normally latent endogenous proviruses are established, but can be activated when the host is exposed to appropriate agents.
The term "lentivirus" is used in its conventional sense to describe a genus of viruses that contain a reverse transcriptase. Lentiviruses include "immunodeficiency viruses," which include Human Immunodeficiency Virus (HIV) types 1 and 2 (HIV-1 and HIV-2) and Simian Immunodeficiency Virus (SIV).
Historically, oncogenic viruses have been further subdivided into A, B, C and group D based on particle morphology as seen under an electron microscope during virus maturation. Type a particles represent immature particles of type B and D viruses seen in the cytoplasm of infected cells. These particles are not infectious. Type B particles bud from the plasma membrane into mature virions through coating of the intracytoplasmic type a particles. On the membrane, they have a75 nm circular core from which long glycoprotein spikes protrude. After budding, type B particles contain eccentrically positioned electron dense nuclei. The prototype type B virus is Mouse Mammary Tumor Virus (MMTV). No intracytoplasmic particles were observed in cells infected with type C virus. Instead, mature granules bud directly from the cell surface by crescent 'C' -shaped coagulation, then close on themselves and are surrounded by the plasma membrane. The envelope glycoprotein spikes can be visible along with a homogeneous electron-dense core. Budding may occur from the surface plasma membrane or directly into the intracellular vacuole. Type C viruses are the most commonly studied and include many avian and Murine Leukemia Viruses (MLVs). Bovine Leukemia Virus (BLV) and human T-cell leukemia viruses type I and II (HTLV-I/II) are similarly classified as type C particles due to their morphology of budding from the cell surface. However, they also have a regular hexagonal morphology and a more complex genomic structure than prototype C-type viruses such as Murine Leukemia Virus (MLV). Type D particles are similar to type B particles in that they exhibit a cyclic structure in the infected cytoplasm, which buds off the cell surface, but the virions incorporate short surface glycoprotein spikes. The electron dense nuclei are also located eccentrically within the particle. The metson-pfeiry monkey virus (MPMV) is the prototype D-type virus.
In many cases where recombinant replication competent retroviruses are used therapeutically, it is advantageous to have a high level of expression of the transgene encoded by the recombinant replication competent retrovirus. For example, for prodrug activating genes such as cytosine deaminase genes, it is advantageous to have a higher level of CD protein expression in the cell so that the conversion of prodrug 5-FC to 5-FU is more efficient. Similarly, high level expression of siRNA or shRNA results in more effective inhibition of target gene expression. Also for cytokine or polypeptide binding domains (e.g., single chain antibodies (scAbs), etc.), it is often advantageous to express high levels of the cytokine or binding domain. Furthermore, in case there are mutations in some copies of the vector that inactivate or impair the activity of the vector or transgene, it is advantageous to have multiple copies of the vector in the target cell, as this provides a high possibility for efficient expression of the complete transgene.
As described above, the integrated DNA intermediate is referred to as a provirus. Methods and retroviruses used by existing gene therapy or gene delivery systems require proviral transcription and simultaneous assembly into infectious virus in the presence of a suitable helper virus or in a cell line containing suitable sequences that enable encapsidation without simultaneous production of contaminating helper virus. Similar approaches (complementary helper virus or cell lines) have been used to generate helper virus-free viral vector preparations, such as those from adenovirus, herpes virus, adeno-associated virus (AAV). As described below, the helper virus is not essential for the production of the recombinant retrovirus of the present invention, because the sequences for encapsidation are provided in the genome, thereby providing a replication-competent retroviral vector for gene delivery or therapy. Similarly, for other replication-competent viral vectors, such as those derived from adenovirus, herpes virus, rhabdovirus, measles, poliovirus, newcastle disease virus, alphavirus, vaccinia or other poxviruses, the viral vector is made by infecting normal host cells without the need for a specific engineered complementary cell line, and the resulting virus is harvested.
The retroviral genome and proviral DNA of the present disclosure have at least three genes: gag, pol and env, which may be flanked by one or two Long Terminal Repeats (LTR), or in proviruses, by two Long Terminal Repeats (LTR) and a sequence containing cis-acting sequences, such as psi. The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes RNA-guided DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes the viral envelope glycoprotein. The 5 'and/or 3' LTRs are used to promote transcription and polyadenylation of virion RNA. The LTR contains all other cis-acting sequences required for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and/or SIV). Those skilled in the art will recognize that a retroviral genome is an RNA genome, and thus reference to any retroviral genome sequence implicitly refers to a sequence in which "T" is "U". Thus, when referring to a retroviral genome, reference to a gag nucleic acid sequence having a particular sequence containing a T implicitly means that T is replaced by U.
Adjacent to the 5' LTR are the sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of the viral RNA into particles (psi site). If the sequence required for encapsidation (or packaging of retroviral RNA into infectious virions) is deleted from the viral genome, the result is a cis-defect that prevents encapsidation of genomic viral RNA. Modified vectors of this type are vectors that are commonly used in existing gene delivery systems (i.e., systems lacking the elements required for virion encapsidation) as "helper" elements, providing for packaging of the trans viral proteins of a non-replicating but packagable RNA genome.
The present disclosure provides modified retroviral vectors. The modified retroviral vector may be derived from a member of the family Retroviridae and engineered to contain an ENV-2A-SSP-transgene cassette. As mentioned above, the family retroviridae consists of three groups: foamy virus (or foamy virus) such as Human Foamy Virus (HFV); lentiviruses and ovine visna virus; and oncogenic viruses (although not all viruses in the group are oncogenic).
In one embodiment, the viral vector may be a replication competent retroviral vector capable of infecting only dividing mammalian cells. In one embodiment, the replication competent retroviral vector comprises a2A peptide or 2A peptide-like sequence located just downstream of and operably linked to the retroviral envelope and just upstream of the coding sequence for a Secretion Signal Peptide (SSP) which in turn is linked to the heterologous nucleic acid sequence to be expressed. In certain embodiments, the vector may additionally include an IRES cassette or a polII (or mini-promoter) or polIII cassette. The heterologous polynucleotide can encode, for example, a cytosine deaminase, a nitroreductase, a cytokine, a receptor, an antibody fragment, a binding domain (e.g., a non-antibody binding domain or a non-Ig polypeptide), and the like. When a polIII promoter is included, the vector may further express miRNA, siRNA or other RNAi sequences.
In another embodiment, the present disclosure provides an ENV-2A-SSP-heterologous gene cassette. The cassette may comprise an envelope selected from one of an amphotropic, pantropic, heterophilic, 10a1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, and influenza virus envelope. The 2A peptide or 2A peptide-like coding sequence may be any of the sequences shown in fig. 1 or 2, operably linked to the C-terminus of the envelope coding sequence. In another embodiment, the 2A peptide or 2A peptide-like coding sequence is linked by a GSG linker sequence, such as ggaagcgga (SEQ ID NO: 3). In another embodiment, the GSG-2A peptide or peptide-like coding sequence is linked to an SSP coding sequence. The heterologous gene is operably linked to the C-terminus of the SSP coding sequence. The heterologous gene can be any desired gene to be delivered to and expressed in the target cell. In one embodiment, the heterologous gene comprises 500 and 1500bp long or any value therebetween (e.g., 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, etc.). In another embodiment, the heterologous gene comprises > 1500bp long. In another embodiment, the cassette comprises two heterologous genes separated by a2A peptide or 2A peptide-like coding sequence upstream of the SSP peptide coding sequence. In yet another embodiment, the cassette may comprise a polynucleotide encoding a2A peptide or 2A peptide-like sequence operably linked between the C-terminus of ENV and the N-terminus of an SSP sequence linked to the N-terminus of a heterologous gene, wherein the heterologous gene is followed by a second cassette comprising an IRES or promoter linked to a second heterologous sequence.
The heterologous nucleic acid sequence is operably linked to a sequence encoding an SSP peptide operably linked to and downstream of a2A peptide or 2A peptide-like sequence. As used herein, the term "heterologous" nucleic acid sequence or transgene refers to (i) a sequence that is not normally found in a wild-type retrovirus, (ii) a sequence derived from a foreign species, or (iii) if from the same species, it may be substantially modified from its original form. Alternatively, the unaltered nucleic acid sequence which is not normally expressed in the cell is a heterologous nucleic acid sequence.
Any number of heterologous polynucleotides or nucleic acid sequences can be inserted into a retroviral vector, depending on the intended use of the retroviral vector of the present disclosure. For example, for in vitro studies, commonly used marker or reporter genes can be used, including antibiotic resistance and fluorescent molecules (e.g., GFP) or luminescent molecules. Additional polynucleotide sequences encoding any desired polypeptide sequence may also be inserted into the vectors of the present disclosure.
When in vivo delivery of heterologous nucleic acid sequences is sought, therapeutic and non-therapeutic sequences may be used. The RRV of the present disclosure may comprise at least one cassette containing an SSP domain. Typically the SSP domain is upstream of the particular polypeptide or protein to be secreted from the RRV-infected cell. In one embodiment, the biological effect of the SSP may be determined by measuring the amount of secreted polypeptide to which the SSP is attached when translated, as compared to the same polypeptide lacking the SSP.
In some embodiments, the-2A-SSP-transgene cassette may be followed by a mini-promoter cassette, a poliII-RNAi cassette, or an IRES cassette. For example, when a mini-promoter or polIII cassette is used, the cassette may comprise heterologous sequences, including mirnas, sirnas, etc., directed to a particular gene associated with a cell proliferative disorder or other gene-related disease or disorder. In other embodiments, the SSP peptide coding sequence or a heterologous gene downstream of the IRES can be a suicide gene (e.g., HSV-tk or PNP or a polypeptide having cytosine deaminase activity; modified or unmodified), a growth factor, or a therapeutic protein (e.g., factor IX, IL2, etc.). Other therapeutic proteins suitable for use in the present disclosure are readily identified in the art. In certain embodiments, where the heterologous gene encodes a protein or polypeptide to be secreted, the heterologous sequence is preceded by a coding sequence for an SSP peptide. For example, where the antibody, antibody fragment, or binding domain is encoded by a heterologous gene, the therapeutic cassette comprises a 2A-peptide or peptide-like coding sequence, followed by an SSP coding sequence, followed by a heterologous polynucleotide sequence encoding a polypeptide or peptide (e.g., an antibody, antibody fragment, or binding domain) to be secreted. In certain embodiments, the polypeptide to be secreted is not a thymidine kinase. In some embodiments, the RRV may comprise two cassettes, one cassette comprising the polypeptide to be secreted and prior to the SSP domain, the second cassette comprising a polypeptide or portion that is not secreted. For example, such a dual cassette may comprise:
-2A-SSP- (polypeptide to be secreted) - (2A or IRES or mini-promoter or poliII) - (polypeptide or miRNA) -.
In one embodiment, the heterologous polynucleotide within the vector comprises a cytosine deaminase or thymidine kinase that has been optimized for expression in human cells. In further embodiments, the cytosine deaminase comprises a sequence that has been optimized for a human codon and comprises a mutation that increases the stability (e.g., reduced degradation or increased thermostability) of the cytosine deaminase and/or a mutation that changes a tryptophan codon to a non-tryptophan-encoding codon as compared to a wild-type cytosine deaminase. In another embodiment, the heterologous polynucleotide encodes a fusion construct comprising a polypeptide having cytosine deaminase activity (human codon optimized or non-optimized, mutated or non-mutated) operably linked to a polynucleotide encoding a polypeptide having UPRT or OPRT activity.
Antibodies (and fragments thereof) are an important class of therapeutic agents. Their specific binding and functional properties determine their mode of action. Most FDA-approved antibodies are antagonists and have high binding affinity for their target. Alternatively, non-immunoglobulin (non-Ig) scaffold proteins derived from native endogenous proteins have begun to be developed in place of antibodies. The advantage of using non-Ig proteins is that they can achieve high binding affinity, and they are relatively small compared to antibodies and therefore can penetrate tissue more efficiently. They may also be engineered to be multivalent and/or multi-target specific.
The present disclosure describes the use of natural or artificial signal peptides in RRVs having a GSG-linked 2A peptide configuration for the production of secreted proteins or polypeptides, including but not limited to prodrug activating genes, cytokines or receptor ligands or their analogs, immunoglobulins (igs) and non-Ig derived proteins. The disclosure also describes other RRV configurations, such as those with IRES or small/mini promoters, for expression of heterologous transgenes with heterologous secretion signal peptides.
Typically, the recombinant replication-competent viral vectors of the present disclosure are modified to include a "cassette" which typically contains a heterologous gene or sequence to be delivered and expressed in a host cell. The heterologous gene or sequence is operably linked to elements that allow for efficient expression (e.g., a promoter, an IRES, or a read-through element that allows for transcription and translation of the heterologous sequence).
The transgene (e.g., a heterologous sequence to be expressed) may be inserted into the retroviral genome at a number of positions, including insertion of a Long Terminal Repeat (LTR), insertion downstream of the envelope and after splicing acceptor, fusion with viral gag or pol proteins, internal IRES sequences or a small internal promoter downstream of the envelope coding sequence. Insertion of the transgene into the LTR and introduction of additional splice acceptors leads to rapid destabilization of the vector genome, while IRES and other approaches have shown greater promise. Expression and constitution of transgenes can be at least partially affected by judicious changes in key sequences, such as elimination of cryptic splice acceptors and humanization of the transgene sequences (see, e.g., U.S. patent No.8,722,867, the disclosure of which is incorporated herein by reference). The size of the transgene may also have an effect on the stability of the vector. For example, in some vectors, as the size of the transgene increases, the virus becomes unstable and rapidly deletes at least a portion of the heterologous gene or sequence. In some cases, this limitation is exacerbated by the need to include expression-enabling sequences such as IRES (typically about 600bp, see, e.g., U.S. Pat. No.8,722,867) or small promoters (typically about 250-300bp, see, e.g., International application publication No. WO2014/066700, which is incorporated herein by reference), possibly leaving only 900 to 1200bp of heterologous gene or sequence insert in, e.g., MLV. Therefore, it would be very useful to be able to maximize the size of the transgene available to include more choices of the transgene or transgenes.
Some examples of retroviruses that replicate efficiently in human cells include the amphotropic, pantropic, heterotropic and 10a1 strains of Murine Leukemia Virus (MLV), as well as Gibbon Ape Leukemia Virus (GALV), baboon endogenous virus and feline virus RD 114. Similarly, ecotropic strains of MLV that have been modified to contain non-ecotropic envelope genes (e.g., amphotropic pseudotyped RRV) can also replicate efficiently in a variety of species and cell types to be treated. However, the retroviral envelope may also be replaced by a non-retroviral envelope (such as a rhabdovirus, alphavirus, measles or influenza virus envelope).
Various viruses, including picornaviruses and encephalomyocarditis viruses, encode 2A or 2A-like peptides in their genomes to mediate multi-protein expression from a single Open Reading Frame (ORF). The 2A peptides are typically about 16-18 amino acids in sequence and share the motif: d [ V/I ] EXNPGP (SEQ ID NO:1), wherein X is any amino acid. When the 2A peptide is encoded between ORFs in an artificial polycistronic mRNA, it causes the ribosome to terminate at the C-terminus of the 2A peptide in the translated polypeptide, resulting in the isolation of polypeptides derived from each ORF (Doronina et al, 2008). The point of separation is at the C-terminus of 2A, and the first amino acid of the downstream ORF is proline (see, e.g., FIG. 1). The unique characteristics of the 2A peptide have led to its use as a molecular tool for expressing polyproteins from a single polycistronic mRNA configuration.
The 2A peptide is present in the viral genome of viruses of the picornaviridae family (e.g., foot and mouth disease virus and equine rhinitis A virus) and other viruses (e.g., porcine Tespidae virus-1 and insect virus, Gloenopsis lanceolata beta-tetrad virus) (FIG. 1). 2A peptides have nearly 100% efficiency of "separation" in their natural environment and generally have lower efficiency of "separation" when they are introduced into non-native sequences. Other 2A-like sequences found in different classes of viruses have also been shown to achieve-85% efficiency of "separation" in non-native sequences (Donnelly et al, 1997). There are a number of 2A-like sequences (fig. 2) that can be used in the methods and compositions of the present disclosure to express transgenes.
Although the 2A sequences have been known to exist for about 20 years, their ability to function in non-native settings has been questioned. In particular, the 2A sequence leaves approximately 17-22 additional amino acids at the C-terminus of the previously translated protein, and adds proline to the N-terminus of the downstream protein, thus potentially affecting the ability of the previous protein to function. If the protein requires post-translational modifications in the endoplasmic and golgi apparatus and/or during virion maturation, as is the case with many viral envelope proteins (t. murakami, Mol Biol int, 2012), there is a further risk of prior loss of protein function.
In general, processing of the native MLV envelope protein involves cleavage of the precursor protein Pr85 into gp70(SU) and p15E (TM) subunits, which occur in infected host cells. During budding from the host cell, Pr85 cleavage is required for efficient incorporation of the viral envelope proteins into the virus particle. As the virion buds from the host cell membrane, the virion undergoes a maturation process to become infectious. One of the processes of MLV virion maturation involves the removal of the R-peptide located at the C-terminus of the TM subunit of the envelope protein by viral proteases. The 2A peptide, except for the last amino acid residue proline (Pro), is expressed downstream of the R-peptide such that the length of the R-peptide is from 16 amino acids to at least 32 amino acids, depending on the sequence of the 2A peptide. Although the length of the R-peptide is extended by the addition of the 2A peptide sequence, theoretically the 2A peptide will be removed simultaneously with the cleavage of the R peptide, resulting in a functional envelope protein.
If the envelope sequence is non-functional or attenuated, the viral vector may be useless. Attempts have been made to use specific 2A sequences (from porcine Tescovirus-1, "P2A") (S.Stavrou et al, PLoS Patholog 10(5): e1004145,2014; and E.P.Brown, J.Virol.89: 155-. However, these viruses do not infect human cells, and it is not expected that the general protein processing problem has been solved. In addition, the viruses thus constructed are designed to express genes that promote replication in vivo, rather than genes that are indicative of achieving therapeutic effects.
In some cases, it is desirable to secrete proteins or polypeptides from the infected cell that are delivered to the host cell by the recombinant retroviral vector. That is, an RRV carrying a cassette containing a heterologous polynucleotide encoding a polypeptide or protein is engineered for secretion from an infected target cell, wherein the proviral DNA of the resulting RRV is incorporated into the genome of the target cell. As described above, a secretion signal peptide can be engineered upstream of a polypeptide or protein in order to cause secretion of the polypeptide or protein from a cell. In this case, the secretion signal peptide coding sequence is engineered to be located between the 2A-or 2A-like peptide and the polypeptide or protein to be secreted. Thus, the cassette in such RRV may be located between the env coding sequence and the 3' LTR having the following general structure: - - (env) - - (2A) - (SSP) - (polypeptide or protein) - - (LTR). As can be seen from the above general structure, the cassette can be regarded as modular and various 2A or 2A-like sequences, SSP sequences and polypeptide or protein sequences can be altered/shuffled.
Monoclonal antibodies remain the mainstay of human therapy in diagnosis and cancer therapy. They have a long serum half-life, bivalent properties and immune effector functions. Although their partially or fully human nature minimizes immunogenicity, monoclonal antibodies are complex proteins with multiple domains that require appropriate disulfide bond formation and glycosylation processes, and therefore their production is limited to eukaryotic cells with limited scalability. Another important potential limitation of monoclonal antibodies is that it is believed that intact antibodies of 150KDa size may also have limited tissue penetration and intracellular accessibility. Some of these limitations have been overcome by the development of fragmented antibodies, such as single chain variable fragments (scFv) or Fab. Further development has also utilized camelid and cartilaginous fish binding proteins that comprise heavy chain only isoforms that lack a light chain.
Non-immunoglobulin (Ig) scaffold proteins for biological therapy have been developed that use a randomization strategy to identify antigen binding sequences (U.H Wiedele et al, Cancer Genomics & Proteomics 10: 155-. non-Ig scaffold proteins are domain-derived subunits of natural proteins from humans and other species, or are artificial, and they range in size from 6-20kDa and can be expressed from a single polypeptide. They have surface exposed loops or amino acids in the alpha-helical or beta-sheet framework that can tolerate insertions, deletions and substitutions that produce antigen binding scaffold proteins that can function as antagonists or agonists through randomization, phage display screening and affinity maturation processes. To date, over 50 different classes of non-Ig scaffold proteins have been identified and developed for therapy as scaffold binding agents. Due to their size, one of the major challenges facing these proteins is rapid renal clearance, resulting in a short circulatory half-life. One common solution to increase the half-life of these non-Ig scaffold proteins involves the use of fusion proteins containing a scaffold protein linked to the Fc region of IgG. Another challenge is that they generally have lower binding affinities (KD 1-100nM) than monoclonal antibodies and are associated with fast off rates. Genetic modification of these scaffold proteins to include multimerization domains may increase steric hindrance-mediated blockade or affinity, which may lead to biological function and therapeutic effects in certain signaling pathways. Various methods have been proposed and tested, at least in part, using fusion proteins containing a scaffold protein linked to the Fc region of IgG, or containing two repeat units of a scaffold protein linked by a linker to produce a dimer. In addition to linking peptides to the Fc region of IgG, dimer-, trimer-, and pentamer-multimerization domains have been utilized to express the extracellular domain of a desired protein in an oligomeric state, or to enhance protein-protein interactions.
The present disclosure provides compositions and methods using binding domains comprising a combination of heavy and/or light chain CDRs connected by a scaffold domain (e.g., an Adhiron scaffold; scaffolds from human Stefin a-see EP22792058B1 and WO2019/008335, the disclosures of which are incorporated herein by reference). In some embodiments, the coding sequence for the binding domain is operably linked downstream of the 2A or 2A-like peptide coding sequence. In another embodiment, the coding sequence for the binding domain is operably linked to the coding sequence for the secretion signal peptide. In yet another embodiment, the coding sequence for the binding domain is operably linked downstream of a2A or 2A-like peptide coding sequence, which in turn is linked to a secretion signal peptide coding sequence, such that the nucleic acid cassette has the following general structure: -2A-SSP-binding domain- -. In other embodiments, the disclosure provides compositions of an Fc region of IgG, portions of Fc regions of IgA and IgM, glycine-serine linkers, and multimerization domains and their use for forming oligomeric antigen binding scaffold proteins. Any of the foregoing may be used in combination with RRV with sequence optimization to minimize Apobec 3-mediated hypermutation, thereby enhancing protein stability and/or avidity and expression for potentially better biological function and therapeutic effect. The present disclosure also provides expression vectors and methods of use, particularly viral vectors with high tumor-targeting specificity, to deliver therapeutic payloads in tumor microenvironments to counteract rapid clearance of these antigen-binding non-Ig scaffold proteins in the circulation and minimize off-target effects and toxicity when administered intravenously. Tables 1,2, 3, 4, and 5 provide sequences useful in the compositions and methods of the present disclosure. Note that since the present disclosure contemplates RNA, in the following nucleic acid sequences, "T" may be "U".
Table 1: amino acid sequences of some non-Ig scaffold proteins that function as antigen binding proteins
Table 2: nucleic acid sequences of non-Ig scaffold proteins that function as antigen binding proteins
Table 3: amino acid and nucleic acid sequences for glycine-serine linkers
Table 4: amino acid sequences of human IgG Fc and IgM C μ 4tp
Table 5: nucleic acid sequences of human IgG Fc and IgM C μ 4tp
Table 6: amino acid sequence of multimerization domain
Table 7: nucleic acid sequences of multimerization domains
RRVs of the present disclosure may be engineered to alter their stability and/or expression. For example, the change in expression may be due to the frequency of accumulation of inactivating or attenuating mutations as the replicating retroviral vector gradually replicates in tumor tissue. Studies have shown that one of the most frequent events is a G to a mutation (corresponding to a C to T characteristic ApoBec-mediated mutation in the negative strand single stranded DNA from the first replication step in the reverse transcription step). This can cause changes in the amino acid composition of the RRV protein and disruptive changes from TGG (tryptophan) to the stop codon (TAG or TGA). In one embodiment, such inactivating changes are avoided by engineering of codons for other amino acids with similar chemical or structural properties (e.g., phenylalanine or tyrosine) instead of tryptophan codons.
Thus, in addition to the 2A-peptide-SSP cassette, the RRV may include a number of additional mutations that improve expression and/or stability of the construct in the host cell. Such mutations may include modification of one or more codons in the GAG, POL, and/or ENV coding sequence that changes the tryptophan codon to an allowable codon that can maintain the biological activity of the GAG, POL, and/or ENV domains. The codon for tryptophan is known in the art as UGG (TGG in DNA). Furthermore, it is known in the art that a "stop codon" is UAA, UAG or UGA (TAA, TAG or TGA in DNA). A single point mutation in a tryptophan codon may result in a non-natural stop codon (e.g., UGG- > UAG or UGG- > UGA). Human APOB EC3GF (hA3G/F) is also known to inhibit retroviral replication by G- > a hypermutation (Neogi et al, j. int. aids soc.,16(1):18472,2013, 2/25). Furthermore, as described below, long term expression and viral stability can be improved by avoiding the use of tryptophan codons in the coding sequence, thereby avoiding the incorporation of unnatural stop codons due to hypermutation by hA 3G/F. For example, in one embodiment, the MLV-derived nucleic acid sequence comprises GAG, POL, and ENV coding regions, which may comprise codon modifications (with reference to nucleotide numbering of SEQ ID NO: 2) comprising the nucleotides identified in Table A, located in the tryptophan codon, may provide for hA3G/F resistant RRV.
Table a summary of recurrent G to a mutations that result in a tryptophan to stop codon change. The nucleotide is SEQ ID NO: 2RRV genome, a "gene" is the gene in which the nucleotide is located, and AA is the amino acid position in the polypeptide.
Nucleotide, its preparation and use
Gene
AA
1306
GAG
35
5299
POL
718
5557
POL
804
5806
POL
887
6193
POL
1016
6232
POL
1029
6298
POL
1051
6801
ENV
148
6978
ENV
207
7578
ENV
407
Thus, in one embodiment of the present disclosure, a recombinant replication competent retrovirus is provided that comprises one or more mutations in a tryptophan codon, wherein the mutation changes the codon to a codon for an amino acid other than tryptophan, and which provides a codon that is biocompatible (i.e., a codon that does not disrupt vector function). Such vectors are sometimes referred to herein as "ApoBec inactivation resistance vectors" or "ApoBec resistance vectors". The recombinant ApoBec inactivation resistance vector may comprise an IRES cassette, a promoter cassette and/or a2A peptide-SSP cassette.
As described above, human APOBEC3G caused a hypermutation in the viral vector sequence that transformed G- > a (Ho gan et al, can. Thus, the tryptophan codon in the heterologous polynucleotide contained in the 2A-SSP peptide cassette is readily converted to a stop codon by hAPOBEC 3. To avoid such mutations, the tryptophan codon may be replaced with a biologically allowable codon for another amino acid. For example, in one embodiment, a 2A-SSP cassette of the disclosure can comprise a polynucleotide encoding a polypeptide having cytosine deaminase activity, wherein the polynucleotide comprises the sequence:
(or the foregoing wherein "t" is "u").
This sequence contains two tryptophan codons (bold/underlined). In one embodiment of the disclosure, the codons are independently changed to provide codons for an amino acid selected from the group consisting of D, M, T, E, S, Q, N, F, Y, A, K, H, P, R, V, L, G, I and C. The resulting polypeptide comprises the sequence:
wherein the polypeptide comprises cytosine deaminase activity, wherein X is any amino acid except tryptophan. In one embodiment, SEQ ID NO: each X in 29 is independently selected from F, D, M, L, S or R.
In another embodiment, a replication-competent retroviral vector may comprise a heterologous polynucleotide encoding a polypeptide comprising a cytosine deaminase (as described herein), and may further comprise a polynucleotide comprising a miRNA or siRNA molecule as part of a primary transcript from a viral promoter or linked to a promoter, which may be cell-type or tissue-specific. In yet another embodiment, the miRNA or siRNA may be preceded by a pol III promoter.
Micrornas (mirnas) are small non-coding RNAs. They are located in introns of coding or non-coding genes, exons or intergenic regions of non-coding genes. The miRNA coding sequence is transcribed by RNA polymerase III, which produces a precursor polynucleotide known as a primary precursor miRNA (pri-miRNA). Pri-mirnas in the nucleus are processed by the ribonuclease Drosha to generate miRNA precursors (pre-mirnas) that form short hairpin structures. Subsequently, the pre-miRNA is transported to the cytoplasm through exporter 5 and further processed by another ribonuclease called Dicer to produce active mature miRNA. The siRNA sequence is not preceded by an SSP coding sequence, but rather the siRNA is part of a second cassette present in a therapeutic cassette in a viral vector.
The length of the mature miRNA is about 21 nucleotides. It functions by binding to the 3' untranslated region of the target gene mRNA and inhibiting protein expression by inhibiting protein translation or mRNA degradation. mirnas are involved in biological processes including development, cell proliferation, differentiation, and cancer progression. miRNA profiling studies have shown that some miRNA expression is tissue specific or enriched in certain tissues. For example, miR-142-3p, miR-181 and miR-223 expression has been shown to be enriched in hematopoietic tissues of humans and mice (Baskerville et al, 2005 RNA 11, 241-flaked 247; Chen et al, 2004Science 303, 83-86).
Some mirnas have been observed to be up-regulated (oncogenic mirnas) or down-regulated (repressors) in a variety of tumors (Spizzo et al, 2009Cell 137,586e 1). For example, miR-21 is overexpressed in glioblastoma, breast, lung, prostate, colon, stomach, esophageal and cervical cancers, uterine leiomyosarcoma, DLBCL, head and neck cancers. In contrast, members of let-7 have been reported to be down-regulated in glioblastoma, lung, breast, gastric, ovarian, prostate, and colon cancers. The remodeling of the homeostasis of miRNA expression in cancer is an essential mechanism to inhibit or reverse cancer progression.
Mirnas that are down-regulated in cancer can be used as anti-cancer agents. Examples include miR-128-1, let-7, miR-26, miR-124 and miR-137 (Equla-Kerscher et al, 2008Cell Cycle 7, 759-764; Kumar et al, 2008Proc Natl Acad Sci USA 105, 3903-3908; Kota et al, 2009Cell 137, 1005-1017; Silber et al, 2008BMC Medicine 6: 141-17). miR-128 expression has been reported to be enriched in the central nervous system and has been observed to be down-regulated in glioblastoma (Sempere et al, 2004Genome Biology 5: R13.5-11; Godlewski et al, 2008Cancer Res 68 (22) 9125-. miR-128 is encoded by two distinct genes, miR-128-1 and miR-128-2. Both are processed into the same mature sequence. Bmi-1 and E2F3a have been reported as direct targets for miR-128 (Godlewski et al, 2008Cancer Res 68 (22) 9125-. In addition, Bmi-1 expression has been observed to be up-regulated in a variety of human cancers, including glioma, mantle cell lymphoma, non-small cell lung cancer, B-cell non-hodgkin lymphoma, breast cancer, colorectal cancer, and prostate cancer. In addition, Bmi-1 has been shown to be necessary for self-renewal of stem cells from different tissues, including neuronal stem cells as well as "stem cell-like" cell populations in gliomas.
Suitable ranges for designing stem lengths for hairpin duplexes include stem lengths of 20-30 nucleotides, 30-50 nucleotides, 50-100 nucleotides, 100-150 nucleotides, 150-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, and 600-700 nucleotides. Suitable ranges for designing the loop length of the hairpin duplex include loop lengths of 4-25 nucleotides, 25-50 nucleotides, or longer if the stem length of the hairpin duplex is substantial. In certain instances, hairpin structures having a duplex region longer than 21 nucleotides can promote effective siRNA-directed silencing regardless of loop sequence and length.
In yet another or further embodiment, the heterologous polynucleotide may comprise a cytokine, such as an interleukin, interferon gamma, or the like. Cytokines that can be expressed from the retroviral vectors of the present disclosure include, but are not limited to, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, 1L-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-13, IL-16, IL-17, IL-6, IL-19, IL-6, IL-7, IL-11, and IL-11, IL-38, anti-CD 40, CD40L, IFN- γ and TNF- α, soluble forms of TNF- α, lymphotoxin- α (LT- α, also known as TNF- β), LT- β (see, Complex heterotrimer LT- α 2- β), OPGL, FasL, CD27L, CD30L, 4-1BBL, DcR3, OX40L, TNF- γ (International publication No. WO 96/14328), AIM-I (International publication No. WO 97/33899), endocrine factor- α (International publication No. WO 98/07880), OPG and neutrokine- α (International publication No. WO 98/18921), OX40 and Nerve Growth Factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-International IBB, TR2 (WO 96/34095), DR3 (publication No. WO 97/33904), International publication No. 36 4 (WO 98/32856), TR5 (International publication No. WO98/30693), TRANK, TR9 (International publication No. WO 98/56892), TR10 (International publication No. WO 98/54202), 312C2 (International publication No. WO 98/06842), and TR12, as well as soluble forms of CD154, CD70, and CD 153. In some embodiments, angiogenic proteins may be used to produce proteins, particularly from cell lines. Such angiogenic factors include, but are not limited to, glioma-derived growth factor (GDGF), platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-B (PDGF-B), placental growth factor (PIGF), placental growth factor-2 (PIGF-2), Vascular Endothelial Growth Factor (VEGF), vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-2 (VEGF-2), vascular endothelial growth factor B (VEGF-3), vascular endothelial growth factor B-186 (VEGF-B186), vascular endothelial growth factor-D (VEGF-D), and vascular endothelial growth factor-E (VEGF-E). Fibroblast growth factors may be delivered by the vectors of the present disclosure and include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15. Hematopoietic growth factors can be delivered using the vectors of the present disclosure, such growth factors including, but not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargrastim), granulocyte colony stimulating factor (G-CSF) (filgrastim), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321(GMCSF/IL-3) fusion protein, and the like.
The heterologous nucleic acid sequence is typically under the control of viral LTR promoter-enhancer elements. Thus, the recombinant retroviral vectors, desired sequences, genes, and/or gene fragments of the present disclosure can be inserted at multiple sites and under different regulatory sequences. For example, the site for insertion may be a viral enhancer/promoter proximal site (i.e., the 5' LTR-driven locus).
In one embodiment, the retroviral genome of the present disclosure contains a2A peptide or 2A peptide-like coding sequence upstream of the SSP coding sequence, wherein the SSP coding sequence is followed by a downstream cloning site for insertion of the desired/heterologous polynucleotide. In one embodiment, the 2A peptide or 2A peptide-like coding sequence is located 3 'to the env gene in the retroviral vector, but 5' to the SSP coding sequence and the desired heterologous polynucleotide. Thus, a heterologous polynucleotide encoding a desired polypeptide is operably linked to a2A peptide or 2A peptide-like-SSP coding sequence.
In another embodiment, the targeting polynucleotide sequence is included as part of a recombinant retroviral vector of the present disclosure. The targeting polynucleotide sequence is a targeting ligand (e.g., a peptide hormone such as a heregulin, a single chain antibody, a receptor, or a ligand for a receptor), a tissue-specific or cell-type specific regulatory element (e.g., a tissue-specific or cell-type specific promoter or enhancer), or a combination of a targeting ligand and a tissue-specific/cell-type specific regulatory element. Preferably, the targeting ligand is operably linked to or present in the env protein of the retrovirus, resulting in a chimeric retroviral env protein. The viral GAG, viral POL and viral ENV proteins may be derived from any suitable retrovirus (e.g. MLV or lentivirus derived). In another embodiment, the viral ENV protein is of non-retroviral origin (e.g., CMV or VSV).
In one embodiment, the recombinant retroviruses of the present disclosure are genetically modified in a manner that allows the virus to be targeted to a particular cell type (e.g., smooth muscle cells, liver cells, kidney cells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrow cells, chondrocytes, epithelial cells, intestinal cells, breast cells, neoplastic cells, glioma cells, neuronal cells, and other cells known in the art) such that the recombinant genome of the retroviral vector is delivered to a target non-dividing cell, a target dividing cell, or a target cell with a cell proliferative disorder.
In one embodiment, the disclosure provides a recombinant retrovirus capable of infecting a non-dividing cell, a dividing cell, or a cell with a cell proliferative disorder. The recombinant replication competent retrovirus of the present disclosure comprises a polynucleotide sequence encoding a viral GAG, viral POL, viral ENV, 2A peptide, or 2A peptide-like coding sequence immediately downstream (e.g., between 1 to 50 nucleotides downstream, e.g., between 1-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, or any integer therebetween) of a viral ENV sequence and an SSP coding sequence operably linked to a heterologous gene and encapsulated within the virus.
The phrase "non-dividing" cells refers to cells that do not undergo mitosis. Non-dividing cells may be at any point in the cell cycle (e.g., G)0/G1、G1/S、G2/M) Blocked as long as the cell is not actively dividing. For ex vivo infection, dividing cells can be treated to block cell division by standard techniques used by those skilled in the art, including irradiation, aphidicolin treatment, serum starvation and contact inhibition. However, it will be appreciated that since many cells have already been arrested (e.g. terminally differentiated cells), ex vivo infection is typically performed without blocking the cells. For example, recombinant lentiviral vectors are capable of infecting non-dividing cells. Examples of pre-existing non-dividing cells in the body include neuronal, muscle, liver, skin, heart, lung and bone marrow cells, and derivatives thereof. For dividing cells, gamma retroviral vectors can be used, since this type of retrovirus only infects dividing cells productively, and this property contributes to the tumor selectivity of this vector class.
"dividing" cells refers to cells that undergo active mitosis or meiosis. Such dividing cells include stem cells, skin cells (e.g., fibroblasts and keratinocytes), endothelial cells, gametes, and other dividing cells known in the art. The term dividing cell is of particular interest and includes cells having a cell proliferative disorder, such as a neoplastic cell. The term "cell proliferative disorder" refers to a condition characterized by an abnormal number of cells dividing. The conditions may include hypertrophy (continued proliferation of cells leading to overgrowth of cell populations within the tissue) and malnutrition (lack or insufficiency of cells within the tissue) cell growth or excessive influx or migration of cells into areas of the body. The cell population need not be transformed, tumorigenic, or malignant cells, but may also include normal cells. Cell proliferative disorders include diseases associated with connective tissue overgrowth, such as various fibrotic conditions, including scleroderma, arthritis, and cirrhosis. Cell proliferative disorders include neoplastic disorders, such as head and neck cancer. Head and neck cancer may include, for example, oral cancer, esophageal cancer, laryngeal cancer, thyroid cancer, tongue cancer, lip cancer, salivary gland cancer, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, upper nasal cavity cancer and sinus tumors, sensory neuroblastoma, squamous cell cancer, malignant melanoma, undifferentiated carcinoma of the Sinuses (SNUC), brain cancer (including glioblastoma, e.g., glioblastoma multiforme), or blood cancer. Also included are regional lymph node cancers including cervical lymph nodes, pre-pharyngeal lymph nodes, lung near esophageal lymph nodes and inframandibular lymph nodes (Harrison's Principles of Internal Medicine (eds., Isselbacher, et al., McGraw-Hill, Inc.,13th Edition, ppl850-1853,1994.) other cancer types including, but not limited to, lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, gastric cancer, skin cancer and ovarian cancer cell proliferative disorders also include rheumatoid arthritis (O' Denll JM 350: 25912004) and other autoimmune disorders (Mackay et al NEJM 345: 3402001) that are generally characterized by proliferation of cells of the immune system.
In one embodiment, the retroviral vector is targeted to a cell by binding to a cell having a molecule on the outer surface of the cell. This method of targeting retroviruses utilizes the expression of targeting ligands on the surface of the retrovirus to assist in targeting the virus to cells or tissues that have receptors or binding molecules that interact with the targeting ligands on the surface of the retrovirus. After a cell is infected with a virus, the virus delivers its nucleic acid into the cell, and upon completion of reverse transcription, the retroviral genetic material can integrate into the host cell genome.
By inserting the heterologous polynucleotide of interest into the viral vectors of the present disclosure along with another gene encoding, for example, a ligand for a receptor on a particular target cell, the vector is now target-specific. Viral vectors can be made target-specific by linking, for example, sugars, glycolipids, or proteins. One skilled in the art will know or be able to readily determine specific polynucleotide sequences that can be inserted into the viral genome or proteins that can be attached to the viral envelope, thereby allowing target-specific delivery of viral vectors containing nucleic acid sequences of interest.
Accordingly, in one embodiment, the present disclosure includes a chimeric ENV protein comprising a retroviral ENV protein operably linked to a targeting polypeptide. The targeting polypeptide can be a cell-specific receptor molecule, a ligand for a cell-specific receptor, an antibody or antibody fragment directed against a cell-specific epitope, or any other ligand readily identified in the art that is capable of binding to or interacting with a target cell. It should be noted that the antibody, antibody fragment or binding domain forming the chimeric ENV is separate and distinct from the heterologous gene downstream of the 2A or 2A-like peptide coding sequence, with or without an SSP that may include the coding sequence of the antibody, antibody fragment or binding domain. Examples of targeting polypeptides or molecules include bivalent antibodies using biotin-streptavidin as linker (Etienne-Julan et al, J.of General virol.,73, 3251-Asca 3255 (1992); Roux et al, Proc.Natl.Acad.Sci USA 86, 9079-Asan 9083(1989)), recombinant viruses containing sequences in the envelope that encode single chain antibody variable regions against haptens (Russell et al, Nucleic Acids Research,21, 1081-Asan 1085(1993)), peptide hormone ligands cloned into the retroviral envelope (Kasahara et al, Science,266, 1373-Asan 1376(1994)), chimeric/env constructs (Kasahara et al, 1994), single chain antibodies against Low Density Lipoprotein (LDL) that cause specific infection of HeLa cells expressing LDL receptors (EPO), Almi ligand, Natl. Wa et al, USA. 7592. Natl. 7574. Natl. V.74, USA. Natl. 7574. Alca. Alfa. 35. et. Natl. Alca. protein, USA. 70, thus, the virus can now cross-species specifically infect rat glioblastoma cells (Valesia-Wittmann et al, J.Virol.68,4609-4619(1994)), and Dornberg and co-workers (Chu and Dornbur g, J.Virol 69,2659-2663 (1995); M.Engelstadter et al Gene Therapy 8, 1202-1206 (2001)) have reported tissue-specific targeting of Spleen Necrosis Virus (SNV) (an avian retrovirus).
The present disclosure provides a method of producing a recombinant retrovirus capable of infecting a target cell, comprising transfecting a suitable host cell with: a vector comprising a polynucleotide sequence encoding viral gag, viral pol and viral env, a2A peptide or 2A peptide-like coding sequence, an SSP coding sequence operably linked between the 2A peptide or 2A peptide-like coding sequence and a heterologous polynucleotide, wherein the 2A peptide or 2A peptide-like coding sequence is downstream of the env, packaging and psi sequences, and recovering the recombinant virus.
The retroviruses and methods of the present disclosure provide a replication competent retrovirus that does not require helper viruses or additional nucleic acid sequences or proteins to propagate and produce virions. For example, the nucleic acid sequences of the retroviruses of the present disclosure encode a set of specific antigens and reverse transcriptases (as well as integrases and protease-enzymes necessary for maturation and reverse transcription), respectively, as described above. The viral gag and pol may be derived from a lentivirus, such as HIV, or an oncogenic retrovirus or gamma retrovirus, such as MoMLV. In addition, the nucleic acid genome of the retroviruses of the present disclosure includes sequences encoding viral Envelope (ENV) proteins. The env gene may be from any retrovirus. env may be an amphotropic envelope protein which allows transduction of cells of human and other species, or may be an ecotropic envelope protein which is only capable of transducing mouse and rat cells. In addition, it may be desirable to target recombinant viruses by linking the envelope proteins to antibodies or specific ligands to target receptors of specific cell types. As described above, retroviral vectors can be made target specific by insertion of, for example, glycolipids or proteins. Targeting is typically accomplished by targeting the retroviral vector to an antigen on a particular cell type (e.g., a cell type found in certain tissues, or a cancer cell type) using an antibody. One skilled in the art will know, or be able to readily determine without undue experimentation, the particular method of delivering a retroviral vector to a particular target. In one embodiment, the env gene is derived from a non-retrovirus (e.g., CMV or VSV). Examples of retroviral-derived env genes include, but are not limited to: moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), Human Immunodeficiency Virus (HIV) and Rous Sarcoma Virus (RSV). Other env genes, such as Vesicular Stomatitis Virus (VSV) (protein G), cytomegalovirus envelope (CMV), or influenza virus Hemagglutinin (HA) may also be used.
In one embodiment, the retroviral genome is derived from an oncogenic retrovirus, and more particularly from a mammalian oncogenic retrovirus. In another embodiment, the retroviral genome is derived from a gammaretrovirus, and more particularly from a mammalian gammaretrovirus. By "derived from" is meant that the parent polynucleotide sequence is a wild-type oncogenic virus that has been modified by insertion or removal of naturally occurring sequences (e.g., insertion of a2A peptide or 2A peptide-like coding sequence, an SSP coding sequence, and a heterologous polynucleotide encoding a polypeptide, and optionally one or more IRES or poliiii promoters linked to another heterologous polynucleotide or inhibitory nucleic acid of interest, respectively).
In another embodiment, the disclosure provides retroviral vectors targeted using regulatory sequences. Cell-or tissue-specific regulatory sequences (e.g., promoters) can be used to target expression of gene sequences in a particular cell population. Suitable mammalian and viral promoters for the present disclosure are described elsewhere herein. Thus, in one embodiment, the present disclosure provides a retrovirus having a tissue-specific promoter element at the 5' end of the retroviral genome. Typically, tissue-specific regulatory elements/sequences are located in the U3 region of the LTR of the retroviral genome, including, for example, cell-or tissue-specific promoters and enhancers (e.g., tumor cell-specific enhancers and promoters) and inducible promoters (e.g., tetracycline) for tumor cells.
The transcriptional control sequences of the present disclosure may also include naturally occurring transcriptional control sequences that are naturally associated with the gene encoding the superantigen, cytokine, or chemokine.
In some cases, it may be desirable to modulate expression. For example, different viral promoters with different strengths of activity may be used, depending on the desired expression level. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. When it is desired to reduce the level of transgene expression, modified versions of the less potent CMV promoter have also been used. When it is desired to express the transgene in hematopoietic cells, retroviral promoters, such as the LTRs from MLV or MMTV, may be used. Other viral promoters that may be used include SV40, RSV LTR, HIV-1 and HIV-2LTR, adenovirus promoters (e.g., promoters from E1A, E2A or MLP regions, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus).
Similarly, tissue-specific or selective promoters can be used to effect transcription in specific tissues or cells in order to reduce potential toxicity or undesirable effects on non-targeted tissues. For example, promoters such as PSA, probasin, prostatic acid phosphatase, or prostate specific gonadal kallikrein (hK2) may be used to target gene expression in the prostate. Whey helper protein (WAP) can be used for breast tissue expression (Andres et al, PNAS 84:1299-1303, 1987). Other promoter/regulatory domains that can be used are described below.
A "tissue-specific regulatory element" is a regulatory element (e.g., a promoter) that is capable of driving gene transcription in one tissue while remaining largely "silent" in other tissue types. However, it is understood that tissue-specific promoters may have detectable amounts of "background" or "base" activity in those tissues where silencing is expected. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in the target tissue/activity in a control tissue). In this regard, tissue-specific promoters useful in the practice of the present disclosure typically have a selectivity ratio of greater than about 5. Preferably, the selectivity ratio is greater than about 15.
In certain indications, it may be desirable to activate transcription at a particular time after administration of a recombinant replication competent retrovirus (RRV) of the disclosure. This can be accomplished using promoters that are regulated by hormones or cytokines. For example, in therapeutic applications where the indication is gonadal tissue, where a particular steroid is produced or delivered, it may be advantageous to use androgen or estrogen regulated promoters. Hormone-regulatable promoters include MMTV, MT-1, ecdysone, and RuBisco. Other hormone-regulated promoters may be used, such as those responsive to thyroid, pituitary and adrenal hormones. Cytokine and inflammatory protein responsive promoters which may be used include K and T kininogen (Kageyama et al, 1987), C-fos, TNF- α, C-reactive protein (Arcone et al, 1988), haptoglobin (Oliviero et al, 1987), serum amyloid A2, C/EBP α, IL-1, IL-6(Poli and Cortese, 1989), complement C3(Wilson et al, 1990), IL-8, α -1 acid glycoprotein (Prowse and Baumann, 1988), α -1 antitrypsin, lipoprotein lipase (Zechner et al, 1988), angiotensinogen (Ron et al, 1990), fibrinogen, C-jun (inducible by phorbol esters, TNF- α, UV radiation, retinoic acid and hydrogen peroxide), collagenase (inducible by phorbol esters and retinoic acid), Metallothioneins (inducible by heavy metals and glucocorticoids), matrilysin (inducible by phorbol esters, interleukin-1 and EGF), alpha-2 macroglobulin, and alpha-1 antichymotrypsin. Tumor specific promoters such as osteocalcin, Hypoxia Response Element (HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP, and tyrosinase can also be used to regulate gene expression in tumor cells.
Furthermore, this list of promoters should not be construed as being exhaustive or limiting, and one skilled in the art will be aware of other promoters that may be used in conjunction with the promoters and methods disclosed herein.
TABLE 8 tissue-specific promoters
It is also understood that certain promoters, while activity is not limited to a single tissue type, may still exhibit selectivity, as they may be active in one group of tissues, while being less active or silent in another group. Such promoters are also referred to as "tissue-specific" and are contemplated for use in the present disclosure. For example, promoters active in various Central Nervous System (CNS) neurons can be therapeutically used to protect against damage due to stroke, which can affect any of a number of different regions of the brain. Thus, the tissue-specific regulatory elements used in the present disclosure are suitable for the regulation of heterologous proteins, as well as for use as targeting polynucleotide sequences in the present retroviral vectors.
In yet another embodiment, the disclosure provides a plasmid comprising a recombinant retrovirus-derived construct. The plasmid may be introduced directly into the target cell or cell culture such as HT1080, NIH 3T3 or other tissue culture cells. The resulting cells release the retroviral vector into the culture medium.
The present disclosure provides a polynucleotide construct comprising, from 5 'to 3': a promoter or regulatory region for initiating transcription; psi package signal; gag-encoding nucleic acid sequence, pol-encoding nucleic acid sequence; env-encoding nucleic acid sequences; a2A peptide or 2A peptide-like coding sequence; SSP coding sequence; a heterologous polynucleotide encoding a marker, therapeutic or diagnostic polypeptide; optionally an IRES or polIII cassette; and LTR nucleic acid sequences. As described above, the gag, pol and env nucleic acid domains may be modified to remove the tryptophan codon, which is converted to a stop codon by ApoBec 3. In certain other embodiments, the vector may further comprise a polIII or IRES cassette downstream of the heterologous polynucleotide and upstream of the 3' LTR. Various segments of the polynucleotide constructs of the disclosure (e.g., recombinant replication-competent retroviral polynucleotides) are engineered, in part, according to the desired host cell, timing or amount of expression, and heterologous polynucleotide, as described elsewhere herein and below. The replication competent retroviral constructs of the present disclosure can be divided into a number of domains that can be individually modified by those skilled in the art.
SEQ ID NO:2, an exemplary DN a sequence for use in producing a recombinant retrovirus of the present disclosure is provided, the promoter may comprise a dna having the sequence set forth in SEQ ID NO:2 from nucleotide 1 to about nucleotide 582, and may include modifications to one or more (e.g., 2-5, 5-10, 10-20, 20-30, 30-50, 50-100 or more) nucleobases, so long as the modified promoter is capable of directing and initiating transcription. In one embodiment, the promoter or regulatory region comprises a CMV-R-U5 domain polynucleotide. The CMV-R-U5 domain contains the immediate early promoter from human cytomegalovirus linked to the MLV R-U5 region. In one embodiment, the CMV-R-U5 domain polynucleotide comprises the sequence of SEQ ID NO:2 from about nucleotide 1 to about nucleotide 1202 or a sequence identical to SEQ ID NO:2, wherein the polynucleotide facilitates transcription of a nucleic acid molecule to which it is operably linked. The gag domain of the polynucleotide may be derived from any number of retroviruses, but is generally derived from an oncogenic retrovirus, and more particularly from a mammalian oncogenic retrovirus such as MLV. In one embodiment, the gag domain comprises SEQ ID NO:2 from about nucleotide number 1203 to about nucleotide 2819, or a sequence having at least 95%, 98%, 99%, or 99.8% (rounded to the nearest tenth) identity thereto. The pol domain of the polynucleotide may be derived from any number of retroviruses, but is typically derived from a gammaretrovirus, and more particularly from a mammalian gammaretrovirus such as MLV. In one embodiment, the pol domain comprises SEQ ID NO:2 from about nucleotide number 2820 to about nucleotide number 6358 or a sequence having at least 95%, 98%, 99% or 99.9% (rounded to the nearest tenth) identity thereto. The env domain of the polynucleotide may be derived from any number of retroviruses, but is typically derived from a gamma-retrovirus, and more particularly from a mammalian gamma-retrovirus such as MLV. In some embodiments, the env-encoding domain comprises an amphotropic env domain. In one embodiment, the env domain comprises SEQ ID NO:2 from about nucleotide number 6359 to about nucleotide 8323 or a sequence having at least 95%, 98%, 99% or 99.8% (rounded to the nearest tenth) identity thereto. The 2A peptide or 2A peptide-like/SSP cassette is inserted after the env domain (e.g., at about nucleotide 8324) and continues to the end of the heterologous polynucleotide. Examples of suitable SSP peptides are provided in tables B and C, and the heterologous domain may be followed by a polypurine-rich domain, or may be an IRES-cassette or a polIII-cassette. The 3' LTR may be derived from any number of retroviruses, typically gamma retroviruses, and more typically mammalian gamma retroviruses, such as MLV. In one embodiment, the 3' LTR comprises the U3-R-U5 domain. In yet another embodiment, the LTR comprises a sequence as set forth in SEQ ID NO:2 or a sequence having at least 95%, 98% or 99.5% (rounded to the nearest tenth) identity thereto, from about nucleotide 9111 to about 11654.
Table B: natural eukaryotic signal peptides were ranked by HMM checks.
Table C: ranking artificial signal peptides by HMM score
Retroviral vectors are useful for The treatment of a variety of diseases and disorders, including a variety of Cell proliferative diseases and disorders (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764, Friedmann,1989, Science,244: 1275. 1281; Mullgan, 1993, Science,260: 926-.
The present disclosure also provides gene therapy for treating cell proliferative disorders. Such treatment may be accomplished by introducing an appropriate therapeutic polynucleotide (e.g., encoding an antigen binding protein/polypeptide, cytokine, ligand, antisense, ribozyme, prodrug activating enzyme, siRNA) into cells of a subject suffering from a proliferative disorder, or into allogeneic Mesenchymal Stem Cells (MSC), Neural Stem Cells (NSC), or other cell types known to be capable of targeting sites of inflammation or tumors. Delivery of the polynucleotide construct can be achieved using the recombinant retroviral vectors of the present disclosure, particularly if it is based on MLV or other gamma retroviruses, capable of infecting dividing cells.
In addition, the treatment methods described herein (e.g., gene therapy or gene delivery methods) can be performed in vivo or ex vivo. Preferably, the majority of the tumor is excised prior to gene therapy, e.g., surgically or by radiation. In some aspects, retroviral therapy may precede or follow surgery, chemotherapy, or radiation therapy.
Accordingly, the present disclosure provides a recombinant retrovirus capable of infecting a non-dividing cell, a dividing cell, or a tumor cell, wherein the recombinant retrovirus comprises a viral GAG; POL, a virus; a virus ENV; a heterologous nucleic acid operably linked to a2A peptide or peptide-like coding sequence; and cis-acting nucleic acid sequences required for packaging, reverse transcription and integration. The recombinant retrovirus may be a lentivirus, such as HIV, or may be a gammaretrovirus.
The present disclosure also provides a method of transferring a nucleic acid to a target cell to provide for expression of a particular nucleic acid (e.g., a heterologous sequence). Thus, in another embodiment, the disclosure provides a method of introducing and expressing a heterologous nucleic acid in a target cell comprising infecting the target cell with a recombinant virus of the disclosure and expressing the heterologous nucleic acid in the target cell, wherein the heterologous nucleic acid is engineered into the recombinant viral vector downstream of the env domain and operably linked to a2A or 2A-like peptide-SSP construct. As noted above, the target cells may be any cell type, including dividing, non-dividing, neoplastic, immortalized, modified and other cell types known to those skilled in the art, so long as they are capable of being infected with a retrovirus.
It may be desirable to transfer a nucleic acid encoding a biological response modifier (e.g., a cytokine) into a cell or subject. Included in this category are immunopotentiators, which include nucleic acids encoding a number of cytokines classified as "interleukins". These include, for example, interleukins 1 to 38, as well as other response modifiers and factors described elsewhere herein. Interferons and in particular gamma interferon, Tumor Necrosis Factor (TNF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) are also included in this class, although not necessarily acting according to the same mechanism. Other polypeptides include, for example, angiogenic factors and anti-angiogenic factors. It may be desirable to deliver such nucleic acids to bone marrow cells or macrophages to treat enzyme deficiencies or immunodeficiency. Nucleic acids encoding growth factors, toxic peptides, ligands, receptors, or other physiologically important proteins may also be introduced into specific target cells. Any of the foregoing biological response modifiers are engineered downstream of the RRV of the present disclosure and operably linked to a2A or 2A-like peptide-SSP construct.
The present disclosure is useful for delivering heterologous polynucleotides that facilitate drug-specific targeting and effects. For example, the EGF receptor family member HER2 is the drug trastuzumab (Herceptin)TMGenentech). Trastuzumab is an antibody-dependent cytotoxicity (ADCC). Activity preferentially targets HER 2-expressing cells with 2+ and 3+ overexpression levels by immunohistochemistry, but not 1+ and non-expressing cells (Herceptin prescription information, Crommelin 2002). Enhancement of HER2 expression by introducing a vector expressing HER2 or truncated HER2 (expressing only the extracellular and transmembrane domains) in HER2 low tumors may facilitate optimal triggering of ADCC and overcome the resistance to rapid development of Herceptin observed in clinical use. In these cases, the heterologous gene may encode HER 2.
In another example, CD20 is the conjugate drug rituximab (Rituxan)TMGenentech). Rituximab is a mediator of Complement Dependent Cytotoxicity (CDC) and ADCC. Cells with higher mean fluorescence intensities as determined by flow cytometry showed enhanced sensitivity to rituximab (van Meerten et al, Clin Cancer Res 2006; 12(13): 4027-. Optimal triggering of ADCC can be facilitated by enhancing expression of CD20 by introducing a CD20 expressing vector in CD20 low B cells. In this case, the heterologous gene encodes CD 20.
The present disclosure provides methods of treating cell proliferative disorders, such as cancers and tumors, comprising administering an RRV vector of the present disclosure followed by treatment with a chemotherapeutic or anti-cancer agent. In one embodiment, the RRV vector is administered to the patient for a period of time prior to administration of the chemotherapeutic or anti-cancer agent to allow the RRV to infect and replicate. The subject is then treated with a chemotherapeutic or anti-cancer agent for a time and dose to reduce cancer cell proliferation or kill cancer cells. In one embodiment, if treatment with a chemotherapeutic or anti-cancer agent reduces but does not kill the cancer/tumor (e.g., partial or transient remission), the subject may be treated with a non-toxic therapeutic agent (e.g., 5-FC) that is converted to a toxic therapeutic agent in cells expressing a cytotoxic gene (e.g., cytosine deaminase) from the RRV.
Using such methods, the RRV vectors of the present disclosure diffuse during tumor cell replication, and these cells can then be killed by treatment with anti-cancer or chemotherapeutic agents, and further killed using the RRV treatment methods described herein.
In yet another embodiment of the present disclosure, the heterologous gene may comprise a coding sequence for a target antigen (e.g., a cancer antigen). In this embodiment, a cell comprising a cell proliferative disorder is infected with an RRV comprising a heterologous polynucleotide encoding a target antigen to provide for expression of the target antigen (e.g., overexpression of a cancer antigen). An anti-cancer agent comprising a targeted cognate moiety that specifically interacts with a target antigen is then administered to the subject. The targeting cognate moiety can be operably linked to a cytotoxic agent, or can itself be an anti-cancer agent. Thus, cancer cells infected with RRV comprising the targeting antigen coding sequence increase expression of the target on the cancer cell, resulting in increased efficiency/efficacy of cytotoxic targeting.
In yet another embodiment, the RRV of the present disclosure comprise a coding sequence comprising a binding domain (e.g., an antibody, antibody fragment, antibody domain, non-antibody binding domain, or receptor ligand) that specifically interacts with a cognate antigen or ligand. The RRV comprising the coding sequence for the binding domain can then be used to infect a cell, such as a cancer cell or a neoplastic cell, in a subject comprising a cell proliferative disorder. The infected cells may then express the binding domain or antibody. An antigen or homolog operably linked to a cytotoxic agent or cytotoxic per se can then be administered to the subject. The cytotoxic homologue will then selectively kill infected cells expressing the binding domain. Alternatively, the binding domain itself may be, for example, an anti-cancer agent that interacts with the immune system, such as anti-PD-L1 or anti-CTLA-4.
The present disclosure provides a method of treating a subject having a cell proliferative disorder. The subject may be any mammal, including a human. Contacting the subject with a recombinant replication competent retroviral vector of the present disclosure. The contacting may be in vivo or ex vivo. Methods of administering the retroviral vectors of the present disclosure are known in the art and include, for example, systemic administration, local administration, intraperitoneal administration, intramuscular administration, intracranial, cerebrospinal administration, and administration directly at the site of a tumor or cell proliferative disorder. Other routes of administration known in the art may also be used.
Accordingly, the present disclosure includes various pharmaceutical compositions for treating cell proliferative disorders. The pharmaceutical compositions according to the present disclosure are prepared by bringing a retroviral vector according to the present disclosure containing a heterologous polynucleotide sequence for use in the treatment or modulation of a cell proliferative disorder into a form suitable for administration to a subject using a carrier, an excipient and an additive or adjuvant. Commonly used carriers or adjuvants include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents such as sterile water, alcohols, glycerol and polyols. Intravenous carriers include fluids and nutritional supplements. Preservatives include antimicrobials, antioxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers and The like, such as described in Remington's Pharmaceutical Sciences,15th ed.Easton: Mack Publishing Co.,1405-1412,1461-1487(1975) and The National Formulary XIV, 14th ed.Washington: American Pharmaceutical Association (1975), The contents of which are incorporated herein by reference. The pH of the pharmaceutical composition and the exact concentrations of the various components are adjusted according to routine techniques in the art. See Goodman and Gilman's The pharmaceutical basic for Therapeutics (7 th edition).
In other embodiments, host cells transfected with replication competent retroviral vectors of the present disclosure are provided. Host cells include eukaryotic cells, such as yeast cells, insect cells, or animal cells. Host cells also include prokaryotic cells, such as bacterial cells.
Also provided are engineered host cells transduced (transformed or transfected) with vectors provided herein (e.g., replication competent retroviral vectors). The engineered host cell may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the encoding polynucleotide. Culture conditions, such as temperature, pH, etc., are those previously used to select for host Cells for expression, and will be apparent to those skilled in the art and in the references cited herein (Sambrook, Ausubel and Berger, and, for example, Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique,3rd ed. (Wiley-Liss, New York) and references cited therein).
Examples of suitable expression hosts include: bacterial cells, such as E.coli, Bacillus subtilis, Streptomyces and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells, such as Drosophila and Spodoptera frugiperda; mammalian cells, such as CHO, COS, BHK, HEK293, or Bowes melanoma cells; or plant cells or explants, etc. Human cells or cell lines are commonly used; however, it may be desirable to clone the vectors and polynucleotides of the present disclosure into non-human host cells for sequencing, amplification, and cloning purposes.
The following examples are intended to illustrate, but not limit, the present disclosure. While these examples are typical examples that may be used, other procedures known to those skilled in the art may alternatively be used.
Examples
Example 1: RRV-2A-GFPm, RRV-GSG-2A, RRV-2A-yCD2 and RRV-GSG-2A-yCD 2.
RRV-yCD2 and RRV-GFP are moloney MLV-based RRVs having an amphotropic envelope gene and an encephalomyocarditis virus Internal Ribosome Entry Site (IRES) -transgene cassette downstream of the env gene (Perez et al, 2012). The RRV-2A-GFP (aka pAC3-2A-GFP) and RRV-2A-yCD2(pAC3-2A-yCD2) vectors are based on RRV-GFP and RRV-yCD2, but the IRES region has been replaced with a variety of different 2A peptides in frame with the amphotropic envelope proteins and transgenes (GFP or yCD 2). Cloning protocols for the RRV-2A-GFP and RRV-yCD2 vectors have been previously described (Hofacre et al hum. Gene ther.29: 437-4512018). Briefly, the pAC3-T2A-GFP construct was first generated using the Gibson assembled cloning kit (NEB) containing 2 DNA fragments and the pAC3-emd backbone digested with BstB I and Not I sites. First, a pair of sense and antisense oligonucleotides (IDT) containing the sequence of the 3 'end of amphotropic evn, 2A peptide from Spodoptera litura beta-tetrad virus (T2A) and 5' of GFP in 5 '-to-3' order were synthesized and hybridized to generate DNA fragments 2A-G. The second DNA fragment in the Gibson assembly is the FP fragment. The FP fragments were generated by PCR using the following primers: GFP-F-Gib (5'-GAAGTTCGAGGGCGACAC-3' (SEQ ID NO:303)) and GFP-R-Gib (5'-TAAAATCTTTTATTTTATCTGCGGCCGCAC-3' (SEQ ID NO: 304)).
In the 2A-G fragment, 5' contains a sequence that overlaps with the BstBI site in the amphotropic env of the pAC3 backbone; the 3 'contains a sequence that overlaps with the 5' of the FP DNA fragment. In addition, the AscI restriction enzyme site was placed at the 3' -end of T2A just upstream of the start codon of the second transgenic GFP. The AscI site was included for subsequent replacement of the T2A peptide with other 2A peptides. Inclusion of the AscI restriction site with an additional nucleotide T, followed by an AscI site, resulted in an additional 3 amino acids (Glycine-Ala-Pro) C-terminus added to the last proline residue in the T2A peptide. During co-translation, the separation of the GFP protein from the envelope protein mediated by the T2A peptide resulted in the addition of an additional 4 amino acids P, G, A and P at the N-terminus of GFP. In the FP fragment, the 5 '-end of the FP fragment contains a sequence that overlaps the 3' -end of the 2A-G fragment by 24 nucleotides, and the 3 '-end of the FP fragment overlaps the 5' -end of the pAC3-GFP backbone spanning the Not I site by 26 nucleotides. The plasmid DNA obtained from the Gibson assembly clone was designated pAC 3-T2A-GFP.
Additional RRV-2A-GFP vectors were subsequently synthesized containing three other commonly used 2A peptides (IDT) derived from porcine teschovirus-1 (P2A), foot and mouth disease virus (F2A) and equine rhinitis a virus (E2A) in two different configurations. Each DNA fragment contained the sequence of the 3' of the amphotropic env gene and the designated 2A peptide replacing T2A of the pAC3-T2A-GFP backbone at the BstBI and AscI sites. The resulting plasmid DNAs were designated pAC3-P2A-GFP, pAC3-F2A-GFP, pAC3-E2A-GFP, pAC3-GSG-T2A-GFP, pAC3-GSG-P2A-GFP, pAC3-GSG-F2A-GFP, and pAC 3-GSG-E2A-GFP.
It was subsequently determined that the described RRV-2A-GFP plasmid DNA (pAC3-E2A-GFP, pAC3-F2A-GFP, pAC3-P2A-GFP, pAC3-T2A-GFP, pAC3-GSG-E2A-GFP, pAC3-GSG-F2A-GFP, pAC3-GSG-P2A-GFP and pAC3-GSG-T2A-GFP) contained a stop codon mutation at the 3' -end of the GFP. When the FP PCR fragment was generated, mutations were introduced into the GFP-R-Gib primer (5'-TAAAATCTTTTATTTTATCTGCGGCCGCAC-3' (SEQ ID NO: 4)). The stop codon mutation in GFP from the PCR resulted in an additional 11 amino acids of the GFP ORF being read through (C-A-A-A-D-K-I-K-D-F-I (SEQ ID NO:5)) before the stop codon was reached. The plasmid DNA was renamed pAC3-E2A-GFPm, pAC3-F2A-GFPm, pAC3-P2A-GFPm, pAC3-T2A-GFPm, pAC3-GSG-E2A-GFPm, pAC3-GSG-F2A-GFPm, pAC3-GSG-P2A-GFPm and pAC 3-GSG-T2A-GFPm. Hereinafter, two nomenclature pAC3-E2A-GFP/pAC3-E2A-GFPm, pAC A-F2A-GFP/pAC A-F2A-GFPm, pAC A-P2A-GFP/pAC A-P2A-GFPm, pAC A-T2A-GFP/pAC A-T2A-GFPm, pAC A-GSG-E2A-GFP 72-GSG-E2A-GFPm, pAC A-GSG-F2A-GSG-P2A-GFP 72-GFP/pAC A-GSG-P2A-GFPm, and pAC A-GSG-pAC A-GFPm can be interchanged with pAC A-GFPm, pAC A-GFP 2A-GFPm, pAC A-GFPm.
An equivalent set of 4 RRV-2A-yCD2 vectors was generated by replacing the GFPm open reading frame with yCD2 ORF in each 2A peptide version of plasmid DNA pAC3-P2A-GFPm, pAC3-GSG-P2A-GFPm, pAC3-T2A-GFPm, and pAC 3-GSG-T2A-GFPm. The AscI-yCD2-NotI PCR fragment was generated from pAC3-yCD2 plasmid DNA using the following primers: AscI-yCD2-F (5'-GATCGGCGCGCCTATGGTGACCGGCGGCATGGC-3' (SEQ ID NO:6) and 3-37 (5'-CCCCTTTTTCTGGAGACTAAATAA-3' (SEQ ID NO: 7). the PCR product and each of the four pAC3-2A-GFPm plasmid DNAs were digested with AscI and NotI restriction enzymes, and the AscI-yCD2-NotI digested PCR product was subcloned in place of GFPm to produce pAC3-P2A-yCD2, pAC3-GSG-P2A-yCD2, pAC3-T2A-yCD2, and pAC3-GSG-T2A-yCD2 (Table D).
Table D: sequence, source of 2A peptide, and RRV plasmid-2A peptide-transgene name.
Example 2: the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors produced from 293T cells are infectious and express GFP proteins.
18 to 20 hours before transfectionHEK293T cells were seeded at 2e6 cells per 10cm plate. The next day, pAC3-2A-GFPm and pAC3-GSG-2A-GFPm plasmids were used to transiently transfect 20. mu.g of plasmid DNA 20 hours after cell inoculation using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with DMEM complete medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-2A-GFPm, RRV-GSG-2A-GFPm, and RRV-IRES-GFP from transient transfection of HEK293T cells as described previously (Perez et al, 2012). Briefly, vector preparation titers were determined on PC3 cells by single cycle infection of the vector. 24 hours post-infection, single-cycle infection was assured by azidothymidine treatment, followed by quantitative PCR (qPCR) of target cell genomic DNA specific for viral vector DNA (MLV LTR primer set; 5-MLV-U3-R (5'-AGCCCACAACCCCTCACTC-3' (SEQ ID NO:20)),3-MLV-Psi (5'-TCTCCCGATCCCGGACGA-3' (SEQ ID NO:21)) and probe (5 '-FAM-CCCCAAATGAAAGACCCCCGCTGACG-BHQ 1-3' (SEQ ID NO:22)) after 48 hours post-infection to quantify viral DNA copy number per cell genome the viral titer (TU/mL) in transduced units per milliliter (TU) was determined by calculating a threshold Cycle (CT) value consisting of 2X 10 of plasmid DNA per 10X 10 of TU7Copy to 2X 101The standard curve of the copies was derived with known amounts of genomic DNA input, cell number and dilution of the virus stock per reaction mixture. Table E shows that the titers of RRV-2A-GFPm and RRV-GSG-2A-GFPm produced by HEK293T cells are comparable to those of RRV-IRES-GFP.
Table E: titres of RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors produced by 293T cells
TU/mL
Stdv
pAC3-E2A-GFP
1.15E+06
2.55E+05
pAC3-F2A-GFP
1.63E+06
2.58E+05
pAC3-P2A-GFP
1.81E+06
3.11E+05
pAC3-T2A-GFP
3.31E+06
1.32E+05
pAC3-GSG-E2A-GFP
1.65E+06
2.76E+05
pAC3-GSG-F2A-GFP
1.32E+06
7.57E+04
pAC3-GSG-P2A-GFP
1.31E+06
1.22E+05
pAC3-GSG-T2A-GFP
2.66E+06
2.14E+05
pAC3emd
1.65E+06
2.12E+05
U87-MG was then infected with RRV-2A-GFPm virus produced by HEK293T cells at a multiplicity of infection (MOI) of 0.01. U87-MG cells were plated at 1X 105Individual cells were seeded in 6-well plates for initial infection. Cells were passaged into new wells of 6-well plates at a dilution of 1-4 per passage, and the remaining cells of each sample were collected and virus transmission was assessed by measuring the percentage of cells expressing GFPm and the mean fluorescence intensity of GFPm using BD FACS Canto II (BD Biosciences). The percentage of GFP-positive cells at each generation was plotted. The length of the assay was performed until all RRV-2A-GFP viruses reached maximum infectivity (-95% or more GFP-positive cells). In infected U87-MG cells, the rate of viral propagation in RRV-2A-GFPm and RRV-GSG-2A-GFPm is similar to RRV-IRES-GFP, except that RRV-P2A-GFPm, RRV-T2A-GFPm and RRV-GSG-F2A-GFPm exhibit hysteresis. Nevertheless, they reached maximum infectivity within 18 days. GFPm expression levels also varied in the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors, but were about 20 to 50% of the expression of RRV-IRES-GFP infected U87-MG cells.
Example 3: the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors are stable in U87-MG cells.
To ensure that reduced GFP expression in RRV-2A-GFPm and RRV-GSG-2A-GFPm infected U87-MG cells was not due to deletion of the GFP gene in the viral genome, the integrity of the 2A-GFPm region was assessed by endpoint PCR using a primer set spanning the 3'env and 3' UTR regions of proviral DNA. At maximal infectivity of U87-MG cells, the cells were subsequently cultured in T75 flasks to confluence, at which time the medium was replaced with fresh medium, and then virus-containing supernatants were collected and subjected to 0.45 μ M filtration 18-24 hours after medium replacement. The collected cell supernatants were aliquoted and stored at-80 ℃ until used in immunoblot and reinfection experiments. At the same time, the cells are divided into two parts; 1/10 were used to isolate genomic DNA and 9/10 was used to isolate total cell lysates. Genomic DNA was extracted from the cell pellet by resuspension in 400. mu.L of 1 XPBS and isolation using Promega Maxwell 16 cell DNA purification kit (Promega). One hundred nanograms of genomic DNA were then used as a template for PCR with the following primer sets: IRES-F (5'-CTGATCTTACTCTTTGGACCTTG-3' (SEQ ID NO:23)) and IRES-R (5'-CCCCTTTTTCTGGAGACTAAATAA-3' (SEQ ID NO: 24)). The resulting PCR products were analyzed on a 1% agarose gel. The data show that the 2A-GFPm and GSG-2A-GFPm regions in proviral DNA of the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors are stable in U87-MG cells during the time of viral replication.
Example 4: RRV-2A-GFPm and RRV-GSG-2A-GFPm produced by maximally infected U87-MG cells remain infectious during subsequent cycles of infection.
Since long-term infectivity is one of many important criteria for maintaining therapeutic efficacy delivered by RRV, infectivity of RRV-2A-GFPm and RRV-GSG-2A-GFPm produced by maximally infected U87-MG cells was assessed by performing additional cycles of infection in naive U87-MG cells. First, virus supernatants collected from maximally infected U87-MG cells were titrated as described, and then re-infected back to the original U87-MG cells at an MOI of 0.01. The titers produced by maximally infected U87-MG cells were similar to those obtained from transiently transfected HEK293T cells, comparable between the RRV-2A-GFPm, RRV-GSG-2A-GFPm and RRV-IRES-GFP vectors.
As described, viral transmission of RRV-2A-GFPm and RRV-GSG-2A-GFPm was monitored at each cell passage. In contrast to the rate of viral transmission observed in the first infection cycle using viral supernatants produced from transiently transfected HEK293T cells, all vectors transmitted at a rate comparable to RRV-IRES-GFP. However, as previously observed, in this infection cycle, the GFP expression levels from U87-MG cells infected with RRV-2A-GFPm and RRV-GSG-2A-GFPm remained 20 to 50% of the levels expressed by RRV-IRES-GFP cells.
Example 5: the viral envelope and GFPm proteins of the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors are processed with different efficiencies in infected U87-MG cells.
To assess GFPm expression, efficiency of GFPm isolation from viral envelope proteins, and proper processing of viral envelope proteins, cell lysates were generated from infected U87-MG cells. At the time U87-MG cells were at maximal infectivity, confluent cell monolayers were washed once in 1 × PBS, dissociated with trpzean (sigma), resuspended in complete DMEM, washed again in 1 × PBS, and subsequently lysed on ice for 30 min in 200 μ L RIPA lysis buffer (Thermo Scientific). Cell debris in the lysate was clarified by centrifugation at 14,000rpm for 15 minutes at 4 ℃, and the supernatant was collected and transferred to a new tube. Protein concentration in cell lysates was then determined using BCA precipitation assay (Thermo Scientific) and 20 μ g of protein was subjected to SDS-PAGE. Proteins were resolved on 4-12% XT-Tris SDS-PAGE gels (BioRad) at 200 volts for 45 min. Subsequently, the proteins were transferred to PVDF membranes (Life Technologies) at 20 volts for 7 minutes using the iBlot dry blotting system. The expression of gp70 subunit and GFPm of the envelope protein of the membrane was determined using anti-gp 70 (rat anti-gp 70, clone 83A 25; 1:500 dilution) and anti-GFP (rabbit anti-GFP; 1:1000 dilution). Protein expression was detected using the corresponding secondary antibody conjugated to horseradish peroxidase. The results showed that the separation efficiency of the GFPm proteins from RRV-F2A-GFPm, RRV-P2A-GFPm, and RRV-T2A-GFPm, RRV-GSG-F2A-GFPm, and RRV-GSG-F2A-GFPm from the viral envelope protein was low as shown by the-120 kDa high molecular weight env-2A-GFPm fusion protein using the anti-GFP antibody. In contrast, GFPm is relatively effective in isolating viral envelope proteins for the RRV-E2A-GFPm, RRV-GSG-P2A-GFPm, and RRV-GSG-T2A-GFPm vectors, as compared to RRV-IRES-GFP. At the same time, processing of viral envelope proteins in infected U87-MG was examined with anti-gp 70 antibody. The results show that virus enveloped in either pro (Pr85) or processed form (gp70) was detected in all RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors, indicating separation of viral envelope proteins from GFPm as seen in anti-GFP immunoblots. Furthermore, the separation efficiency observed in the anti-gp 70 blot was somewhat consistent with that observed in the anti-GFP immunoblot. Although protein expression of the fusion polyprotein Env-GFPm differs between the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors, RRV-GSG-P2A-GFPm and RRV-T2A-GFPm appear to have the most efficient separation, as indicated by the absence of detection of the viral envelope-GFPm fusion polyprotein in both anti-GFP and anti-gp 70 immunoblots.
Example 6: the level of incorporation of correctly processed viral envelope proteins correlates with the efficiency of separation between the viral envelope and the GFPm protein.
Viral supernatants from RRV-2A-GFPm and RRV-GSG-2A-GFPm maximally infected U87-MG cells were pelleted for 30 min at 14000rpm by a 20% sucrose gradient at 4 ℃ and then resuspended in 20. mu.L of 1 Laemmli buffer containing 5% 2-mercaptoethanol and SDS PAGE was performed on 4-20% Tris glycine gel (BioRad). Electrophoresis and protein transfer were performed as described. Correctly processed virion-associated viral envelope protein expression was tested with anti-gp 70 (rat-produced anti-gp 70, clone 83A 25; 1:500 dilution) and anti-p 15E (mouse-produced anti-TM, clone 372; 1:250 dilution). Protein expression was detected using the corresponding secondary antibody conjugated to horseradish peroxidase. The data indicate that, in addition to the RRV-P2A-GFPm and RRV-T2A-GFPm vectors, the correctly processed envelope proteins gp70 and P12E/P15E of the RRV-2A-GFPm and RRV-GSG-2A-GFPm vectors were detected in virions at levels comparable to RRV-IRES-GFP. As expected, RRV-GSG-P2A-GFPm and RRV-T2A-GFPm, which showed the lowest levels of virion-associated envelope proteins, expressed the highest levels of the fusion polyprotein in cell lysates. Consistent with the published data, this data supports the following insights: the unprocessed envelope protein precursor protein Pr85, or in this case the viral envelope-GFPm fusion polyprotein, is not incorporated into the virion. Furthermore, cleavage of the R peptide carrying the 2A peptide resulted in "fusion promoting" p12E, which also appeared to be sufficient to produce infectious viral particles during virion maturation, as indicated by the titers produced by maximally infected U87-MG cells. The nature of the p15E/p12E ratio and its role in membrane fusion during infection is not clear. In summary, the data indicate that the level of viral envelope protein incorporation is not correlated with titer values measured in the target cells. The unexpected lack of difference in titer values between the vectors (particularly the RRV-GSG-P2A-GFPm and RRV-T2A-GFPm vectors) indicates that a range of envelope expression levels can be tolerated on the RRV particles without affecting the titer of these cells.
Example 7: the RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 vectors produced by 293T cells are infectious and express yCD2 proteins.
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The next day, 20. mu.g of plasmid DNA was transiently transfected using pAC3-P2A-yCD2, pAC3-T2A-yCD2, pAC3-GSG-P2A-yCD2, and pAC3-GSG-T2A-yCD2 plasmids, 20 hours after cell inoculation, using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with DMEM complete medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers of RRV-P2A-yCD2, RRV-T2A-yCD2, RRV-GSG-P2A-yCD2, and RRV-GSG-T2A-yCD2 from transiently transfected HEK293T cells were determined as previously described (Perez et al, 2012). Briefly, vector preparation titers were determined on PC3 cells by single cycle infection of the vector. Single cycle infection was confirmed by azidothymidine treatment 24 hours post infection, followed by quantitative PCR (qPCR) of target cell genomic DNA specific for viral vector DNA (MLV LTR primer set; 5-MLV-U3-R (5'-AGCCCACAACCCCTCACTC-3' (SEQ ID NO:20)),3-MLV-Psi (5'-TCTCCCGATCCCGGACGA-3' (SEQ ID NO:21)) and probe (5 '-FAM-CCCCAAATGAAAGACCCCCGCTGACG-BHQ 1-3' (SEQ ID NO:22)) at 48 hours post infection to quantify viral DNA copy number per cell genome the viral titer (TU/mL) reported in transduced units per milliliter (TU) was determined by calculating a threshold Cycle (CT) value from 2X 10 (TU)7Copy to 2X 101Standard curves for individual copies of plasmid DNA were derived with known amounts of genomic DNA input, cell number, and virus stock dilutions per reaction mixture. Table F shows that the titers of RRV-P2A-yCD2, RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 produced by HEK293T cells are comparable to RRV-IRES-yCD 2.
Table F: titres of RRV-P2A-yCD2, RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 vectors produced by 293T cells
In addition, virus supernatants collected from maximally infected U87-MG cells were titrated as described to ensure that they remained infectious. The primer set used for the titer had similar priming efficiency to the primer set containing 5-MLV-U3-R, 3-MLV-Psi primers and probes. The primer sets used to titrate RRV-P2A-yCD2, RRV-T2A-yCD2, RRV-GSG-P2A-yCD2, and RRV-GSG-T2A-yCD2 vectors from infected U87-MG cells were: env2 is: 5'-ACCCTCAACCTCCCCTACAAGT-3' (SEQ ID NO:25), Env2 Rev: 5'-GTTAAGCGCCTGATAGGCTC-3' (SEQ ID NO:26) and probe 5 '-FAM-CCCCAAATGAAAGACCCCCGCTGACG-BHQ 1-3' (SEQ ID NO: 27). The titers generated from maximally infected U87-MG cells were similar to those obtained from transiently transfected HEK293T cells and were comparable between the RRV-IRES-yCD2 vectors.
Example 8: in infected U87-MG cells, the viral envelope of the RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 vectors and the yCD2 protein were treated with different efficiencies.
To evaluate the expression of yCD2, the efficiency of separation of the yCD2 protein from the viral envelope protein, and the correct processing of the viral envelope protein, cell lysates were generated from infected U87-MG cells. When U87-MG cells were at maximal infectivity, confluent cell monolayers were washed once in 1X PBS, dissociated with TrpZ ean (Sigma), resuspended in complete DMEM, washed again in 1X PBS, and subsequently lysed in 200 μ L RIPA lysis buffer (Thermo Scientific) on ice for 30 min. Cell debris from the lysate was clarified by centrifugation at 14,000rpm for 15 minutes at 4 ℃, and the supernatant was collected and transferred to a new tube. The protein concentration of the cell lysates was then determined using BCA precipitation assay (Thermo Scientific) and 20 μ g of protein was subjected to SDS-PAGE. Proteins were resolved on 4-12% XT-Tris SDS-PAGE gels (BioRad) at 200 volts for 45 min. Subsequently, the proteins were transferred to PVDF membranes (Life Technologies) at 20 volts for 7 minutes using the iBlot dry blotting system. The expression of gp70 subunit and yCD2 of the envelope proteins of the membranes was determined using anti-gp 70 (rat anti-gp 70, clone 83A 25; 1:500 dilution) and anti-yCD 2 (mouse anti-yCD 2; 1:1000 dilution). Protein expression was detected using the corresponding secondary antibody conjugated to horseradish peroxidase. The results show that the separation of the yCD2 proteins from RRV-P2A-yCD2 and RRV-T2A-yCD2 from the viral envelope proteins was inefficient, as indicated by the high molecular weight of the env-2A-yCD2 fusion polyprotein at-110 kDa using the anti-yCD 2 antibody. In contrast, isolation of the yCD2 protein from the viral envelope protein was relatively effective against RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2, as compared to RRV-IRES-yCD 2. At the same time, processing of viral envelope proteins in infected U87-MG was examined with anti-gp 70 antibody. The results show that viruses coated in either precursor (Pr85) or processed form (gp70) can be readily detected in the RRV-GSG-P2A-yCD2, RRV-GSG-T2A-yCD2 vectors, but at much lower levels in the RRV-P2A-yCD2 and RRV-T2A-yCD2 vectors. In addition, the levels of Pr85/gp70 virus envelope proteins were somewhat consistent with those observed in anti-yCD 2 immunoblots. However, unlike the RRV-2A-GFPm or RRV-GSG-2A-GFPm vector, the viral envelope-yCD 2 fusion polyprotein could not be detected using anti-gp 70 antibody or anti-2A antibody (Cat # ABS31, EMD Millipore). Of the 4 vectors, the RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 vectors showed the most efficient isolation of the fusion polyprotein, as shown by the absence of the viral envelope-yCD 2 fusion polyprotein detected in the anti-yCD 2 immunoblot. Taken together, the data indicate that the GSG-P2A and GSG-T2A configurations produce the most efficient multi-protein separations in the context of the RRV envelope protein open reading frame.
Example 9: RRV-G2G-P2A-YCD2 and RRV-GSG-T2A-yCD2 have long-term stability in U87-MG cells.
Sequential infections were performed to evaluate the long-term vector stability of RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 in U87-MG cells. About 10 of the cells to be seeded in 6-well plates5An initial U87-MG cell was initially infected with the viral vector at an MOI of 0.1 and cultured for 1 week to complete a single infection cycle. 100 μ L of 2ml viral supernatant from fully infected cells was used for infection 105One initial cell and repeated up to 16 cycles. Genomic DNA was extracted from the pellet by resuspension in 400. mu.L of 1 XPBS and isolation using the Promega Maxwell 16 cell DNA purification kit (Promega). One hundred nanograms of genomic DNA were then used as a template with a spanning transgeneCarrying out PCR on the primer pair of the kit; IRES-F (5'-CTGATCTTACTCTTTGGACCTTG-3' (SEQ ID NO:23)) and IRES-R (5'-CCCCTTTTTCTGGAGACTAAATAA-3' (SEQ ID NO: 24)). Vector stability of the 2A-yCD2 region was assessed by PCR amplification of the integrated provirus from infected cells. The expected size of the PCR product was approximately 0.73 kb. The presence of any band smaller than 0.73kb indicates a deletion in the 2A-yCD2 region. The IRES-yCD2(1.2Kb) region in RRV-yCD2 stabilized to the infection cycle 16 as previously reported (Perez et al, 2012). Similarly, the 2A-yCD2 region in RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 also remained stable to the infection cycle 16. However, the 2A-yCD2 region in RRV-GSG-T2A-yCD2 is slightly less stable than RRV-GSG-P2A-yCD2 because of the deletion (0.4kb) that occurs in the infectious cycle 13, but remains stable throughout cycle 16.
Example 10: incorporation of correctly processed viral envelope proteins correlated with the efficiency of separation between viral envelope and yCD2 proteins in U87-MG cells infected with RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2, and RRV-GSG-T2A-yCD2 vectors.
Viral supernatants produced by RRV-2A-yCD2 and RRV-GSG-2A-yCD2 maximally infected U87-MG cells were pelleted at 14,000rpm for 30 min at 4 ℃ by a 20% sucrose gradient, then resuspended in 20. mu.L of 1 Laemmli buffer containing 5% 2-mercaptoethanol and SDS PAGE was performed on 4-20% Tris glycine gel (BioRad, Hercules CA). Electrophoresis and protein transfer were performed as described. Correctly processed virion viral envelope protein expression and maturation were determined using anti-gp 70 (rat-produced anti-gp 70, clone 83a 25; 1:500 dilution) and anti-p 15E (mouse-produced anti-TM, clone 372; 1:250 dilution). Protein expression was detected using the corresponding secondary antibody conjugated to horseradish peroxidase. The data show that in virions, correctly processed envelope proteins, i.e., gp70 of RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2, but not gp70 of RRV-P2A-yCD2 and RRV-T2A-yCD2, were detected at levels comparable to RRV-IRES-yCD 2.
Importantly, the data indicate that the level of incorporation of correctly processed viral envelope proteins is not correlated with titer values.
Example 11: the expression level of yCD2 protein was different in RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 infected U87-MG cells, but showed 5-FC sensitivity comparable to RRV-IRES-yCD2 infected U87-MG cells
Since immunoblotting of RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 showed that the amount of yCD2 protein expressed as a protein separated from the viral envelope protein or as a fusion polyprotein was different in infected U87-MG cells, LD was performed50Their 5-FC sensitivity was experimentally measured. U87-MG cells maximally infected with the RRV-P2A-yCD2 and RRV-T2A-yCD2, RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 vectors were used to determine their 5-FC LD by MTS assay50. For each infected or uninfected U87-MG cell line, 1X 10 cells were used3Individual cells/well/100 μ L of medium were plated in triplicate in 96-well plates. Cells were treated with 5-FC (cat # F7129, Sigma) at 1:10 serial dilutions from 0.00001mM to 1 mM. 5-FC treatment was not included as a control. 5-FC was added 1 day after plating, and then complete medium supplemented with 5-FC every 2 days. Initial U87-MG cells were included as controls to determine the non-5-FU mediated cytotoxic effects of 5-FC. Cells were monitored over a7 day incubation period and cell death was measured every 2 days by using the CellTiter 96AQueous single solution cell proliferation assay system (Promega). After addition of MTS, OD values at 490nm were obtained using an Infinite M200(Tecan) plate reader at 60 minutes of MTS incubation. The mean OD values for each sample in triplicate were converted to percent cell survival relative to untreated but RRV-infected cells. Subsequently, percent values were plotted against 5-FC concentration in a logarithmic scale using GraphPad Prim to generate LD50And (4) mapping. Calculating LD by software using non-linear four-parameter fitting of acquired data points50The value is obtained. The data indicate that, although the level of "isolated" yCD2 protein is higher in RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 infected U87-MG cells than in RRV-P2A-yCD2 and RRV-T2A-yCD2 infected U87-MG cells, the viral envelope-yCD 2 fusion polyprotein observed in RRV-P2A-yCD2 and RRV-T2A-yCD2 infected U87-MG cells has enzymatic activity in converting 5-FC to 5-FU, thereby having similar LC activity to RRV-IRES-yCD250Lower extreme of concentrationThe cytotoxic effect is present.
Example 12: tu2449 cells infected with RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 exhibited 5-FC sensitivity comparable to RRV-IRES-yCD2
Tu2449 cells maximally infected with RRV-GSG-P2A-GMCSF-T2A-yCD2 were used to determine their 5-FC LD50 by MTS assay as described. RRV-IRES-yCD2 was included as a control. Treatment was performed with 5-FC (cat # F7129, Sigma) at 1:10 serial dilutions from 0.00001mM to 1 mM. 5-FC treatment was not included as a control. 5-FC was added 1 day after plating, and then complete medium supplemented with 5-FC every 2 days. Initial Tu2449 cells were included as a control to determine the non-5-FU mediated cytotoxic effect of 5-FC. Cells were monitored over a7 day incubation period and cell death was measured every 2 days by using the CellTiter 96Aqueous single solution cell proliferation assay system (Promega). After addition of MTS, OD values at 490nm were obtained using an Infinite M200(Tecan) plate reader at 60 minutes of MTS incubation. The mean OD values for each sample in triplicate were converted to percent cell survival relative to untreated but RRV-infected cells. Percent values were plotted against 5-FC concentration in a logarithmic scale using GraphPad Prim to generate LD50And (4) mapping. Calculating LD by software using non-linear four-parameter fitting of acquired data points50The value is obtained. The data indicate that the yCD2 protein expressed by Tu-2449 cells infected with RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 has enzymatic activity in converting 5-FC into 5-FU, and thus in LC similar to RRV-IRES-yCD250Cytotoxic effects are achieved at concentrations.
Example 13: RRV-GSG-T2A-yCD2 treated subcutaneous syngeneic glioma mice showed a delayed tumor growth comparable to RRV-IRES-yCD 2.
The homologous cell line Tu-2449 was used as an orthotopic brain tumor model in B6C3F1 mice (Ostertag et al, 2012). A Tu-2449 cell subline (Tu-2449SQ) for subcutaneous tumor modeling was established. A mixture of 98% naive Tu-2449SQ cells and 2% RRV-GSG-T2A-yCD2 infected Tu-2449SQ cells was prepared in vitro and resuspended in phosphate buffered saline (PBS; Hyclone) for subcutaneous tumor implantation. Including 98% of initial Tu-2449SQ cells and 2% of RRV-IRES-mixture of yCD2 infected Tu-2449SQ cells as positive control and comparator. On day 0, B6C3F1 mice in each group (n-10 per group) were implanted subcutaneously by 1 × 106Tumor cells, on day 12 after tumor implantation (when about > 75% of tumors were infected with RRV), mice were administered either PBS or 5-FC (500mg/kg body weight/dose, i.p. twice daily) for 45 consecutive days, followed by 2 days without drug to allow diffusion of vehicle from the remaining infected cells. The cycle of 5 more days drug treatment, 2 days no drug treatment was repeated. Tumor volume measurements were taken daily. The results show that mice bearing tumors bearing RRV-IRES-yCD2 or RRV-GSG-T2A but not treated with 5-FC continue to grow. In contrast, mice bearing tumors bearing RRV-GSG-T2A treated with 5-FC delayed the growth of pre-established tumors and were comparable to mice treated with RRV-IRES-yCD2+ 5-FC. The data indicate that RRV-GSG-T2A-yCD2 has comparable therapeutic efficacy to RRV-IRES-yCD2 in a mouse model of subcutaneous synglioma.
Example 14: RRV-GSG-T2A-GMCSF-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2-GSG-PS2-GMCSF vectors produced by HEK293T cells express GMCSF and yCD2 proteins and are infectious.
pAC3-GSG-T2A-GMCSF-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2-GSG-P2A-GMCSF are generated by cloning chemically synthesized human GMCSF-GSG-P2A-yCD2 and yCD2-GSG-P2A-GMCSF cassettes (Genewiz) with AscI and NotI restriction sites present at the 5 'and 3' ends, respectively, into the pAC3-GSG-T2A-yCD2 backbone digested with AscI and NotI restriction enzymes. The resulting GMCSF-GSG-P2A-yCD2 and yCD2-GSG-P2A-GMCSF cassettes are in frame with GSG-T2A at the N-terminus of the cassette (5' upstream of the AscI restriction site).
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The following day, 20 hours after cell inoculation, 20. mu.g of pAC3-GSG-T2A-GMCSF-GSG-P2A-yCD2 or pAC3-GSG-T2A-yCD2-GSG-P2A-GMCSF plasmid was used for transient transfection using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with CMEM medium and incubated with fresh complete medium. Approximately 42 hours after transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-GSG-T2A-GMCSF-GSG-P2A-yCD2 from transient transfection of HEK293T cells as described. The data show that the titers (. about.2E 6 TU/mL) of RRV-GSG-T2A-GMCSF-GSG-P2A-yCD2 and pAC3-GSG-T2A-yCD2-GSG-P2A-GMCSF are comparable to RRV-IRES-yCD 2.
To assess yCD2 protein expression, cell lysates were generated from 293T cells transiently transfected with pAC3-GSG-P2A-GMCSF-GSG-T2A-yCD2 or pAC3-GSG-T2A-yCD 2-GSG-P2A-GMCSF. In this experiment, pAC3-IRES-yCD2 and pAC3-IRES-GMCSF were also included as controls. For GMCSF expression, supernatants from transiently transfected 293T cells were collected for measurement by ELISA (Cat # DGM00, R & D Systems). Whole cell lysates were assayed for expression of yCD2 protein as described. anti-yCD 2 results indicated that yCD2 protein from pAC3-GSG-P2A-GMCSF-GSG-T2A-yCD2 or pAC3-GSG-T2A-yCD2-GSG-P2A-GMCSF was effectively separated from GMCSF as shown by the-15 KDa band. However, in both configurations, separation of yCD2 mediated by the 2A peptide was significantly different from GMCSF (pAC3-GSG-P2A-GMCSF-GSG-T2A-yCD2) or from the viral envelope protein (pAC3-GSG-T2A-yCD2-GSG-P2A-GMCSF), with the yCD2 protein being correctly separated from GMCSF as indicated by the size of yCD2 compared to yCD2 from RRV-IRES-yCD 2. In contrast, the yCD2 protein isolated from viral env has a slightly higher molecular weight, consistent with the RRV-GSG-P2A-GFP, RRV-GSG-T2A-GFP, RRV-GSG-P2A-yCD2, and RRV-GSG-T2A-yCD2 constructs. The data indicate that separation of yCD2 from env may not occur exactly at the theoretically expected amino acid sequence. However, when yCD2 was placed downstream of another secreted protein (i.e., GMCSF), correct isolation of the yCD2 protein was observed. However, it is important to note that the enzymatic activity of the 2A-yCD2 protein expressed by RRV-GSG-P2A-yCD2 and RRV-GSG-T2A-yCD2 does not appear to affect the sensitivity and cytotoxic effects of 5-FC in vitro and in vivo.
Although the efficiency of separation of GMCSF protein from viral envelope protein in the pAC3-GSG-P2A-GMCSF-GSG-T2A-yCD2 construct, or from yCD2 in the pAC3-GSG-T2A-yCD2-GSG-P2A-GMCSF construct, was not determined, GMCSF ELISA results indicated that the amount of GMCSF secreted was-500 ng/mL for RRV-GSG-P2A-GMCSF-GSG-T2A-yCD2, and-760 ng/mL for RRV-GSG-T2A-yCD 2-GSG-P2A-GMCSF. In both cases, GMCSF is expressed in an amount about 20-30 times greater than RRV-IRES-GMCSF (25 ng/mL). At the same time, processing of viral envelope proteins in infected U87-MG was examined using anti-gp 70 antibody. The results show that the viral envelope protein in precursor (Pr85) or processed form (gp70) can be easily detected. Taken together, these data indicate that both the Env-GSG-T2A-GMCSF-GSG-P2A-yCD2 and the Env-GSG-T2A-yCD2-GSG-P2A-GMCSF polyprotein configuration can express GMCSF and yCD2 proteins.
In addition, virus supernatants collected from maximally infected U87-MG cells were titrated as described to ensure that the virus remained infectious. The data show that the titers produced by maximally infected U87-MG cells (-3E 6 TU/mL) are similar to those obtained by transiently transfected HEK293T cells and comparable to RRV-IRES-yCD 2.
Example 15: RRV-GSG-T2A-GMCSF-P2A-yCD2 and RRV-GSG-T2A-yCD2-P2A-GMCSF vectors exhibit 5-FC sensitivity comparable to RRV-IRES-yCD2 infected U87-MG cells.
As described, U87-MG cells maximally infected with RRV-GSG-T2A-GMCSF-GSG-P2A-yCD2 or RRV-GSG-T2A-yCD2-GSG-P2A-GMCSF were used for their determination of 5-FCLD by MTS assay50. RRV-IRES-yCD2 was included as a control. The data indicate LD at 0.008mM for "isolated" yCD2 protein detected in infected U87-MG cells50Cytotoxic effects were achieved at concentrations similar to those of RRV-IRES-yCD 2.
Example 16: RRV-GSG-T2A-GMCSF-RSV-yCD2 and vectors produced by HEK293T cells and maximally infected U87-MG cells were infectious and expressed GMCSF and yCD2 proteins.
pAC3-GSG-T2A-GMCSF-RSV-yCD2 was generated by cloning a chemically synthesized human GM CSF-RSV-yCD2 cassette (Genewiz) with AscI and NotI restriction sites at the 5 'and 3' ends, respectively, into the AscI and NotI restriction enzyme digested pAC3-GSG-T2A-yCD2 backbone. The chemically synthesized GMCSF-RSV-yCD2 cassette contained a stop codon at the 3' end of the GMCSF ORF.
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The following day, 20 hours after cell inoculation, 20 μ g of pAC3-GSG-T2A-GMCSF-RSV-yCD2 plasmid was used for transient transfection using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with DMEM medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-GSG-T2A-GMCSF-RSV-yCD2 from transient transfection of HEK293T cells as described. The data show that the titer (. about.2E 6 TU/mL) of RRV-GSG-T2A-GMCSF-RSV-yCD2 is comparable to RRV-IRES-yCD 2.
In addition, virus supernatants collected from maximally infected U87-MG cells were titrated to ensure that the virus remained infectious. The data show that the titers produced by maximally infected U87-MG cells (. about.2E 6 TU/mL) are similar to those obtained by transiently transfected HEK293T cells and comparable to RRV-IRES-yCD 2.
To assess GMCSF and yCD2 protein expression, cell lysates were generated from RRV-GSG-T2A-GMCSF-RSV-yCD2 infected U87-MG cells. In this experiment, RRV-IRES-yCD2 and RRV-IRES-GMCSF were included as controls. Supernatants from maximally infected U87-MG cells were collected and protein expression levels of GMCSF were measured by ELISA (R & D Systems). Whole cell lysates were assayed for expression of yCD2 protein as described. anti-yCD 2 immunoblot results indicated that the expression level of yCD2 protein from U87-MG cells infected with RRV-GSG-T2A-GMCSF-RSV-yCD2 was about 2-3 fold lower than RRV-IRES-yCD 2. At the same time, processing of viral envelope proteins in infected U87-MG was examined using anti-gp 70 antibody. The results show that the virus envelope protein in precursor (Pr85) or processed form (gp70) can be easily detected. As expected, the viral envelope-GMCSF fusion polyprotein was also detected in cell lysates using anti-gp 70 antibody. Although the separation of GMCSF protein from viral envelope protein was not determined, the GMCSF ELISA results indicated that the amount of secreted GMCSF was-300 ng/mL and about 10-fold greater than RRV-IRES-GMCSF (30 ng/mL). Taken together, these data indicate that the viral envelope protein, GSG-T2A-GMCSF-RSV-yCD2 polyprotein configuration, can produce infectious virus as well as GMCSF and yCD2 proteins in an RRV context.
Example 17: the RRV-GSG-T2A-GMCSF-RSV-yCD2 vector exhibits 5-FC sensitivity comparable to U87-MG cells infected with RRV-IRES-yCD 2.
As has been described in the above-mentioned publication,U87-MG cells maximally infected with RRV-GSG-T2A-GMCSF-RSV-yCD2 vector were used for their 5-FC LD50 determination by MTS assay. In this experiment, RRV-IRES-yCD2 was included as a control. The data indicate LD at 0.010mM for the amount of yCD2 protein expressed in infected U87-MG cells50The cytotoxic effect can be achieved at the concentration, and is equivalent to that of RRV-IRES-yCD 2.
Example 18: RRV-GSG-P2A-yCD2-RSV-PDL1miR30shRNA vector produced by 293T cells and infected U87-MG cells is infectious and expresses yCD2 protein.
pAC3-GSG-T2A-yCD2-RSV-miRPDL1 was generated by cloning a chemically synthesized human yCD2-RSV-miRPDL1 cassette (Genewiz) with AscI and NotI restriction sites present at the 5 'and 3' ends, respectively, into the AscI and NotI restriction enzyme digested pAC3-GSG-T2A-yCD2 backbone. The chemically synthesized yCD2-RSV-mirPDL1 cassette contained a stop codon at the end of the yCD2 ORF.
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The following day, 20 hours after cell inoculation, 20 μ g of pAC3-GSG-T2A-yCD2-RSV-mirPDL1 plasmid was used for transient transfection using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with DMEM medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-GSG-T2A-yCD2-RSV-mrRPDL1 from transient transfection of HEK293T cells as described. The data show that the titer (. about.2E 6 TU/mL) of RRV-GSG-T2A-yCD2-RSV-mirPDL1 is comparable to that of RRV-IRES-yCD 2.
In addition, virus supernatants collected from maximally infected U87-MG cells were titrated to ensure that the virus remained infectious. The data show that the titers produced by maximally infected U87-MG cells (-2E 6 TU/mL) are similar to those obtained from transiently transfected HEK293T cells and comparable to RRV-IRES-yCD 2.
To measure expression of the yCD2 protein and PDL1 cell surface expression, maximally infected U87-MG cells were harvested and whole cell lysates were assayed for yCD2 protein expression as described. anti-yCD 2 immunoblot results showed that yCD2 protein from U87-MG cells infected with RRV-GSG-T2A-yCD2-RSV-mirPDL1 was efficiently separated from viral envelope proteins as indicated by the-15 kDa band using anti-yCD 2 antibody. As expected, the viral envelope-yCD 2 fusion polyprotein was also detected in cell lysates using anti-yCD 2 and anti-gp 70 antibodies. At the same time, processing of viral envelope proteins in infected U87-MG was examined using anti-gp 70 antibody. The results show that the virus envelope protein in precursor (Pr85) or processed form (gp70) can be easily detected. In addition, the fusion polyprotein was detected as seen in the anti-yCD 2 immunoblot.
Example 19: RRV-GSG-T2A-yCD2-RSV-mirPDL1 infected U87-MG cells showed 5-FC sensitivity comparable to RRV-IRES-yCD2 infected U87-MG cells.
U87-MG cells maximally infected with RRV-GSG-T2A-yCD2-RSV-mirPDL1 vector were used for their 5-FC LD determination by MTS assay as described50. In this experiment, RRV-IRES-yCD2 was included as a control. The data indicate that the amount of "isolated" yCD2 protein detected in infected U87-MG cells is at an LD comparable to RRV-IRES-yCD250Cytotoxic effects were achieved at concentrations (0.008 mM).
Example 20: RRV-GSG-P2A-yCD2-RSV-mirpDL1 infected MDA-MB231 cells showed potent PD-L1 knockdown on the cell surface.
To evaluate the PDL1 knockdown activity of RRV-GSG-T2A-yCD2-RSV-miRPDL1, MDA-MB231 cells that have been shown to express significant levels of PDL1 were infected with an MOI of 0.1. In this experiment, RRV-RSV-mirPDL1 was included as a positive control to evaluate PDL1 knockdown activity. At approximately day 14 post-infection, cells were harvested and cell surface stained to measure the level of PDL1 protein by FACS. The data show that cell surface expression of PDL1 in MDA-MB231 cells infected with RRV-GSG-T2A-yCD2-RSV-miRPDL1 is reduced by about 75% and is comparable to RRV-RSV-miRPDL 1. Taken together, these data indicate that the viral envelope protein, GSG-T2A-yCD2-RSV-mirPDL1 configuration, can produce infectious virus, yCD2 protein and mirPDL1 in an RRV context.
Example 21: RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO vectors produced by HEK293T cells and maximally infected U87-MG cells are infectious and express TKO protein
pAC3-P2A-TKO, pAC3-GSG-P2A-TKO, pAC3-T2A-TKO, and pAC3-GSG-T2A-TKO were generated by cloning Sr39-tk (Black et al, Cancer Res.,61: 3022-3026, 2001; Kokoris et al, Protein Science 11: 2267-2272, 2002) having the human codon optimized (TKO) (see International application publication No. WO2014/066700, incorporated herein by reference) cassette into the pAC3-2A backbone. The sequence of TKO is chemically synthesized (Genewiz) with AscI and NotI restriction sites at the 5 'and 3' ends, respectively, cloned into the backbone pAC3-GSG-P2A-yCD2 or pAC3-GSG-T2A-yCD2 digested with AscI and NotI restriction enzymes.
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The next day, 20 hours after cell inoculation, 20 μ g of pAC3-GSG-P2A-TKO or pAC3-GSG-T2A-TKO plasmid was used for transient transfection using the calcium phosphate method. Eighteen hours after transfection, cells were washed three times with DMEM medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO, and RRV-GSG-T2A-TKO from transient transfection of HEK293T cells as described. The data show that the titers are comparable to those of RRV-IRES-yCD2 (Table G).
Table G: titers of RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO vectors produced from HER293T cells
In addition, virus supernatants collected from maximally infected U87-MG cells were titrated as described to ensure that the virus remained infectious. The data show that the maximally infected U87-MG cells produced titers comparable to those obtained from transiently transfected HEK293T cells.
To assess TKO protein expression, cell lysates were generated from RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO, and RRV-GSG-T2A-TKO infected U87-MG cells. TKO protein expression of whole cell lysates was determined using an anti-HSV-tk antibody (Cat # sc28037, Santa Cruz Biotech Inc) at 1: 200. The results show that the isolation efficiency of TKO proteins from U87-MG cells infected with RRV-P2A-TKO and RRV-T2A-TKO was lower than that of RRV-GSG-P2A-TKO and RRV-GSG-T2A-TKO, as previously seen with the GFP and yCD2 transgenes.
Example 22: the RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO vectors are stable in U87-MG cells.
To evaluate the stability of the vector in maximally infected U87-MG cells, genomic DNA was extracted from the cells using the Promega Maxwell 16 cell DNA purification kit (Promega). Then one hundred nanograms of genomic DNA were used as template for PCR with primer pairs spanning the transgene cassette; IRES-F (5'-CTGATCTTACTCTTTGGACCTTG-3' (SEQ ID NO:23)) and IRES-R (5'-CCCCTTTTTCTGGAGACTAAATAA-3' (SEQ ID NO:24)) as previously described. The expected PCR product for all RRV-2A-TKO constructs was 1.4 kb. The data indicate that the 2A-TKO and GSG-2A-TKO regions in the proviral DNA RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO vectors are stable in U87-MG cells during the time of viral replication.
Example 23: RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO infected U87-MG cells showed better GCV sensitivity than RRV-S1-TKO
Maximal infection of U87-MG cells with RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO, the GCV LD was determined by MTS assay50. RRV-S1-TKO was included as a control, with TKO expression driven by a synthetic minimal promoter (see International patent publication No. WO2014/066700, incorporated herein by reference). Treatment with GCV (cat #345700-50MG, EMD Millipore) was performed at 0.0001. mu.M-0.5. mu.M of a series of 1:2 dilutions. GCV treatment was not included as a control. GCV was added 1 day after plating, and then complete medium supplemented with GCV every 2 days. Initial U87-MG cells were included as controls to determine the cytotoxic effects of GCV. Cells were monitored over a7 day incubation period and cell death was measured every 2 days by using the CellTiter 96Aqueous single solution cell proliferation assay system (Promega). After addition of MTS, Infinite M20 was used at 60 min MTS incubationA0 (Tecan) plate reader gave OD values at 490 nm. The mean OD values for each sample in triplicate were converted to percent survival relative to untreated but RRV infected cells. Percentage values were plotted against GCV concentration in a logarithmic scale using GraphPad Prim to generate LD50Figure (a). Calculating LD by software using non-linear four-parameter fitting of acquired data points50The value is obtained. The data indicate that the TKO proteins expressed by RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO are enzymatically active in converting GCV into cytotoxic GCV in the tenth millimolar range, thus achieving cytotoxic effects. RRV-P2A-TKO, RRV-GSG-P2A-TKO, RRV-T2A-TKO and RRV-GSG-T2A-TKO show 12.5-20 times higher GCV sensitivity compared to RRV-S1-TKO. Furthermore, GCV LD despite differences in the separation between TKO and Env-TKO fusion polyproteins50There was no significant difference between RRV-P2A-TKO versus RRV-GSG-P2A-TKO or RRV-T2A-TKO versus RRV-GSG-T2A-TKO. Similar to 2A-yCD2, the data indicate that the amount of TKO protein expressed in the cells is sufficient to convert GCV to cytotoxic GCV.
Example 24: the RRV-GSG-P2A-TKO and RRV-GSG-T2A-TKO subcutaneously treated syngeneic glioma mice showed tumor growth delay comparable to RRV-IRES-yCD 2.
In B6C3F1 mice, the homologous cell line Tu-2449 was used as an orthotopic brain tumor model (Ostertag et al, 2012). A Tu-2449 cell subline for a subcutaneous tumor model was established in Tocagen (Tu-2449 SQ). A mixture of 98% of the original Tu-2449SQ cells and 2% of RRV-GSG-P2A-TKO, RRV-GSG-T2A-TKO or RRV-S1-TKO infected Tu-2449SQ cells was prepared in vitro and resuspended in phosphate buffered saline (PBS; Hyclone) for subcutaneous tumor implantation. A mixture of 98% of initial Tu-2449SQ cells and 2% RRV-IRES-yCD2 infected Tu-2449SQ cells was included as a positive control as well as a comparator. B6C3F1 mice in each group (n ═ 10 per group) were implanted subcutaneously at day 0 by 1 × 106And (4) tumor cells. On day 12 after tumor implantation (at which time, approximately > 75% of tumors were infected with RRV), mice were administered PBS, 5-FC (500mg/kg body weight/dose, intraperitoneal) or GCV (50mg/kg body weight/dose, intraperitoneal, twice daily) for 5 consecutive days, followed by 2 days without drug to allow vehicle to follow the remaining timeSpread among infected cells. The cycle of 5 more days administration, 2 days without drug treatment was repeated twice. Tumor volume measurements were taken daily. The results show that mice bearing tumors bearing RRV-GSG-P2A-TKO, RRV-GSG-T2A-TKO or RRV-S1-TKO without GCV or RRV-IRES-yCD2 continued to grow without 5-FC treatment. In contrast, RRV-GSG-P2A-TKO, RRV-GSG-T2A-TKO + GCV treated tumor-bearing mice delayed tumor growth of pre-established tumors. Furthermore, tumor-bearing mice treated with RRV-S1-TKO + GCV also exhibited a delay in tumor growth, but to a lesser extent and for a longer time than tumors treated with RRV-GSG-P2A-TKO, RRV-GSG-T2A-TKO + GCV, probably due to decreased expression of TKO. Taken together, the data indicate that the tumor growth delays of RRV-GSG-P2A-TKO + GCV and RRV-GSG-T2A-TKO + GCV are comparable to those treated with RRV-IRES-yCD2+ 5-FC. The data indicate that RRV-GSG-P2A-TKO and RRV-GSG-T2A-TKO have comparable therapeutic efficacy to RRV-IRES-yCD2 in a subcutaneous synglioma mouse model.
Example 25: RRV-GSG-T2A-PDL1scFv and RRV-GSG-T2A-PDL1scFvFc vectors produced by HEK293T cells and most infected U87-MG cells were infectious and expressed scFv and scFvFc proteins.
pAC3-T2A-PDL1scFv, pAC3-T2A-PDL1 scFv-tag, pAC3-T2A-PDL1scFvFc and pAC3-T2A-PDL1 scFvFc-tag were generated to act as blocking single chain variable fragments (scFv) against human and mouse PDL 1. Designed PDL1scFv cassettes with or without human IgG1A fragment crystallizable (Fc) region of (2). Furthermore, a matching cassette with HA and Flag epitope tags incorporated into the C-terminus of scFv or scfvffc was also generated for detection of scFv or scfvffc protein expression. The sequence of each cassette (PDL1scFv, PDL1 scFv-tag, PDL1scFvFc and PDL1 scFvFC-tag) was chemically synthesized (Genewiz), with AscI and NotI restriction sites present at the 5 'and 3' ends, respectively, and cloned into the pAC3-GSG-T2A-yCD2 backbone digested with AscI and NotI restriction enzymes.
HEK293T cells were seeded at 2e6 cells per 10cm plate 18 to 20 hours prior to transfection. The next day, 20 μ g of pAC3-T2A-PDL1scFv, pAC3-T2A-PDL1 scFv-tag, pAC3-T2A-PDL1scFvFc, and pAC3-T2A-PDL1 scFvFc-tag plasmids were used for transient transfection using the calcium phosphate method 20 hours after cell inoculation. Eighteen hours after transfection, cells were washed three times with DMEM medium and incubated with fresh complete medium. Approximately 42 hours post transfection, virus supernatants were collected and filtered through a 0.45 μm syringe filter. Viral titers were determined for RRV-GSG-T2A-GMCSF-GSG-P2A-yCD2 from transient transfection of HEK293T cells as described. The data show that the titer values of RRV-GSG-T2A-PDL1scFv, RRV-GSG-T2A-PDL1scFvFc, RRV-GSG-T2A-PDL1 scFv-tag, RRV-GSG-T2A-PDL1 scFvFc-tag are comparable to RRV-IRES-yCD2 (Table H).
Table H: titer values for RRV-GSG-T2A-PDL1scFv, RRV-GSG-T2A-PDL1scFvFc, RRV-GSG-T2A-PDL1 scFv-tag, RRV-GSG-T2A-PDL1 scFvFc-tag from transiently transfected HEK293T cells
TU/mL
Std Dev
RRV-PDL 1scFv
2.09E+06
4.80E+05
RRV-PDL 1scFv Fc
1.98E+06
4.38E+05
RRV-PDL 1scFv-Tag
2.08E+06
6.73E+05
RRV-PDL 1scFv Fc-Tag
1.29E+06
1.87E+05
To evaluate scFv protein expression, cell lysates were generated from HEK293T cells transfected with RRV-GSG-T2A-PDL1scFv and RRV-GSG-T2A-PDL1 scFvFc. Whole cell lysates were assayed for scFv protein expression using anti-Flag and anti-HA antibodies (Cat #1804 and Cat # H3663, Sigma Aldrich) at 1:1,000. The results show that PDL1 scFv-tag and PDL1 scfvffc-tag protein expression from HEK293T cells transiently transfected with RRV-GSG-T2A-PDL1 scFv-tag, RRV-GSG-T2A-PDL1 scffc-tag was separated from Env-scFv polyprotein as previously seen with GFP and yCD2 and TKO transgenes (fig. 4A).
At the same time, the processing of viral envelope proteins in HEK293T cells was examined with anti-2A antibodies. The results showed that virus coated in either precursor (Pr85) or processed form (p15E) containing the 2A peptide sequence was detected in all 4 vectors (fig. 4B), indicating that the viral envelope protein was separated from the scFv and scfvffc proteins as seen in anti-Flag and anti-HA immunoblots. Although expression of the fusion polyprotein Env-scFv or Env-scfvffc was detected in cell lysates, significant amounts of PDL1scFv and PDL1 scfvffc protein were separated from the fusion polyprotein as shown by immunoblotting from cell lysates and supernatants.
Similarly, abundant scFv-tag and scfvffc-tag protein expression was also detected in the supernatant of transiently transfected HEK293T cells by immunoprecipitation with anti-Flag antibody followed by detection with anti-HA, and vice versa. In addition, cell lysates and supernatants from maximally infected MDA-MB231 (human breast cancer cell line) and CT26 (murine colorectal cancer cell line) cells were also tested for scFv-tag and scFvFc-tag protein expression at levels approximately 2-3 fold lower than transiently transfected HEK293T cells.
Example 26: RRV-GSG-T2A-PDL1scFv and RRV-GSG-T2A-PDL1scFvFc restored PHA-stimulated T cell activation and showed the equivalence of PDL1 blocking antibodies in vitro.
To determineWhether PDL1 blockade of tumor cells by RRV-GSG-T2A-PDL1scFv or RRV-GSG-T2A-PDL1scFvFc could alleviate PDL 1-mediated T cell inhibition, we performed a PDL 1-mediated trans-inhibition co-culture experiment. Here, we evaluated whether modulating PDL1 expression on various tumor cell lines could alter PHA-stimulated activation of healthy donor PBMCs as measured by intracellular expression of IFN γ or release of IFN γ into the supernatant. To eliminate the potential pleiotropic effects of IFN γ pretreatment in the trans-suppression co-culture assay, we established a co-culture system using the human breast cancer cell line MDA-MB-231 with high PDL1 basal cell surface expression level. To confirm that PDL1 is required to be involved in this assay, an anti-PDL 1 blocking antibody was also included. PDL1 in the presence of anti-PDL 1 blocking antibodies+Tumor cells MDA-MB-231 cells failed to inhibit activation of CD8+ T cells, as indicated by an increased frequency of IFN γ +/CD8+ T cells. Similarly, MDA-MB-231 cells infected with RRV-GSG-T2A-scFv or RRV-GSG-T2A-scFvFc similarly restored CD8+T cell activation. The data indicate that disruption of the PDL1 on tumor cells and lymphocytes by PDL1 blocking scFv the PD1 axis shows comparable activity to anti-PDL 1 blocking antibodies and provides evidence demonstrating substantial immunological benefit of RRV-GSG-T2A-PDL1scFv and RRV-GSG-T2A-PDL1 scfvffc.
Example 27: RRV, TOCA-511, mutation profile.
A variety of tumor types are variably able to support rapid replication of RRV, and this variability may alter the sensitivity of different tumors to RRV-based therapeutic treatments, such as RRV Toca 511 (aka T5.0002) and prodrug Toca FC treatment of high grade glioma (t.f. cloughsey et al, Sci trans med.,8(341):341ra75, June 1,2016, doi: 10.1126/sciitranslmed. aad 9784). This variability can be attributed to a variety of factors, but from the sequencing data we have recovered RRV encoding the modified yeast cytosine deaminase from patients' blood or tumors, it appears to be relevant to changes in the function of APOBEC, particularly APOBEC3B and APOBEC3G (B.P.Doehle et al, J.Virol.79:8201-8207, 2005). The alteration in expression is deduced from the frequency with which inactivating or attenuating mutations accumulate in the replicating retroviral vector when it is progressively replicated in tumour tissue. Studies have shown that one of the most frequent events is a G to a mutation, which corresponds to the C to T transition characteristic of APOBEC-mediated mutations on the negative strand single stranded DNA of the first replication step in the reverse transcription step. These mutations can cause changes in the amino acid composition of the RRV protein, such as a disruptive change from TGG (tryptophan) to a stop codon (TAG, TGA or TAA). It has been shown that some tumors (especially bladder, cervical, lung (adenocarcinoma and squamous cell), head and neck and breast, APOBEC3B activity is upregulated and that this upregulation is associated with increased mutation load, these changes being consistent with APOBEC3B activity (mb. burns et al, Nature Geneti cs 45:977-, toca 511 is susceptible to mutations due to reverse transcription errors and cellular antiviral defense mechanisms (e.g., APOBEC-mediated cytidine deaminase). The APOBEC protein targets single-stranded DNA, primarily during reverse transcription of the Toca 511RNA genome, as represented by the G to a site.
The Toca 511 sequence mutation profile was plotted by high throughput sequencing of Toca 511 from clinical samples isolated from tumors and blood. The G to a point mutation is the most common type of mutation in Toca 511, consistent with APOBEC activity. This is the first characterization of the gamma-retroviral gene therapy mutation profile from human samples by high throughput sequencing. Analysis of G to a mutations shows that these mutations often result in non-synonymous changes in the coding sequence. Within the gene encoding the cytosine deaminase polypeptide, there are two positions with recurrent G to a mutations in samples from multiple patients (table I). These mutations convert the codon TGG encoding tryptophan to a TGA, TAG or TAA stop codon, thereby terminating CD translation after only nine amino acids. These results highlight that tryptophan codons are a potential source of retroviral gene therapy inactivation.
TABLE I summary of point mutations in recombinant cytosine deaminase of Toca 511 (SEQ ID NO: 28-29). Positions are amino acid positions within the CD protein. The sample indicates the number of clinical samples from blood or tumors that show mutations. Codons and changes show the original codon sequence and subsequent changes. AA is the original amino acid encoded by the original codon, and the change shows what the amino acid became after the codon was mutated.
Thus, altering a tryptophan codon to a replacement codon that encodes an amino acid compatible with protein function may mitigate APOBEC-mediated inactivation of retroviral gene therapy.
To test the effect of mutations on stability, the Toca 511 genomic sequence (see, e.g., U.S. patent No.8,722,867, SEQ ID NOs 19, 20, and 22 of the' 867 patent, which are incorporated herein by reference) was engineered to change codons that show ApoBec hypermutation to codons that encode alternative amino acids that maintain stability and function (e.g., to change the codon for tryptophan to some other allowed amino acid). The Toca 511 polypeptide having cytosine deaminase activity (see SEQ ID NO: 29) is closely related to the naturally occurring fungal cytosine deaminase protein and a high resolution structure of this cytosine deaminase can be obtained. Thus, combinations of structures and multiple sequence alignments from phylogenetically diverse fungal CD proteins can be used to identify potential amino acid substitutions that do not have an adverse effect on biological function, for example using ROSETTA, Provean, PSIpred, or similar programs. A set of putative amino acid substitutions was then tested by altering the To ca 511 genome and measuring enzyme and biological activity, solubility, thermostability in solution, and ability To function in cell culture assays and mouse tumor models (e.g., 5-FC conversion To 5-FU, initiation of cell death, and activation of an immune response against the tumor To achieve a sustained response). Similar analysis can be applied to GAG, POL and ENV sequences to modify these sequences to remove codons susceptible to ApoBec hypermutation.
Example 28: the APOBEC-resistant yCD viral vector is therapeutic in intracranial human xenografts in nude mice (T98G).
An intracranial xenograft model using a T98G human glioma cell line highly expressing APOBEC was established to test RRV vector spread and biodistribution and therapeutic efficacy of APOBEC-resistant RCR-vector mediated cytosine deaminase suicide gene therapy in nude mouse hosts under conditions of high APOBEC activity.
After acclimation, mice were randomly assigned to one of 9 treatment groups (see group description below). Eight groups 1X 10 per mouse on day 05Right striatum of individual T98G cells was administered intracranial. Group 9 mice were not tumor implanted. On day 5, mice were injected with 9X10 only5TU/5. mu.l of formulation buffer, 9X105TU/5. mu.l of T5.0002 (APOBEC-sensitive RRV expressing yCD; group 3), or 9X105TU/5μl、9x104TU/5. mu.l or 9X103Tu/5. mu.l of APOBEC-resistant RCR vector (T5.002A). Randomized 5-FC administration was performed at 500 mg/kg/day as a single IP injection administration, starting on day 19, or some groups were not administered 5-FC (groups 1, 4, 8). Mice receiving the medium dose of vehicle all received 5-FC (i.e., the dose did not have a separate control group). 5-FC was administered daily for 7 consecutive days, followed by 15 days of no treatment. The drug plus rest cycle was repeated until 4 cycles. 10 mice from each group, except group 8, were randomly assigned to the survival analysis category. The remaining mice were sacrificed according to a predetermined schedule.
Component dispensing and dosage levels
Intravenous administration was by injection into the tail vein. Intraperitoneal administration is performed by injection into the abdomen, taking care to avoid the bladder. For intracranial injection, mice were anesthetized with isoflurane and placed in a stereotaxic device with blunt ear stems. The skin was shaved and the scalp was treated with biot iodine to prepare the surgical site. Animals were placed on a heating pad and a scalpel was used under sterile conditions to make a midline incision through the skin. The contraction of the skin and the reflection of the fascia at the incision site will allow visualization of the skull. A guide cannula with a 3mm protrusion was mated with a cap with a 3.5mm protrusion, inserted through a small bore hole in the skull and attached to the skull with dental cement and three small screws. After the cement has hardened, the skin is closed with sutures. The projected stereotactic coordinate is AP ═ 0.5-1.0mm, ML ═ 1.8-2.0mm, DV ═ 3.0 mm. In pilot experiments (2-3 animals), accurate stereotactic coordinates of groups of animals were determined by injecting dye and determining its position. Animals were monitored during recovery from anesthesia. The analgesic buprenorphine is administered Subcutaneously (SC) prior to the end of the procedure, and then approximately every 12 hours for up to 3 days. Animals were monitored daily. Cells or vectors were infused intracranially through an injection cannula, where a 3.5mm protrusion was inserted through a guide cannula. The rate was controlled with a syringe pump equipped with a Hamilton syringe and flexible tubing. For cell injection, 1 microliter of cells was delivered at a flow rate of 0.2 microliter per minute (5 minutes total). For vehicle injections, 5 microliters of vehicle was delivered at a flow rate of 0.33 microliters/minute (15 minutes total).
The APOBEC-resistant vector was delivered to mice and calculated as Transformation Units (TU) per gram brain weight. Using this calculation, the dose conversion can be calculated for other mammals, including humans. The APOBEC-resistant vectors showed an effective dose response, while vectors sensitive to APOBEC activity showed a diminished effective response. The same experiment was performed in a U87 cell line transplanted into a xenograft model, transfected with expression vectors for human APOBEC3G or APOBEC3B, expressing these proteins at a level at least 3-fold higher than the native level of U87. These experiments indicate that the modified codon virus designed to be APOBEC-resistant has a replication and/or therapeutic response advantage in line U87 with increased levels of APOBEC over the original RRV without APOBEC resistance codon modification.
Example 29: the APOBEC-resistant yCD viral vector is therapeutic in an isogenic mouse model of brain cancer.
Additional experiments were performed to demonstrate the methods and compositions of the present disclosure in syngeneic animal models.
An intracranial implant model was established to generate murine APOBEC3 using stably transfected CT26 colorectal cancer cell line in syngeneic BALB/c mice to test the spread and biodistribution of the APOBEC-resistant RRV vector and the therapeutic efficacy of RRV-vector mediated cytosine deaminase suicide gene therapy and its immunological impact.
The study included 129 animals, 0 males, 119 females and 10 sporadic animals (10 females). After acclimation, mice were randomly assigned to one of 9 treatment groups (see group description below). Eight groups 1X 10 per mouse on day 04APOBEC-right striatum expressing CT26 cells was administered intracranial. Group 9 mice were not tumor implanted. On day 4, mice were injected with formulation buffer alone, 9X105TU/5. mu.l of control vector still sensitive to APOBEC (T5.0002), or 9X105TU/5μl、9×104TU/5. mu.l or 9X103TU/5. mu.l of APOBEC-resistant vector (T5.0002A). Mice that did not receive the vector, or that received 9X105TU/5. mu.l or 9X103Mice with TU/5. mu.l of vector received 5-FC (500mg/kg/BID) randomly, beginning on day 13 by IP injection, or no 5-FC (PBS) administration as indicated. Mice receiving the medium dose of vehicle received 5-FC (i.e., the dose did not have a separate control group). 5-FC was administered daily for 7 consecutive days, followed by 10 days of no treatment. The drug plus rest cycle was repeated until 4 cycles. 10 mice from each group, except group 9, were randomly assigned to the survival analysis category. The remaining mice were sacrificed according to a predetermined schedule.
Initial sentinel mice were co-housed with pre-assigned sacrificed animals and removed at the same time point to assess the delivery of the vector by shedding.
Component dispensing and dosage levels
Intravenous administration was by injection into the tail vein. Intraperitoneal administration is performed by injection into the abdomen, taking care to avoid the bladder. For intracranial administration, mice with a guide cannula implanted with a 3.2mm protrusion of the right striatum were used, and a cap with a 3.7mm protrusion was used. The projected stereotactic coordinates are AP-0.5-1.0 mm, ML-1.8-2.0 mm, DV-3.2 mm (from bregma). Cells or vectors were infused intracranially through an injection cannula, where a 3.7mm protrusion was inserted through a guide cannula. The rate was controlled with a syringe pump equipped with a Hamilton syringe and flexible tubing.
For cell injection, 1 microliter of cells was delivered at a flow rate of 0.2 microliter/minute (5 minutes total). For vehicle injections, 5 microliters of vehicle was delivered at a flow rate of 0.33 microliters/minute (15 minutes total).
Vectors were delivered to mice and calculated as Transformation Units (TU) per gram of brain weight. Using this calculation, the dose conversion can be calculated for other mammals, including humans. The results of this study indicate that APOBEC-resistant virus spread throughout the tumor, maintained yCD integrity, and was more effective in treating tumors in combination with 5FC when compared to APOBEC-sensitive RRV. APOBEC-resistant RRV also did not diffuse horizontally to the original cage partner.
As noted above, RRV contains the "2A cassette". For example, SEQ ID NO: 2. 43-53 and 54 provide general constructs containing the 2A cassette. The cartridge may be replaced with a number of different cartridges. For example, the following cassettes can be prepared and cloned into SEQ ID NO: 2. 43-53 or 54 in any of the vector backbones, the cassette in those particular constructs is replaced.
Using the methods and sequences provided herein, a number of vectors were designed as follows:
pAC3-T2A-GFPm(SEQ ID NO:43)
pAC3-GSG-T2A-GFPm(SEQ ID NO:44)
pAC3-P2A-GFPm(SEQ ID NO:45)
pAC3-GSG-P2A-GFPm(SEQ ID NO:46)
pAC3-E2A-GFP(SEQ ID NO:47)
pAC3-GSG-E2A-GFPm(SEQ ID NO:48)
pAC3-F2A-GFPm(SEQ ID NO:49)
pAC3-GSG-F2A-GFPm(SEQ ID NO:50)
pAC3-T2A-yCD2(SEQ ID NO:51)
pAC3-GSG-T2A-yCD2(SEQ ID NO:52)
pAC3-P2A-yCD2(SEQ ID NO:53)
pAC3-GSG-P2A-yCD2(SEQ ID NO:54)
example 30: secretion of scFv PD-L1 lacking the signal peptide sequence can be achieved by insertion of a heterologous signal peptide at the N-terminus.
Construction of RRV-scFv-PDL1 plasmid DNA. Two pairs of single chain variable fragments (scFv) of two different configurations were designed for PD-L1. A pair consisting of scFv with and without Fc from human IgG1 was named scFv-PDL1 and scFvFc-PDL1, respectively. The other pair consisted of scFv-PDL1 and ScFvFc-PDL1, named scFv-HF-PDL1 and scFvFc-HF-PDL1, which incorporated HA and Flag epitopes at the C-terminus. The coding sequence for each configuration contained the 3' coding sequence of the viral envelope gene, followed by the gT2A peptide sequence and synthesized with Asc I and Not I restriction sites for subcloning into pAC3-gT2A-yCD2 at the corresponding sites to replace the g2A-yCD2 transgene cassette, yielding pAC3-scFv-PDL1, pAC3-scFvFc-PDL1, pAC3-scFv-HF-PDL1, pAC3-scFvFc-HF-PDL 1. For all scFv-PDL1 variants, a signal peptide from human IL-2 was incorporated at the N-terminus to allow secretion of scFv PDL 1.
Expression and correct processing of scFv PD-L1 encoded in the RRV-2A configuration. As shown in FIG. 3, scFv PD-L1 with a heterologous signal peptide can be expressed in a manner different from the 2A sequence, for example using an IRES sequence or a small promoter, and a vector expressing the secretable form of scFv PD-L1 is obtained. However, we describe here the RRV configuration, which utilizes virus-derived "self-cleaving" 2A peptides for transgene expression, demonstrating that the RRV-2A configuration can tolerate transgene insertions up to 1.2 kb. In this study, we designed two different configurations of single-chain variable fragments (scFv) against PD-L1. One consisted of scFv alone and the other consisted of scFv with Fc from human IgG1, designated pAC3-scFvP DL1 and pAC 3-scfvffc-PDL 1, respectively. In the absence of antibodies to scFv PD-L1 protein, we generated a matched pair of constructs with HA and FLAG epitopes incorporated at the C-terminus of the transgene, designated pAC3-scFv-HF-PDL1 and pAC3-scFvFc-HF-PDL1 (FIG. 3).
Transgenes targeting different cellular compartments are encoded in frame with the viral envelope (Env) protein of the RRV-2A configuration, effectively separated from the Env-transgenic polyprotein (Hofacre et al, 2018). Since both the epitope-tagged and unlabeled scFv PD-L1 and scfvffc PD-L1 proteins were designed to be separated from the viral Env protein and secreted by the cells, we used a transient transfection system to highly overexpress the transgenic proteins to help detect the epitope-tagged scFv PD-L1 and scfvffc proteins. Cell lysates of transiently transfected 293T cells were analyzed on SDS-PAGE and detected with anti-HA and anti-Flag antibodies to confirm the presence of scFv PD-L1 and its efficiency of separation mediated by the 2A peptide, respectively. In addition, anti-2A antibodies were included to confirm proper processing of viral Env protein from the polyprotein. Figure 5 shows that both scFv-HF PD-L1 and scfvffc-HF PD-L1 were detected and separated from the polyprotein as expected, and that the viral Env protein was correctly processed into its subunits as shown by detection of 15E-2A. The detection of residual, unseparated polyprotein is also expected, since the cell lysate comes from a transient transfection system, where the protein is highly overexpressed, and it has been shown previously that such unseparated polyprotein is not incorporated into the virion. In addition, Western detection of intracellular epitope-tagged scFv PD-L1 suggested that the protein may not reach maximal secretion.
scFv PD-L1 and scFvFc PD-L1 secreted by RRV-scFv-PDL1 and RRV-scFvFc-PDL1 infected cells competed with PD-1 for PD-L1 binding. After confirming transgene protein expression and viral function of RRV-scFv-PDL1 and RRV-scFvFc-PDL1, we evaluated the binding characteristics of scFv PD-L1 and scFvFc PD-L1. The potency of scFv PD-L1 and scFvFc PD-L1 proteins to block the PD-1/PD-L1 interaction was evaluated using an ELISA-based competition assay to quantify the conservation following co-incubation of PD-1 with scFv PD-L1 or scFvFc PD-L1Amount of His-tagged PD-1 that binds to PD-L1. Although the concentrations of scFv PD-L1 and scFvFc PD-L1 in the supernatant were not clear, they bound specifically to human PD-L1 and mouse PD-L1 in a dose-dependent manner. The level of inhibition using 100 μ L of supernatant was comparable to that of blocking antibody control, with no significant difference between scFv PD-L1 and scffc PD-L1 (figure 6A). The potency of scFv PD-L1 and scffc PD-L1 in blocking mouse PD-1/PD-L1 interaction appeared to be effective, although slightly less than the human counterpart, but more than the anti-mouse PD-L1 antibody control (fig. 6B). We further evaluated the binding kinetics of scFv PD-L1 to human and mouse PD-L1 using a surface plasmon resonance system. The scFv PD-L1 cDNA was cloned into a CMV-driven expression vector for transient transfection, followed by purification to achieve > 85% purity. Equilibrium dissociation constant (K) for scFv PD-L1 for recombinant human PD-L1 and mouse PD-L1D) Determined as 0.426nM and 4.78nM, respectively, Table J. Due to slower KoffResulting in about 10-fold higher binding affinity for human PD-L1, which may explain the higher potency of scFv PD-L1 to block human PD-1/PD-L1 interaction observed in competitive ELISA, despite the fact that human and mouse PD-L1 have close to 80% homology in their amino acid sequences.
TABLE J
Example 31: scFv PD-L1 secreted by RRV-scFv-PDL1 infected cells showed bystander trans-binding activity to PD-L1 on the cell surface. Since none of the current virus-based therapies (including RRV) can infect 100% of patient tumor cells in situ, we designed a secreted transgene product with the ability to bind PD-L1 on neighboring uninfected cells. Here, we confirmed antigen-specific binding of scFv PD-L1 or scffc PD-L1 by flow cytometry using a cell-based assay. In this experiment, we used epitope-tagged scFv PD-L1 and scfvffc PD-L1(scFv-HF PD-L1 and scfvcr-HF PD-L1), followed by anti-HA antibody, due to the lack of antibodies to detect the presence of bound scFv PD-L1 and scfvffc PD-L1 on the cell surface. These data show that scFv-HF PD-L1 and scfvcfc-HF PD-L1 bind to PD-L1 expressed on the cell surface in human and mouse cell lines, as shown by a significant shift in Mean Fluorescence Intensity (MFI) with anti-HA antibodies. The higher shift in MFI observed with scffc-HF PD-L1 in the tested human and mouse cell lines was likely due to dimer formation of the bivalent dimer of scffc-HF PD-L1 by disulfide bond formation between Fc regions, thus reflecting only more anti-HA antibody binding to scffc-HF PD-L1 on the cell surface, rather than increased binding affinity, as scffc PD-L1 did not compete more efficiently than scFv PDL1 in ELISA (fig. 5). Furthermore, when co-incubated with anti-HA antibodies, antigen binding specificity was demonstrated by blocking the accessibility of the anti-PD-L1 blocking antibodies to cell surface PD-L1, resulting in a significant decrease in MFI with the anti-PD-L1 antibody. Consistent with the data observed in the competitive ELISA, scFv-HF PD-L1 and scfvffc-HF PD-L1 specifically bound PD-L1 on the cell surface and blocked the binding of anti-PD-L1 antibody to PD-L1, indicating that the epitope of scFv-HF PD-L1 and scfvffc-HF PD-L1 overlaps or is close to the epitope of anti-PD-L1 antibody. Furthermore, a significant reduction in MFI using the anti-PD-L1 antibody also indicates complete receptor (PD-L1) occupancy on the cell surface.
To evaluate the bystander effect of RRV-scFv-PD-L1 in vitro, we tested the minimum level of transduction required to achieve complete receptor occupancy on tumor cells. In this experiment, EMT6 mouse breast cancer cells maximally infected with RRV-scFv-HF-PD-L1 were co-cultured in a mixture with EMT6 cells maximally infected with RRV-GFP at different ratios to measure bound scFv-HF PD-L1 and unbound PD-L1 on the cell surface using anti-HA and anti-PD-L1 antibodies. Our data show that when only 5% of the cells expressed scFv-HF PD-L1, bound scFv-HF PD-L1 was detected on all cell surfaces, fig. 7A. Complete occupancy of PD-L1 was inversely correlated with a decrease in PD-L1 signal on the cell surface in a dose-dependent manner (fig. 7B), indicating that scFv PD-L1 could achieve a 100% bystander effect with minimal levels of transduction.
Example 32: treatment with scFv PD-L1 and scffc PD-L1 resulted in tumor growth inhibition in a dose-dependent manner and elicited immune memory responses in syngeneic tumor models. We have demonstrated that in vitro scFv PD-L1 secreted from as low as 5% of pre-transduced cells exhibits bystander trans-binding activity, resulting in complete PD-L1 occupancy on the cell surface of non-scFv PD-L1 expressing cells. Next, we evaluated the dose response of scFv PD-L1 to anti-tumor activity in an isogenic in situ EMT6 breast cancer model, which has been reported to be responsive to checkpoint inhibitors. To evaluate the antitumor activity of scFv PD-L1 and scfvffc PD-L1 in more clinically relevant contexts, we attempted to determine the minimum level of transduction required for scFv PD-L1 to achieve antitumor activity using varying proportions of EMT6 cells maximally pre-transduced with RRV-scFv-PDL1, RRV-scffc-PDL 1, or RRV-GFP vectors. These cells are resistant to further RRV infection mediated via amphotropic envelope proteins due to receptor downregulation. In this experiment, a mixture of EMT6 tumor cells transduced with RRV-scFv-PDL1 or RRV-GFP was implanted into the mammary fat pad of BALB/c mice in the proportions indicated. Survival was monitored for 90 days and a Kaplan-Meier survival assay was performed to evaluate the anti-tumor activity of scFv PD-L1. Mice bearing necrotic tumors were euthanized and the results examined according to animal use protocols (indicated by a tick in fig. 6A; these mice were not scored as dead and not excluded from the figure). Mice bearing tumors expressing the same proportion of either scFv PD-L1 or scffc PD-L1 were grouped together for survival analysis. These data show that mice bearing tumors with 2%, 30% and 100% scFv PD-L1 or scFv Fc PD-L1 expressing tumor cells had a trend of survival benefit, although statistically not significant (figure 8A) (0% scFv/scfvffc versus anti-PD-1, p-0.2529; 0% versus 2%, p-0.2529; 0% versus 30%, p-0.0919; 0% versus 100%, p-0.1674) compared to untreated animals. We further attempted to investigate whether mice surviving the primary tumor established an anti-tumor immune memory response by re-challenging them with initial EM T6 tumor cells on the flank. Figure 8B shows that tumor-cleared mice treated with scFv/scfvffc in the primary setting exhibited moderately delayed tumor growth in the re-challenge setting, indicating that an anti-tumor immune response was established in these mice. In summary, the data indicate that tumor cells expressing either scFv PD-L1 or scffc PD-L1 can result in anti-tumor activity, which appears to be superior to treatment with commercial antibodies.
The Tu-2449SC tumor model was tested in B6C3F1 mice to determine the minimum transduction level required for scFv PD-L1 to exert anti-tumor activity. Figure 8C shows that in the Tu-2449SC tumor model, mice bearing tumors with as low as 2% of Tu-2449SC cells expressing scFv PD-L1 resulted in a delay in tumor progression comparable to anti-PD-1 antibody treatment and showed a strong trend towards dominance when compared to control mice (figure 8C). Tumor progression was completely inhibited using 30% pre-transduced cells, as also seen in mice with tumors of 100% pre-transduced cells.
Example 33: intracranial injection of RRV-scFv-PDL1 prolonged survival in the homologous orthotopic glioma model.
scFv PD-L1 antitumor activity was studied in a previously reported orthologous glioma model responsive to treatment with Toca 511 and Toca FC. Previously established intratumoral RRV delivery methods were employed (Ostertag et al, 2012). RRV-scF v-PDL1 virus function and genomic stability were confirmed in vitro in maximally infected Tu-2449 cells. In this experiment, two different doses of RRV-scFv-PDL1(1E5 and 1E6 TU) were delivered by a single intratumoral injection 4 days after tumor implantation. The data show that a single administration of RRV-scFv-PDL1 of 1E6 TU was equally effective as TU-2449 cells maximally pre-transduced with RRV-scFv-PDL1, which was included as a control and as a comparator (fig. 9A). Consistent with observations made in previous experiments, re-challenging Tu-2449SC tumor cells subcutaneously at distant sites from the primary tumor showed a systemic anti-tumor immune response, resulting in a significant delay in tumor growth compared to the initial mice (fig. 9B). Taken together, these findings indicate that scFv PD-L1 has anti-tumor activity in the glioma tumor model and represents a second glioma mouse model that responds as a monotherapy checkpoint inhibitor.
Example 34: replacement of the IL-2 signal peptide in scFvPD-L1 encoded in RRV-scFv-PDL1 with the signal peptide from cystatin S and the artificial signal peptide AP1 increased the in vitro secretion of scFv PD-L1 protein and enhanced bystander effects and tumor activity in various murine tumor models.
To further increase the bystander effect of scFv PD-L1, which may lead to enhanced anti-tumor efficacy, the IL-2 signal peptide was replaced with a signal peptide from cystatin S and an artificial signal peptide predicted to have high levels of secretion (ASP 1 from table B). In vitro bystander experiments revealed that infected cells expressing epitope-tagged scFv PD-L1 carrying signal peptides from cystatin S (RRV-CSscFv-PDL1) and ASP1(RRV-AP1scFv-PDL1) showed higher trans-binding activity to PD-L1 on neighboring bystander cells. While 5-10% of the RRV-scFv-PDL1 cells were required to saturate all cell surface PD-L1 on the bystander cells, only 2-4% of the RRV-CSscFv-PDL1 infected or RRV-AP1scFv-PDL1 infected cells were required to achieve complete PD-L1 receptor occupancy on the bystander cells.
The Tu2449SC tumor model with 2% pre-transduced tumor was used to compare antitumor activity between tumors infected with RRV-scFv-PD-L1, RRV-CSscFv-PD-L1, and RRV-AP1 scFv-PD-L1. Since 2% transduction levels have previously been shown to be less effective than 30% pre-transduction of tumors infected with RRV-scFv-PD-L1, we expected that the greater bystander effect observed in vitro with RRV-CSscFv-PD-L1 and RRV-AP1scFv-PD-L1 would show greater anti-tumor activity in the 2% pre-transduction setting. Our data revealed that the anti-tumor effect of scFv PD-L1 produced by RRV-CSscFv-PD-L1 and RRV-AP1scFv-PD-L1 infected tumors was significantly higher than that of scFv PD-L1 produced by RRV-scFv-PDL 1. Our data support the annotation that the selection of signal peptides can also modulate protein secretion levels, leading to enhanced antitumor activity.
Example 35: incorporation of an effective signal peptide at the N-terminus of an antigen-specific binding Agent (ASB) derived from a scaffold protein may also be expressed by RRV.
Construction of plasmid DNA RRV-ASB-PDL 1. A pair of ASBs of the same configuration were designed for PD-L1. One consisting of ASB and the other having HA and FLAG epitopes incorporated at the C-terminus, designated ASB-HF-PDL1 and ASB-HF-PDL 1. The coding sequence for each configuration contained the 3' coding sequence of the viral envelope gene, followed by the gT2A peptide sequence, and was synthesized with Asc I and Not I restriction sites for subcloning into the corresponding sites in pAC3-gT2A-yCD2 to replace the g2A-yCD2 transgene cassette, yielding pAC3-ASB-PDL1 and pAC3-ASB-HF-PDL 1. For all ASB-PDL1 variants, a signal peptide from human IL-2 was incorporated at the N-terminus to allow secretion of either ASB PD-L1 or ASB-HF PD-L1.
In vitro bystander experiments showed that infected cells expressing epitope-tagged ASB PD-L1 exhibited trans-binding activity to PD-L1 on adjacent bystander cells, comparable to scFv PD-L1, where 5% RRV-scFv-PDL1 infected cells or 5% RRV-ABS-PDL1 infected cells were required to saturate all cell surface PD-L1 on the bystander cells.
Subsequently, the dose response of the anti-tumor activity of ASB PD-L1 was evaluated in parallel to scFv PD-L1 in an isogenic Tu2449SC subcutaneous model. In vivo data indicate that ASB PD-L1 has anti-tumor activity. Mice bearing tumors with as low as 2% Tu-2449SC cells expressing ASB PD-L1 resulted in a delay in tumor progression comparable to 2% Tu-2449SC cells expressing scFv PD-L1 or anti-PD-1 antibody treatment, but not statistically significant compared to control mice. With 30% of pre-transduced cells, tumor progression was completely inhibited, as also seen in mice bearing tumors with 100% of pre-transduced cells.
Example 36: intracranial injection of RRV-scFv-PDL1-yCD2 prolonged survival in an isogenic orthotopic glioma model. The combination of scFv PD-L1 anti-tumor activity with yCD2 and 5FC was studied to evaluate their synergy in an in situ syngeneic glioma model. A binary vector was designed with a cassette consisting of the human IL-2 signal peptide, scFv-PDL1 linked to gP2A-yCD 2. This fragment was synthesized and cloned into the AscI and NotI sites of the RRV-gT2A backbone. The resulting vector was named pAC3-scFv-PDL1-yCD 2. In vitro characterization data showed that the scFv PDL1 and yCD2 proteins were expressed by RRV-scFv-PDL1-yCD2 infected cells and retained their biological function (i.e., scFv PD-L1 bound PD-L1, and yCD2 converted 5-FC into 5-FU). Purified RRV-scFv-PDL1 and RRV-scFv-PDL1-yCD2 vectors were generated for in vivo studies. In this experiment, 1E5TU was delivered as monotherapy by a single intratumoral injection 4 days after tumor implantation, RRV-scFv-PDL1 (fig. 9A) and RRVscFvPDL1-yCD2 of 1E5TU, which showed suboptimal anti-tumor activity. After allowing viral transmission and anti-tumor activity of scFv PD-L1 for 10 days, mice were then treated once daily with PBS or 5-FC (500mg/kg) IP for 7 days of treatment and 7 days of no treatment. Our data show that RRV-scFv-PDL-yCD2 treated with 5-FC for a single administration of 1E5TU outperformed RRV-scFv-PDL1 and RRV-scFv-PDL-yCD2 treated with PBS. Consistent with what was observed in the previous experiment, re-challenging Tu-2449SC tumor cells subcutaneously at sites distant from the primary tumor showed a systemic anti-tumor immune response, resulting in a significant delay in tumor growth compared to the original mice. Some re-challenged mice were tumor-free for up to 90 days. These data indicate that combination therapy of scFv PD-L1 and yCD2/5FC has better anti-tumor activity than scFv PD-L1 monotherapy in glioma tumor models.
Example 37: RRV-g T2A-Affimer-SQT produced by 293T cells is infectious and expresses Affimer-SQT protein in a secreted form.
The coding region for the SQT variant of Affimer was obtained from Stadler et al (Protein Engineering, Design and Selection,24(9)751-763, 2011). To detect Affimer-SQT protein expression, HA, AU1 and Myc epitopes were inserted at the N-terminus (before the signal peptide), L1 and L2 of Affimer-SQT, respectively. The signal peptide from human IL-2 was placed N-terminal to the Affimer-SQT coding region. DNA fragments were synthesized and cloned into the AscI and Not I sites in the RRV gT2A backbone. The resulting construct was designated pAC3-gT 2A-Affimer-SQT.
HEK293T cells were seeded at 2e6 cells per 10cm plate the day before transfection. The next day, calcium phosphate transfection was performed using 20. mu.g of plasmid DNA. Eighteen hours after transfection, cells were washed twice with DMEM and replaced with complete medium. Approximately 24 hours after media change, virus supernatant was collected and filtered through a 0.45 μm syringe filter. The viral titer of RRV-g T2A-Affimer-SQT was determined as previously described (Perez et al, 2012). Table K shows that the titer of RRV-g T2A-Affimer-SQT produced by HEK293T cells was comparable to that of RRV-GFP.
The Affimer-SQT protein encoded in pAC3-gT2A-Affimer-SQT was designed to be secreted into the supernatant. Due to the uncertainty of the amount of Affimer-SQT protein present in the supernatant, detection of Affimer-SQT protein in the supernatant was performed either by direct immunoblotting of 15. mu.L of the supernatant using an anti-HA antibody (Sigma Cat # H6908, 1:1000), or by immunoblotting with an anti-HA antibody and a HPR-conjugated secondary antibody after immunoprecipitation by incubating 1mL of the supernatant with 10. mu.g of an anti-myc antibody (Abcam Cat # ab206486) for 1618 hours at 4 ℃. FIG. 10 shows that Affimer SQT is expressed in large amounts in the supernatant, with the expected molecular weight of 15 kDa.
Table K: the titer of RRV-gT2A-Affimer-SQT produced by transiently transfected 293T cells.
TU/mL
RRV-GFP
3.36E+6
RRV-gT2A-Affimer-SQT
3.70E+6
Example 38: RRV-gT2A-Hck and RRV-IRES-Hck produced by 293T cells are infectious and express Hck protein.
The coding region for Hck was obtained from WO2017009533A 1. To detect Hck protein expression, Flag and His epitope tags were inserted into the C-terminus of Hck, and a signal peptide derived from human IL-2 was placed N-terminal to the Hck coding region. DNA fragments with AscI and Not I sites were synthesized and cloned into the AscI and Not I sites in the RRV-gT2A backbone, and DNA fragments with PsiI and Not I sites were synthesized and cloned into the PsiI and Not I sites in the RRV-IRES backbone, resulting in constructs designated pAC3-gT2A-Hck and pAC3-IRES-Hck, respectively.
RRV virus supernatant and Hck protein were produced in HEK293T cells as described. Table L shows that the titer of RRV-gT2A-Hck produced by HEK293T cells was comparable to that of RRV-GFP.
Table L: the titers of RRV-gT2A-Hck and RRV-IRES-Hck produced by transiently transfected 293T cells.
TU/mL
RRV-GFP
3.36E+6
RRV-gT2A-Hck
6.07E+6
RRV-IRES-Hck
2.00E+6
The Hck protein encoded in pAC3-gT2A-Hck was designed to be secreted into the supernatant. Detection of Hck protein in the supernatant was performed by direct immunoblotting of 15. mu.L of the supernatant using an anti-Flag M2 antibody (Sigma Cat # F1804, 1:1000) and an HPR-conjugated secondary antibody. FIG. 11 shows that Hck protein is expressed in large amounts in the supernatant, with the expected molecular weight of 7 kDa.
Example 39: RRV-gT 2A-antiporter produced by 293T cells is infectious and expresses antiporter proteins.
The coding region for the anti-transporter-Lcn 2 was obtained from Gebauer et al, 2013(JMB 425(4) 780-802). To detect anti-transporter-Lcn 2 protein expression, Flag and His epitope tags were inserted at the C-terminus of anti-transporter-Lcn 2, and a signal peptide derived from human IL-2 was placed at the N-terminus of the anti-transporter-Lcn 2 coding region. DNA fragments were synthesized and cloned into the AscI and Not I sites in the RRV-gT2A backbone. The resulting construct was designated pAC3-gT 2A-anti-transporter-Lcn 2.
The anti-transporter-Lcn 2 protein encoded in pAC3-gT 2A-anti-transporter-Lcn 2 was designed to be secreted into the supernatant. Detection of anti-transportan-Lcn 2 protein in the supernatant was performed by direct immunoblotting of 15. mu.L of the supernatant using an anti-Flag M2 antibody (Sigma Cat # F1804, 1:1000) and an HPR-conjugated secondary antibody. The data show that the anti-transporter-Lcn 2 protein supernatant is expressed in large quantities and the expected molecular weight is 20 kDa.
Example 40: the backbone framework amino acid residues and surface exposed amino acid residues involved in antigen binding as well as amino acid residues in the oligomerization domain can be optimized to become Apobec-resistant.
An important aspect of a scaffold protein is to maintain the overall integrity or structure of the scaffold. To avoid Apobec 3-mediated mutations that could lead to coding nonsense/stop codons (nucleic acids TGA, TAA and TAG) during viral infection, nucleic acid substitutions were introduced that confer resistance to the therapeutic transgene coding sequence Apobec 3-by replacing the selective or all tryptophan residues present in the scaffold backbone framework and/or surface exposed amino acids involved in antigen binding with other 19 amino acids to avoid hypermutation of the nonsense/stop codon mediated by Apobec 3.
The anti-transporter protein derived from Lcn2 (Gebauer et al, 2012J Mol Biol 425(4):780-802) contains two tryptophan residues: one present in beta-strand A and the other present in beta-strand D. In addition, the ED-B binding agent, anti-transporter N7A, contains 3 additional tryptophan residues in beta-strand D and loop 3/beta-strand F. A computational algorithm (Parthiban et al, BMC statistical Biology 20077: 54; Bywate, PLoS 201611 (3): e150769) was used and a combinatorial mutagenesis library of 19 amino Acids for the selected tryptophan residues was generated (Y a ň ez et al, Nucleic Acids research 32(20) e158,2004) to evaluate and test its expression, antigen binding affinity. Our data show that tryptophan residues involved in structural integrity present in the backbone framework and antigen binding loop of the anti-transporter N7A can be replaced by conserved amino acid residues (e.g., tyrosine and phenylalanine). When encoded in the RRV-gT2A backbone, the Apobec-resistant N7A variant showed comparable protein expression levels to the parent N7A protein. Most importantly, the purified Apobec-resistant N7A protein expressed from pcdna3.1 vector in 293F cells showed comparable secondary structure when analyzed by far-ultraviolet circular dichroism spectroscopy, and showed similar binding affinity to EB-D by SPR-based biosensor analysis.
The tolerance of replacement of tryptophan with tyrosine or phenylalanine in the scaffold framework has also been demonstrated in Hck proteins, where two consecutive tryptophan residues present adjacent to the src-loop can be replaced with two phenylalanine (FF), two tyrosine (YY), tyrosine-phenylalanine (YF) or phenylalanine-tyrosine (FY) without impairing its expression. Furthermore, we also show that the tryptophan residue in the type I deiodinase dimerization motif can be substituted with phenylalanine and tyrosine without compromising its dimerization function.
Example 41: using the Fc region of human IgG, epitope-tagged Affimer-SQT can be expressed as homodimers in the RRV-gT2A backbone.
To express the homodimer of Affimer-SQT, in addition to the incorporation of the human IL-2 signal peptide at the N-terminus, the coding sequence of Affimer-SQT was linked to a (G4S)3 glycine-serine linker, followed by an IgG4 Fc region. The design of vectors encoding this type of non-IG binding protein is shown in figure 12, along with other types of modifications that comprise genes encoding binding proteins that allow multimer formation, or other types of modifications that multiplex binding specificities to form bispecific antibodies or antibody-like bispecific or trispecific molecules. The synthesized fragments were cloned into AscI and NotI sites in the backbone of RRV gT 2A. One of the resulting constructs was designated pAC3-gT 2A-Affimer-SQT-Fc. The data show that under non-reducing conditions, the dimeric form of Affimer-SQT was detected using the anti-human IgG4 Fc antibody, with an expected molecular size of-50 kDa.
Example 42: epitope tagged Affimer-SQT can be expressed as homodimers using the dimerization domain.
To express the homodimer of Affimer-SQT, in addition to the incorporation of the human IL-2 signal peptide at the N-terminus of Affimer-SQT and the epitope tag at the C-terminus of Affimer-SQT, a dimerization domain of a type I deiodinase (table 6) linked to a ggggg glycine linker at both the N-and C-termini was placed downstream of the signal peptide, followed by Affimer-SQT. In another configuration, the human IL-2 signal peptide and epitope tag are placed N-terminal to Affimer-SQT, and the dimerization domain, which is linked to a GGGG glycine linker at both the N-and C-terminals, is placed C-terminal to Affimer-SQT. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRVgT 2A. The resulting constructs were designated pAC3-gT2A-2Affimer-SQT and pAC3-gT2A-Affimer-SQT2, respectively.
Protein expression data showed that under non-reducing conditions, more than 85% of the 2Affimer SQT and Affimer SQT2 proteins were detected in dimeric form, with an expected molecular size of 32 kDa.
Example 43: epitope tagged Affimer-SQT can be expressed in a homotrimeric form using a trimerization domain.
To express the homotrimer of Affimer-SQT, in addition to the incorporation of the human IL-2 signal peptide at the N-terminus of Affimer-SQT and the epitope tag at the C-terminus of Affimer-SQT, the trimerization domain of crown protein 1a (Table 6), which has a GGGG glycine linker at both the N-and C-termini, was placed downstream of the signal peptide and subsequently placed in Affimer-SQT. In another configuration, the human IL-2 signal peptide and epitope tag are placed at the N-terminus of Affimer-SQT, and the trimerization domain linked at the N-and C-termini to a GGGG glycine-linker is placed at the C-terminus of Affimer-SQT. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT2A-3Affimer-SQT and pAC3-gT2A-Affimer-SQT3, respectively.
Protein expression data indicated that more than 85% of the 3Affimer SQT and Affimer SQT3 proteins were detected as trimers under non-reducing conditions, with an expected molecular size of 56 kDa.
Example 44: using the tetramerization domain, epitope-tagged Affimer-SQT can be expressed in a homotetrameric form.
To express the homotetrameric form of Affimer-SQT, in addition to the incorporation of a human IL-2 signal peptide at the N-terminus of Affimer-SQT and an epitope tag at the C-terminus of Affimer-SQT, a Cartilage Matrix Protein (CMP) CMP (R27Q) tetramerization domain (Table 6) linked at both the N-and C-termini to a GGGG glycine linker was placed downstream of the signal peptide, followed by Affimer-SQT. In another configuration, the human IL-2 signal peptide and epitope tag are placed at the N-terminus of Affimer-SQT, and the tetramerization domain, which is linked at both the N-and C-termini to a GGGGG glycine-linker, is placed at the C-terminus of Affimer-SQT. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT2A-4Affimer-SQT and pAC3-gT2A-Affimer-SQT4, respectively.
Protein expression data showed that under non-reducing conditions, more than 85% of the 4Affimer SQT and Affimer SQT4 proteins were detected in tetrameric form with an expected molecular size of 56 kDa.
Example 45: using the pentameric domain, epitope tagged Affimer-SQT can be expressed in a homologous pentameric form.
To express the homologous pentamer of Affimer-SQT, in addition to the incorporation of the human IL-2 signal peptide at the N-terminus of Affimer-SQT and the epitope tag at the C-terminus of Affimer-SQT, a Cartilage Oligomeric Matrix Protein (COMP) pentamer domain (Table 6) linked to a GGGG glycine linker at the N-and C-termini was placed downstream of the signal peptide, followed by Affimer-SQT. In another configuration, the human IL-2 signal peptide and epitope tag were placed at the N-terminus of Affimer-SQT and the pentameric domain linked to a GGGG glycine-linker at both the N-and C-termini was placed at the C-terminus of Affimer-SQT. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT2A-5Affimer-SQT and pAC3-gT2A-Affimer-SQT5, respectively.
Protein expression data showed that under non-reducing conditions, more than 85% of the 5Affimer SQT and Affimer SQT5 proteins were detected in tetrameric form with an expected molecular size of 100 kDa.
Example 46: epitope-tagged Affimer-SQT can be expressed as a homo-hexamer using IgM-derived hexamer domains.
To express the homologous hexamer of Affimer-SQT, in addition to the incorporation of the human IL-2 signal peptide at the N-terminus of Affimer-SQT and the epitope tag at the C-terminus of Affimer-SQT, a human IgM C μ 4tp hexamer domain (Table 4) linked to a GGGG glycine linker at both the N-and C-termini was placed downstream of the signal peptide, followed by Affimer-SQT. In another configuration, the human IL-2 signal peptide and epitope tag are placed at the N-terminus of Affimer-SQT, and the hexameric domain linked to a GGGG glycine-linker at both the N-and C-termini is placed at the C-terminus of Affimer-SQT. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT2A-6Affimer-SQT and pAC3-gT2A-Affimer-SQT6, respectively.
Protein expression data showed that under non-reducing conditions, more than 95% of the 6Affimer SQT and Affimer SQT6 proteins were detected in tetrameric form with an expected molecular size of 175 kDa.
Example 47: epitope tagged Affimer-SQT and Hck can be expressed as heterodimers in the RRV gT2A backbone using (G4)3 glycine-serine linker 4S.
To express heterodimeric Affimer-SQT and Hck, the coding sequences for Affimer-SQT and Hck were linked in two possible configurations (Affimer-SQT-g-Hck and Hck-g-Affimer-SQT) with a (GGGGS)3 glycine-serine linker, incorporating a human IL-2 signal peptide at the N-terminus and an epitope tag at the C-terminus of the "fusion" protein. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT2A-Affimer-SQT-g-Hck and pAC3-gT2A-Hck-g-Affimer-SQT, respectively.
Protein expression data showed that heterodimeric forms of Affimer-SQT-g-Hck and Hck-g-Affimer-SQT were detected, with an expected molecular size of 23 kDa.
Example 48: epitope tagged Affimer-SQT and the anti-transporter can be expressed as heterodimers in the RRV gT2A backbone using a (G4S)3 glycine-serine linker.
To express heterodimeric Affimer-SQT and antiporter, the coding sequences for Affimer-SQT and antiporter were linked in two possible configurations (Affimer-SQT-g-antiporter and antiporter-g-Affimer-SQT) with a (GGGGS)3 glycine-serine linker, in which the human IL-2 signal peptide was incorporated at the N-terminus and the epitope tag at the C-terminus of the "fusion" protein. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT 2A-Affimer-SQT-g-antiporter and pAC3-gT 2A-antiporter-g-Affimer-SQT, respectively.
Protein expression data showed that heterodimeric forms of Affimer-SQT-g-antiporter and antiporter-g-Affimer-SQT were detected, with an expected molecular size of-36 kDa.
Example 49: using a (G4S)3 glycine-serine linker, epitope-tagged transporter and Hck can be expressed as heterodimers in the RRV gT2A backbone.
To express heterodimeric Hck and anti-transporter, the coding sequences for Hck and anti-transporter were linked in two possible configurations (Hck-g-anti-transporter and anti-transporter-g-Hck) with a (GGGGS)3 glycine-serine linker, incorporating a human IL-2 signal peptide at the N-terminus and an epitope tag at the C-terminus of the "fusion" protein. The synthesized fragments were cloned into the AscI site and Not I site in the backbone of RRV gT 2A. The resulting constructs were designated pAC3-gT 2A-Hck-g-anti-transporter and pAC3-gT 2A-anti-transporter-g-Hck, respectively.
Protein expression data showed that heterodimeric forms of Hck-g-antiporter and antiporter-g-Hck were detected with an expected molecular size of 28 kDa.
Example 50: epitope-tagged Affimer-SQT, Hck and anti-transporter proteins can be expressed as heterotrimers in the RRV gT2A backbone using (G4S)3 glycine-serine linkers.
To express heterotrimeric AFfimer-SQT, Hck and anti-carrier proteins, the coding sequences for Affimer-SQT, Hck and anti-carrier proteins were linked with a (GGGGS)3 glycine-serine linker, incorporating a human IL-2 signal peptide at the N-terminus of the "fusion" protein and an epitope tag at the C-terminus. Fragments with six possible combinations (Hck-g-Affimer-SQT-g-antiporter, Hck-g-antiporter-g-Affimer-SQT, Affimer-SQT-g-Hck-g-antiporter, Affimer-SQT-g-antiporter-g-Hck, antiporter-g-Hck-g-Affimer-SQT, and antiporter-g-Affimer-SQT-g-Hck) were synthesized and cloned into AscI and Not I sites in the RRV gT2A backbone. The resulting constructs were designated pAC3-gT 2A-Hck-g-Affimer-SQT-g-antiporter and pAC3-gT 2A-Hck-g-antiporter-g-Affimer-SQT, pAC3-gT 2A-Affimer-SQT-g-Hck-g-antiporter, pAC3-gT 2A-Affimer-SQT-g-antiporter-g-Hck, pAC3-gT 2A-antiporter-g-Hck-g-Affimer-SQT and pAC3-gT 2A-antiporter-g-Affimer-SQT-g-Hck, respectively.
Protein expression data showed that heterotrimeric forms of Hck-g-Affimer-SQT-g-antiporter, Hck-g-antiporter-g-Affimer-SQT, Affimer-SQT-g-Hck-g-antiporter, Affimer-SQT-g-antiporter-g-Hck, antiporter-g-Hck-g-Affimer-SQT, and antiporter-g-Affimer-SQT-g-Hck were detected, with an expected molecular size of 43 kDa.
Example 51: RRV-S1-antiporter protein produced by 293T cells is infectious and expresses the antiporter protein mediated by the core promoter.
The coding region for the anti-transporter-Lcn 2 was obtained from Gebauer et al, 2013(JMB 425(4) 780-802). To detect anti-transporter-Lcn 2 protein expression, Flag and His epitope tag were inserted into the C-terminus of anti-transporter-Lcn 2 and a signal peptide derived from human IL-2 was placed at the N-terminus of the anti-transporter-Lcn 2 coding region and downstream of the core promoter. These core promoters are based on, but are not limited to, adenovirus major late (AdML) and Cytomegalovirus (CMV) major immediate early genes, and a synthetic "super core promoter" SCP1 (see also U.S. patent publication No.2015/0273029a1, the disclosure of which is incorporated herein by reference in its entirety). DNA fragments containing the core promoters AdML-antiporter-Lcn 2, CMV-antiporter-Lcn 2 and SCP 1-antiporter-Lcn 2 were synthesized and cloned into pAC 3-derived RRV backbone to give constructs designated pAC 3-A1-antiporter-Lcn 2, pAC 3-C1-antiporter-Lcn 2 and pAC 3-S1-antiporter-Lcn 2, respectively.
The anti-transporter-Lcn 2 protein encoded in pAC 3-A1-anti-transporter-Lcn 2, pAC 3-C1-anti-transporter-Lcn 2, and pAC 3-S1-anti-transporter-Lcn 2 were designed to be secreted into the supernatant. Detection of anti-transportan-Lcn 2 protein in the supernatant was performed by direct immunoblotting of 15. mu.L of the supernatant using an anti-Flag M2 antibody (Sigma Cat # F1804, 1:1000) and an HPR-conjugated secondary antibody. Our data show that the anti-transporter-Lcn 2 protein is expressed in large amounts in the supernatant with the expected molecular weight of-20 kDa.
Example 52: tu2449-MG cells infected with RRV-GSG-T2A-syCD2 (a secreted modified yeast cytosolic protein deaminase) showed delayed 5FU cytotoxicity but had greater bystander effect than RRV-GSG-T2A-yCD 2.
pAC3-IRES-syCD2 and pAC3-GSG-T2A-syCD2 were generated to express secreted yCD2(syCD 2). Cytosine deaminase secreted by bacteria in non-replicating adenoviral vectors (Rehemmulla et al. anti xcan Res.,23: 1393-. There are several significant differences between Rehemtulla and the study described herein. These include: 1) rehemtulla is investigating bacterial cytosine deaminase (bCD) which has a 20-fold lower affinity for 5-FC compared to the wild-type yeast cytosine (Kievet et al. Can Res.59: 1417-14211999); animal model data showed that tumor suppression efficiency of secreted and cytoplasmic bCD was low compared to yeast-derived yCD2 (Rhemtulla et al; Ostertag et al NeuroOnc 2012); the vector used by rehemdulate is non-replicating, unlike the RRV-encoded yCD2, which spreads between cells. Thus, the effects on cell killing and bystander effects are more complex and ydd cannot be predicted from bCD data of rehemalla.
IRES-scyCD2 and GSG-T2A-syCD2 cassettes were designed so that SSPs derived from human IL-2 were placed in-frame at the N-terminus of yCD2 for pAC3-IRES-syCD2 or between GSG-T2A and yCD2 for pAC3-GSG-T2A-syCD 2. Cassettes were chemically synthesized (Genewiz) with the PsiI and NotI sites of pAC3-IRES-syCD2 and the AscI and NotI sites of pAC3-GSG-T2A-syCD2 and cloned into pAC3 as pAC3-IRES-syCD2 and pAC3-GSG-T2A-yCD2 backbones, respectively, in place of yCD 2. syCD2 protein expression was evaluated using anti-yCD 2 antibody from cell lysates and supernatants collected from transfected HEK293T cells. In contrast to the intracellular expression of yCD2 derived from IRES-yCD2 and GSG-T2A-yCD2, the results demonstrate that the inclusion of human IL2SSP in IRES-syCD2 and GSG-T2A-syCD2 results in the detection of strong expression of syCD2 in the supernatant, while lower or no detection was detected in the cell lysate. Secretion of syCD2 was effective in both constructs as shown by a minimum input of 10 μ L supernatant in the immunoblot assay. In addition, the extracellular form of syCD2 is similar in size compared to its parent constructs (pAC3-IRES-yCD2 and pAC3-GSG-T2A-yCD 2). In addition, viral supernatants of RRV-IRES-syCD2 and RRV-GSG-T2A-syCD2 collected from transiently transfected HEK293T cells showed titer values of 0.5-5E6 TU/mL and were comparable to RRV-IRES-syCD2(1.5E6 TU/mL) and RRV-GSG-T2A-yCD2(2E6 TU/mL), respectively.
Extracellular 5-FU concentrations in Tu2449 cells maximally infected with Tu2449/RRV-IRES-syCD2 and Tu2449/RRV-GSG-T2A-syCD2 were compared to Tu24449/RRV-IRES-yCD2 and Tu2449/RRV-GSG-T2A-yCD2, respectively. The data show that after 1 hour of reaction with excess 5-FC, the concentration of 5-FU present in the supernatant from Tu2449/RRV-IRES-syCD2 and Tu2449/RRV-GSG-T2A-syCD2 cells in the culture medium after 5-FC addition increased with cell growth time and reached maximum levels from initial cell inoculation to day 2-6. The concentration of 5-FU present in the supernatant of Tu2449/RRV-IRES-syCD2 and Tu2449/RRV-GSG-T2A-syCD2 is 4-log orders of magnitude higher than the concentration of 5-FU in the supernatant of Tu2449/RRV-IRES-yCD2 and Tu2449/RRV-GSG-T2A-yCD 2. Subsequently, the effectiveness of the 5FU bystander effect was evaluated in tissue culture by generating matched pairs of RRV-IRES-yCD2/RRV-IRES-GFP and RRV-IRES-syCD2/RRV-IRES-GFP, RRV-GSG-T2A-yCD2/RRV-GSG-T2A-GFP, and RRV-GSG-T2A-syCD2/RRV-GSG-T2A-GFP infected RRV-transduced Tu2449 cells at ratios of 3/97, 15/85 and 30/70 and treating the cultures with 5-FC. In these experiments, GFP vector infected cells were blocked from further infection, and thus no further viral transmission of the CD encoding vector occurred. In vitro cell killing data at set ratios of 3/97 and 15/85 indicated that both RRV-IRES-syCD2 and RRV-GSG-T2A-syCD2 had more bystander-mediated cytotoxic effects than RRV-IRES-yCD2 and RRV-GSG-T2A-yCD 2. IRES-syCD2 and RRV-GSG-T2A-syCD2 show more efficient cell killing compared to RRV-IRES-yCD2 and RRV-GSG-T2A-yCD2, respectively.
Example 53: subcutaneous syngeneic glioma tumours of mice treated with RRV-GSG-T2A-syCD2 or RRV-IRES-syCD2 show delayed tumour growth, comparable to RRV-GSG-T2A-yCD2 or RRV-GSG-T2A-yCD2, respectively.
To test whether syCD2 secreted by infected tumor cells resulted in an increased anti-tumor response in vivo, an isogenic orthotopic glioma model was established in B6C3F1 mice using Tu2449 cells. Matched pairs of RRV-transduced Tu2449 cells infected with RRV-IRES-yCD2/RRV-IRES-GFP and RRV-IRES-syCD2/RRV-IRES-GFP, RRV-GSG-T2A-yCD2/RRV-GSG-T2A-GFP and RRV-GSG-T2A-syCD2/RRV-GSG-T2A-GFP were generated at ratios of 3/97, 15/85 and 30/70 as previously described. Dose-dependent survival benefits compared to animals without 5-FC treatment were observed within subgroups RRV-IRES-yCD2/RRV-IRES-GFP, RRV-IRES-syCD2/RRV-IRES-GFP, RRV-GSG-T2A-yCD2/RRV-GSG-T2A-GFP, and RRV-GSG-T2A-syCD 2/RRV-GSG-T2A-GFP. However, when comparing survival data between the RRV-IRES-yCD2 and RRV-IRES-syCD2 groups and between the RRV-GSG-T2A-yCD2 and RRV-GSG-T2A-syCD2 groups over a 90 day period at ratios 3/97 and 15/85 and 30/70, the data indicate that in both cases mice bearing tumors transduced with the syCD2 variant had significantly higher survival benefit than mice bearing tumors transduced with the yCD2 version. This is seen more clearly when a lower rate of syCD infects cells. Our data indicate that expression of the secreted prodrug-activating enzyme is advantageous. This may be due to a number of factors, including: avoiding immediate high concentrations of intracellular 5-FC leading to early depletion of virus-producing cells, thereby hindering further viral spread; and/or further diffusion of CD protein and thus further diffusion of lethal concentrations of 5-FU.
Sequence listing
<110> Tokagen Inc. (Tocagen Inc.)
<120> recombinant vector comprising gene for binding domain and secretable peptide
<130> 00014-034WO1
<140> not yet allocated
<141> 2019-11-13
<150> US 62/760,912
<151> 2018-11-13
<150> US 62/893,673
<151> 2019-08-29
<160> 304
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> 2A peptide consensus sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa is V or I
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa is any amino acid
<400> 1
Asp Xaa Glu Xaa Asn Pro Gly Pro
1 5
<210> 2
<211> 11654
<212> DNA
<213> Artificial sequence
<220>
<223> 2A-cassette-containing RRV vector
<400> 2
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gtgaaacaga ctttgaattt 8340
tgaccttctc aagttggcgg gagacgtgga gtccaaccct ggacctggcg cgcctatggc 8400
cagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 8460
cgtaaacggc cacaagttca gcgtgtccgg cgaaggagag ggcgatgcca cctacggcaa 8520
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 8580
gaccaccttg acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca 8640
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 8700
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 8760
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 8820
ggagtacaac tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat 8880
caaggtgaac ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 8940
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 9000
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 9060
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtgtgcggc 9120
cgcagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca 9180
cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat 9240
aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc 9300
caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa 9360
cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc 9420
caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga 9480
tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc 9540
agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac 9600
aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc 9660
caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc 9720
ctctgagtga ttgactaccc gtcagcgggg gtctttcatt acatgtgagc aaaaggccag 9780
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9840
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9900
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9960
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc 10020
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10080
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10140
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10200
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10260
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10320
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 10380
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10440
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10500
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10560
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 10620
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 10680
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 10740
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 10800
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 10860
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 10920
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 10980
cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11040
ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11100
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11160
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 11220
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11280
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11340
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11400
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11460
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11520
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 11580
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 11640
cttcaagaat tcat 11654
<210> 3
<211> 9
<212> DNA
<213> Artificial sequence
<220>
<223> GSG linker sequence
<400> 3
ggaagcgga 9
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> GFP-R-Gib primer
<400> 4
taaaatcttt tattttatct gcggccgcac 30
<210> 5
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> peptide read-through sequence
<400> 5
Cys Ala Ala Ala Asp Lys Ile Lys Asp Phe Ile
1 5 10
<210> 6
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> Ascl-yCD2 Forward primer
<400> 6
gatcggcgcg cctatggtga ccggcggcat ggc 33
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> 3-37 primer
<400> 7
cccctttttc tggagactaa ataa 24
<210> 8
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 8
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
<210> 9
<211> 63
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 9
ggaagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60
cct 63
<210> 10
<211> 57
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 10
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57
<210> 11
<211> 66
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 11
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 12
<211> 66
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 12
gtgaaacaga ctttgaattt tgaccttctc aagttggcgg gagacgtgga gtccaaccct 60
ggacct 66
<210> 13
<211> 75
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 13
ggaagcggag tgaaacagac tttgaatttt gaccttctca agttggcggg agacgtggag 60
tccaaccctg gacct 75
<210> 14
<211> 60
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 14
cagtgtacta attatgctct cttgaaattg gctggagatg ttgagagcaa ccctggacct 60
<210> 15
<211> 69
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 15
ggaagcggac agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaac 60
cctggacct 69
<210> 16
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 16
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
<210> 17
<211> 63
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 17
ggaagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60
cct 63
<210> 18
<211> 57
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 18
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57
<210> 19
<211> 66
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide sequence
<400> 19
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 20
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> 5-MLV-U3-R primer
<400> 20
agcccacaac ccctcactc 19
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> 3-MLV-Psi primer sequences
<400> 21
tctcccgatc ccggacga 18
<210> 22
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> Probe sequence
<400> 22
ccccaaatga aagacccccg ctgacg 26
<210> 23
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> IRES Forward primer
<400> 23
ctgatcttac tctttggacc ttg 23
<210> 24
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> IRES reverse primer
<400> 24
cccctttttc tggagactaa ataa 24
<210> 25
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> ENV Forward primer
<400> 25
accctcaacc tcccctacaa gt 22
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> ENV reverse primer
<400> 26
gttaagcgcc tgataggctc 20
<210> 27
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> Env Probe sequences
<400> 27
ccccaaatga aagacccccg ctgacg 26
<210> 28
<211> 477
<212> DNA
<213> Artificial sequence
<220>
<223> human codon optimized thermostable CD coding sequence
<400> 28
atggtgaccg gcggcatggc ctccaagtgg gatcaaaagg gcatggatat cgcttacgag 60
gaggccctgc tgggctacaa ggagggcggc gtgcctatcg gcggctgtct gatcaacaac 120
aaggacggca gtgtgctggg caggggccac aacatgaggt tccagaaggg ctccgccacc 180
ctgcacggcg agatctccac cctggagaac tgtggcaggc tggagggcaa ggtgtacaag 240
gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gtaccggcgc tatcatcatg 300
tacggcatcc ctaggtgtgt gatcggcgag aacgtgaact tcaagtccaa gggcgagaag 360
tacctgcaaa ccaggggcca cgaggtggtg gttgttgacg atgagaggtg taagaagctg 420
atgaagcagt tcatcgacga gaggcctcag gactggttcg aggatatcgg cgagtaa 477
<210> 29
<211> 158
<212> PRT
<213> Artificial sequence
<220>
<223> thermostable APOBEC-modified CD polypeptide
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is any amino acid
<220>
<221> MISC_FEATURE
<222> (152)..(152)
<223> Xaa is any amino acid
<400> 29
Met Val Thr Gly Gly Met Ala Ser Lys Xaa Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Xaa Phe Glu Asp Ile Gly Glu
145 150 155
<210> 30
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> 2A peptide coding sequence
<400> 30
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
<210> 31
<211> 1062
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-T2A-GFPm of pAC3-T2A-GFPm
<400> 31
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagag ggcagaggaa gtcttctaac atgcggtgac 240
gtggaggaga atcccggccc tggcgcgcct atggccagca agggcgagga gctgttcacc 300
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 360
tccggcgaag gagagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 420
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttgaccta cggcgtgcag 480
tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 540
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 600
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 660
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaag 720
gtctatatca ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa gacccgccac 780
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 840
gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa 900
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 960
actctcggca tggacgagct gtacaagtgt gcggccgcag ataaaataaa agattttatt 1020
tagtctccag aaaaaggggg gaatgaaaga ccccacctgt ag 1062
<210> 32
<211> 1026
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-P2A-GFPm of pAC3-P2A-GFPm
<400> 32
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagct actaacttca gcctgctgaa gcaggctgga 240
gacgtggagg agaaccctgg acctggcgcg cctatggcca gcaagggcga ggagctgttc 300
accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 360
gtgtccggcg aaggagaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 420
accaccggca agctgcccgt gccctggccc accctcgtga ccaccttgac ctacggcgtg 480
cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 540
cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 600
cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 660
gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 720
aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc 780
cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 840
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 900
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 960
atcactctcg gcatggacga gctgtacaag tgtgcggccg cagataaaat aaaagatttt 1020
atttag 1026
<210> 33
<211> 1029
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-E2A-GFPm of pAC3-E2A-GFPm
<400> 33
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccacag tgtactaatt atgctctctt gaaattggct 240
ggagatgttg agagcaaccc tggacctggc gcgcctatgg ccagcaaggg cgaggagctg 300
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 360
agcgtgtccg gcgaaggaga gggcgatgcc acctacggca agctgaccct gaagttcatc 420
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt gacctacggc 480
gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 540
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 600
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 660
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 720
cacaaggtct atatcaccgc cgacaagcag aagaacggca tcaaggtgaa cttcaagacc 780
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 840
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 900
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 960
gggatcactc tcggcatgga cgagctgtac aagtgtgcgg ccgcagataa aataaaagat 1020
tttatttag 1029
<210> 34
<211> 1035
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-F2A-GFPm of pAC3-F2A-GFPm
<400> 34
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagtg aaacagactt tgaattttga ccttctcaag 240
ttggcgggag acgtggagtc caaccctgga cctggcgcgc ctatggccag caagggcgag 300
gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 360
aagttcagcg tgtccggcga aggagagggc gatgccacct acggcaagct gaccctgaag 420
ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttgacc 480
tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag 540
tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 600
tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 660
aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 720
aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc 780
aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 840
acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 900
gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 960
gccgccggga tcactctcgg catggacgag ctgtacaagt gtgcggccgc agataaaata 1020
aaagatttta tttag 1035
<210> 35
<211> 1032
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-GSG-T2A-GFPm of pAC3-GSG-T2A-GFPm
<400> 35
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagga agcggagagg gcagaggaag tcttctaaca 240
tgcggtgacg tggaggagaa tcccggccct ggcgcgccta tggccagcaa gggcgaggag 300
ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 360
ttcagcgtgt ccggcgaagg agagggcgat gccacctacg gcaagctgac cctgaagttc 420
atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttgacctac 480
ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 540
gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 600
aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 660
ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 720
agccacaagg tctatatcac cgccgacaag cagaagaacg gcatcaaggt gaacttcaag 780
acccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 840
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 900
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 960
gccgggatca ctctcggcat ggacgagctg tacaagtgtg cggccgcaga taaaataaaa 1020
gattttattt ag 1032
<210> 36
<211> 1035
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-GSG-P2A-GFPm of pAC3-GSG-P2A-GFPm
<400> 36
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagga agcggagcta ctaacttcag cctgctgaag 240
caggctggag acgtggagga gaaccctgga cctggcgcgc ctatggccag caagggcgag 300
gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 360
aagttcagcg tgtccggcga aggagagggc gatgccacct acggcaagct gaccctgaag 420
ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttgacc 480
tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag 540
tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 600
tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 660
aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 720
aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc 780
aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 840
acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 900
gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 960
gccgccggga tcactctcgg catggacgag ctgtacaagt gtgcggccgc agataaaata 1020
aaagatttta tttag 1035
<210> 37
<211> 1044
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-GSG-F2A-GFPm of pAC3-GSG-F2A-GFPm
<400> 37
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagga agcggagtga aacagacttt gaattttgac 240
cttctcaagt tggcgggaga cgtggagtcc aaccctggac ctggcgcgcc tatggccagc 300
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 360
aacggccaca agttcagcgt gtccggcgaa ggagagggcg atgccaccta cggcaagctg 420
accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 480
accttgacct acggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac 540
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 600
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 660
atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 720
tacaactaca acagccacaa ggtctatatc accgccgaca agcagaagaa cggcatcaag 780
gtgaacttca agacccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 840
cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 900
acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 960
ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg tgcggccgca 1020
gataaaataa aagattttat ttag 1044
<210> 38
<211> 1038
<212> DNA
<213> Artificial sequence
<220>
<223> BstBI-env-GSG-E2A-GFPm of pAC3-GSG-E2A-GFPm
<400> 38
ttcgaagggc tgtttaatag atccccctgg tttaccacct taatctccac catcatggga 60
cctctaatag tactcttact gatcttactc tttggacctt gcattctcaa tcgattggtc 120
caatttgtta aagacaggat ctcagtggtc caggctctgg ttttgactca gcaatatcac 180
cagctaaaac ccatagagta cgagccagga agcggacagt gtactaatta tgctctcttg 240
aaattggctg gagatgttga gagcaaccct ggacctggcg cgcctatggc cagcaagggc 300
gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 360
cacaagttca gcgtgtccgg cgaaggagag ggcgatgcca cctacggcaa gctgaccctg 420
aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttg 480
acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc 540
aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 600
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 660
ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 720
tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat caaggtgaac 780
ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 840
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 900
tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 960
accgccgccg ggatcactct cggcatggac gagctgtaca agtgtgcggc cgcagataaa 1020
ataaaagatt ttatttag 1038
<210> 39
<211> 548
<212> DNA
<213> Artificial sequence
<220>
<223> T2A-AscI-yCD2 of pAC3-T2A-yCD2
<400> 39
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctggcgcg 60
cctatggtga ccggcggcat ggcctccaag tgggatcaaa agggcatgga tatcgcttac 120
gaggaggccc tgctgggcta caaggagggc ggcgtgccta tcggcggctg tctgatcaac 180
aacaaggacg gcagtgtgct gggcaggggc cacaacatga ggttccagaa gggctccgcc 240
accctgcacg gcgagatctc caccctggag aactgtggca ggctggaggg caaggtgtac 300
aaggacacca ccctgtacac caccctgtcc ccttgtgaca tgtgtaccgg cgctatcatc 360
atgtacggca tccctaggtg tgtgatcggc gagaacgtga acttcaagtc caagggcgag 420
aagtacctgc aaaccagggg ccacgaggtg gtggttgttg acgatgagag gtgtaagaag 480
ctgatgaagc agttcatcga cgagaggcct caggactggt tcgaggatat cggcgagtaa 540
gcggccgc 548
<210> 40
<211> 551
<212> DNA
<213> Artificial sequence
<220>
<223> P2A-AscI-yCD2 of pAC3-P2A-yCD2
<400> 40
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctggc 60
gcgcctatgg tgaccggcgg catggcctcc aagtgggatc aaaagggcat ggatatcgct 120
tacgaggagg ccctgctggg ctacaaggag ggcggcgtgc ctatcggcgg ctgtctgatc 180
aacaacaagg acggcagtgt gctgggcagg ggccacaaca tgaggttcca gaagggctcc 240
gccaccctgc acggcgagat ctccaccctg gagaactgtg gcaggctgga gggcaaggtg 300
tacaaggaca ccaccctgta caccaccctg tccccttgtg acatgtgtac cggcgctatc 360
atcatgtacg gcatccctag gtgtgtgatc ggcgagaacg tgaacttcaa gtccaagggc 420
gagaagtacc tgcaaaccag gggccacgag gtggtggttg ttgacgatga gaggtgtaag 480
aagctgatga agcagttcat cgacgagagg cctcaggact ggttcgagga tatcggcgag 540
taagcggccg c 551
<210> 41
<211> 557
<212> DNA
<213> Artificial sequence
<220>
<223> GSG-T2A-AscI-yCD2 of pAC3-GSG-T2A-yCD2
<400> 41
ggaagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60
cctggcgcgc ctatggtgac cggcggcatg gcctccaagt gggatcaaaa gggcatggat 120
atcgcttacg aggaggccct gctgggctac aaggagggcg gcgtgcctat cggcggctgt 180
ctgatcaaca acaaggacgg cagtgtgctg ggcaggggcc acaacatgag gttccagaag 240
ggctccgcca ccctgcacgg cgagatctcc accctggaga actgtggcag gctggagggc 300
aaggtgtaca aggacaccac cctgtacacc accctgtccc cttgtgacat gtgtaccggc 360
gctatcatca tgtacggcat ccctaggtgt gtgatcggcg agaacgtgaa cttcaagtcc 420
aagggcgaga agtacctgca aaccaggggc cacgaggtgg tggttgttga cgatgagagg 480
tgtaagaagc tgatgaagca gttcatcgac gagaggcctc aggactggtt cgaggatatc 540
ggcgagtaag cggccgc 557
<210> 42
<211> 560
<212> DNA
<213> Artificial sequence
<220>
<223> GSG-P2A-AscI-yCD2 of pAC3-GSG-P2A-yCD2
<400> 42
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacctggcg cgcctatggt gaccggcggc atggcctcca agtgggatca aaagggcatg 120
gatatcgctt acgaggaggc cctgctgggc tacaaggagg gcggcgtgcc tatcggcggc 180
tgtctgatca acaacaagga cggcagtgtg ctgggcaggg gccacaacat gaggttccag 240
aagggctccg ccaccctgca cggcgagatc tccaccctgg agaactgtgg caggctggag 300
ggcaaggtgt acaaggacac caccctgtac accaccctgt ccccttgtga catgtgtacc 360
ggcgctatca tcatgtacgg catccctagg tgtgtgatcg gcgagaacgt gaacttcaag 420
tccaagggcg agaagtacct gcaaaccagg ggccacgagg tggtggttgt tgacgatgag 480
aggtgtaaga agctgatgaa gcagttcatc gacgagaggc ctcaggactg gttcgaggat 540
atcggcgagt aagcggccgc 560
<210> 43
<211> 11642
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-T2A-GFPm
<400> 43
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gagggcagag gaagtcttct 8340
aacatgcggt gacgtggagg agaatcccgg ccctggcgcg cctatggcca gcaagggcga 8400
ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 8460
caagttcagc gtgtccggcg aaggagaggg cgatgccacc tacggcaagc tgaccctgaa 8520
gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttgac 8580
ctacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa 8640
gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 8700
ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 8760
gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 8820
caacagccac aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt 8880
caagacccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 8940
cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 9000
cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 9060
cgccgccggg atcactctcg gcatggacga gctgtacaag tgtgcggccg cagataaaat 9120
aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg 9180
gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag 9240
agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat 9300
ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg 9360
ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 9420
gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt 9480
gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 9540
gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 9600
ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc 9660
ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt 9720
gactacccgt cagcgggggt ctttcattac atgtgagcaa aaggccagca aaaggccagg 9780
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 9840
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 9900
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 9960
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 10020
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 10080
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 10140
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 10200
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 10260
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 10320
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 10380
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 10440
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 10500
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 10560
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 10620
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 10680
tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 10740
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 10800
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 10860
ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg 10920
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 10980
aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 11040
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 11100
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 11160
ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta 11220
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 11280
ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 11340
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 11400
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 11460
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 11520
ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 11580
atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc 11640
at 11642
<210> 44
<211> 11651
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-T2A-GFPm
<400> 44
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggag agggcagagg 8340
aagtcttcta acatgcggtg acgtggagga gaatcccggc cctggcgcgc ctatggccag 8400
caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 8460
aaacggccac aagttcagcg tgtccggcga aggagagggc gatgccacct acggcaagct 8520
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 8580
caccttgacc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga 8640
cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 8700
cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 8760
catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 8820
gtacaactac aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa 8880
ggtgaacttc aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 8940
ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 9000
cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 9060
gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt gtgcggccgc 9120
agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct 9180
gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac 9240
tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa 9300
acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag 9360
ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 9420
gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt 9480
ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt 9540
tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac 9600
ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa 9660
taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc 9720
tgagtgattg actacccgtc agcgggggtc tttcattaca tgtgagcaaa aggccagcaa 9780
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9840
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9900
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9960
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 10020
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 10080
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 10140
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 10200
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 10260
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10320
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10380
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10440
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10500
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10560
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10620
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10680
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10740
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10800
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10860
gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg 10920
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 10980
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 11040
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 11100
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 11160
atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc 11220
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11280
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11340
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11400
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11460
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11520
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11580
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt 11640
caagaattca t 11651
<210> 45
<211> 11645
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-P2A-GFPm
<400> 45
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gctactaact tcagcctgct 8340
gaagcaggct ggagacgtgg aggagaaccc tggacctggc gcgcctatgg ccagcaaggg 8400
cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 8460
ccacaagttc agcgtgtccg gcgaaggaga gggcgatgcc acctacggca agctgaccct 8520
gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt 8580
gacctacggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt 8640
caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg 8700
caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 8760
gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 8820
ctacaacagc cacaaggtct atatcaccgc cgacaagcag aagaacggca tcaaggtgaa 8880
cttcaagacc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca 8940
gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 9000
gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 9060
gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtgtgcgg ccgcagataa 9120
aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt 9180
ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca taactgagaa 9240
tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg ccaaacagga 9300
tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga acagctgaat 9360
atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag 9420
atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag atgtttccag 9480
ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt 9540
ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca 9600
ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc 9660
ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg 9720
attgactacc cgtcagcggg ggtctttcat tacatgtgag caaaaggcca gcaaaaggcc 9780
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 9840
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 9900
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 9960
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 10020
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 10080
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 10140
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 10200
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 10260
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 10320
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 10380
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 10440
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 10500
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 10560
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 10620
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 10680
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 10740
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 10800
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 10860
agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt 10920
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 10980
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 11040
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 11100
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 11160
cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact 11220
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 11280
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 11340
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 11400
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 11460
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 11520
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 11580
attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa 11640
ttcat 11645
<210> 46
<211> 11654
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-P2A-GFPm
<400> 46
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggag ctactaactt 8340
cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctggcg cgcctatggc 8400
cagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 8460
cgtaaacggc cacaagttca gcgtgtccgg cgaaggagag ggcgatgcca cctacggcaa 8520
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 8580
gaccaccttg acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca 8640
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 8700
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 8760
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 8820
ggagtacaac tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat 8880
caaggtgaac ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 8940
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 9000
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 9060
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtgtgcggc 9120
cgcagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca 9180
cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat 9240
aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc 9300
caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa 9360
cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc 9420
caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga 9480
tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc 9540
agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac 9600
aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc 9660
caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc 9720
ctctgagtga ttgactaccc gtcagcgggg gtctttcatt acatgtgagc aaaaggccag 9780
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9840
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9900
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9960
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc 10020
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10080
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10140
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10200
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10260
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10320
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 10380
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10440
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10500
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10560
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 10620
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 10680
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 10740
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 10800
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 10860
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 10920
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 10980
cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11040
ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11100
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11160
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 11220
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11280
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11340
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11400
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11460
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11520
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 11580
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 11640
cttcaagaat tcat 11654
<210> 47
<211> 11648
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-E2A-GFP
<400> 47
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca cagtgtacta attatgctct 8340
cttgaaattg gctggagatg ttgagagcaa ccctggacct ggcgcgccta tggccagcaa 8400
gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 8460
cggccacaag ttcagcgtgt ccggcgaagg agagggcgat gccacctacg gcaagctgac 8520
cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 8580
cttgacctac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt 8640
cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 8700
cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 8760
cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 8820
caactacaac agccacaagg tctatatcac cgccgacaag cagaagaacg gcatcaaggt 8880
gaacttcaag acccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 8940
gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 9000
ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 9060
cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgtg cggccgcaga 9120
taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta 9180
ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga 9240
gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca 9300
ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg 9360
aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 9420
cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc 9480
cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg 9540
cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc 9600
tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa 9660
accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga 9720
gtgattgact acccgtcagc gggggtcttt cattacatgt gagcaaaagg ccagcaaaag 9780
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 9840
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 9900
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 9960
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 10020
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 10080
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 10140
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 10200
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 10260
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 10320
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 10380
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 10440
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 10500
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 10560
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 10620
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 10680
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 10740
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 10800
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 10860
aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 10920
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 10980
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 11040
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 11100
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 11160
cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 11220
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 11280
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 11340
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 11400
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 11460
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 11520
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 11580
attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa 11640
gaattcat 11648
<210> 48
<211> 11657
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-E2A-GFPm
<400> 48
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggac agtgtactaa 8340
ttatgctctc ttgaaattgg ctggagatgt tgagagcaac cctggacctg gcgcgcctat 8400
ggccagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 8460
cgacgtaaac ggccacaagt tcagcgtgtc cggcgaagga gagggcgatg ccacctacgg 8520
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 8580
cgtgaccacc ttgacctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca 8640
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 8700
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 8760
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 8820
gctggagtac aactacaaca gccacaaggt ctatatcacc gccgacaagc agaagaacgg 8880
catcaaggtg aacttcaaga cccgccacaa catcgaggac ggcagcgtgc agctcgccga 8940
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 9000
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 9060
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtgtgc 9120
ggccgcagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 9180
ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 9240
cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 9300
ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 9360
gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 9420
ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 9480
agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 9540
atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 9600
cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 9660
atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 9720
ctcctctgag tgattgacta cccgtcagcg ggggtctttc attacatgtg agcaaaaggc 9780
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 9840
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 9900
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 9960
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 10020
tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 10080
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 10140
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 10200
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 10260
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 10320
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 10380
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 10440
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 10500
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 10560
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 10620
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 10680
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 10740
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 10800
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 10860
tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 10920
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 10980
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 11040
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 11100
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 11160
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 11220
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 11280
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 11340
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 11400
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 11460
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 11520
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 11580
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 11640
cgtcttcaag aattcat 11657
<210> 49
<211> 11654
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-F2A-GFPm
<400> 49
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gtgaaacaga ctttgaattt 8340
tgaccttctc aagttggcgg gagacgtgga gtccaaccct ggacctggcg cgcctatggc 8400
cagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 8460
cgtaaacggc cacaagttca gcgtgtccgg cgaaggagag ggcgatgcca cctacggcaa 8520
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 8580
gaccaccttg acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca 8640
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 8700
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 8760
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 8820
ggagtacaac tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat 8880
caaggtgaac ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 8940
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 9000
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 9060
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtgtgcggc 9120
cgcagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca 9180
cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat 9240
aactgagaat agagaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc 9300
caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa 9360
cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc 9420
caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga 9480
tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc 9540
agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac 9600
aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc 9660
caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc 9720
ctctgagtga ttgactaccc gtcagcgggg gtctttcatt acatgtgagc aaaaggccag 9780
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9840
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9900
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9960
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc 10020
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10080
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10140
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10200
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10260
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10320
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 10380
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10440
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10500
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10560
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 10620
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 10680
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 10740
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 10800
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 10860
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 10920
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 10980
cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11040
ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11100
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11160
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 11220
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11280
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11340
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11400
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11460
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11520
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 11580
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 11640
cttcaagaat tcat 11654
<210> 50
<211> 11663
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-F2A-GFPm
<400> 50
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggag tgaaacagac 8340
tttgaatttt gaccttctca agttggcggg agacgtggag tccaaccctg gacctggcgc 8400
gcctatggcc agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 8460
ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gaaggagagg gcgatgccac 8520
ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 8580
caccctcgtg accaccttga cctacggcgt gcagtgcttc gcccgctacc ccgaccacat 8640
gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 8700
cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 8760
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 8820
gcacaagctg gagtacaact acaacagcca caaggtctat atcaccgccg acaagcagaa 8880
gaacggcatc aaggtgaact tcaagacccg ccacaacatc gaggacggca gcgtgcagct 8940
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 9000
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 9060
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 9120
gtgtgcggcc gcagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 9180
aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 9240
aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg 9300
aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 9360
cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 9420
gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 9480
accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 9540
taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 9600
agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 9660
ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 9720
gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatta catgtgagca 9780
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 9840
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 9900
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 9960
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 10020
tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 10080
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 10140
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 10200
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 10260
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 10320
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 10380
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 10440
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 10500
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 10560
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 10620
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 10680
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 10740
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 10800
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 10860
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg 10920
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 10980
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 11040
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 11100
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 11160
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc 11220
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 11280
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 11340
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 11400
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 11460
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 11520
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 11580
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 11640
ccctttcgtc ttcaagaatt cat 11663
<210> 51
<211> 11399
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-T2A-yCD2
<400> 51
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gagggcagag gaagtcttct 8340
aacatgcggt gacgtggagg agaatcccgg ccctggcgcg cctatggtga ccggcggcat 8400
ggcctccaag tgggatcaaa agggcatgga tatcgcttac gaggaggccc tgctgggcta 8460
caaggagggc ggcgtgccta tcggcggctg tctgatcaac aacaaggacg gcagtgtgct 8520
gggcaggggc cacaacatga ggttccagaa gggctccgcc accctgcacg gcgagatctc 8580
caccctggag aactgtggca ggctggaggg caaggtgtac aaggacacca ccctgtacac 8640
caccctgtcc ccttgtgaca tgtgtaccgg cgctatcatc atgtacggca tccctaggtg 8700
tgtgatcggc gagaacgtga acttcaagtc caagggcgag aagtacctgc aaaccagggg 8760
ccacgaggtg gtggttgttg acgatgagag gtgtaagaag ctgatgaagc agttcatcga 8820
cgagaggcct caggactggt tcgaggatat cggcgagtaa gcggccgcag ataaaataaa 8880
agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca 8940
agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga 9000
agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg 9060
tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc 9120
caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc 9180
cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc 9240
ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct 9300
tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg 9360
gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg 9420
cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac 9480
tacccgtcag cgggggtctt tcattacatg tgagcaaaag gccagcaaaa ggccaggaac 9540
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 9600
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 9660
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 9720
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 9780
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 9840
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 9900
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 9960
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 10020
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 10080
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 10140
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 10200
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 10260
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 10320
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 10380
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 10440
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 10500
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 10560
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 10620
cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct 10680
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 10740
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 10800
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 10860
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 10920
agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa 10980
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 11040
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 11100
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 11160
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 11220
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 11280
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 11340
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcat 11399
<210> 52
<211> 11408
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-T2A-yCD2
<400> 52
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggag agggcagagg 8340
aagtcttcta acatgcggtg acgtggagga gaatcccggc cctggcgcgc ctatggtgac 8400
cggcggcatg gcctccaagt gggatcaaaa gggcatggat atcgcttacg aggaggccct 8460
gctgggctac aaggagggcg gcgtgcctat cggcggctgt ctgatcaaca acaaggacgg 8520
cagtgtgctg ggcaggggcc acaacatgag gttccagaag ggctccgcca ccctgcacgg 8580
cgagatctcc accctggaga actgtggcag gctggagggc aaggtgtaca aggacaccac 8640
cctgtacacc accctgtccc cttgtgacat gtgtaccggc gctatcatca tgtacggcat 8700
ccctaggtgt gtgatcggcg agaacgtgaa cttcaagtcc aagggcgaga agtacctgca 8760
aaccaggggc cacgaggtgg tggttgttga cgatgagagg tgtaagaagc tgatgaagca 8820
gttcatcgac gagaggcctc aggactggtt cgaggatatc ggcgagtaag cggccgcaga 8880
taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta 8940
ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga 9000
gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca 9060
ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg 9120
aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 9180
cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc 9240
cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg 9300
cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc 9360
tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa 9420
accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga 9480
gtgattgact acccgtcagc gggggtcttt cattacatgt gagcaaaagg ccagcaaaag 9540
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 9600
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 9660
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 9720
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 9780
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 9840
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 9900
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 9960
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 10020
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 10080
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 10140
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 10200
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 10260
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 10320
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 10380
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 10440
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 10500
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 10560
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 10620
aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt 10680
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 10740
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 10800
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 10860
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 10920
cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 10980
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 11040
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 11100
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 11160
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 11220
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 11280
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 11340
attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa 11400
gaattcat 11408
<210> 53
<211> 11402
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-P2A-yCD2
<400> 53
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca gctactaact tcagcctgct 8340
gaagcaggct ggagacgtgg aggagaaccc tggacctggc gcgcctatgg tgaccggcgg 8400
catggcctcc aagtgggatc aaaagggcat ggatatcgct tacgaggagg ccctgctggg 8460
ctacaaggag ggcggcgtgc ctatcggcgg ctgtctgatc aacaacaagg acggcagtgt 8520
gctgggcagg ggccacaaca tgaggttcca gaagggctcc gccaccctgc acggcgagat 8580
ctccaccctg gagaactgtg gcaggctgga gggcaaggtg tacaaggaca ccaccctgta 8640
caccaccctg tccccttgtg acatgtgtac cggcgctatc atcatgtacg gcatccctag 8700
gtgtgtgatc ggcgagaacg tgaacttcaa gtccaagggc gagaagtacc tgcaaaccag 8760
gggccacgag gtggtggttg ttgacgatga gaggtgtaag aagctgatga agcagttcat 8820
cgacgagagg cctcaggact ggttcgagga tatcggcgag taagcggccg cagataaaat 8880
aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg 8940
gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag 9000
agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat 9060
ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg 9120
ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 9180
gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt 9240
gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 9300
gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 9360
ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc 9420
ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt 9480
gactacccgt cagcgggggt ctttcattac atgtgagcaa aaggccagca aaaggccagg 9540
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 9600
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 9660
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 9720
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 9780
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 9840
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 9900
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 9960
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 10020
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 10080
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 10140
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 10200
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 10260
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 10320
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 10380
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 10440
tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 10500
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 10560
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 10620
ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg 10680
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 10740
aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 10800
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 10860
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 10920
ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta 10980
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 11040
ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 11100
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 11160
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 11220
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 11280
ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 11340
atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc 11400
at 11402
<210> 54
<211> 11411
<212> DNA
<213> Artificial sequence
<220>
<223> pAC3-GSG-P2A-yCD2
<400> 54
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg 600
actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660
ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc 720
tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca 780
ccgggaggta agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac 840
tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg 900
tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960
cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg 1020
gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080
tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140
tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga 1200
atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260
agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct 1320
ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380
tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg 1440
tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct 1500
ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560
ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg 1620
ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680
aagacccccc gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg 1740
gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg 1800
ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860
gaggaaacgg acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa 1920
ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc gagtctgttc 1980
tcatcaccca tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg 2040
gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc 2100
gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160
attacaccac ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg 2220
gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga ataacacaag 2280
ggcccaatga gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340
ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc 2400
agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa aacaagacgc 2460
ttggagattt ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa 2520
gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580
agcagaaaga gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca 2640
ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg 2700
atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760
aaccacgagg acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg 2820
gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880
ccgtcacctt cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac 2940
ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga 3000
ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060
cagactgtcc ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc 3120
actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg caagtgttga 3180
ccctaaatat agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc 3240
tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac 3300
tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360
taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac 3420
tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480
ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540
gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac 3600
cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660
accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag 3720
gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780
aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac 3840
agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc 3900
gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960
tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga 4020
ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa 4080
gggagttcct agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140
cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac 4200
aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag 4260
atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc 4320
taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380
cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440
aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag 4500
aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc 4560
aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg 4620
ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680
aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct 4740
ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga 4800
ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860
ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt 4920
atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc 4980
gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040
taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg 5100
gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160
tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag 5220
aacattttca ttacacagtg actgatataa aggacctaac caagttgggg gccatttatg 5280
ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340
ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc 5400
tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460
tcactgagac ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg 5520
gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc accgagataa 5580
agcccggatt gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga 5640
tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg 5700
agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760
tctccaaggt gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg 5820
catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880
taactaaatt aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc 5940
tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg 6000
gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc 6060
cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc 6120
tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg 6180
gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240
cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga 6300
tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgacat 6360
ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 6420
agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480
aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540
aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 6600
gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660
acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 6720
tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780
ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840
cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 6900
cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960
aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 7020
aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080
ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 7140
tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 7200
tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 7260
tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 7320
gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380
cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 7440
taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 7500
tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 7560
acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 7620
gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 7680
ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740
atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 7800
agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 7860
ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 7920
gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980
aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040
gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 8100
aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160
caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 8220
caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc tggttttgac 8280
tcagcaatat caccagctaa aacccataga gtacgagcca ggaagcggag ctactaactt 8340
cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctggcg cgcctatggt 8400
gaccggcggc atggcctcca agtgggatca aaagggcatg gatatcgctt acgaggaggc 8460
cctgctgggc tacaaggagg gcggcgtgcc tatcggcggc tgtctgatca acaacaagga 8520
cggcagtgtg ctgggcaggg gccacaacat gaggttccag aagggctccg ccaccctgca 8580
cggcgagatc tccaccctgg agaactgtgg caggctggag ggcaaggtgt acaaggacac 8640
caccctgtac accaccctgt ccccttgtga catgtgtacc ggcgctatca tcatgtacgg 8700
catccctagg tgtgtgatcg gcgagaacgt gaacttcaag tccaagggcg agaagtacct 8760
gcaaaccagg ggccacgagg tggtggttgt tgacgatgag aggtgtaaga agctgatgaa 8820
gcagttcatc gacgagaggc ctcaggactg gttcgaggat atcggcgagt aagcggccgc 8880
agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct 8940
gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac 9000
tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa 9060
acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag 9120
ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 9180
gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt 9240
ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt 9300
tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac 9360
ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa 9420
taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc 9480
tgagtgattg actacccgtc agcgggggtc tttcattaca tgtgagcaaa aggccagcaa 9540
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9600
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9660
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9720
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 9780
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 9840
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 9900
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 9960
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 10020
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10080
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10140
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10200
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10260
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10320
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10380
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10440
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10500
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10560
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10620
gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg 10680
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 10740
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 10800
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 10860
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 10920
atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc 10980
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11040
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11100
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11160
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11220
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11280
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 11340
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt 11400
caagaattca t 11411
<210> 55
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> equine rhinitis A Virus 2A peptide
<400> 55
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 56
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease 2A peptide
<400> 56
Pro Val Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp
1 5 10 15
Val Glu Ser Asn Pro Gly Pro
20
<210> 57
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> porcine Tespidae virus-12A peptide
<400> 57
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 58
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Thosea asigna virus 2A peptide
<400> 58
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 59
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> encephalomyocarditis virus-B2A peptide
<400> 59
Gly Ile Phe Asn Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile
1 5 10 15
His Asp Ile Glu Thr Asn Pro Gly Pro
20 25
<210> 60
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> encephalomyocarditis virus-D2A peptide
<400> 60
Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu Thr Asn Pro Gly
1 5 10 15
Pro
<210> 61
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> encephalomyocarditis virus-PV 212A peptide
<400> 61
Arg Ile Phe Asn Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile
1 5 10 15
His Asp Ile Glu Thr Asn Pro Gly Pro
20 25
<210> 62
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> mengo virus 2A peptide
<400> 62
His Val Phe Glu Thr His Tyr Ala Gly Tyr Phe Ser Lys Leu Leu Ile
1 5 10 15
His Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 63
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of Taylor encephalomyelitis virus-GD 72A
<400> 63
Lys Ala Val Arg Gly Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu Ile
1 5 10 15
His Asp Val Glu Met Asn Pro Gly Pro
20 25
<210> 64
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of Taylor encephalomyelitis virus-DA 2A
<400> 64
Arg Ala Val Arg Ala Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu Ile
1 5 10 15
His Asp Val Glu Met Asn Pro Gly Pro
20 25
<210> 65
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of Taylor encephalomyelitis virus-BEAN 2A
<400> 65
Lys Ala Val Arg Gly Tyr His Ala Asp Tyr Tyr Arg Gln Arg Leu Ile
1 5 10 15
His Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 66
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of Taylor's-like virus 2A
<400> 66
Lys His Val Arg Glu Tyr His Ala Ala Tyr Tyr Lys Gln Arg Leu Met
1 5 10 15
His Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 67
<211> 26
<212> PRT
<213> Artificial sequence
<220>
<223> Ljungan Virus-174F 2A peptide
<400> 67
Met His Ser Asp Glu Met Asp Phe Ala Gly Gly Lys Phe Leu Asn Gln
1 5 10 15
Cys Gly Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 68
<211> 26
<212> PRT
<213> Artificial sequence
<220>
<223> Ljungan Virus-145 SL 2A peptide
<400> 68
Met His Asn Asp Glu Met Asp Tyr Ser Gly Gly Lys Phe Leu Asn Gln
1 5 10 15
Cys Gly Asp Val Glu Ser Asn Pro Gly Pro
20 25
<210> 69
<211> 26
<212> PRT
<213> Artificial sequence
<220>
<223> Ljungan Virus- (87-012) 2A peptide
<400> 69
Met His Ser Asp Glu Met Asp Phe Ala Gly Gly Lys Phe Leu Asn Gln
1 5 10 15
Cys Gly Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 70
<211> 26
<212> PRT
<213> Artificial sequence
<220>
<223> Ljungan Virus- (M1146) 2A peptide
<400> 70
Tyr His Asp Lys Asp Met Asp Tyr Ala Gly Gly Lys Phe Leu Asn Gln
1 5 10 15
Cys Gly Asp Val Glu Thr Asn Pro Gly Pro
20 25
<210> 71
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus 2A peptide
<400> 71
Ala Pro Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 72
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus-A122A peptide
<400> 72
Ala Pro Gly Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 73
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus-C12A peptide
<400> 73
Ala Pro Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 74
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus-O1G 2A peptide
<400> 74
Ala Pro Val Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Met Glu Ser Asn Pro Gly Pro
20
<210> 75
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus O1K 2A peptide
<400> 75
Ala Pro Val Lys Gln Leu Thr Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 76
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus-O (Taiwan) 2A peptide
<400> 76
Ala Pro Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 77
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease virus-O/SK 2A peptide
<400> 77
Ala Pro Val Lys Gln Leu Leu Ser Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 78
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease Virus-SAT 32A peptide
<400> 78
Lys Pro Asp Lys Gln Met Cys Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 79
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> foot-and-mouth disease Virus-SAT 22A peptide
<400> 79
Gly Val Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 80
<211> 24
<212> PRT
<213> Artificial sequence
<220>
<223> equine rhinitis A Virus 2A peptide
<400> 80
Asn Ile Asn Lys Gln Cys Thr Asn Tyr Ser Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 81
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> equine rhinitis B virus 2A peptide
<400> 81
Thr Ile Leu Ser Glu Gly Ala Thr Asn Phe Ser Leu Leu Lys Leu Ala
1 5 10 15
Gly Asp Val Glu Leu Asn Pro Gly Pro
20 25
<210> 82
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> endogenous retrovirus-32A peptides
<400> 82
Asn Leu Leu Ser Gln Gly Ala Thr Asn Phe Asp Leu Leu Lys Leu Ala
1 5 10 15
Gly Asp Val Glu Ser Asn Pro Gly Pro
20 25
<210> 83
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-12A
<400> 83
Val Met Ala Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 84
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> Petasium virus-22A peptide
<400> 84
Thr Met Met Leu Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 85
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> Petasium virus-32A peptide
<400> 85
Thr Met Ser Phe Gln Gly Pro Gly Ala Ser Ser Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 86
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-42A
<400> 86
Thr Met Met Leu Gln Gly Pro Gly Ala Ser Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 87
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-52A
<400> 87
Thr Met Leu Phe Gln Gly Pro Gly Ala Ala Asn Phe Ser Leu Leu Arg
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 88
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-62A
<400> 88
Thr Met Ser Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 89
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-72A
<400> 89
Val Val Ser Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 90
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> Petasium virus-82A peptide
<400> 90
Thr Met Ser Leu Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Ile Glu Glu Asn Pro Gly Pro
20 25
<210> 91
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> Petasium virus-92A peptide
<400> 91
Thr Met Ala Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 92
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-102A
<400> 92
Thr Leu Ser Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 93
<211> 27
<212> PRT
<213> Artificial sequence
<220>
<223> peptide of botatoro virus-112A
<400> 93
Arg Met Ser Phe Gln Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys
1 5 10 15
Arg Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 94
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Gryllus palustris virus 2A peptide
<400> 94
Phe Leu Arg Lys Arg Thr Gln Leu Leu Met Ser Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 95
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Drosophila C virus 2A peptide
<400> 95
Glu Ala Ala Arg Gln Met Leu Leu Leu Leu Ser Gly Asp Val Glu Thr
1 5 10 15
Asn Pro Gly Pro
20
<210> 96
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> acute bee paralytic virus 2A peptide
<400> 96
Gly Ser Trp Thr Asp Ile Leu Leu Leu Leu Ser Gly Asp Val Glu Thr
1 5 10 15
Asn Pro Gly Pro
20
<210> 97
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> acute bee paralytic virus Poland 1 isolate 2A peptide
<400> 97
Gly Ser Trp Thr Asp Ile Leu Leu Leu Leu Ser Gly Asp Val Glu Thr
1 5 10 15
Asn Pro Gly Pro
20
<210> 98
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> acute bee paralytic virus Hungarian 1 isolate 2A peptide
<400> 98
Gly Ser Trp Thr Asp Ile Leu Leu Leu Trp Ser Gly Asp Val Glu Thr
1 5 10 15
Asn Pro Gly Pro
20
<210> 99
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> infectious osteomalacia virus 2A peptide
<400> 99
Thr Arg Ala Glu Ile Glu Asp Glu Leu Ile Arg Ala Gly Ile Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 100
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> tomato sterility virus 2A peptide
<400> 100
Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 101
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> equine encephalopathy virus 2A peptide
<400> 101
Gln Gly Ala Gly Arg Gly Ser Leu Val Thr Cys Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 102
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> avian polyoma virus 2A peptide
<400> 102
Asn Tyr Pro Met Pro Glu Ala Leu Gln Lys Ile Ile Asp Leu Glu Ser
1 5 10 15
Asn Pro Pro Pro
20
<210> 103
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Klishimil bee virus 2A peptide
<400> 103
Gly Thr Trp Glu Ser Val Leu Asn Leu Leu Ala Gly Asp Ile Glu Leu
1 5 10 15
Asn Pro Gly Pro
20
<210> 104
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Polychaenochaeta small RNA-like virus (a) 2A peptide
<400> 104
Ala Gln Gly Trp Val Pro Asp Leu Thr Val Asp Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 105
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Polychaenochaeta small RNA-like virus (b) 2A peptide
<400> 105
Ile Gly Gly Gly Gln Lys Asp Leu Thr Gln Asp Gly Asp Ile Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 106
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> tea geometrid small RNA-like virus (a) 2A peptide
<400> 106
Ala Gln Gly Trp Ala Pro Asp Leu Thr Gln Asp Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 107
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> tea geometrid small RNA-like virus (b) 2A peptide
<400> 107
Ile Gly Gly Gly Gln Arg Asp Leu Thr Gln Asp Gly Asp Ile Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 108
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Provedenus virus (a) 2A peptide
<400> 108
Val Gly Asp Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Ser Asn
1 5 10 15
Pro Gly Pro
<210> 109
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Provedenus virus (b) 2A peptide
<400> 109
Gly Asp Pro Ile Glu Asp Leu Thr Asp Asp Gly Asp Ile Glu Lys Asn
1 5 10 15
Pro Gly Pro
<210> 110
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Provedenus virus (c) 2A peptide
<400> 110
Ser Gly Gly Arg Gly Ser Leu Leu Thr Ala Gly Asp Val Glu Lys Asn
1 5 10 15
Pro Gly Pro
<210> 111
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> bovine rotavirus 2A peptide
<400> 111
Ser Lys Phe Gln Ile Asp Arg Ile Leu Ile Ser Gly Asp Ile Glu Leu
1 5 10 15
Asn Pro Gly Pro
20
<210> 112
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> porcine rotavirus 2A peptide
<400> 112
Ala Lys Phe Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp Val Glu Leu
1 5 10 15
Asn Pro Gly Pro
20
<210> 113
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> human rotavirus 2A peptide
<400> 113
Ser Lys Phe Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp Ile Glu Leu
1 5 10 15
Asn Pro Gly Pro
20
<210> 114
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> cultivated silkworm reovirus 2A peptide
<400> 114
Phe Arg Ser Asn Tyr Asp Leu Leu Lys Leu Cys Gly Asp Ile Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 115
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Lymantria dispar reovirus 2A peptide
<400> 115
Phe Arg Ser Asn Tyr Asp Leu Leu Lys Leu Cys Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 116
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> punctate Trichinella pinicola reovirus 2A peptide
<400> 116
Phe Arg Ser Asn Tyr Asp Leu Leu Lys Leu Cys Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 117
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Trypanosoma brucei TSR 12A peptide
<400> 117
Ser Ser Ile Ile Arg Thr Lys Met Leu Val Ser Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 118
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Trypanosoma CAB95325.12A peptide
<400> 118
Ser Ser Ile Ile Arg Thr Lys Met Leu Leu Ser Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 119
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Trypanosoma CAB95559.12A peptide
<400> 119
Ser Ser Ile Ile Arg Thr Lys Ile Leu Leu Ser Gly Asp Val Glu Glu
1 5 10 15
Asn Pro Gly Pro
20
<210> 120
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Trypanosoma cruzi 2A peptides
<400> 120
Cys Asp Ala Gln Arg Gln Lys Leu Leu Leu Ser Gly Asp Ile Glu Gln
1 5 10 15
Asn Pro Gly Pro
20
<210> 121
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> T. maritima aguA 2A peptide
<400> 121
Tyr Ile Pro Asp Phe Gly Gly Phe Leu Val Lys Ala Asp Ser Glu Phe
1 5 10 15
Asn Pro Gly Pro
20
<210> 122
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Bordetella bronchiseptica 2A peptide
<400> 122
Val His Cys Ala Gly Arg Gly Gly Pro Val Arg Leu Leu Asp Lys Glu
1 5 10 15
Gly Asn Pro Gly Pro
20
<210> 123
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> murine mor-1F 2A peptide
<400> 123
Asp Leu Glu Leu Glu Thr Val Gly Ser His Gln Ala Asp Ala Glu Thr
1 5 10 15
Asn Pro Gly Pro
20
<210> 124
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Drosophila melanogaster mod (mdg4) 2A peptide
<400> 124
Thr Ala Ala Asp Lys Ile Gln Gly Ser Trp Lys Met Asp Thr Glu Gly
1 5 10 15
Asn Pro Gly Pro
20
<210> 125
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Aspergillus nidulans Ca through MID 12A peptide
<400> 125
Pro Ile Thr Asn Arg Pro Arg Asn Ser Gly Leu Ile Asp Thr Glu Ile
1 5 10 15
Asn Pro Gly Pro
20
<210> 126
<211> 288
<212> DNA
<213> Artificial sequence
<220>
<223> Adnectins (10Fn3) sequence
<220>
<221> CDS
<222> (1)..(288)
<400> 126
gtg agc gac gtg ccc aga aag ctg gag gtg gtg gcc gcc acc ccc acc 48
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
agc ctg ctg atc agc tgg gac gcc ccc gcc gtg acc gtg aga tac tac 96
Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr
20 25 30
aga atc acc tac ggc gag acc ggc ggc aac agc ccc gtg cag gag ttc 144
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
acc gtg ccc ggc agc aag agc acc gcc acc atc agc ggc ctg aag ccc 192
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
ggc gtg gac tac acc atc acc gtg tac gcc gtg acc ggc aga ggc gac 240
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp
65 70 75 80
agc ccc gcc agc agc aag ccc atc agc aac tac aga acc gcc ctg gag 288
Ser Pro Ala Ser Ser Lys Pro Ile Ser Asn Tyr Arg Thr Ala Leu Glu
85 90 95
<210> 127
<211> 96
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 127
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr
20 25 30
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp
65 70 75 80
Ser Pro Ala Ser Ser Lys Pro Ile Ser Asn Tyr Arg Thr Ala Leu Glu
85 90 95
<210> 128
<211> 303
<212> DNA
<213> Artificial sequence
<220>
<223> Adnectin 1
<220>
<221> CDS
<222> (1)..(303)
<400> 128
gtg agc gac gtg ccc aga aag ctg gag gtg gtg gcc gcc acc ccc acc 48
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
agc ctg ctg atc agc tgg gac agc ggc aga ggc agc tac aga tac tac 96
Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Arg Tyr Tyr
20 25 30
aga atc acc tac ggc gag acc ggc ggc aac agc ccc gtg cag gag ttc 144
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
acc gtg ccc ggc ccc gtg cac acc gcc acc atc agc ggc ctg aag ccc 192
Thr Val Pro Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
ggc gtg gac tac acc atc acc gtg tac gcc gtg acc gac cac aag ccc 240
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His Lys Pro
65 70 75 80
cac gcc gac ggc ccc cac acc tac cac gag agc ccc atc agc aac tac 288
His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Asn Tyr
85 90 95
aga acc gcc ctg gag 303
Arg Thr Ala Leu Glu
100
<210> 129
<211> 101
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 129
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Arg Tyr Tyr
20 25 30
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
Thr Val Pro Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His Lys Pro
65 70 75 80
His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Asn Tyr
85 90 95
Arg Thr Ala Leu Glu
100
<210> 130
<211> 288
<212> DNA
<213> Artificial sequence
<220>
<223> Adnectin 2
<220>
<221> CDS
<222> (1)..(288)
<400> 130
gtg agc gac gtg ccc aga aag ctg gag gtg gtg gcc gcc acc ccc acc 48
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
agc ctg ctg atc agc tgg gag cac gac tac ccc tac aga aga tac tac 96
Ser Leu Leu Ile Ser Trp Glu His Asp Tyr Pro Tyr Arg Arg Tyr Tyr
20 25 30
aga atc acc tac ggc gag acc ggc ggc aac agc ccc gtg cag gag ttc 144
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
acc gtg ccc aag gac gtg gac acc gcc acc atc agc ggc ctg aag ccc 192
Thr Val Pro Lys Asp Val Asp Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
ggc gtg gac tac acc atc acc gtg tac gcc gtg acc agc agc tac aag 240
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Ser Tyr Lys
65 70 75 80
tac gac atg cag tac agc ccc atc agc aac tac aga acc gcc ctg gag 288
Tyr Asp Met Gln Tyr Ser Pro Ile Ser Asn Tyr Arg Thr Ala Leu Glu
85 90 95
<210> 131
<211> 96
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 131
Val Ser Asp Val Pro Arg Lys Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15
Ser Leu Leu Ile Ser Trp Glu His Asp Tyr Pro Tyr Arg Arg Tyr Tyr
20 25 30
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
35 40 45
Thr Val Pro Lys Asp Val Asp Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Ser Tyr Lys
65 70 75 80
Tyr Asp Met Gln Tyr Ser Pro Ile Ser Asn Tyr Arg Thr Ala Leu Glu
85 90 95
<210> 132
<211> 276
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (1Fn3)
<220>
<221> CDS
<222> (1)..(276)
<400> 132
agc ggc ccc gtg gag gtg ttc atc acc gag acc ccc agc cag ccc aac 48
Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn
1 5 10 15
agc cac ccc atc cag tgg aac gcc ccc cag ccc agc cac atc agc aag 96
Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys
20 25 30
tac atc ctg aga tgg aga ccc aag aac agc gtg ggc aga tgg aag gag 144
Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu
35 40 45
gcc acc atc ccc ggc cac ctg aac agc tac acc atc aag ggc ctg aag 192
Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys
50 55 60
ccc ggc gtg gtg tac gag ggc cag ctg atc agc atc cag cag tac ggc 240
Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly
65 70 75 80
cac cag gag gtg acc aga ttc gac ttc acc acc acc 276
His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr
85 90
<210> 133
<211> 92
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 133
Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn
1 5 10 15
Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys
20 25 30
Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu
35 40 45
Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys
50 55 60
Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly
65 70 75 80
His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr
85 90
<210> 134
<211> 270
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (2Fn3)
<220>
<221> CDS
<222> (1)..(270)
<400> 134
agc ccc ctg gtg gcc acc agc gag agc gtg acc gag atc acc gcc agc 48
Ser Pro Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser
1 5 10 15
agc ttc gtg gtg agc tgg gtg agc gcc agc gac acc gtg agc ggc ttc 96
Ser Phe Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe
20 25 30
aga gtg gag tac gag ctg agc gag gag ggc gac gag ccc cag tac ctg 144
Arg Val Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu
35 40 45
gac ctg ccc agc acc gcc acc agc gtg aac atc ccc gac ctg ctg ccc 192
Asp Leu Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro
50 55 60
ggc aga aag tac atc gtg aac gtg tac cag agc gag gac ggc gag cag 240
Gly Arg Lys Tyr Ile Val Asn Val Tyr Gln Ser Glu Asp Gly Glu Gln
65 70 75 80
agc ctg atc ctg agc acc agc cag acc acc 270
Ser Leu Ile Leu Ser Thr Ser Gln Thr Thr
85 90
<210> 135
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 135
Ser Pro Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser
1 5 10 15
Ser Phe Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe
20 25 30
Arg Val Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu
35 40 45
Asp Leu Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro
50 55 60
Gly Arg Lys Tyr Ile Val Asn Val Tyr Gln Ser Glu Asp Gly Glu Gln
65 70 75 80
Ser Leu Ile Leu Ser Thr Ser Gln Thr Thr
85 90
<210> 136
<211> 282
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (3Fn3)
<220>
<221> CDS
<222> (1)..(282)
<400> 136
gcc ccc gac gcc ccc ccc gac ccc acc gtg gac cag gtg gac gac acc 48
Ala Pro Asp Ala Pro Pro Asp Pro Thr Val Asp Gln Val Asp Asp Thr
1 5 10 15
agc atc gtg gtg aga tgg agc aga ccc cag gcc ccc atc acc ggc tac 96
Ser Ile Val Val Arg Trp Ser Arg Pro Gln Ala Pro Ile Thr Gly Tyr
20 25 30
aga atc gtg tac agc ccc agc gtg gag ggc agc agc acc gag ctg aac 144
Arg Ile Val Tyr Ser Pro Ser Val Glu Gly Ser Ser Thr Glu Leu Asn
35 40 45
ctg ccc gag acc gcc aac agc gtg acc ctg agc gac ctg cag ccc ggc 192
Leu Pro Glu Thr Ala Asn Ser Val Thr Leu Ser Asp Leu Gln Pro Gly
50 55 60
gtg cag tac aac atc acc atc tac gcc gtg gag gag aac cag gag agc 240
Val Gln Tyr Asn Ile Thr Ile Tyr Ala Val Glu Glu Asn Gln Glu Ser
65 70 75 80
acc ccc gtg gtg atc cag cag gag acc acc ggc acc ccc aga 282
Thr Pro Val Val Ile Gln Gln Glu Thr Thr Gly Thr Pro Arg
85 90
<210> 137
<211> 94
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 137
Ala Pro Asp Ala Pro Pro Asp Pro Thr Val Asp Gln Val Asp Asp Thr
1 5 10 15
Ser Ile Val Val Arg Trp Ser Arg Pro Gln Ala Pro Ile Thr Gly Tyr
20 25 30
Arg Ile Val Tyr Ser Pro Ser Val Glu Gly Ser Ser Thr Glu Leu Asn
35 40 45
Leu Pro Glu Thr Ala Asn Ser Val Thr Leu Ser Asp Leu Gln Pro Gly
50 55 60
Val Gln Tyr Asn Ile Thr Ile Tyr Ala Val Glu Glu Asn Gln Glu Ser
65 70 75 80
Thr Pro Val Val Ile Gln Gln Glu Thr Thr Gly Thr Pro Arg
85 90
<210> 138
<211> 270
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (4Fn3)
<220>
<221> CDS
<222> (1)..(270)
<400> 138
acc gtg ccc agc ccc aga gac ctg cag ttc gtg gag gtg acc gac gtg 48
Thr Val Pro Ser Pro Arg Asp Leu Gln Phe Val Glu Val Thr Asp Val
1 5 10 15
aag gtg acc atc atg tgg acc ccc ccc gag agc gcc gtg acc ggc tac 96
Lys Val Thr Ile Met Trp Thr Pro Pro Glu Ser Ala Val Thr Gly Tyr
20 25 30
aga gtg gac gtg atc ccc gtg aac ctg ccc ggc gag cac ggc cag aga 144
Arg Val Asp Val Ile Pro Val Asn Leu Pro Gly Glu His Gly Gln Arg
35 40 45
ctg ccc atc agc aga aac acc ttc gcc gag gtg acc ggc ctg agc ccc 192
Leu Pro Ile Ser Arg Asn Thr Phe Ala Glu Val Thr Gly Leu Ser Pro
50 55 60
ggc gtg acc tac tac ttc aag gtg ttc gcc gtg agc cac ggc aga gag 240
Gly Val Thr Tyr Tyr Phe Lys Val Phe Ala Val Ser His Gly Arg Glu
65 70 75 80
agc aag ccc ctg acc gcc cag cag acc acc 270
Ser Lys Pro Leu Thr Ala Gln Gln Thr Thr
85 90
<210> 139
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 139
Thr Val Pro Ser Pro Arg Asp Leu Gln Phe Val Glu Val Thr Asp Val
1 5 10 15
Lys Val Thr Ile Met Trp Thr Pro Pro Glu Ser Ala Val Thr Gly Tyr
20 25 30
Arg Val Asp Val Ile Pro Val Asn Leu Pro Gly Glu His Gly Gln Arg
35 40 45
Leu Pro Ile Ser Arg Asn Thr Phe Ala Glu Val Thr Gly Leu Ser Pro
50 55 60
Gly Val Thr Tyr Tyr Phe Lys Val Phe Ala Val Ser His Gly Arg Glu
65 70 75 80
Ser Lys Pro Leu Thr Ala Gln Gln Thr Thr
85 90
<210> 140
<211> 270
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (5Fn3)
<220>
<221> CDS
<222> (1)..(270)
<400> 140
aag ctg gac gcc ccc acc aac ctg cag ttc gtg aac gag acc gac agc 48
Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu Thr Asp Ser
1 5 10 15
acc gtg ctg gtg aga tgg acc ccc ccc aga gcc cag atc acc ggc tac 96
Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln Ile Thr Gly Tyr
20 25 30
aga ctg acc gtg ggc ctg acc aga aga ggc cag ccc aga cag tac aac 144
Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln Pro Arg Gln Tyr Asn
35 40 45
gtg ggc ccc agc gtg agc aag tac ccc ctg aga aac ctg cag ccc gcc 192
Val Gly Pro Ser Val Ser Lys Tyr Pro Leu Arg Asn Leu Gln Pro Ala
50 55 60
agc gag tac acc gtg agc ctg gtg gcc atc aag ggc aac cag gag agc 240
Ser Glu Tyr Thr Val Ser Leu Val Ala Ile Lys Gly Asn Gln Glu Ser
65 70 75 80
ccc aag gcc acc ggc gtg ttc acc acc ctg 270
Pro Lys Ala Thr Gly Val Phe Thr Thr Leu
85 90
<210> 141
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 141
Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu Thr Asp Ser
1 5 10 15
Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln Ile Thr Gly Tyr
20 25 30
Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln Pro Arg Gln Tyr Asn
35 40 45
Val Gly Pro Ser Val Ser Lys Tyr Pro Leu Arg Asn Leu Gln Pro Ala
50 55 60
Ser Glu Tyr Thr Val Ser Leu Val Ala Ile Lys Gly Asn Gln Glu Ser
65 70 75 80
Pro Lys Ala Thr Gly Val Phe Thr Thr Leu
85 90
<210> 142
<211> 258
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (6Fn3)
<220>
<221> CDS
<222> (1)..(258)
<400> 142
cag ccc ggc agc agc atc ccc ccc tac aac acc gag gtg acc gag acc 48
Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val Thr Glu Thr
1 5 10 15
acc atc gtg atc acc tgg acc ccc gcc ccc aga ctg ggc ttc aag ctg 96
Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Leu Gly Phe Lys Leu
20 25 30
ggc gtg aga ccc agc cag ggc ggc gag gcc ccc aga gag gtg acc agc 144
Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro Arg Glu Val Thr Ser
35 40 45
gac agc ggc agc gtg gtg agc ggc ctg acc ccc ggc gtg gag tac gtg 192
Asp Ser Gly Ser Val Val Ser Gly Leu Thr Pro Gly Val Glu Tyr Val
50 55 60
tac acc atc cag gtg ctg aga gac ggc cag gag aga gac gcc ccc atc 240
Tyr Thr Ile Gln Val Leu Arg Asp Gly Gln Glu Arg Asp Ala Pro Ile
65 70 75 80
gtg aac aag gtg gtg acc 258
Val Asn Lys Val Val Thr
85
<210> 143
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 143
Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val Thr Glu Thr
1 5 10 15
Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Leu Gly Phe Lys Leu
20 25 30
Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro Arg Glu Val Thr Ser
35 40 45
Asp Ser Gly Ser Val Val Ser Gly Leu Thr Pro Gly Val Glu Tyr Val
50 55 60
Tyr Thr Ile Gln Val Leu Arg Asp Gly Gln Glu Arg Asp Ala Pro Ile
65 70 75 80
Val Asn Lys Val Val Thr
85
<210> 144
<211> 282
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (7Fn3)
<220>
<221> CDS
<222> (1)..(282)
<400> 144
ccc ctg agc ccc ccc acc aac ctg cac ctg gag gcc aac ccc gac acc 48
Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr
1 5 10 15
ggc gtg ctg acc gtg agc tgg gag aga agc acc acc ccc gac atc acc 96
Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile Thr
20 25 30
ggc tac aga atc acc acc acc ccc acc aac ggc cag cag ggc aac agc 144
Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly Asn Ser
35 40 45
ctg gag gag gtg gtg cac gcc gac cag agc agc tgc acc ttc gac aac 192
Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr Phe Asp Asn
50 55 60
ctg agc ccc ggc ctg gag tac aac gtg agc gtg tac acc gtg aag gac 240
Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr Thr Val Lys Asp
65 70 75 80
gac aag gag agc gtg ccc atc agc gac acc atc atc ccc tga 282
Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile Ile Pro
85 90
<210> 145
<211> 93
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 145
Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr
1 5 10 15
Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile Thr
20 25 30
Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly Asn Ser
35 40 45
Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr Phe Asp Asn
50 55 60
Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr Thr Val Lys Asp
65 70 75 80
Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile Ile Pro
85 90
<210> 146
<211> 276
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (8Fn3)
<220>
<221> CDS
<222> (1)..(276)
<400> 146
gcc gtg ccc ccc ccc acc gac ctg aga ttc acc aac atc ggc ccc gac 48
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
1 5 10 15
acc atg aga gtg acc tgg gcc ccc ccc ccc agc atc gac ctg acc aac 96
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
20 25 30
ttc ctg gtg aga tac agc ccc gtg aag aac gag gag gac gtg gcc gag 144
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
35 40 45
ctg agc atc agc ccc agc gac aac gcc gtg gtg ctg acc aac ctg ctg 192
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
50 55 60
ccc ggc acc gag tac gtg gtg agc gtg agc agc gtg tac gag cag cac 240
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His
65 70 75 80
gag agc acc ccc ctg aga ggc aga cag aag acc tga 276
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr
85 90
<210> 147
<211> 91
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 147
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
1 5 10 15
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
20 25 30
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
35 40 45
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
50 55 60
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His
65 70 75 80
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr
85 90
<210> 148
<211> 273
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (9Fn3)
<220>
<221> CDS
<222> (1)..(273)
<400> 148
ggc ctg gac agc ccc acc ggc atc gac ttc agc gac atc acc gcc aac 48
Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn
1 5 10 15
agc ttc acc gtg cac tgg atc gcc ccc aga gcc acc atc acc ggc tac 96
Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr
20 25 30
aga atc aga cac cac ccc gag cac ttc agc ggc aga ccc aga gag gac 144
Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp
35 40 45
aga gtg ccc cac agc aga aac agc atc acc ctg acc aac ctg acc ccc 192
Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro
50 55 60
ggc acc gag tac gtg gtg agc atc gtg gcc ctg aac ggc aga gag gag 240
Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu
65 70 75 80
agc ccc ctg ctg atc ggc cag cag agc acc tga 273
Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr
85 90
<210> 149
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 149
Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn
1 5 10 15
Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr
20 25 30
Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp
35 40 45
Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro
50 55 60
Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu
65 70 75 80
Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr
85 90
<210> 150
<211> 279
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (10Fn3)
<220>
<221> CDS
<222> (1)..(279)
<400> 150
gtg agc gac gtg ccc aga gac ctg gtg gtg gcc gcc acc ccc acc agc 48
Val Ser Asp Val Pro Arg Asp Leu Val Val Ala Ala Thr Pro Thr Ser
1 5 10 15
ctg ctg atc agc tgg gac gcc ccc gcc gtg acc gtg aga tac tac aga 96
Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg
20 25 30
atc acc tac ggc gag acc ggc ggc aac agc ccc gtg cag gag ttc acc 144
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr
35 40 45
gtg ccc ggc agc aag agc acc gcc acc atc agc ggc ctg aag ccc ggc 192
Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
50 55 60
gtg gac tac acc atc acc gtg tac gcc gtg acc ggc aga ggc gac agc 240
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser
65 70 75 80
ccc gcc agc agc aag ccc atc agc atc aac tac aga acc 279
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr
85 90
<210> 151
<211> 93
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 151
Val Ser Asp Val Pro Arg Asp Leu Val Val Ala Ala Thr Pro Thr Ser
1 5 10 15
Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg
20 25 30
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr
35 40 45
Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
50 55 60
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser
65 70 75 80
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr
85 90
<210> 152
<211> 270
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (11Fn3)
<220>
<221> CDS
<222> (1)..(270)
<400> 152
gag atc gac aag ccc agc cag atg cag gtg acc gac gtg cag gac aac 48
Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val Gln Asp Asn
1 5 10 15
agc atc agc gtg aag tgg ctg ccc agc agc agc ccc gtg acc ggc tac 96
Ser Ile Ser Val Lys Trp Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr
20 25 30
aga gtg acc acc acc ccc aag aac ggc ccc ggc ccc acc aag acc aag 144
Arg Val Thr Thr Thr Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys
35 40 45
acc gcc ggc ccc gac cag acc gag atg acc atc gag ggc ctg cag ccc 192
Thr Ala Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro
50 55 60
acc gtg gag tac gtg gtg agc gtg tac gcc cag aac ccc agc ggc gag 240
Thr Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu
65 70 75 80
agc cag ccc ctg gtg cag acc gcc gtg acc 270
Ser Gln Pro Leu Val Gln Thr Ala Val Thr
85 90
<210> 153
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 153
Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val Gln Asp Asn
1 5 10 15
Ser Ile Ser Val Lys Trp Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr
20 25 30
Arg Val Thr Thr Thr Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys
35 40 45
Thr Ala Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro
50 55 60
Thr Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu
65 70 75 80
Ser Gln Pro Leu Val Gln Thr Ala Val Thr
85 90
<210> 154
<211> 273
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (12Fn3)
<220>
<221> CDS
<222> (1)..(273)
<400> 154
aac atc gac aga ccc aag ggc ctg gcc ttc acc gac gtg gac gtg gac 48
Asn Ile Asp Arg Pro Lys Gly Leu Ala Phe Thr Asp Val Asp Val Asp
1 5 10 15
agc atc aag atc gcc tgg gag agc ccc cag ggc cag gtg agc aga tac 96
Ser Ile Lys Ile Ala Trp Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr
20 25 30
aga gtg acc tac agc agc ccc gag gac ggc atc cac gag ctg ttc ccc 144
Arg Val Thr Tyr Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro
35 40 45
gcc ccc gac ggc gag gag gac acc gcc gag ctg cag ggc ctg aga ccc 192
Ala Pro Asp Gly Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro
50 55 60
ggc agc gag tac acc gtg agc gtg gtg gcc ctg cac gac gac atg gag 240
Gly Ser Glu Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu
65 70 75 80
agc cag ccc ctg atc ggc acc cag agc acc tga 273
Ser Gln Pro Leu Ile Gly Thr Gln Ser Thr
85 90
<210> 155
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 155
Asn Ile Asp Arg Pro Lys Gly Leu Ala Phe Thr Asp Val Asp Val Asp
1 5 10 15
Ser Ile Lys Ile Ala Trp Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr
20 25 30
Arg Val Thr Tyr Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro
35 40 45
Ala Pro Asp Gly Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro
50 55 60
Gly Ser Glu Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu
65 70 75 80
Ser Gln Pro Leu Ile Gly Thr Gln Ser Thr
85 90
<210> 156
<211> 276
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (13Fn3)
<220>
<221> CDS
<222> (1)..(276)
<400> 156
gcc atc ccc gcc ccc acc gac ctg aag ttc acc cag gtg acc ccc acc 48
Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr
1 5 10 15
agc ctg agc gcc cag tgg acc ccc ccc aac gtg cag ctg acc ggc tac 96
Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr
20 25 30
aga gtg aga gtg acc ccc aag gag aag acc ggc ccc atg aag gag atc 144
Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile
35 40 45
aac ctg gcc ccc gac agc agc agc gtg gtg gtg agc ggc ctg atg gtg 192
Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val
50 55 60
gcc acc aag tac gag gtg agc gtg tac gcc ctg aag gac acc ctg acc 240
Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr
65 70 75 80
agc aga ccc gcc cag ggc gtg gtg acc acc ctg gag 276
Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu
85 90
<210> 157
<211> 92
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 157
Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr
1 5 10 15
Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr
20 25 30
Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile
35 40 45
Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val
50 55 60
Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr
65 70 75 80
Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu
85 90
<210> 158
<211> 264
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (14Fn3)
<220>
<221> CDS
<222> (1)..(264)
<400> 158
aac gtg agc ccc ccc aga aga gcc aga gtg acc gac gcc acc gag acc 48
Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr
1 5 10 15
acc atc acc atc agc tgg aga acc aag acc gag acc atc acc ggc ttc 96
Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe
20 25 30
cag gtg gac gcc gtg ccc gcc aac ggc cag acc ccc atc cag aga acc 144
Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr
35 40 45
atc aag ccc gac gtg aga agc tac acc atc acc ggc ctg cag ccc ggc 192
Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly
50 55 60
acc gac tac aag atc tac ctg tac acc ctg aac gac aac gcc aga agc 240
Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
65 70 75 80
agc gtg gtg atc gac gcc agc acc 264
Ser Val Val Ile Asp Ala Ser Thr
85
<210> 159
<211> 88
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 159
Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr
1 5 10 15
Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe
20 25 30
Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr
35 40 45
Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly
50 55 60
Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
65 70 75 80
Ser Val Val Ile Asp Ala Ser Thr
85
<210> 160
<211> 270
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (15Fn3)
<220>
<221> CDS
<222> (1)..(270)
<400> 160
gcc atc gac gcc ccc agc aac ctg aga ttc ctg gcc acc acc ccc aac 48
Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn
1 5 10 15
agc ctg ctg gtg agc tgg cag ccc ccc aga gcc aga atc acc ggc tac 96
Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr
20 25 30
atc atc aag tac gag aag ccc ggc agc ccc ccc aga gag gtg gtg ccc 144
Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro
35 40 45
aga ccc aga ccc ggc gtg acc gag gcc acc atc acc ggc ctg gag ccc 192
Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro
50 55 60
ggc acc gag tac acc atc tac gtg atc gcc ctg aag aac aac cag aag 240
Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys
65 70 75 80
agc gag ccc ctg atc ggc aga aag aag acc 270
Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr
85 90
<210> 161
<211> 90
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 161
Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn
1 5 10 15
Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr
20 25 30
Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro
35 40 45
Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro
50 55 60
Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys
65 70 75 80
Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr
85 90
<210> 162
<211> 264
<212> DNA
<213> Artificial sequence
<220>
<223> Pronectins (16Fn3)
<220>
<221> CDS
<222> (1)..(264)
<400> 162
ccc ggc ctg aac ccc aac gcc agc acc ggc cag gag gcc ctg agc cag 48
Pro Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser Gln
1 5 10 15
acc acc atc agc tgg gcc ccc ttc cag gac acc agc gag tac atc atc 96
Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile Ile
20 25 30
agc tgc cac ccc gtg ggc acc gac gag gag ccc ctg cag ttc aga gtg 144
Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe Arg Val
35 40 45
ccc ggc acc agc acc agc gcc acc ctg acc ggc ctg acc aga ggc gcc 192
Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr Arg Gly Ala
50 55 60
acc tac aac atc atc gtg gag gcc ctg aag gac cag cag aga cac aag 240
Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln Gln Arg His Lys
65 70 75 80
gtg aga gag gag gtg gtg acc gtg 264
Val Arg Glu Glu Val Val Thr Val
85
<210> 163
<211> 88
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 163
Pro Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser Gln
1 5 10 15
Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile Ile
20 25 30
Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe Arg Val
35 40 45
Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr Arg Gly Ala
50 55 60
Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln Gln Arg His Lys
65 70 75 80
Val Arg Glu Glu Val Val Thr Val
85
<210> 164
<211> 276
<212> DNA
<213> Artificial sequence
<220>
<223> Adhiron
<220>
<221> CDS
<222> (1)..(276)
<400> 164
gcc acc ggc gtg aga gcc gtg ccc ggc aac gag aac agc ctg gag atc 48
Ala Thr Gly Val Arg Ala Val Pro Gly Asn Glu Asn Ser Leu Glu Ile
1 5 10 15
gag gag ctg gcc aga ttc gcc gtg gac gag cac aac aag aag gag aac 96
Glu Glu Leu Ala Arg Phe Ala Val Asp Glu His Asn Lys Lys Glu Asn
20 25 30
gcc ctg ctg gag ttc gtg aga gtg gtg aag gcc aag gag cag gtg gtg 144
Ala Leu Leu Glu Phe Val Arg Val Val Lys Ala Lys Glu Gln Val Val
35 40 45
gcc ggc acc atg tac tac ctg acc ctg gag gcc aag gac ggc ggc aag 192
Ala Gly Thr Met Tyr Tyr Leu Thr Leu Glu Ala Lys Asp Gly Gly Lys
50 55 60
aag aag ctg tac gag gcc aag gtg tgg gtg aag ccc tgg gag aac ttc 240
Lys Lys Leu Tyr Glu Ala Lys Val Trp Val Lys Pro Trp Glu Asn Phe
65 70 75 80
aag gag ctg cag gag ttc aag ccc gtg ggc gac gcc 276
Lys Glu Leu Gln Glu Phe Lys Pro Val Gly Asp Ala
85 90
<210> 165
<211> 92
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 165
Ala Thr Gly Val Arg Ala Val Pro Gly Asn Glu Asn Ser Leu Glu Ile
1 5 10 15
Glu Glu Leu Ala Arg Phe Ala Val Asp Glu His Asn Lys Lys Glu Asn
20 25 30
Ala Leu Leu Glu Phe Val Arg Val Val Lys Ala Lys Glu Gln Val Val
35 40 45
Ala Gly Thr Met Tyr Tyr Leu Thr Leu Glu Ala Lys Asp Gly Gly Lys
50 55 60
Lys Lys Leu Tyr Glu Ala Lys Val Trp Val Lys Pro Trp Glu Asn Phe
65 70 75 80
Lys Glu Leu Gln Glu Phe Lys Pro Val Gly Asp Ala
85 90
<210> 166
<211> 177
<212> DNA
<213> Artificial sequence
<220>
<223> affinity body
<220>
<221> CDS
<222> (1)..(177)
<400> 166
gtg gac aac aag ttc aac aag gag cag cag aac gcc ttc tac gag atc 48
Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
ctg cac ctg ccc aac ctg aac gag gag cag aga aac gcc ttc atc cag 96
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
agc ctg aag gac gac ccc agc cag agc gcc aac ctg ctg gcc gag gcc 144
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
aag aag ctg aac gac gcc cag gcc ccc aag tga 177
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 167
<211> 58
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 167
Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
1 5 10 15
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln
20 25 30
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
<210> 168
<211> 522
<212> DNA
<213> Artificial sequence
<220>
<223> Affilins (gamma-B-crystallin)
<220>
<221> CDS
<222> (1)..(522)
<400> 168
ggc aag atc acc ttc tac gag gac aga gcc ttc cag ggc aga agc tac 48
Gly Lys Ile Thr Phe Tyr Glu Asp Arg Ala Phe Gln Gly Arg Ser Tyr
1 5 10 15
gag tgc acc acc gac tgc ccc aac ctg cag ccc tac ttc agc aga tgc 96
Glu Cys Thr Thr Asp Cys Pro Asn Leu Gln Pro Tyr Phe Ser Arg Cys
20 25 30
aac agc atc aga gtg gag agc ggc tgc tgg atg atc tac gag aga ccc 144
Asn Ser Ile Arg Val Glu Ser Gly Cys Trp Met Ile Tyr Glu Arg Pro
35 40 45
aac tac cag ggc cac cag tac ttc ctg aga aga ggc gag tac ccc gac 192
Asn Tyr Gln Gly His Gln Tyr Phe Leu Arg Arg Gly Glu Tyr Pro Asp
50 55 60
tac cag cag tgg atg ggc ctg agc gac agc atc aga agc tgc tgc ctg 240
Tyr Gln Gln Trp Met Gly Leu Ser Asp Ser Ile Arg Ser Cys Cys Leu
65 70 75 80
atc ccc ccc cac agc ggc gcc tac aga atg aag atc tac gac aga gac 288
Ile Pro Pro His Ser Gly Ala Tyr Arg Met Lys Ile Tyr Asp Arg Asp
85 90 95
gag ctg aga ggc cag atg agc gag ctg acc gac gac tgc atc agc gtg 336
Glu Leu Arg Gly Gln Met Ser Glu Leu Thr Asp Asp Cys Ile Ser Val
100 105 110
cag gac aga ttc cac ctg acc gag atc cac agc ctg aac gtg ctg gag 384
Gln Asp Arg Phe His Leu Thr Glu Ile His Ser Leu Asn Val Leu Glu
115 120 125
ggc agc tgg atc ctg tac gag atg ccc aac tac aga ggc aga cag tac 432
Gly Ser Trp Ile Leu Tyr Glu Met Pro Asn Tyr Arg Gly Arg Gln Tyr
130 135 140
ctg ctg aga ccc ggc gag tac aga aga ttc ctg gac tgg ggc gcc ccc 480
Leu Leu Arg Pro Gly Glu Tyr Arg Arg Phe Leu Asp Trp Gly Ala Pro
145 150 155 160
aac gcc aag gtg ggc agc ctg aga aga gtg atg gac ctg tac 522
Asn Ala Lys Val Gly Ser Leu Arg Arg Val Met Asp Leu Tyr
165 170
<210> 169
<211> 174
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 169
Gly Lys Ile Thr Phe Tyr Glu Asp Arg Ala Phe Gln Gly Arg Ser Tyr
1 5 10 15
Glu Cys Thr Thr Asp Cys Pro Asn Leu Gln Pro Tyr Phe Ser Arg Cys
20 25 30
Asn Ser Ile Arg Val Glu Ser Gly Cys Trp Met Ile Tyr Glu Arg Pro
35 40 45
Asn Tyr Gln Gly His Gln Tyr Phe Leu Arg Arg Gly Glu Tyr Pro Asp
50 55 60
Tyr Gln Gln Trp Met Gly Leu Ser Asp Ser Ile Arg Ser Cys Cys Leu
65 70 75 80
Ile Pro Pro His Ser Gly Ala Tyr Arg Met Lys Ile Tyr Asp Arg Asp
85 90 95
Glu Leu Arg Gly Gln Met Ser Glu Leu Thr Asp Asp Cys Ile Ser Val
100 105 110
Gln Asp Arg Phe His Leu Thr Glu Ile His Ser Leu Asn Val Leu Glu
115 120 125
Gly Ser Trp Ile Leu Tyr Glu Met Pro Asn Tyr Arg Gly Arg Gln Tyr
130 135 140
Leu Leu Arg Pro Gly Glu Tyr Arg Arg Phe Leu Asp Trp Gly Ala Pro
145 150 155 160
Asn Ala Lys Val Gly Ser Leu Arg Arg Val Met Asp Leu Tyr
165 170
<210> 170
<211> 294
<212> DNA
<213> Artificial sequence
<220>
<223> Affimers
<220>
<221> CDS
<222> (1)..(294)
<400> 170
atg atc ccc aga ggc ctg agc gag gcc aag ccc gcc acc ccc gag atc 48
Met Ile Pro Arg Gly Leu Ser Glu Ala Lys Pro Ala Thr Pro Glu Ile
1 5 10 15
cag gag atc gtg gac aag gtg aag ccc cag ctg gag gag aag acc aac 96
Gln Glu Ile Val Asp Lys Val Lys Pro Gln Leu Glu Glu Lys Thr Asn
20 25 30
gag acc tac ggc aag ctg gag gcc gtg cag tac aag acc cag gtg ctg 144
Glu Thr Tyr Gly Lys Leu Glu Ala Val Gln Tyr Lys Thr Gln Val Leu
35 40 45
gcc agc acc aac tac tac atc aag gtg aga gcc ggc gac aac aag tac 192
Ala Ser Thr Asn Tyr Tyr Ile Lys Val Arg Ala Gly Asp Asn Lys Tyr
50 55 60
atg cac ctg aag gtg ttc aac ggc ccc ccc ggc cag aac gcc gac aga 240
Met His Leu Lys Val Phe Asn Gly Pro Pro Gly Gln Asn Ala Asp Arg
65 70 75 80
gtg ctg acc ggc tac cag gtg gac aag aac aag gac gac gag ctg acc 288
Val Leu Thr Gly Tyr Gln Val Asp Lys Asn Lys Asp Asp Glu Leu Thr
85 90 95
ggc ttc 294
Gly Phe
<210> 171
<211> 98
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 171
Met Ile Pro Arg Gly Leu Ser Glu Ala Lys Pro Ala Thr Pro Glu Ile
1 5 10 15
Gln Glu Ile Val Asp Lys Val Lys Pro Gln Leu Glu Glu Lys Thr Asn
20 25 30
Glu Thr Tyr Gly Lys Leu Glu Ala Val Gln Tyr Lys Thr Gln Val Leu
35 40 45
Ala Ser Thr Asn Tyr Tyr Ile Lys Val Arg Ala Gly Asp Asn Lys Tyr
50 55 60
Met His Leu Lys Val Phe Asn Gly Pro Pro Gly Gln Asn Ala Asp Arg
65 70 75 80
Val Leu Thr Gly Tyr Gln Val Asp Lys Asn Lys Asp Asp Glu Leu Thr
85 90 95
Gly Phe
<210> 172
<211> 462
<212> DNA
<213> Artificial sequence
<220>
<223> antiporter protein (lipocalin Lcn1)
<220>
<221> CDS
<222> (1)..(462)
<400> 172
atc gcc agc gac gag gag atc cag gac gtg agc ggc acc tgg tac ctg 48
Ile Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu
1 5 10 15
aag gcc atg acc gtg gac aga gag ttc ccc gag atg aac ctg gag agc 96
Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn Leu Glu Ser
20 25 30
gtg acc ccc atg acc ctg acc acc ctg gag ggc ggc aac ctg gag gcc 144
Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala
35 40 45
aag gtg acc atg ctg atc agc ggc aga tgc cag gag gtg aag gcc gtg 192
Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln Glu Val Lys Ala Val
50 55 60
ctg gag aag acc gac gag ccc ggc aag tac acc gcc gac ggc ggc aag 240
Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys
65 70 75 80
cac gtg gcc tac atc atc aga agc cac gtg aag gac cac tac atc ttc 288
His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr Ile Phe
85 90 95
tac agc gag ggc gag ctg cac ggc aag ccc gtg aga ggc gtg aag ctg 336
Tyr Ser Glu Gly Glu Leu His Gly Lys Pro Val Arg Gly Val Lys Leu
100 105 110
gtg ggc aga gac ccc aag aac aac ctg gag gcc ctg ctg gac ttc gag 384
Val Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Leu Asp Phe Glu
115 120 125
aag gcc gcc ggc gcc aga ggc ctg agc acc gag agc atc ctg atc ccc 432
Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro
130 135 140
aga cag agc gag acc tgc agc ccc ggc agc 462
Arg Gln Ser Glu Thr Cys Ser Pro Gly Ser
145 150
<210> 173
<211> 154
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 173
Ile Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu
1 5 10 15
Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn Leu Glu Ser
20 25 30
Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala
35 40 45
Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln Glu Val Lys Ala Val
50 55 60
Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys
65 70 75 80
His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr Ile Phe
85 90 95
Tyr Ser Glu Gly Glu Leu His Gly Lys Pro Val Arg Gly Val Lys Leu
100 105 110
Val Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Leu Asp Phe Glu
115 120 125
Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro
130 135 140
Arg Gln Ser Glu Thr Cys Ser Pro Gly Ser
145 150
<210> 174
<211> 534
<212> DNA
<213> Artificial sequence
<220>
<223> antiporter protein (lipocalin Lcn2)
<220>
<221> CDS
<222> (1)..(534)
<400> 174
cag gac agc acc agc gac ctg atc ccc gcc ccc ccc ctg agc aag gtg 48
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
ccc ctg cag cag aac ttc cag gac aac cag ttc cag ggc aag tgg tac 96
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr
20 25 30
gtg gtg ggc ctg gcc ggc aac gcc atc ctg aga gag gac aag gac ccc 144
Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro
35 40 45
cag aag atg tac gcc acc atc tac gag ctg aag gag gac aag agc tac 192
Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
aac gtg acc agc gtg ctg ttc aga aag aag aag tgc gac tac tgg atc 240
Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile
65 70 75 80
aga acc ttc gtg ccc ggc tgc cag ccc ggc gag ttc acc ctg ggc aac 288
Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn
85 90 95
atc aag agc tac ccc ggc ctg acc agc tac ctg gtg aga gtg gtg agc 336
Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110
acc aac tac aac cag cac gcc atg gtg ttc ttc aag aag gtg agc cag 384
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln
115 120 125
aac aga gag tac ttc aag atc acc ctg tac ggc aga acc aag gag ctg 432
Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
acc agc gag ctg aag gag aac ttc atc aga ttc agc aag agc ctg ggc 480
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
ctg ccc gag aac cac atc gtg ttc ccc gtg ccc atc gac cag tgc atc 528
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
gac ggc 534
Asp Gly
<210> 175
<211> 178
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 175
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr
20 25 30
Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro
35 40 45
Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn
85 90 95
Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln
115 120 125
Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 176
<211> 255
<212> DNA
<213> Artificial sequence
<220>
<223> (C426) C-MET targeted Avimers
<220>
<221> CDS
<222> (1)..(255)
<400> 176
tgc gag agc ggc gag ttc cag tgc cac agc acc ggc aga tgc atc ccc 48
Cys Glu Ser Gly Glu Phe Gln Cys His Ser Thr Gly Arg Cys Ile Pro
1 5 10 15
cag gag tgg gtg tgc gac ggc gac aac gac tgc gag gac agc agc gac 96
Gln Glu Trp Val Cys Asp Gly Asp Asn Asp Cys Glu Asp Ser Ser Asp
20 25 30
gag gcc ccc gac ctg tgc gcc agc gcc gag ccc acc tgc ccc agc ggc 144
Glu Ala Pro Asp Leu Cys Ala Ser Ala Glu Pro Thr Cys Pro Ser Gly
35 40 45
gag ttc cag tgc aga agc acc aac aga tgc atc ccc gag acc tgg ctg 192
Glu Phe Gln Cys Arg Ser Thr Asn Arg Cys Ile Pro Glu Thr Trp Leu
50 55 60
tgc gac ggc gac aac gac tgc gag gac ggc agc gac gag gag agc tgc 240
Cys Asp Gly Asp Asn Asp Cys Glu Asp Gly Ser Asp Glu Glu Ser Cys
65 70 75 80
acc ccc ccc acc tga 255
Thr Pro Pro Thr
<210> 177
<211> 84
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 177
Cys Glu Ser Gly Glu Phe Gln Cys His Ser Thr Gly Arg Cys Ile Pro
1 5 10 15
Gln Glu Trp Val Cys Asp Gly Asp Asn Asp Cys Glu Asp Ser Ser Asp
20 25 30
Glu Ala Pro Asp Leu Cys Ala Ser Ala Glu Pro Thr Cys Pro Ser Gly
35 40 45
Glu Phe Gln Cys Arg Ser Thr Asn Arg Cys Ile Pro Glu Thr Trp Leu
50 55 60
Cys Asp Gly Asp Asn Asp Cys Glu Asp Gly Ser Asp Glu Glu Ser Cys
65 70 75 80
Thr Pro Pro Thr
<210> 178
<211> 267
<212> DNA
<213> Artificial sequence
<220>
<223> Centyrins (Fn 3 domain of tenascin)
<220>
<221> CDS
<222> (1)..(267)
<400> 178
ctg ccc gcc ccc aag aac ctg gtg gtg agc gag gtg acc gag gac agc 48
Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15
gcc aga ctg agc tgg acc gcc ccc gac gcc gcc ttc gac agc ttc ctg 96
Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu
20 25 30
atc ggc tac ggc gag agc gag aag gtg ggc gag gcc atc gtg ctg acc 144
Ile Gly Tyr Gly Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr
35 40 45
gtg ccc ggc agc gag aga agc tac gac ctg acc ggc ctg aag ccc ggc 192
Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60
acc gag tac acc gtg agc atc tac ggc gtg aag ggc ggc cac aga agc 240
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser
65 70 75 80
aac ccc ctg agc gcc atc ttc acc acc 267
Asn Pro Leu Ser Ala Ile Phe Thr Thr
85
<210> 179
<211> 89
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 179
Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser
1 5 10 15
Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu
20 25 30
Ile Gly Tyr Gly Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr
35 40 45
Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly
50 55 60
Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser
65 70 75 80
Asn Pro Leu Ser Ala Ile Phe Thr Thr
85
<210> 180
<211> 171
<212> DNA
<213> Artificial sequence
<220>
<223> Kunitz Domain/BPTI
<220>
<221> CDS
<222> (1)..(171)
<400> 180
gtg aga gag gtg tgc agc gag cag gcc gag acc ggc ccc tgc aga gcc 48
Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala
1 5 10 15
atg atc agc aga tgg tac ttc gac gtg acc gag ggc aag tgc gcc ccc 96
Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro
20 25 30
ttc ttc tac ggc ggc tgc tgc ggc ggc aac aga aac aac ttc gac acc 144
Phe Phe Tyr Gly Gly Cys Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr
35 40 45
gag gag tac tgc atg gcc gtg tgc ggc 171
Glu Glu Tyr Cys Met Ala Val Cys Gly
50 55
<210> 181
<211> 57
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 181
Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala
1 5 10 15
Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro
20 25 30
Phe Phe Tyr Gly Gly Cys Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr
35 40 45
Glu Glu Tyr Cys Met Ala Val Cys Gly
50 55
<210> 182
<211> 516
<212> DNA
<213> Artificial sequence
<220>
<223> Obodies (human AspRS)
<220>
<221> CDS
<222> (1)..(516)
<400> 182
gag atc atg gac gcc gcc gag gac tac gcc aag gag aga tac ggc atc 48
Glu Ile Met Asp Ala Ala Glu Asp Tyr Ala Lys Glu Arg Tyr Gly Ile
1 5 10 15
agc agc atg atc cag agc cag gag aag ccc gac aga gtg ctg gtg aga 96
Ser Ser Met Ile Gln Ser Gln Glu Lys Pro Asp Arg Val Leu Val Arg
20 25 30
gtg aga gac ctg acc atc cag aag gcc gac gag gtg gtg tgg gtg aga 144
Val Arg Asp Leu Thr Ile Gln Lys Ala Asp Glu Val Val Trp Val Arg
35 40 45
gcc aga gtg cac acc agc aga gcc aag ggc aag cag tgc ttc ctg gtg 192
Ala Arg Val His Thr Ser Arg Ala Lys Gly Lys Gln Cys Phe Leu Val
50 55 60
ctg aga cag cag cag ttc aac gtg cag gcc ctg gtg gcc gtg ggc gac 240
Leu Arg Gln Gln Gln Phe Asn Val Gln Ala Leu Val Ala Val Gly Asp
65 70 75 80
cac gcc agc aag cag atg gtg aag ttc gcc gcc aac atc aac aag gag 288
His Ala Ser Lys Gln Met Val Lys Phe Ala Ala Asn Ile Asn Lys Glu
85 90 95
agc atc gtg gac gtg gag ggc gtg gtg aga aag gtg aac cag aag atc 336
Ser Ile Val Asp Val Glu Gly Val Val Arg Lys Val Asn Gln Lys Ile
100 105 110
ggc agc tgc acc cag cag gac gtg gag ctg cac gtg cag aag atc tac 384
Gly Ser Cys Thr Gln Gln Asp Val Glu Leu His Val Gln Lys Ile Tyr
115 120 125
gtg atc agc ctg gcc gag ccc aga ctg ccc ctg cag ctg gac gac gcc 432
Val Ile Ser Leu Ala Glu Pro Arg Leu Pro Leu Gln Leu Asp Asp Ala
130 135 140
gtg aga ccc gag gcc gag ggc gag gag gag ggc aga gcc acc gtg aac 480
Val Arg Pro Glu Ala Glu Gly Glu Glu Glu Gly Arg Ala Thr Val Asn
145 150 155 160
cag gac acc aga ctg gac aac aga gtg atc gac ctg 516
Gln Asp Thr Arg Leu Asp Asn Arg Val Ile Asp Leu
165 170
<210> 183
<211> 172
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 183
Glu Ile Met Asp Ala Ala Glu Asp Tyr Ala Lys Glu Arg Tyr Gly Ile
1 5 10 15
Ser Ser Met Ile Gln Ser Gln Glu Lys Pro Asp Arg Val Leu Val Arg
20 25 30
Val Arg Asp Leu Thr Ile Gln Lys Ala Asp Glu Val Val Trp Val Arg
35 40 45
Ala Arg Val His Thr Ser Arg Ala Lys Gly Lys Gln Cys Phe Leu Val
50 55 60
Leu Arg Gln Gln Gln Phe Asn Val Gln Ala Leu Val Ala Val Gly Asp
65 70 75 80
His Ala Ser Lys Gln Met Val Lys Phe Ala Ala Asn Ile Asn Lys Glu
85 90 95
Ser Ile Val Asp Val Glu Gly Val Val Arg Lys Val Asn Gln Lys Ile
100 105 110
Gly Ser Cys Thr Gln Gln Asp Val Glu Leu His Val Gln Lys Ile Tyr
115 120 125
Val Ile Ser Leu Ala Glu Pro Arg Leu Pro Leu Gln Leu Asp Asp Ala
130 135 140
Val Arg Pro Glu Ala Glu Gly Glu Glu Glu Gly Arg Ala Thr Val Asn
145 150 155 160
Gln Asp Thr Arg Leu Asp Asn Arg Val Ile Asp Leu
165 170
<210> 184
<211> 267
<212> DNA
<213> Artificial sequence
<220>
<223> Tn3A
<220>
<221> CDS
<222> (1)..(267)
<400> 184
gcc atc gag gtg aag gac gtg acc gac acc acc gcc ctg atc acc tgg 48
Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15
agc gac gag ttc ggc cac gac tac gac ggc tgc gag ctg acc tac ggc 96
Ser Asp Glu Phe Gly His Asp Tyr Asp Gly Cys Glu Leu Thr Tyr Gly
20 25 30
atc aag gac gtg ccc ggc gac aga acc acc atc gac ctg tgg tgg cac 144
Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His
35 40 45
agc gcc tgg tac agc atc ggc aac ctg aag ccc gac acc gag gac gtg 192
Ser Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Asp Val
50 55 60
agc ctg atc tgc tac acc gac cag gag gcc ggc aac ccc gcc aag gag 240
Ser Leu Ile Cys Tyr Thr Asp Gln Glu Ala Gly Asn Pro Ala Lys Glu
65 70 75 80
acc ttc acc acc ggc ctg gtg ccc aga 267
Thr Phe Thr Thr Gly Leu Val Pro Arg
85
<210> 185
<211> 89
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 185
Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15
Ser Asp Glu Phe Gly His Asp Tyr Asp Gly Cys Glu Leu Thr Tyr Gly
20 25 30
Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His
35 40 45
Ser Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Asp Val
50 55 60
Ser Leu Ile Cys Tyr Thr Asp Gln Glu Ala Gly Asn Pro Ala Lys Glu
65 70 75 80
Thr Phe Thr Thr Gly Leu Val Pro Arg
85
<210> 186
<211> 276
<212> DNA
<213> Artificial sequence
<220>
<223> Tn3B
<220>
<221> CDS
<222> (1)..(276)
<400> 186
gcc atc gag gtg gag gac gtg acc gac acc acc gcc ctg atc acc tgg 48
Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15
acc aac aga agc agc tac agc aac ctg cac ggc tgc gag ctg gcc tac 96
Thr Asn Arg Ser Ser Tyr Ser Asn Leu His Gly Cys Glu Leu Ala Tyr
20 25 30
ggc atc aag gac gtg ccc ggc gac aga acc acc atc gac ctg aac cag 144
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln
35 40 45
ccc tac gtg cac tac agc atc ggc aac ctg aag ccc gac acc gag tac 192
Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
50 55 60
gag gtg agc ctg atc tgc ctg acc acc gac ggc acc tac aac aac ccc 240
Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Asn Asn Pro
65 70 75 80
gcc aag gag acc ttc acc acc ggc ctg gtg ccc aga 276
Ala Lys Glu Thr Phe Thr Thr Gly Leu Val Pro Arg
85 90
<210> 187
<211> 92
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 187
Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp
1 5 10 15
Thr Asn Arg Ser Ser Tyr Ser Asn Leu His Gly Cys Glu Leu Ala Tyr
20 25 30
Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln
35 40 45
Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
50 55 60
Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Asn Asn Pro
65 70 75 80
Ala Lys Glu Thr Phe Thr Thr Gly Leu Val Pro Arg
85 90
<210> 188
<211> 177
<212> DNA
<213> Artificial sequence
<220>
<223> Hckomers
<220>
<221> CDS
<222> (1)..(177)
<400> 188
acc ctg ttc gtg gcc ctg tac gac tac gag gcc aga acc gag gac gag 48
Thr Leu Phe Val Ala Leu Tyr Asp Tyr Glu Ala Arg Thr Glu Asp Glu
1 5 10 15
ctg agc ttc cac aag ggc gag aag ttc cag atc ctg aac agc agc gag 96
Leu Ser Phe His Lys Gly Glu Lys Phe Gln Ile Leu Asn Ser Ser Glu
20 25 30
ggc gac tgg tgg gag gcc aga gac agc ctg acc acc ggc gag acc ggc 144
Gly Asp Trp Trp Glu Ala Arg Asp Ser Leu Thr Thr Gly Glu Thr Gly
35 40 45
tac atc ccc agc aac tac gtg gcc ccc gtg gac 177
Tyr Ile Pro Ser Asn Tyr Val Ala Pro Val Asp
50 55
<210> 189
<211> 59
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 189
Thr Leu Phe Val Ala Leu Tyr Asp Tyr Glu Ala Arg Thr Glu Asp Glu
1 5 10 15
Leu Ser Phe His Lys Gly Glu Lys Phe Gln Ile Leu Asn Ser Ser Glu
20 25 30
Gly Asp Trp Trp Glu Ala Arg Asp Ser Leu Thr Thr Gly Glu Thr Gly
35 40 45
Tyr Ile Pro Ser Asn Tyr Val Ala Pro Val Asp
50 55
<210> 190
<211> 174
<212> DNA
<213> Artificial sequence
<220>
<223> NPHP1
<220>
<221> CDS
<222> (1)..(174)
<400> 190
gag gag tac atc gcc gtg ggc gac ttc gac acc gcc cag cag gtg ggc 48
Glu Glu Tyr Ile Ala Val Gly Asp Phe Asp Thr Ala Gln Gln Val Gly
1 5 10 15
gac ctg acc ttc aag aag ggc gag atc ctg ctg gtg atc gag aag aag 96
Asp Leu Thr Phe Lys Lys Gly Glu Ile Leu Leu Val Ile Glu Lys Lys
20 25 30
ccc gac ggc tgg tgg atc gcc aag gac gcc aag ggc aac gag ggc ctg 144
Pro Asp Gly Trp Trp Ile Ala Lys Asp Ala Lys Gly Asn Glu Gly Leu
35 40 45
gtg ccc aga acc tac ctg gag ccc tac agc 174
Val Pro Arg Thr Tyr Leu Glu Pro Tyr Ser
50 55
<210> 191
<211> 58
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 191
Glu Glu Tyr Ile Ala Val Gly Asp Phe Asp Thr Ala Gln Gln Val Gly
1 5 10 15
Asp Leu Thr Phe Lys Lys Gly Glu Ile Leu Leu Val Ile Glu Lys Lys
20 25 30
Pro Asp Gly Trp Trp Ile Ala Lys Asp Ala Lys Gly Asn Glu Gly Leu
35 40 45
Val Pro Arg Thr Tyr Leu Glu Pro Tyr Ser
50 55
<210> 192
<211> 171
<212> DNA
<213> Artificial sequence
<220>
<223> Tec
<220>
<221> CDS
<222> (1)..(171)
<400> 192
gag atc gtg gtg gcc atg tac gac ttc cag gcc gcc gag ggc cac gac 48
Glu Ile Val Val Ala Met Tyr Asp Phe Gln Ala Ala Glu Gly His Asp
1 5 10 15
ctg aga ctg gag aga cag gag tac ctg atc ctg gag aag aac gac gtg 96
Leu Arg Leu Glu Arg Gln Glu Tyr Leu Ile Leu Glu Lys Asn Asp Val
20 25 30
cac tgg tgg aga gcc aga gac aag tac ggc aac gag ggc tac atc ccc 144
His Trp Trp Arg Ala Arg Asp Lys Tyr Gly Asn Glu Gly Tyr Ile Pro
35 40 45
agc aac tac gtg acc ggc aag aag tga 171
Ser Asn Tyr Val Thr Gly Lys Lys
50 55
<210> 193
<211> 56
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 193
Glu Ile Val Val Ala Met Tyr Asp Phe Gln Ala Ala Glu Gly His Asp
1 5 10 15
Leu Arg Leu Glu Arg Gln Glu Tyr Leu Ile Leu Glu Lys Asn Asp Val
20 25 30
His Trp Trp Arg Ala Arg Asp Lys Tyr Gly Asn Glu Gly Tyr Ile Pro
35 40 45
Ser Asn Tyr Val Thr Gly Lys Lys
50 55
<210> 194
<211> 171
<212> DNA
<213> Artificial sequence
<220>
<223> Hck
<220>
<221> CDS
<222> (1)..(171)
<400> 194
atc atc gtg gtg gcc ctg tac gac tac gag gcc atc cac cac gag gac 48
Ile Ile Val Val Ala Leu Tyr Asp Tyr Glu Ala Ile His His Glu Asp
1 5 10 15
ctg agc ttc cag aag ggc gac cag atg gtg gtg ctg gag gag agc ggc 96
Leu Ser Phe Gln Lys Gly Asp Gln Met Val Val Leu Glu Glu Ser Gly
20 25 30
gag tgg tgg aag gcc aga agc ctg gcc acc aga aag gag ggc tac atc 144
Glu Trp Trp Lys Ala Arg Ser Leu Ala Thr Arg Lys Glu Gly Tyr Ile
35 40 45
ccc agc aac tac gtg gcc aga gtg gac 171
Pro Ser Asn Tyr Val Ala Arg Val Asp
50 55
<210> 195
<211> 57
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 195
Ile Ile Val Val Ala Leu Tyr Asp Tyr Glu Ala Ile His His Glu Asp
1 5 10 15
Leu Ser Phe Gln Lys Gly Asp Gln Met Val Val Leu Glu Glu Ser Gly
20 25 30
Glu Trp Trp Lys Ala Arg Ser Leu Ala Thr Arg Lys Glu Gly Tyr Ile
35 40 45
Pro Ser Asn Tyr Val Ala Arg Val Asp
50 55
<210> 196
<211> 213
<212> DNA
<213> Artificial sequence
<220>
<223> Amph
<220>
<221> CDS
<222> (1)..(213)
<400> 196
tac aag gtg gag acc ctg cac gac ttc gag gcc gcc aac agc gac gag 48
Tyr Lys Val Glu Thr Leu His Asp Phe Glu Ala Ala Asn Ser Asp Glu
1 5 10 15
ctg acc ctg cag aga ggc gac gtg gtg ctg gtg gtg ccc agc gac agc 96
Leu Thr Leu Gln Arg Gly Asp Val Val Leu Val Val Pro Ser Asp Ser
20 25 30
gag gcc gac cag gac gcc ggc tgg ctg gtg ggc gtg aag gag agc gac 144
Glu Ala Asp Gln Asp Ala Gly Trp Leu Val Gly Val Lys Glu Ser Asp
35 40 45
tgg ctg cag tac aga gac ctg gcc acc tac aag ggc ctg ttc ccc gag 192
Trp Leu Gln Tyr Arg Asp Leu Ala Thr Tyr Lys Gly Leu Phe Pro Glu
50 55 60
aac ttc acc aga aga ctg gac 213
Asn Phe Thr Arg Arg Leu Asp
65 70
<210> 197
<211> 71
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 197
Tyr Lys Val Glu Thr Leu His Asp Phe Glu Ala Ala Asn Ser Asp Glu
1 5 10 15
Leu Thr Leu Gln Arg Gly Asp Val Val Leu Val Val Pro Ser Asp Ser
20 25 30
Glu Ala Asp Gln Asp Ala Gly Trp Leu Val Gly Val Lys Glu Ser Asp
35 40 45
Trp Leu Gln Tyr Arg Asp Leu Ala Thr Tyr Lys Gly Leu Phe Pro Glu
50 55 60
Asn Phe Thr Arg Arg Leu Asp
65 70
<210> 198
<211> 192
<212> DNA
<213> Artificial sequence
<220>
<223> RIMBP#3
<220>
<221> CDS
<222> (1)..(192)
<400> 198
aag atc atg atc gcc gcc ctg gac tac gac ccc ggc gac ggc cag atg 48
Lys Ile Met Ile Ala Ala Leu Asp Tyr Asp Pro Gly Asp Gly Gln Met
1 5 10 15
ggc ggc cag ggc aag ggc aga ctg gcc ctg aga gcc ggc gac gtg gtg 96
Gly Gly Gln Gly Lys Gly Arg Leu Ala Leu Arg Ala Gly Asp Val Val
20 25 30
atg gtg tac ggc ccc atg gac gac cag ggc ttc tac tac ggc gag ctg 144
Met Val Tyr Gly Pro Met Asp Asp Gln Gly Phe Tyr Tyr Gly Glu Leu
35 40 45
ggc ggc cac aga ggc ctg gtg ccc gcc cac ctg ctg gac cac atg agc 192
Gly Gly His Arg Gly Leu Val Pro Ala His Leu Leu Asp His Met Ser
50 55 60
<210> 199
<211> 64
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 199
Lys Ile Met Ile Ala Ala Leu Asp Tyr Asp Pro Gly Asp Gly Gln Met
1 5 10 15
Gly Gly Gln Gly Lys Gly Arg Leu Ala Leu Arg Ala Gly Asp Val Val
20 25 30
Met Val Tyr Gly Pro Met Asp Asp Gln Gly Phe Tyr Tyr Gly Glu Leu
35 40 45
Gly Gly His Arg Gly Leu Val Pro Ala His Leu Leu Asp His Met Ser
50 55 60
<210> 200
<211> 180
<212> DNA
<213> Artificial sequence
<220>
<223> IRIKS
<220>
<221> CDS
<222> (1)..(180)
<400> 200
cag aag gtg aag acc atc ttc ccc cac acc gcc ggc agc aac aag acc 48
Gln Lys Val Lys Thr Ile Phe Pro His Thr Ala Gly Ser Asn Lys Thr
1 5 10 15
ctg ctg agc ttc gcc cag ggc gac gtg atc acc ctg ctg atc ccc gag 96
Leu Leu Ser Phe Ala Gln Gly Asp Val Ile Thr Leu Leu Ile Pro Glu
20 25 30
gag aag gac ggc tgg ctg tac ggc gag cac gac gtg agc aag gcc aga 144
Glu Lys Asp Gly Trp Leu Tyr Gly Glu His Asp Val Ser Lys Ala Arg
35 40 45
ggc tgg ttc ccc agc agc tac acc aag ctg ctg gag 180
Gly Trp Phe Pro Ser Ser Tyr Thr Lys Leu Leu Glu
50 55 60
<210> 201
<211> 60
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 201
Gln Lys Val Lys Thr Ile Phe Pro His Thr Ala Gly Ser Asn Lys Thr
1 5 10 15
Leu Leu Ser Phe Ala Gln Gly Asp Val Ile Thr Leu Leu Ile Pro Glu
20 25 30
Glu Lys Asp Gly Trp Leu Tyr Gly Glu His Asp Val Ser Lys Ala Arg
35 40 45
Gly Trp Phe Pro Ser Ser Tyr Thr Lys Leu Leu Glu
50 55 60
<210> 202
<211> 174
<212> DNA
<213> Artificial sequence
<220>
<223> SNX33
<220>
<221> CDS
<222> (1)..(174)
<400> 202
ctg aag ggc aga gcc ctg tac gac ttc cac agc gag aac aag gag gag 48
Leu Lys Gly Arg Ala Leu Tyr Asp Phe His Ser Glu Asn Lys Glu Glu
1 5 10 15
atc agc atc cag cag gac gag gac ctg gtg atc ttc agc gag acc agc 96
Ile Ser Ile Gln Gln Asp Glu Asp Leu Val Ile Phe Ser Glu Thr Ser
20 25 30
ctg gac ggc tgg ctg cag ggc cag aac agc aga ggc gag acc ggc ctg 144
Leu Asp Gly Trp Leu Gln Gly Gln Asn Ser Arg Gly Glu Thr Gly Leu
35 40 45
ttc ccc gcc agc tac gtg gag atc gtg aga 174
Phe Pro Ala Ser Tyr Val Glu Ile Val Arg
50 55
<210> 203
<211> 58
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 203
Leu Lys Gly Arg Ala Leu Tyr Asp Phe His Ser Glu Asn Lys Glu Glu
1 5 10 15
Ile Ser Ile Gln Gln Asp Glu Asp Leu Val Ile Phe Ser Glu Thr Ser
20 25 30
Leu Asp Gly Trp Leu Gln Gly Gln Asn Ser Arg Gly Glu Thr Gly Leu
35 40 45
Phe Pro Ala Ser Tyr Val Glu Ile Val Arg
50 55
<210> 204
<211> 168
<212> DNA
<213> Artificial sequence
<220>
<223> Eps8L1
<220>
<221> CDS
<222> (1)..(168)
<400> 204
aag tgg gtg ctg tgc aac tac gac ttc cag gcc aga aac agc agc gag 48
Lys Trp Val Leu Cys Asn Tyr Asp Phe Gln Ala Arg Asn Ser Ser Glu
1 5 10 15
ctg agc gtg aag cag aga gac gtg ctg gag gtg ctg gac gac agc aga 96
Leu Ser Val Lys Gln Arg Asp Val Leu Glu Val Leu Asp Asp Ser Arg
20 25 30
aag tgg tgg aag gtg aga gac ccc gcc ggc cag gag ggc tac gtg ccc 144
Lys Trp Trp Lys Val Arg Asp Pro Ala Gly Gln Glu Gly Tyr Val Pro
35 40 45
tac aac atc ctg acc ccc tac ccc 168
Tyr Asn Ile Leu Thr Pro Tyr Pro
50 55
<210> 205
<211> 56
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 205
Lys Trp Val Leu Cys Asn Tyr Asp Phe Gln Ala Arg Asn Ser Ser Glu
1 5 10 15
Leu Ser Val Lys Gln Arg Asp Val Leu Glu Val Leu Asp Asp Ser Arg
20 25 30
Lys Trp Trp Lys Val Arg Asp Pro Ala Gly Gln Glu Gly Tyr Val Pro
35 40 45
Tyr Asn Ile Leu Thr Pro Tyr Pro
50 55
<210> 206
<211> 177
<212> DNA
<213> Artificial sequence
<220>
<223> FISH#5
<220>
<221> CDS
<222> (1)..(177)
<400> 206
gac gtg tac gtg agc atc gcc gac tac gag ggc gac gag gag acc gcc 48
Asp Val Tyr Val Ser Ile Ala Asp Tyr Glu Gly Asp Glu Glu Thr Ala
1 5 10 15
ggc ttc cag gag ggc gtg agc atg gag gtg ctg gag aga aac ccc aac 96
Gly Phe Gln Glu Gly Val Ser Met Glu Val Leu Glu Arg Asn Pro Asn
20 25 30
ggc tgg tgg tac tgc cag atc ctg gac ggc gtg aag ccc ttc aag ggc 144
Gly Trp Trp Tyr Cys Gln Ile Leu Asp Gly Val Lys Pro Phe Lys Gly
35 40 45
tgg gtg ccc agc aac tac ctg gag aag aag aac 177
Trp Val Pro Ser Asn Tyr Leu Glu Lys Lys Asn
50 55
<210> 207
<211> 59
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 207
Asp Val Tyr Val Ser Ile Ala Asp Tyr Glu Gly Asp Glu Glu Thr Ala
1 5 10 15
Gly Phe Gln Glu Gly Val Ser Met Glu Val Leu Glu Arg Asn Pro Asn
20 25 30
Gly Trp Trp Tyr Cys Gln Ile Leu Asp Gly Val Lys Pro Phe Lys Gly
35 40 45
Trp Val Pro Ser Asn Tyr Leu Glu Lys Lys Asn
50 55
<210> 208
<211> 171
<212> DNA
<213> Artificial sequence
<220>
<223> CMS#1
<220>
<221> CDS
<222> (1)..(171)
<400> 208
gtg gac tac atc gtg gag tac gac tac gac gcc gtg cac gac gac gag 48
Val Asp Tyr Ile Val Glu Tyr Asp Tyr Asp Ala Val His Asp Asp Glu
1 5 10 15
ctg acc atc aga gtg ggc gag atc atc aga aac gtg aag aag ctg cag 96
Leu Thr Ile Arg Val Gly Glu Ile Ile Arg Asn Val Lys Lys Leu Gln
20 25 30
gag gag ggc tgg ctg gag ggc gag ctg aac ggc aga aga ggc atg ttc 144
Glu Glu Gly Trp Leu Glu Gly Glu Leu Asn Gly Arg Arg Gly Met Phe
35 40 45
ccc gac aac ttc gtg aag gag atc aag 171
Pro Asp Asn Phe Val Lys Glu Ile Lys
50 55
<210> 209
<211> 57
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 209
Val Asp Tyr Ile Val Glu Tyr Asp Tyr Asp Ala Val His Asp Asp Glu
1 5 10 15
Leu Thr Ile Arg Val Gly Glu Ile Ile Arg Asn Val Lys Lys Leu Gln
20 25 30
Glu Glu Gly Trp Leu Glu Gly Glu Leu Asn Gly Arg Arg Gly Met Phe
35 40 45
Pro Asp Asn Phe Val Lys Glu Ile Lys
50 55
<210> 210
<211> 168
<212> DNA
<213> Artificial sequence
<220>
<223> OSTF1
<220>
<221> CDS
<222> (1)..(168)
<400> 210
aag gtg ttc aga gcc ctg tac acc ttc gag ccc aga acc ccc gac gag 48
Lys Val Phe Arg Ala Leu Tyr Thr Phe Glu Pro Arg Thr Pro Asp Glu
1 5 10 15
ctg tac ttc gag gag ggc gac atc atc tac atc acc gac atg agc gac 96
Leu Tyr Phe Glu Glu Gly Asp Ile Ile Tyr Ile Thr Asp Met Ser Asp
20 25 30
acc aac tgg tgg aag ggc acc agc aag ggc aga acc ggc ctg atc ccc 144
Thr Asn Trp Trp Lys Gly Thr Ser Lys Gly Arg Thr Gly Leu Ile Pro
35 40 45
agc aac tac gtg gcc gag cag gcc 168
Ser Asn Tyr Val Ala Glu Gln Ala
50 55
<210> 211
<211> 56
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 211
Lys Val Phe Arg Ala Leu Tyr Thr Phe Glu Pro Arg Thr Pro Asp Glu
1 5 10 15
Leu Tyr Phe Glu Glu Gly Asp Ile Ile Tyr Ile Thr Asp Met Ser Asp
20 25 30
Thr Asn Trp Trp Lys Gly Thr Ser Lys Gly Arg Thr Gly Leu Ile Pro
35 40 45
Ser Asn Tyr Val Ala Glu Gln Ala
50 55
<210> 212
<211> 34
<212> PRT
<213> Artificial sequence
<220>
<223> Cys-knots/kink bacterin (SOTI Var. 1)
<400> 212
Cys Ser Pro Ser Gly Ala Ile Cys Ser Gly Phe Gly Pro Pro Glu Gln
1 5 10 15
Cys Cys Ser Ala Gly Cys Val Leu Asn Arg Arg Ala Arg Ser Trp Arg
20 25 30
Cys Gln
<210> 213
<211> 35
<212> PRT
<213> Artificial sequence
<220>
<223> Cys-knotts/kink bacterin (SOTI-III)
<400> 213
Cys Ser Pro Ser Gly Ala Ile Cys Ser Gly Phe Gly Pro Pro Glu Gln
1 5 10 15
Cys Cys Ser Ala Gly Ala Cys Val Pro His Pro Ile Leu Arg Ile Phe
20 25 30
Val Cys Gln
35
<210> 214
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> Kalata B1
<400> 214
Gly Leu Pro Val Cys Gly Glu Thr Cys Val Gly Gly Thr Cys Asn Thr
1 5 10 15
Pro Gly Cys Thr Cys Ser Trp Pro Val Cys Thr Arg Asn
20 25
<210> 215
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> Kalata B1
<400> 215
Gly Leu Pro Val Cys Gly Glu Thr Cys Val Gly Gly Thr Cys Asn Thr
1 5 10 15
Pro Gly Cys Thr Cys Ser Trp Pro Val Cys Thr Arg Asn
20 25
<210> 216
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> Kalata B2
<400> 216
Gly Leu Pro Val Cys Gly Glu Thr Cys Phe Gly Gly Thr Cys Asn Thr
1 5 10 15
Pro Gly Cys Ser Cys Thr Trp Pro Ile Cys Thr Arg Asp
20 25
<210> 217
<211> 34
<212> PRT
<213> Artificial sequence
<220>
<223> MCoTI-I
<400> 217
Gly Gly Val Cys Pro Lys Ile Leu Gln Arg Cys Arg Arg Asp Ser Asp
1 5 10 15
Cys Pro Gly Ala Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly Ser Gly
20 25 30
Ser Asp
<210> 218
<211> 34
<212> PRT
<213> Artificial sequence
<220>
<223> MCoTI-II
<400> 218
Gly Gly Val Cys Pro Lys Ile Leu Lys Lys Cys Arg Arg Asp Ser Asp
1 5 10 15
Cys Pro Gly Ala Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly Ser Gly
20 25 30
Ser Asp
<210> 219
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(12)
<400> 219
ggc ggc ggc ggc 12
Gly Gly Gly Gly
1
<210> 220
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 220
Gly Gly Gly Gly
1
<210> 221
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(12)
<400> 221
ggc ggc ggc agc 12
Gly Gly Gly Ser
1
<210> 222
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 222
Gly Gly Gly Ser
1
<210> 223
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(24)
<400> 223
ggc ggc ggc agc ggc ggc ggc aga 24
Gly Gly Gly Ser Gly Gly Gly Arg
1 5
<210> 224
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 224
Gly Gly Gly Ser Gly Gly Gly Arg
1 5
<210> 225
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(36)
<400> 225
ggc ggc ggc agc ggc ggc ggc agc ggc ggc ggc aga 36
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg
1 5 10
<210> 226
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 226
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg
1 5 10
<210> 227
<211> 48
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(48)
<400> 227
ggc ggc ggc agc ggc ggc ggc agc ggc ggc ggc aga ggc ggc ggc aga 48
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg Gly Gly Gly Arg
1 5 10 15
<210> 228
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 228
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg Gly Gly Gly Arg
1 5 10 15
<210> 229
<211> 60
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(60)
<400> 229
ggc ggc ggc agc ggc ggc ggc agc ggc ggc ggc aga ggc ggc ggc aga 48
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg Gly Gly Gly Arg
1 5 10 15
ggc ggc ggc aga 60
Gly Gly Gly Arg
20
<210> 230
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 230
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Arg Gly Gly Gly Arg
1 5 10 15
Gly Gly Gly Arg
20
<210> 231
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(15)
<400> 231
ggc ggc ggc ggc agc 15
Gly Gly Gly Gly Ser
1 5
<210> 232
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 232
Gly Gly Gly Gly Ser
1 5
<210> 233
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(30)
<400> 233
ggc ggc ggc ggc agc ggc ggc ggc ggc agc 30
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 234
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 234
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 235
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(45)
<400> 235
ggc ggc ggc ggc agc ggc ggc ggc ggc agc ggc ggc ggc ggc agc 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 236
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 236
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 237
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(36)
<400> 237
ggc ggc ggc agc ggc ggc ggc ggc agc ggc ggc agc 36
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser
1 5 10
<210> 238
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 238
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser
1 5 10
<210> 239
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(12)
<400> 239
ggc ggc agc ggc 12
Gly Gly Ser Gly
1
<210> 240
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 240
Gly Gly Ser Gly
1
<210> 241
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(24)
<400> 241
ggc ggc agc ggc ggc ggc agc ggc 24
Gly Gly Ser Gly Gly Gly Ser Gly
1 5
<210> 242
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 242
Gly Gly Ser Gly Gly Gly Ser Gly
1 5
<210> 243
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(36)
<400> 243
ggc ggc agc ggc ggc ggc agc ggc ggc ggc agc ggc 36
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
1 5 10
<210> 244
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 244
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly
1 5 10
<210> 245
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(21)
<400> 245
agc ggc ggc ggc ggc atc ggc 21
Ser Gly Gly Gly Gly Ile Gly
1 5
<210> 246
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 246
Ser Gly Gly Gly Gly Ile Gly
1 5
<210> 247
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(36)
<400> 247
agc ggc ggc ggc ggc agc ggc ggc ggc ggc atc ggc 36
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ile Gly
1 5 10
<210> 248
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 248
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ile Gly
1 5 10
<210> 249
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> joint
<220>
<221> CDS
<222> (1)..(15)
<400> 249
agc ggc ggc ggc ggc 15
Ser Gly Gly Gly Gly
1 5
<210> 250
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 250
Ser Gly Gly Gly Gly
1 5
<210> 251
<211> 681
<212> DNA
<213> human
<220>
<221> CDS
<222> (1)..(681)
<400> 251
gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 48
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 96
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 144
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 192
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 240
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 288
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 336
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 384
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc 432
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 480
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 528
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 576
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 624
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
cac gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 672
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
ccg ggt aaa 681
Pro Gly Lys
225
<210> 252
<211> 227
<212> PRT
<213> human
<400> 252
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 253
<211> 669
<212> DNA
<213> human
<220>
<221> CDS
<222> (1)..(669)
<400> 253
gtg gag tgc cca cct tgc cca gca cca cct gtg gca gga cct tca gtc 48
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
1 5 10 15
ttc ctc ttc ccc cca aaa ccc aag gac acc ctg atg atc tcc aga acc 96
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
20 25 30
cct gag gtc acg tgc gtg gtg gtg gac gtg agc cac gaa gac ccc gag 144
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45
gtc cag ttc aac tgg tac gtg gac ggc atg gag gtg cat aat gcc aag 192
Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His Asn Ala Lys
50 55 60
aca aag cca cgg gag gag cag ttc aac agc acg ttc cgt gtg gtc agc 240
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
65 70 75 80
gtc ctc acc gtc gtg cac cag gac tgg ctg aac ggc aag gag tac aag 288
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95
tgc aag gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa acc atc 336
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
100 105 110
tcc aaa acc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc 384
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125
cca tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg 432
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140
gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat 480
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160
ggg cag ccg gag aac aac tac aag acc aca cct ccc atg ctg gac tcc 528
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175
gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg 576
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190
tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg 624
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205
cac aac cac tac aca cag aag agc ctc tcc ctg tct ccg ggt aaa 669
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 254
<211> 223
<212> PRT
<213> human
<400> 254
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
1 5 10 15
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
20 25 30
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45
Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His Asn Ala Lys
50 55 60
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
65 70 75 80
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
100 105 110
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 255
<211> 681
<212> DNA
<213> human
<220>
<221> CDS
<222> (1)..(681)
<400> 255
gac aca cct ccc ccg tgc cca agg tgc cca gca cct gaa ctc ctg gga 48
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gat acc ctt atg 96
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
att tcc cgg acc cct gag gtc acg tgc gtg gtg gtg gac gtg agc cac 144
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
gaa gac ccc gag gtc cag ttc aag tgg tac gtg gac ggc gtg gag gtg 192
Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val
50 55 60
cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg ttc 240
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe
65 70 75 80
cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aac ggc 288
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 336
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
gag aaa acc atc tcc aaa acc aaa gga cag ccc cga gaa cca cag gtg 384
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc 432
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag 480
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
tgg gag agc agc ggg cag ccg gag aac aac tac aac acc acg cct ccc 528
Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro
165 170 175
atg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 576
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
gac aag agc agg tgg cag cag ggg aac atc ttc tca tgc tcc gtg atg 624
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met
195 200 205
cat gag gct ctg cac aac cgc ttc acg cag aag agc ctc tcc ctg tct 672
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
ccg ggt aaa 681
Pro Gly Lys
225
<210> 256
<211> 227
<212> PRT
<213> human
<400> 256
Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro
165 170 175
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 257
<211> 672
<212> DNA
<213> human
<220>
<221> CDS
<222> (1)..(672)
<400> 257
ccc cca tgc cca tca tgc cca gca cct gag ttc ctg ggg gga cca tca 48
Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
1 5 10 15
gtc ttc ctg ttc ccc cca aaa ccc aag gac act ctc atg atc tcc cgg 96
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
acc cct gag gtc acg tgc gtg gtg gtg gac gtg agc cag gaa gac ccc 144
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
35 40 45
gag gtc cag ttc aac tgg tac gtg gat ggc gtg gag gtg cat aat gcc 192
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
aag aca aag ccg cgg gag gag cag ttc aac agc acg tac cgt gtg gtc 240
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
65 70 75 80
agc gtc ctc acc gtc ctg cac cag gac tgg ctg aac ggc aag gag tac 288
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
aag tgc aag gtc tcc aac aaa ggc ctc ccg tcc tcc atc gag aaa acc 336
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
100 105 110
atc tcc aaa gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg 384
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
ccc cca tcc cag gag gag atg acc aag aac cag gtc agc ctg acc tgc 432
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
130 135 140
ctg gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc 480
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac 528
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
tcc gac ggc tcc ttc ttc ctc tac agc agg cta acc gtg gac aag agc 576
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
180 185 190
agg tgg cag gag ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct 624
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
ctg cac aac cac tac aca cag aag agc ctc tcc ctg tct ccg ggt aaa 672
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 258
<211> 224
<212> PRT
<213> human
<400> 258
Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
35 40 45
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 259
<211> 387
<212> DNA
<213> Artificial sequence
<220>
<223> human IgM C-mu-4tp
<220>
<221> CDS
<222> (1)..(387)
<400> 259
aag cac ccc ccc gcc gtg tac ctg ctg ccc ccc gcc aga gag cag ctg 48
Lys His Pro Pro Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu
1 5 10 15
aac ctg aga gag agc gcc acc gtg acc tgc ctg gtg aag ggc ttc agc 96
Asn Leu Arg Glu Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser
20 25 30
ccc gcc gac atc agc gtg cag tgg ctg cag aga ggc cag ctg ctg ccc 144
Pro Ala Asp Ile Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro
35 40 45
cag gag aag tac gtg acc agc gcc ccc atg ccc gag ccc ggc gcc ccc 192
Gln Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro
50 55 60
ggc ttc tac ttc acc cac agc atc ctg acc gtg acc gag gag gag tgg 240
Gly Phe Tyr Phe Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp
65 70 75 80
aac agc ggc gag acc tac acc tgc gtg gtg ggc cac gag gcc ctg ccc 288
Asn Ser Gly Glu Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro
85 90 95
cac ctg gtg acc gag aga acc gtg gac aag agc acc ggc aag ccc acc 336
His Leu Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr
100 105 110
ctg tac aac gtg agc ctg atc atg agc gac acc ggc ggc acc tgc tac 384
Leu Tyr Asn Val Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr
115 120 125
tga 387
<210> 260
<211> 128
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 260
Lys His Pro Pro Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu
1 5 10 15
Asn Leu Arg Glu Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser
20 25 30
Pro Ala Asp Ile Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro
35 40 45
Gln Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro
50 55 60
Gly Phe Tyr Phe Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp
65 70 75 80
Asn Ser Gly Glu Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro
85 90 95
His Leu Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr
100 105 110
Leu Tyr Asn Val Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr
115 120 125
<210> 261
<211> 390
<212> DNA
<213> Artificial sequence
<220>
<223> human IgA C-alpha-3 tp
<220>
<221> CDS
<222> (1)..(390)
<400> 261
acc ttc ccc ccc cag gtg cac ctg ctg ccc ccc ccc agc gag gag ctg 48
Thr Phe Pro Pro Gln Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu
1 5 10 15
gcc ctg aac gag ctg ctg agc ctg acc tgc ctg gtg aga gcc ttc aac 96
Ala Leu Asn Glu Leu Leu Ser Leu Thr Cys Leu Val Arg Ala Phe Asn
20 25 30
ccc aag gag gtg ctg gtg aga tgg ctg cac ggc aac gag gag ctg agc 144
Pro Lys Glu Val Leu Val Arg Trp Leu His Gly Asn Glu Glu Leu Ser
35 40 45
ccc gag agc tac ctg gtg ttc gag ccc ctg aag gag ccc ggc gag ggc 192
Pro Glu Ser Tyr Leu Val Phe Glu Pro Leu Lys Glu Pro Gly Glu Gly
50 55 60
gcc acc acc tac ctg gtg acc agc gtg ctg aga gtg agc gcc gag acc 240
Ala Thr Thr Tyr Leu Val Thr Ser Val Leu Arg Val Ser Ala Glu Thr
65 70 75 80
tgg aag cag ggc gac cag tac agc tgc atg gtg ggc cac gag gcc ctg 288
Trp Lys Gln Gly Asp Gln Tyr Ser Cys Met Val Gly His Glu Ala Leu
85 90 95
ccc atg aac ttc acc cag aag acc atc gac aga ctg agc ggc aag ccc 336
Pro Met Asn Phe Thr Gln Lys Thr Ile Asp Arg Leu Ser Gly Lys Pro
100 105 110
acc aac gtg agc gtg agc gtg atc atg agc gag ggc gac ggc atc tgc 384
Thr Asn Val Ser Val Ser Val Ile Met Ser Glu Gly Asp Gly Ile Cys
115 120 125
tac tga 390
Tyr
<210> 262
<211> 129
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 262
Thr Phe Pro Pro Gln Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu
1 5 10 15
Ala Leu Asn Glu Leu Leu Ser Leu Thr Cys Leu Val Arg Ala Phe Asn
20 25 30
Pro Lys Glu Val Leu Val Arg Trp Leu His Gly Asn Glu Glu Leu Ser
35 40 45
Pro Glu Ser Tyr Leu Val Phe Glu Pro Leu Lys Glu Pro Gly Glu Gly
50 55 60
Ala Thr Thr Tyr Leu Val Thr Ser Val Leu Arg Val Ser Ala Glu Thr
65 70 75 80
Trp Lys Gln Gly Asp Gln Tyr Ser Cys Met Val Gly His Glu Ala Leu
85 90 95
Pro Met Asn Phe Thr Gln Lys Thr Ile Asp Arg Leu Ser Gly Lys Pro
100 105 110
Thr Asn Val Ser Val Ser Val Ile Met Ser Glu Gly Asp Gly Ile Cys
115 120 125
Tyr
<210> 263
<211> 60
<212> DNA
<213> Artificial sequence
<220>
<223> dimerization motif
<220>
<221> CDS
<222> (1)..(60)
<400> 263
gtg gcc gac ttc ctg atc atc tac atc gag gag gcc cac gcc acc gac 48
Val Ala Asp Phe Leu Ile Ile Tyr Ile Glu Glu Ala His Ala Thr Asp
1 5 10 15
ggc tgg gcc ctg 60
Gly Trp Ala Leu
20
<210> 264
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 264
Val Ala Asp Phe Leu Ile Ile Tyr Ile Glu Glu Ala His Ala Thr Asp
1 5 10 15
Gly Trp Ala Leu
20
<210> 265
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif GCN4
<220>
<221> CDS
<222> (1)..(84)
<400> 265
atc aag cag atc gag gac aag atc gag gag atc ctg agc aag atc tac 48
Ile Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr
1 5 10 15
cac atc gag aac gag atc gcc aga atc aag aag ctg 84
His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu
20 25
<210> 266
<211> 28
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 266
Ile Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr
1 5 10 15
His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu
20 25
<210> 267
<211> 117
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif maternal protein 1
<220>
<221> CDS
<222> (1)..(117)
<400> 267
tgc gcc tgc gag agc ctg gtg aag ttc cag gcc aag gtg gag ggc ctg 48
Cys Ala Cys Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu
1 5 10 15
ctg cag gcc ctg acc aga aag ctg gag gcc gtg agc aag aga ctg gcc 96
Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Arg Leu Ala
20 25 30
atc ctg gag aac acc gtg gtg 117
Ile Leu Glu Asn Thr Val Val
35
<210> 268
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 268
Cys Ala Cys Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu
1 5 10 15
Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Arg Leu Ala
20 25 30
Ile Leu Glu Asn Thr Val Val
35
<210> 269
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif crown protein 1a
<220>
<221> CDS
<222> (1)..(96)
<400> 269
gtg agc aga ctg gag gag gag atg aga aag ctg cag gcc acc gtg cag 48
Val Ser Arg Leu Glu Glu Glu Met Arg Lys Leu Gln Ala Thr Val Gln
1 5 10 15
gag ctg cag aag aga ctg gac aga ctg gag gag acc gtg cag gcc aag 96
Glu Leu Gln Lys Arg Leu Asp Arg Leu Glu Glu Thr Val Gln Ala Lys
20 25 30
<210> 270
<211> 32
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 270
Val Ser Arg Leu Glu Glu Glu Met Arg Lys Leu Gln Ala Thr Val Gln
1 5 10 15
Glu Leu Gln Lys Arg Leu Asp Arg Leu Glu Glu Thr Val Gln Ala Lys
20 25 30
<210> 271
<211> 108
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization Biochemical motif CMP
<220>
<221> CDS
<222> (1)..(108)
<400> 271
gag agc ctg gtg aag ttc cag gcc aag gtg gag ggc ctg ctg cag gcc 48
Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu Leu Gln Ala
1 5 10 15
ctg acc aga aag ctg gag gcc gtg agc aag aga ctg gcc atc ctg gag 96
Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Arg Leu Ala Ile Leu Glu
20 25 30
aac acc gtg gtg 108
Asn Thr Val Val
35
<210> 272
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 272
Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu Leu Gln Ala
1 5 10 15
Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Arg Leu Ala Ile Leu Glu
20 25 30
Asn Thr Val Val
35
<210> 273
<211> 210
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif DMPK
<220>
<221> CDS
<222> (1)..(210)
<400> 273
gag gcc gag gcc gag gtg acc ctg aga gag ctg cag gag gcc ctg gag 48
Glu Ala Glu Ala Glu Val Thr Leu Arg Glu Leu Gln Glu Ala Leu Glu
1 5 10 15
gag gag gtg ctg acc aga cag agc ctg agc aga gag atg gag gcc atc 96
Glu Glu Val Leu Thr Arg Gln Ser Leu Ser Arg Glu Met Glu Ala Ile
20 25 30
aga acc gac aac cag aac ttc gcc agc cag ctg aga gag gcc gag gcc 144
Arg Thr Asp Asn Gln Asn Phe Ala Ser Gln Leu Arg Glu Ala Glu Ala
35 40 45
aga aac aga gac ctg gag gcc cac gtg aga cag ctg cag gag aga atg 192
Arg Asn Arg Asp Leu Glu Ala His Val Arg Gln Leu Gln Glu Arg Met
50 55 60
gag ctg ctg cag gcc gag 210
Glu Leu Leu Gln Ala Glu
65 70
<210> 274
<211> 70
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 274
Glu Ala Glu Ala Glu Val Thr Leu Arg Glu Leu Gln Glu Ala Leu Glu
1 5 10 15
Glu Glu Val Leu Thr Arg Gln Ser Leu Ser Arg Glu Met Glu Ala Ile
20 25 30
Arg Thr Asp Asn Gln Asn Phe Ala Ser Gln Leu Arg Glu Ala Glu Ala
35 40 45
Arg Asn Arg Asp Leu Glu Ala His Val Arg Gln Leu Gln Glu Arg Met
50 55 60
Glu Leu Leu Gln Ala Glu
65 70
<210> 275
<211> 99
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif Langerin
<220>
<221> CDS
<222> (1)..(99)
<400> 275
gcc agc gcc ctg aac acc aag atc aga gcc ctg cag ggc agc ctg gag 48
Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly Ser Leu Glu
1 5 10 15
aac atg agc aag ctg ctg aag aga cag aac gac atc ctg cag gtg gtg 96
Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu Gln Val Val
20 25 30
agc 99
Ser
<210> 276
<211> 33
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 276
Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly Ser Leu Glu
1 5 10 15
Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu Gln Val Val
20 25 30
Ser
<210> 277
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> trimerization motif Surfectin protein SP-D
<220>
<221> CDS
<222> (1)..(87)
<400> 277
gac gtg gcc agc ctg aga cag cag gtg gag gcc ctg cag ggc cag gtg 48
Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val
1 5 10 15
cag cac ctg cag gcc gcc ttc agc cag tac aag aag gtg 87
Gln His Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val
20 25
<210> 278
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 278
Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val
1 5 10 15
Gln His Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val
20 25
<210> 279
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> trimeric group tenascin-C
<220>
<221> CDS
<222> (1)..(90)
<400> 279
gcc tgc ggc tgc gcc gcc gcc ccc gac gtg aag gag ctg ctg agc aga 48
Ala Cys Gly Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg
1 5 10 15
ctg gag gag ctg gag aac ctg gtg agc agc ctg aga gag cag 90
Leu Glu Glu Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln
20 25 30
<210> 280
<211> 30
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 280
Ala Cys Gly Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg
1 5 10 15
Leu Glu Glu Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln
20 25 30
<210> 281
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> trimeric group tenascin-R
<220>
<221> CDS
<222> (1)..(93)
<400> 281
gcc tgc ccc tgc gcc agc agc gcc cag gtg ctg cag gag ctg ctg agc 48
Ala Cys Pro Cys Ala Ser Ser Ala Gln Val Leu Gln Glu Leu Leu Ser
1 5 10 15
aga atc gag atg ctg gag aga gag gtg agc gtg ctg aga gac cag 93
Arg Ile Glu Met Leu Glu Arg Glu Val Ser Val Leu Arg Asp Gln
20 25 30
<210> 282
<211> 31
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 282
Ala Cys Pro Cys Ala Ser Ser Ala Gln Val Leu Gln Glu Leu Leu Ser
1 5 10 15
Arg Ile Glu Met Leu Glu Arg Glu Val Ser Val Leu Arg Asp Gln
20 25 30
<210> 283
<211> 111
<212> DNA
<213> Artificial sequence
<220>
<223> trimeric group tenascin-X
<220>
<221> CDS
<222> (1)..(111)
<400> 283
ggc tgc ggc tgc ccc ccc ggc acc gag ccc ccc gtg ctg gcc agc gag 48
Gly Cys Gly Cys Pro Pro Gly Thr Glu Pro Pro Val Leu Ala Ser Glu
1 5 10 15
gtg cag gcc ctg aga gtg aga ctg gag atc ctg gag gag ctg gtg aag 96
Val Gln Ala Leu Arg Val Arg Leu Glu Ile Leu Glu Glu Leu Val Lys
20 25 30
ggc ctg aag gag cag 111
Gly Leu Lys Glu Gln
35
<210> 284
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 284
Gly Cys Gly Cys Pro Pro Gly Thr Glu Pro Pro Val Leu Ala Ser Glu
1 5 10 15
Val Gln Ala Leu Arg Val Arg Leu Glu Ile Leu Glu Glu Leu Val Lys
20 25 30
Gly Leu Lys Glu Gln
35
<210> 285
<211> 108
<212> DNA
<213> Artificial sequence
<220>
<223> tetramerization motif CMP (R27Q)
<220>
<221> CDS
<222> (1)..(108)
<400> 285
gag agc ctg gtg aag ttc cag gcc aag gtg gag ggc ctg ctg cag gcc 48
Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu Leu Gln Ala
1 5 10 15
ctg acc aga aag ctg gag gcc gtg agc aag cag ctg gcc atc ctg gag 96
Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Gln Leu Ala Ile Leu Glu
20 25 30
aac acc gtg gtg 108
Asn Thr Val Val
35
<210> 286
<211> 36
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 286
Glu Ser Leu Val Lys Phe Gln Ala Lys Val Glu Gly Leu Leu Gln Ala
1 5 10 15
Leu Thr Arg Lys Leu Glu Ala Val Ser Lys Gln Leu Ala Ile Leu Glu
20 25 30
Asn Thr Val Val
35
<210> 287
<211> 135
<212> DNA
<213> Artificial sequence
<220>
<223> pentameric motif (COMP)
<220>
<221> CDS
<222> (1)..(135)
<400> 287
gac ctg gcc ccc cag atg ctg aga gag ctg cag gag acc aac gcc gcc 48
Asp Leu Ala Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala
1 5 10 15
ctg cag gac gtg aga gag ctg ctg aga cag cag gtg aag gag atc acc 96
Leu Gln Asp Val Arg Glu Leu Leu Arg Gln Gln Val Lys Glu Ile Thr
20 25 30
ttc ctg aag aac acc gtg atg gag tgc gac gcc tgc ggc 135
Phe Leu Lys Asn Thr Val Met Glu Cys Asp Ala Cys Gly
35 40 45
<210> 288
<211> 45
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 288
Asp Leu Ala Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala
1 5 10 15
Leu Gln Asp Val Arg Glu Leu Leu Arg Gln Gln Val Lys Glu Ile Thr
20 25 30
Phe Leu Lys Asn Thr Val Met Glu Cys Asp Ala Cys Gly
35 40 45
<210> 289
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP1
<400> 289
Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp
1 5 10 15
Pro Met Val Ala
20
<210> 290
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP2
<400> 290
Met Arg Pro Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu Ala
1 5 10 15
Leu Trp Ala Pro Gly
20
<210> 291
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP3
<400> 291
Met Lys Val Gln Trp Leu Leu Leu Trp Val Leu Leu Leu Leu Val Leu
1 5 10 15
Phe Cys Ser Arg Gly
20
<210> 292
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP4
<400> 292
Met Arg Pro Trp Thr Trp Val Leu Leu Leu Leu Leu Leu Ile Cys Ala
1 5 10 15
Pro Ser Tyr Ala
20
<210> 293
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP5
<400> 293
Met Met Trp Leu Trp Leu Val Leu Leu Leu Leu Cys Leu Ala Gly Asn
1 5 10 15
Val Gln Ala
<210> 294
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP6
<400> 294
Met Pro Pro Lys Lys Cys Leu Leu Leu Leu Leu Thr Leu Leu Leu Leu
1 5 10 15
Ile Ser Thr Thr Phe Gly
20
<210> 295
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP7
<400> 295
Met Ala Gly Gly Val Ala Gly Leu Leu Leu Ala Leu Leu Leu Pro Ser
1 5 10 15
Ala Leu Ser
<210> 296
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP8
<400> 296
Met Lys Leu Leu Leu Ile Phe Phe Val Leu Val Val Trp Met Gly Pro
1 5 10 15
Ala His Arg
<210> 297
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP 9
<400> 297
Met Val Arg Gly Val Leu Ala Leu Leu Leu Met Ala Leu Gln Met Asp
1 5 10 15
Ala Ser Ser Gly
20
<210> 298
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP10
<400> 298
Met Ser Ala Asp Cys Ser Trp Gly Ala Ala Phe Gly Ala Leu Leu Pro
1 5 10 15
Leu Ala Ala Gly
20
<210> 299
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP11
<400> 299
Met Thr Lys His Leu Gly Val Leu Phe Ala Gly Phe Thr Ser Ala Asp
1 5 10 15
Val Ser Ala
<210> 300
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP12
<400> 300
Met Ile Phe Asn Pro Met Val Val Phe Leu Phe Cys Val Ser Asn His
1 5 10 15
Ala Leu Arg
<210> 301
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP13
<400> 301
Met Asp Leu Val Ser Trp Thr Phe Met Glu Val Ser Thr Leu Val Leu
1 5 10 15
Pro Lys Arg Pro
20
<210> 302
<211> 25
<212> PRT
<213> Artificial sequence
<220>
<223> Signal peptide ASP14
<400> 302
Met Leu Ala Ala Leu Arg Arg Ala Cys Thr Ser Ala Cys Arg Val Pro
1 5 10 15
Ile Lys Pro Thr His Leu Ala Gln Gly
20 25
<210> 303
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> GFP forward primer
<400> 303
gaagttcgag ggcgacac 18
<210> 304
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> GFP reverse primer
<400> 304
taaaatcttt tattttatct gcggccgcac 30
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