阅读说明：本技术 腺病毒载体AdC68XY、由其包装的病毒及应用 (Adc68XY adenovirus vector, virus packaged by same and application ) 是由 周东明 邢嫚 祝丰 于 2019-08-22 设计创作，主要内容包括：本发明提供了一种新型腺病毒载体AdC68XY、由其包装的病毒及应用。本发明的腺病毒表达载体能够高效地包装,病毒产量高,且获得的病毒感染能力强。同时,本发明的腺病毒表达载体可容纳较大的外源基因,具有很大的应用价值。(The invention provides a novel adenovirus vector AdC68XY, a virus packaged by the same and application thereof. The adenovirus expression vector can be efficiently packaged, the virus yield is high, and the obtained virus has strong infection capacity. Meanwhile, the adenovirus expression vector can contain larger exogenous genes and has great application value.)

1. A method of making an adenoviral expression vector comprising: based on the genome sequence of the chimpanzee adenovirus AdC68, the E1 sequence and the E3 sequence are deleted, and the E4 sequence is modified; the modification comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum adenovirus type 5 AdHu5 genome.

2. A recombinant adenoviral vector comprising sequences based on the genome of chimpanzee adenovirus AdC68, wherein the E1 and E3 sequences are deleted and the E4 sequence is engineered; the modification comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum adenovirus type 5 AdHu5 genome.

3. The method of claim 1 or the recombinant adenoviral vector of claim 2, wherein the engineering is selected from the group consisting of:

(a) replacing the AdC68 genomic E4 sequence with an AdHu5 genomic E4 sequence; more preferably, the deletion region is the 33518-36105 bit sequence of the AdC68 genome, and the 32914-35641 bit sequence of the AdHu5 genome is inserted into the deletion region;

(b) replacing the sequences of ORF6 and ORF6/7 regions in the sequence of AdC68 genome E4 by the sequences of ORF6 and ORF6/7 regions in the sequence of AdHu5 genome E4; more preferably, the deletion region is the 33518-34671 bit sequence of the AdC68 genome, and the 32914-34077 bit sequence of the AdHu5 genome is inserted into the deletion region; or

(c) Replacing ORF 1-ORF 6/7 sequences in the AdC68 genome E4 sequence by ORF 3-ORF 6 sequences in the AdHu5 genome E4 sequence; more preferably, the deletion region is the 33518-36105 bit sequence of the AdC68 genome, into which the 33193-34703 bit sequence of the AdHu5 genome was inserted.

4. The method of claim 1 or the recombinant adenoviral vector of claim 2, further comprising an element or gene selected from the group consisting of:

an exogenous gene expression cassette;

an expression cassette for a reporter gene;

an inverted terminal repeat sequence; and/or

A restriction enzyme site; preferably comprising: HpaI, I-CeuI, PI-SceI, SwaI and PacI.

5. The method of claim 1 or the recombinant adenoviral vector of claim 2, which has the nucleotide sequence shown in SEQ ID NO 1, but,

wherein positions 29278-31865 are replaced by SEQ ID NO 3;

wherein the 29278-30431 position is replaced by SEQ ID NO. 4; or

Wherein positions 29278-31865 are replaced by SEQ ID NO 5.

6. Use of a recombinant adenoviral vector according to any one of claims 1 to 5 for preparing a recombinant adenovirus; or, for insertion of a foreign gene, thereby expressing the foreign gene.

7. A recombinant adenovirus obtained by packaging the recombinant adenovirus vector according to any one of claims 1 to 5.

8. A method of producing the recombinant adenovirus of claim 7, comprising:

(1) preparing a recombinant adenoviral vector according to any one of claims 1 to 5;

(2) transfecting the recombinant adenovirus vector in (1) into a virus production cell, and packaging to obtain the recombinant adenovirus.

9. Use of the recombinant adenovirus according to claim 7, for: expressing the exogenous gene to obtain the target protein; preferably, the target protein comprises: proteins with immunogenicity, proteins with pharmaceutical activity, proteins with diagnostic activity, structural proteins, enzymes, fusion peptides, reporter proteins.

10. A kit, comprising:

a recombinant adenoviral vector according to any one of claims 1 to 5;

the recombinant adenovirus of claim 7; and/or

A virus-producing cell.

Technical Field

The invention belongs to the field of biomedicine and virology, and particularly relates to a novel adenovirus vector AdC68XY, a virus packaged by the same, a preparation method and application thereof; preferably, the adenoviral vector AdC68XY is constructed based on synthetic biology techniques.

Background

Adenovirus vectors are one of the most promising viral vectors currently. Adenovirus is a non-enveloped, linear, double-stranded DNA virus with a genome size of approximately 36 kb. Since the first discovery of adenoviruses in 1953, to date, 57 human serotypes (classified into subgenera A-G7) and a number of adenoviruses from other species have been isolated, the most common of which is the human serum type 5 adenovirus, AdHu 5. Since adenovirus has high transduction efficiency, can carry foreign genes, is easy to amplify and purify, is regarded as an excellent gene delivery vehicle, and is widely applied to various basic researches or clinical applications, including the development of vaccines for diseases such as HIV, influenza, Ebola, malaria, rabies, prostate cancer, melanoma and the like, and the oncolytic therapy. However, since pre-existing neutralizing antibodies against AdHu5 are present in 75-80% of individuals, the vaccine with AdHu5 as a vector has immune effects affected by the pre-existing neutralizing antibodies, not only impairing the immune effects, but also possibly enhancing side effects. To avoid this problem, the inventors have previously selected as a vaccine vector an adenovirus of a rare serotype or other species origin, such as a chimpanzee type adenovirus or the like. The chimpanzee type adenovirus is only popular in chimpanzees in Africa, and people generally do not contain neutralizing antibodies for resisting the chimpanzee type adenovirus, so the chimpanzee type adenovirus is taken as a vaccine vector, and the comprehensive immune effect of the vaccine vector is superior to that of an AdHu5 vector.

Because the adenovirus genome is relatively large, the construction of the recombinant adenovirus vector is difficult to complete by common molecular biology methods, and therefore, the construction of the adenovirus vector is still a technical bottleneck. Traditional adenovirus vector construction strategies mainly include homologous recombination and direct genome cloning, and are time-consuming and labor-consuming, and take more than 2 months at the fastest speed. A further strategy to improve on this is isothermal assembly, which, although claimed to require only about 1 week, only refers to the time required to join multiple fragments together into one clone by isothermal assembly, not including the time and validation process required to obtain multiple small fragments with complementary sequences at the linker sites. Furthermore, these strategies must first obtain the wild-type virus and amplify and purify the virus to obtain its genomic DNA. If only the adenovirus gene sequence is known, the construction of the adenovirus vector cannot be completed.

In conclusion, there is a need in the art for further research and exploration to obtain improved adenoviral vectors and adenoviruses to enhance their clinical applicability.

Disclosure of Invention

The invention aims to provide a novel adenovirus vector AdC68XY constructed based on synthetic biology technology.

In a first aspect of the invention, there is provided a method of preparing an adenoviral expression vector comprising: based on the genome sequence of the chimpanzee adenovirus AdC68, the E1 sequence and the E3 sequence are deleted, and the E4 sequence is modified; the modification comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum adenovirus type 5 AdHu5 genome.

In another aspect of the invention, there is provided a recombinant adenoviral vector comprising sequences based on the genome of the adenovirus AdC68 of chimpanzee type, wherein the E1 sequence and the E3 sequence are deleted and the E4 sequence is engineered; the modification comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum adenovirus type 5 AdHu5 genome.

In a preferred embodiment, in the aforementioned method or vector, the modification is selected from:

(a) replacing the AdC68 genomic E4 sequence with an AdHu5 genomic E4 sequence; more preferably, the deletion region is the 33518-36105 bit sequence of the AdC68 genome, and the 32914-35641 bit sequence of the AdHu5 genome is inserted into the deletion region;

(b) replacing the sequences of ORF6 and ORF6/7 regions in the sequence of AdC68 genome E4 by the sequences of ORF6 and ORF6/7 regions in the sequence of AdHu5 genome E4; more preferably, the deletion region is the 33518-34671 bit sequence of the AdC68 genome, and the 32914-34077 bit sequence of the AdHu5 genome is inserted into the deletion region; or

(c) Replacing ORF 1-ORF 6/7 sequences in the AdC68 genome E4 sequence by ORF 3-ORF 6 sequences in the AdHu5 genome E4 sequence; more preferably, the deletion region is the 33518-36105 bit sequence of the AdC68 genome, into which the 33193-34703 bit sequence of the AdHu5 genome was inserted.

In another preferred embodiment, the recombinant adenovirus vector further comprises the following operably linked elements or genes: an exogenous gene expression cassette; an expression cassette for a reporter gene; an inverted terminal repeat sequence; and/or a cleavage site; preferably including (but not limited to): HpaI, I-CeuI, PI-SceI, SwaI and PacI.

In another preferred embodiment, the expression cassette of the reporter gene has the nucleotide sequence shown in SEQ ID NO. 2.

In another preferred example, the enzyme cleavage site includes an enzyme cleavage site for inserting a foreign gene.

In another preferred example, the exogenous gene expression cassette can be expressed to obtain the target protein; preferably, the protein of interest includes (but is not limited to): proteins with immunogenicity, proteins with pharmaceutical activity (e.g. antibodies), proteins with diagnostic activity (e.g. antibodies), structural proteins, enzymes, fusion peptides, reporter proteins.

In another preferred embodiment, the foreign gene is a foreign gene having a length of 30bp to 8.5kb, such as 50bp, 100bp, 200bp, 500bp, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8 kb.

In another preferred embodiment, the adenoviral expression vector is constructed using synthetic techniques.

In another preferred embodiment, the recombinant adenovirus vector has the nucleotide sequence shown in SEQ ID NO. 1(AdC68XY1-EGFP), but wherein the 29278-31865 positions are replaced by SEQ ID NO. 3; wherein the 29278-30431 position is replaced by SEQ ID NO. 4; or wherein positions 29278-31865 are replaced by SEQ ID NO 5.

In another aspect of the invention there is provided the use of a recombinant adenoviral vector as described in any one of the preceding paragraphs for the preparation of a recombinant adenovirus; or, for insertion of a foreign gene, thereby expressing the foreign gene.

In another aspect of the present invention, there is provided a recombinant adenovirus obtained by packaging the recombinant adenovirus vector described in any one of the above.

In another aspect of the present invention, there is provided a method for preparing the recombinant adenovirus, the method comprising: (1) preparing a recombinant adenoviral vector as described in any one of the preceding paragraphs; and (2) transfecting the recombinant adenovirus vector of (1) into a virus production cell, thereby packaging to obtain the recombinant adenovirus.

In a preferred embodiment, the virus-producing cell is a cell that can effect viral packaging; preferably comprising: HEK293 cells.

In another aspect of the present invention, there is provided a use of the recombinant adenovirus for: expressing the exogenous gene to obtain the target protein; preferably, the protein of interest includes (but is not limited to): proteins with immunogenic properties, proteins with pharmaceutical activity (such as antibodies, proteins with killing activity (such as tumoricidal proteins)), proteins with diagnostic activity (such as antibodies), structural proteins, enzymes, fusion peptides, reporter proteins.

In a preferred embodiment, the immunogenic proteins include, but are not limited to: protein vaccine for HIV, influenza, Ebola, malaria, rabies, prostate cancer, melanoma, etc.

In another aspect of the present invention, there is provided a kit comprising: a recombinant adenoviral vector as described in any one of the preceding; the recombinant adenovirus as described above; and/or virus producing cells.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a schematic diagram showing the construction of the recombinant adenovirus vector pAdC68XY1-EGFP in which the E1 region has been completely deleted; the E3 region was partially deleted, and a SwaI cleavage site was added for subsequent foreign gene insertion.

FIG. 2 shows the results of the restriction and identification of recombinant adenovirus vectors pAdC68XY1-EGFP, pAdC68XY2-EGFP, pAdC68XY3-EGFP and pAdC68XY 4-EGFP. Wherein, M is 1kb plus DNA ladder; 1-3: BglII, Xho I and MfeI.

FIG. 3A viral plaque formation of recombinant adenovirus AdC68XY 1-EGFP.

FIG. 3B viral plaque formation of recombinant adenovirus AdC68XY 2-EGFP.

FIG. 3C viral plaque formation of recombinant adenovirus AdC68XY 3-EGFP.

FIG. 3 viral plaque formation of recombinant adenovirus AdC68XY 4-EGFP.

FIG. 4 shows the results of genome restriction and identification of recombinant adenovirus AdC68XY1-EGFP, AdC68XY2-EGFP, AdC68XY3-EGFP and AdC68XY 4-EGFP. Wherein, M is 1kb plus DNA ladder; 1-3: BglII, Xho I and MfeI.

Detailed Description

Adenovirus has a large genome size, and it is difficult to express foreign genes. Therefore, the present inventors have conducted intensive studies to disclose a novel adenovirus vector constructed based on synthetic biology techniques, which is called AdC68XY series vector. On one hand, the adenovirus expression vector can be efficiently packaged, the virus yield is high, and the obtained virus has strong infection capacity; on the other hand, the adenovirus expression vector can contain larger exogenous genes and has larger application value.

As used herein, the term "element" refers to a series of functional nucleic acid sequences useful for the expression of a protein, and in the present invention, is systematically constructed to form an expression construct. The sequence of the "element" may be those provided in the present invention, and also includes variants thereof, as long as the variants substantially retain the function of the "element", which are obtained by inserting or deleting some bases (e.g., 1 to 50 bp; preferably 1 to 30bp, more preferably 1 to 20bp, still more preferably 1 to 10bp), or by random or site-directed mutagenesis, or the like.

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "expression cassette" refers to a gene expression system comprising all the necessary elements required for expression of a gene of interest, typically comprising the following elements: promoter, target gene sequence, terminator; in addition, a signal peptide coding sequence and the like can be optionally included. These elements are operatively connected.

As used herein, the term "construct" refers to a single-or double-stranded DNA molecule that has been artificially manipulated to contain DNA segments combined and arranged according to sequences not found in nature. The "construct" includes an expression vector; alternatively, the "construct" is included in an expression vector as part of the expression vector.

As used herein, the term "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a nucleic acid from a different source and a host cell. For example, a nucleic acid is heterologous to a host cell if the combination of the nucleic acid and the host cell does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

Adenoviral vectors

The genome of the adenovirus is relatively large, and in the prior art, the inventor tries more modification strategies, including carrying out total or partial modification on E1 and E3 in the genome and deleting partial genes, but in view of the important role of the E4 region, the prior art has not been precedent for carrying out successful modification on E4. The present inventors have found that a virus having enhanced replication and infection abilities can be obtained by modifying E4 wholly or partially, replacing the corresponding region in the human serum adenovirus type 5 AdHu5 genome, or deleting the sequence to a limited extent, and that even an increase in the insertion capacity of a foreign gene can be achieved.

In the invention, a novel replication-defective recombinant adenovirus vector deleted by E1/E3 is designed by using chimpanzee adenovirus AdC68 as a template and using the genome sequence of the chimpanzee adenovirus AdC 68. The adenovirus genome contains the E1-E4 early genes, and E1A can activate the promoters of E1B, E2A, E2B, E3, and E4. The adenovirus E4 region includes 7 open reading frames, the products of which are ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7, respectively. Among them, E4ORF6 is a multifunctional protein involved in many processes of viral replication and is extremely important for replication of adenovirus. ORF6 can bind to E1B 55-kDa to form an ORF6-E1B complex that promotes export of major late viral mRNA during the post-transcriptional phase, thereby enhancing expression of late viral genes while inhibiting synthesis of host proteins. In adenovirus-infected cells, the ORF6-E1B complex is also involved in inhibiting E1A-induced p53 activation, thereby degrading p53 and inhibiting apoptosis of host cells. Furthermore, ORF6 can indirectly increase the efficiency of viral DNA synthesis. ORF3 is functionally similar to ORF6 and can partially or completely compensate for each other deletion. For improved safety, adenoviruses used as vaccine vectors are usually replication-defective, i.e., the E1 gene is deleted. Thus, non-replicating adenoviruses are typically packaged and amplified in HEK293 cells incorporating the AdHu5E1 gene. Although the interaction between AdHu5E1B 55k and E4ORF6 proteins is relatively conserved, binding of chimpanzee adenovirus E4ORF6 protein to AdHu5E1B 55k may be unstable or less efficient, resulting in reduced non-replicating chimpanzee adenovirus production. In order to avoid the problems and improve the yield of the recombinant adenovirus AdC68, the inventor replaces the E4 sequence of AdC68 with the E4 sequence of AdHu5 in whole or in part, designs a plurality of recombinant AdC68 vectors with different E4 sequences respectively, and names AdC68XY1, AdC68XY2, AdC68XY3 and AdC68XY4 respectively; in the E1 deletion region of the recombinant vector, the green fluorescent protein gene was inserted. Then, the inventor adopts synthetic biology technology, the whole gene is combined into a novel recombinant adenosis AdC68XY1 with green fluorescence label, the linear recombinant adenosis AdC68XY1 is successfully packaged in HEK293 cells, and the genetically stable recombinant adenosis AdC68XY1 is amplified. On the basis of obtaining the recombinant adenovirus vector AdC68XY1, the inventors make other various substitutions on the E4 sequence of AdC68 according to the established design scheme to obtain vectors such as AdC68XY2, AdC68XY3, AdC68XY4, and the like. The method is time-saving and labor-saving, and does not depend on wild adenovirus genome DNA.

Based on the new findings of the present inventors, there is provided a recombinant adenovirus vector (plasmid) comprising: an engineered chimpanzee adenovirus AdC68 genomic sequence; wherein the whole sequence of E1 and the partial sequence of E3 are deleted, and the sequence of E4 is modified; the modification comprises replacing the E4 sequence or fragment thereof in the AdC68 genome with the corresponding E4 sequence or fragment thereof in the human serum adenovirus type 5 AdHu5 genome.

As a preferred mode of the present invention, the modification is selected from: (a) the AdHu5 genomic E4 sequence replaces the AdC68 genomic E4 sequence; (b) replacing the sequences of ORF6 and ORF6/7 regions in the sequence of AdC68 genome E4 by the sequences of ORF6 and ORF6/7 regions in the sequence of AdHu5 genome E4; or (c) replacing the sequences of ORF 1-ORF 6/7 in the sequence of AdC68 genome E4 by the sequences of ORF 3-ORF 6 in the sequence of AdHu5 genome E4.

Preferred embodiments of the present invention further comprise an element or gene selected from the group consisting of: an exogenous gene expression cassette; an expression cassette for a reporter gene; inverted terminal repeats and/or cleavage sites. It is understood that the design of the reporter gene, the restriction enzyme site, and the like is sometimes designed to provide a convenient way to observe or detect when performing research, but is not necessary when the recombinant adenovirus vector or the recombinant adenovirus of the present invention is applied to the field of clinical medicine; alternatively, the reporter gene may be replaced at a position where it is capable of expressing the protein of interest having pharmaceutical activity.

The expression vector usually further contains an origin of replication and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention, as suggested by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter (e.g., CMV) in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

The expression vector of the invention can be inserted with an expression cassette of a foreign gene, thereby expressing a target protein of interest. The adenovirus expression vector of the invention can be used as an expression vector platform and is suitable for expressing a plurality of target proteins, and the target proteins are not particularly limited. As some alternative embodiments, the protein of interest includes (but is not limited to): proteins with immunogenicity, proteins with pharmaceutical activity (e.g. antibodies), proteins with diagnostic activity (e.g. antibodies), structural proteins, enzymes, fusion peptides, reporter proteins. The target protein may be a single protein or two or more proteins, so long as the length of the gene encoding the target protein is within the packaging capacity that the adenovirus can accommodate.

In the expression vector of the present invention, the insertion position of the foreign gene expression cassette is not unique, and preferably includes (but is not limited to): the E1 region, the E3 region, the E4 region.

The elements of the expression vectors of the present invention are operably linked to facilitate viral packaging or efficient expression of the protein of interest.

As a preferred mode of the present invention, the novel adenoviral vector of the present invention is constructed using synthetic biology-based techniques. In contrast to the research direction of traditional biology, synthetic biology is a step-by-step building of parts starting from the most basic elements, in the process of which genetic material is designed and modified to finally obtain ideal new organisms. However, it is understood that, in addition to synthetic biology techniques, other methods of constructing the adenoviral vectors of the invention are also encompassed by the invention, once the technical solution of the invention has been obtained.

The invention provides a rapid and effective method for constructing a novel adenovirus vector, which is beneficial to expanding the application of the adenovirus vector and simultaneously obtaining a novel recombinant adenovirus AdC68XY series vector.

Adenoviral vectors

After obtaining the adenovirus expression vector, transfecting the adenovirus expression vector into a virus production cell to propagate viruses. After a period of time following transfection, the virus may be harvested. The virus-producing cell may be any cell known in the art that is capable of propagating adenovirus, such as HEK293 cells and the like.

As a preferred mode of the present invention, the harvested virus can be repeatedly infected with virus-producing cells and passaged continuously. Viral Titer (TCID)₅₀) The determination of (b) can be performed according to a method conventional in the art. The obtained recombinant adenovirus is also included in the present invention.

The adenovirus obtained by the adenovirus vector expression of the invention can form clear and obvious plaques on the surface of a cell culture 4 days after transfecting virus production cells, and the quantity of the plaques is more, obviously because of the adenovirus vector before modification.

The recombinant adenovirus generated by the recombinant adenovirus vector can transfect mammalian cells efficiently, is easy to amplify and purify, has low toxicity, high expression efficiency and long term. The adenovirus vector expression recombination process of the invention is simple, low in price and lasting in curative effect.

Composition, kit or kit

The invention also provides a composition comprising an effective amount of the adenovirus, and a pharmaceutically acceptable carrier. Preferably, the adenovirus expresses a protein of interest. For example, the protein of interest includes, but is not limited to: proteins with immunogenicity, proteins with pharmaceutical activity (such as antibodies, proteins with activity of killing tumor cells), proteins with diagnostic activity (such as antibodies), structural proteins, enzymes, fusion peptides.

As used herein, "comprising" means that the various ingredients can be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

As used herein, "effective amount" or "effective dose" refers to an amount that produces a function or activity in a human and/or animal and is acceptable to the human and/or animal as used herein.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

Typically, the adenovirus will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5 to about 8, preferably a pH of about 6 to about 8.

The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The amount of active ingredient (adenovirus) administered is a therapeutically effective amount, for example from about 0.1 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Based on the new discovery of the invention, the invention also provides a kit/kit, wherein the kit/kit comprises the adenovirus expression vector or the adenovirus. The kit/kit may also include virus producing cells (e.g., 293 cells), culture media, and the like. In addition, the kit/kit may further comprise instructions for using the adenovirus after the expression method.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Cells

The HEK293 cell line was purchased from Shanghai cell bank of Chinese academy of sciences, and the culture medium was DMEM containing 10% fetal bovine serum.

Restriction enzyme, competent cell, genomic sequence

All restriction enzymes were purchased from New England Biolabs; stbl2 strain was purchased from Suzhou New Saimei Biotechnology Inc. (Suzhou).

Chimpanzee adenovirus AdC68 sequences are referenced to GenBank published sequences, i.e. GenBank accession numbers: AC _ 000011.1.

The E4 sequence of adenovirus human serotype 5 (AdHu5) is referenced to GenBank accession No.: AC _ 000008.1.

Example 1 preparation of adenovirus vector AdC68XY and recombinant adenovirus

1. Design of AdC68XY AdC

First, the inventors deleted part of the E1 and E3 sequences in the AdC68 genomic sequence. The E1 deletion was 2552bp (deletion region was 459bp-3010bp), and thus a replication-defective adenovirus vector was obtained. The E3 deletion was 3721bp (deletion region AC _000011.1, 27615bp-31335 bp). The deletion of E1/E3 can make the insertion capacity of the foreign gene about 7.5 kb.

Secondly, the inventors transformed or replaced the E4 sequence of AdC68 with the E4 sequence of AdHu5 in a different way to obtain different recombinant vectors, and some optimized recombinant vectors are as follows:

AdC68XY1 vector: wherein E1/E3 is deleted, enzyme cutting sites HpaI are added at 33517bp and 36106bp (corresponding to AC _000011.1), and the sequence of E4 is not changed. Meanwhile, rare enzyme cutting sites I-CeuI and PI-SceI are inserted into the deletion region of E1 by the inventor, so that the insertion of a foreign gene into the region is facilitated. The restriction site SwaI was inserted in the deletion region of E3, so that a foreign gene could also be inserted in this region. Enzyme cutting sites HpaI are added at two ends of the E4 region so as to be convenient for carrying out subsequent modification on the E4 region. The ITRs of the modified AdC68 genome are added with PacI restriction sites on both sides (so that the recombinant adenovirus plasmid is linearized by PacI), and the recombinant adenovirus vector AdC68XY1 is obtained. In order to facilitate the observation of the packaging of the novel recombinant adenovirus, the inventors inserted a CMV-EGFP sequence (CMV promoter driving EGFP expression, SEQ ID NO:2) between the I-CeuI and PI-SceI cleavage sites of the deletion region of E1, so that the obtained recombinant adenovirus carries a green fluorescent marker (AdC68XY 1-EGFP). AdC68XY1-EGFP has the sequence shown in SEQ ID NO 1, and the components are shown in figure 1.

AdC68XY2 vector: wherein E1/E3 is deleted and the E4 sequence is completely replaced by the E4 sequence of AdHu 5: the deletion region of the AdC68E4 sequence is 33518bp-36105bp (corresponding to AC _ 000011.1); the insertion region (SEQ ID NO:3) of the AdHu5E4 sequence was 32914bp-35641bp (corresponding to AC _ 000008.1).

AdC68XY3 vector: wherein E1/E3 is deleted and the E4 regions ORF6/7 and ORF6 are replaced with AdHu 5: the deletion region of the AdC68E4 sequence is 33518bp-34671bp (corresponding to AC _ 000011.1); the insertion region (SEQ ID NO:4) of the AdHu5E4 sequence was 32914bp-34077bp (corresponding to AC-000008.1). In which the ORF1-4 sequence of AdC68 was retained.

AdC68XY4 vector: wherein E1/E3 was deleted and the E4 region was replaced with ORF3-6 of AdHu5 (i.e., ORFs 1-2 and 6/7 were deleted): the deletion region of the AdC68E4 sequence was 33518bp-36105bp (corresponding to AC _000011.1), and the insertion region of the AdHu5E4 sequence (SEQ ID NO:5) was 33193bp-34703bp (corresponding to AC _ 000008.1).

2. Synthesis of recombinant adenovirus pAdC68XY1-EGFP molecular clone

The above-designed recombinant adenovirus pAdC68XY1-EGFP molecular clone sequence was sent to general biological systems (Anhui) Inc., and the molecular clone was synthesized by the company.

On the basis of the sequence of the synthesized pAdC68XY1-EGFP vector, the vector is further modified to obtain an AdC68XY2 vector, an AdC68XY3 vector and an AdC68XY4 vector.

3. Recombinant adenovirus AdC68XY1-EGFP packaging and amplification

The recombinant adenovirus pAdC68XY1-EGFP molecular clone (plasmid DNA) obtained by the synthesis is linearized by PacI digestion, and the digested AdC68XY1-EGFP plasmid DNA is transfected into HEK293 cells (6-well plates) with the density of 60-70% according to the instructions of a Lipofectamine 2000 transfection kit. Before transfection, the medium was changed to serum-free medium, the DNA/liposome complex was added, and after 5 hours of transfection, the medium was changed to DMEM medium containing 10% FBS and 1% double antibody. EGFP fluorescence intensity and cytopathic effect are observed every other day under a fluorescence microscope, cells are collected until a great amount of plaques appear in HEK293 cells, the cells are repeatedly frozen and thawed 3 times between room temperature and an ultra-low temperature refrigerator at minus 80 ℃, centrifugation is carried out for 10min at 3500rpm, and supernatant is collected and placed in the ultra-low temperature refrigerator at minus 80 ℃ for seed preservation.

4. Molecular cloning and virus packaging of pAdC68XY2-EGFP, pAdC68XY3-EGFP and pAdC68XY4-EGFP

On the basis of obtaining the recombinant adenovirus vector pAdC68XY1-EGFP, the inventor carries out other various substitutions on an E4 sequence of AdC68 according to a set design scheme, firstly enzyme-cuts the pAdC68XY1-EGFP vector by HpaI, then respectively obtains pAdC68XY2-EGFP, pAdC68XY3-EGFP and pAdC68XY4-EGFP vectors by an isothermal recombinant molecular cloning method, and packages, amplifies and purifies the viruses according to the method.

5. Identification of recombinant adenovirus AdC68XY-EGFP

Amplifying each recombinant adenovirus, purifying virus liquid by cesium chloride density gradient centrifugation, measuring the titer of the virus, and subpackaging in a refrigerator at the ultralow temperature of-80 ℃ for storage. Each recombinant adenovirus AdC68XY-EGFP was partially purified and isolated from genomic DNA of each recombinant adenovirus according to the instructions of DNeasy Tissue Kit (QIAGEN, Valencia, CA) and then identified by multiple-enzyme cutting.

Example 2 identification of recombinant adenovirus pAdC68XY-EGFP molecular clones

The plasmid DNA of the recombinant adenovirus pAdC68XY1-EGFP is cut by a plurality of enzymes (MfeI, BglII and XhoI) and subjected to electrophoresis by 1% agarose gel, and the molecular clone of the pAdC68XY1-EGFP is proved to be complete and correct by DNA bands, which is shown in figure 2.

Plasmid DNA of other recombinant adenoviruses pAdC68XY2-EGFP, pAdC68XY3-EGFP and pAdC68XY4-EGFP obtained by further modification are subjected to the same enzyme digestion identification, and the plasmid DNA is shown in figure 2.

Example 3 viral packaging and rescue of recombinant adenovirus AdC68XY-EGFP

Lipofefection pAdC68XY1-EGFP, pAdC68XY2-EGFP, pAdC68XY3-EGFP, pAdC68XY4-EGFP plasmid DNA linearized by PacI was transfected into HEK293 cells (cultured in 6-well plates) at a density of about 70%, and green fluorescent protein expression was observed periodically and recorded by photography. Over time, green fluorescence expression changes, fluorescence gradually enriches and forms clusters, and plaques are finally formed, which indicates that the chimpanzee-derived replication-defective adenoviruses AdC68XY1-EGFP, AdC68XY2-EGFP and AdC68XY3-EGFP are successfully packaged and replicated to obtain infectious virus particles in HEK293 cells, and the figure is shown in figure 3.

According to FIG. 3, the adenovirus AdC68XY1-EGFP, which was not modified in the E4 region, showed plaques at day 10, with a plaque number of 3.

AdC68XY2-EGFP with deletion of E4 region showed plaques at day 4, with 11 plaques.

The E4 region-engineered AdC68XY3-EGFP showed plaques at day 4, with a 12 plaque number.

The E4 region-engineered AdC68XY4-EGFP showed plaques at day 4, with a plaque number of 15.

According to the result, the time required for adenovirus rescue after the E4 region is modified is obviously shorter than that of an unmodified group, and the plaque number is obviously more than that of the unmodified group, so that the replication capacity is enhanced after the E4 region is partially or completely replaced by the corresponding region of the AdHu5 adenovirus.

In addition, for the group of the foreign gene insertion capacity AdC68XY4-EGFP, after the deletion of ORF1, ORF2 and ORF6/7 in the E4 region, the insertion capacity of the foreign gene is reduced by 970bp compared with that of AdC68XY1, so that the insertion capacity of the foreign gene is increased and can reach about 8.5 kb. Meanwhile, the inserting capacity of AdC68XY4 is increased, the replication capacity of adenovirus, virus package and the like are not affected, and the yield is not affected.

Example 4 identification of recombinant adenovirus AdC68XY-EGFP

Purifying the amplified recombinant adenoviruses AdC68XY1-EGFP, AdC68XY2-EGFP, AdC68XY3-EGFP and AdC68XY4-EGFP by cesium chloride gradient centrifugation, wherein the titer of each purified recombinant virus is 6 multiplied by 10¹²vp/L、2×10¹³vp/L、1×10¹³vp/L、2×10¹³vp/L. Higher titer can be obtained.

In the experimental result, the E4 region is partially or completely replaced by the Hu5 source recombinant adenovirus, the virus yield is relatively highest, the expected effect is achieved, and the virus yield is also obviously improved by the other partially modified recombinant adenovirus with the E4 region. Therefore, the invention obtains various recombinant adenoviruses which are more beneficial to growth in HEK293 cells and improves the yield.

Separating virus genome DNA, carrying out enzyme digestion by MfeI, BglII and XhoI, carrying out 1% agarose gel electrophoresis, and proving that recombinant adenoviruses AdC68XY1-EGFP, AdC68XY2-EGFP, AdC68XY3-EGFP and AdC68XY4-EGFP are correct virus genome through the bands of the genome DNA subjected to enzyme digestion. See fig. 3.

Conclusion

In the invention, the adenovirus AdC68 genome sequence is analyzed and designed, and a biosynthesis technology is utilized to synthesize the recombinant adenovirus vector AdC68XY1 as a whole. In order to embody the effect, the inventor adds green fluorescent protein into the plasmid, and can directly obtain the recombinant adenovirus AdC68XY1-EGFP after transfection, virus packaging, amplification and purification. The correct recombinant adenovirus vector AdC68XY is obtained by enzyme digestion identification analysis. On the basis, the inventor further replaces E4 of AdC68XY1 by conventional molecular cloning to obtain recombinant adenoviruses AdC68XY2-EGFP, AdC68XY3-EGFP and AdC68XY4-EGFP, and obtains corresponding recombinant adenoviruses. The vectors can be used in a variety of basic research and clinical applications, such as expression vectors, vaccine development, gene therapy, and the like.

The invention not only obtains a series of novel vectors AdC68XY based on chimpanzee adenovirus, the modified vector is more suitable for growth and propagation in a packaging cell HEK293 compared with the traditional AdC68 vector, and the yield is high, thereby reducing the production cost of the product; in addition, the invention establishes a new strategy for preparing the recombinant adenovirus vector by synthetic biology, and the strategy does not need to obtain wild adenovirus, thereby avoiding complex cloning process, directly synthesizing molecular cloning of the recombinant adenovirus and saving time and labor. The strategy solves the technical bottleneck of constructing the recombinant adenovirus vector, and provides solid theoretical and technical support for the development, popularization and application of the recombinant adenovirus vector.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Suzhou gambling biotechnology, Inc

<120> AdC68XY adenovirus vector, virus packaged by it and use

<130> 196276

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35048

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(35048)

<223> AdC68XY1-EGFP nucleotide sequence of recombinant adenovirus

<400> 1

gtcgagggat gagcgaccgt taggggcggg gcgagtgacg ttttgatgac gtggttgcga 60

ggaggagcca gtttgcaagt tctcgtggga aaagtgacgt caaacgaggt gtggtttgaa 120

cacggaaata ctcaattttc ccgcgctctc tgacaggaaa tgaggtgttt ctgggcggat 180

gcaagtgaaa acgggccatt ttcgcgcgaa aactgaatga ggaagtgaaa atctgagtaa 240

tttcgcgttt atggcaggga ggagtatttg ccgagggccg agtagacttt gaccgattac 300

gtgggggttt cgattaccgt gtttttcacc taaatttccg cgtacggtgt caaagtccgg 360

tgtttttacg tacgatatca tttccccgaa aagtgccacc tgaccgtaac tataacggtc 420

ctaaggtagc gaaagctcag atctcccgat cccctatggt gcactctcag tacaatctgc 480

tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 540

tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 600

aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 660

tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 720

ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 780

aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 840

actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat 900

caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 960

tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 1020

ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 1080

cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 1140

tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 1200

atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga 1260

gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct 1320

ggctagcgtt taaacgggcc cgcccgggct tataagccac catggtgagc aagggcgagg 1380

agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 1440

agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 1500

tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 1560

acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 1620

ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 1680

acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 1740

agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 1800

acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 1860

agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 1920

cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 1980

ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 2040

ccgccgggat cactctcggc atggacgagc tgtacaagta attataagcc cgggcaagct 2100

taagtttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 2160

gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 2220

aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 2280

tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 2340

tgggctctat ggcttctgag gcggaaagaa ccagcagatc tgcagatctg aattcatcta 2400

tgtcgggtgc ggagaaagag gtaatgaaat ggcattatgg gtattatggg tctgcattaa 2460

tgaatcggcc agatatcgac atatgctggc caccgtacat gtggcttccc atgctcgcaa 2520

gccctggccc gagttcgagc acaatgtcat gaccaggtgc aatatgcatc tggggtcccg 2580

ccgaggcatg ttcatgccct accagtgcaa cctgaattat gtgaaggtgc tgctggagcc 2640

cgatgccatg tccagagtga gcctgacggg ggtgtttgac atgaatgtgg aggtgtggaa 2700

gattctgaga tatgatgaat ccaagaccag gtgccgagcc tgcgagtgcg gagggaagca 2760

tgccaggttc cagcccgtgt gtgtggatgt gacggaggac ctgcgacccg atcatttggt 2820

gttgccctgc accgggacgg agttcggttc cagcggggaa gaatctgact agagtgagta 2880

gtgttctggg gcgggggagg acctgcatga gggccagaat aactgaaatc tgtgcttttc 2940

tgtgtgttgc agcagcatga gcggaagcgg ctcctttgag ggaggggtat tcagccctta 3000

tctgacgggg cgtctcccct cctgggcggg agtgcgtcag aatgtgatgg gatccacggt 3060

ggacggccgg cccgtgcagc ccgcgaactc ttcaaccctg acctatgcaa ccctgagctc 3120

ttcgtcgttg gacgcagctg ccgccgcagc tgctgcatct gccgccagcg ccgtgcgcgg 3180

aatggccatg ggcgccggct actacggcac tctggtggcc aactcgagtt ccaccaataa 3240

tcccgccagc ctgaacgagg agaagctgtt gctgctgatg gcccagctcg aggccttgac 3300

ccagcgcctg ggcgagctga cccagcaggt ggctcagctg caggagcaga cgcgggccgc 3360

ggttgccacg gtgaaatcca aataaaaaat gaatcaataa ataaacggag acggttgttg 3420

attttaacac agagtctgaa tctttatttg atttttcgcg cgcggtaggc cctggaccac 3480

cggtctcgat cattgagcac ccggtggatc ttttccagga cccggtagag gtgggcttgg 3540

atgttgaggt acatgggcat gagcccgtcc cgggggtgga ggtagctcca ttgcagggcc 3600

tcgtgctcgg gggtggtgtt gtaaatcacc cagtcatagc aggggcgcag ggcatggtgt 3660

tgcacaatat ctttgaggag gagactgatg gccacgggca gccctttggt gtaggtgttt 3720

acaaatctgt tgagctggga gggatgcatg cggggggaga tgaggtgcat cttggcctgg 3780

atcttgagat tggcgatgtt accgcccaga tcccgcctgg ggttcatgtt gtgcaggacc 3840

accagcacgg tgtatccggt gcacttgggg aatttatcat gcaacttgga agggaaggcg 3900

tgaaagaatt tggcgacgcc tttgtgcccg cccaggtttt ccatgcactc atccatgatg 3960

atggcgatgg gcccgtgggc ggcggcctgg gcaaagacgt ttcgggggtc ggacacatca 4020

tagttgtggt cctgggtgag gtcatcatag gccattttaa tgaatttggg gcggagggtg 4080

ccggactggg ggacaaaggt accctcgatc ccgggggcgt agttcccctc acagatctgc 4140

atctcccagg ctttgagctc ggaggggggg atcatgtcca cctgcggggc gataaagaac 4200

acggtttccg gggcggggga gatgagctgg gccgaaagca agttccggag cagctgggac 4260

ttgccgcagc cggtggggcc gtagatgacc ccgatgaccg gctgcaggtg gtagttgagg 4320

gagagacagc tgccgtcctc ccggaggagg ggggccacct cgttcatcat ctcgcgcacg 4380

tgcatgttct cgcgcaccag ttccgccagg aggcgctctc cccccaggga taggagctcc 4440

tggagcgagg cgaagttttt cagcggcttg agtccgtcgg ccatgggcat tttggagagg 4500

gtttgttgca agagttccag gcggtcccag agctcggtga tgtgctctac ggcatctcga 4560

tccagcagac ctcctcgttt cgcgggttgg gacggctgcg ggagtagggc accagacgat 4620

gggcgtccag cgcagccagg gtccggtcct tccagggtcg cagcgtccgc gtcagggtgg 4680

tctccgtcac ggtgaagggg tgcgcgccgg gctgggcgct tgcgagggtg cgcttcaggc 4740

tcatccggct ggtcgaaaac cgctcccgat cggcgccctg cgcgtcggcc aggtagcaat 4800

tgaccatgag ttcgtagttg agcgcctcgg ccgcgtggcc tttggcgcgg agcttacctt 4860

tggaagtctg cccgcaggcg ggacagagga gggacttgag ggcgtagagc ttgggggcga 4920

ggaagacgga ctcgggggcg taggcgtccg cgccgcagtg ggcgcagacg gtctcgcact 4980

ccacgagcca ggtgaggtcg ggctggtcgg ggtcaaaaac cagtttcccg ccgttctttt 5040

tgatgcgttt cttacctttg gtctccatga gctcgtgtcc ccgctgggtg acaaagaggc 5100

tgtccgtgtc cccgtagacc gactttatgg gccggtcctc gagcggtgtg ccgcggtcct 5160

cctcgtagag gaaccccgcc cactccgaga cgaaagcccg ggtccaggcc agcacgaagg 5220

aggccacgtg ggacgggtag cggtcgttgt ccaccagcgg gtccaccttt tccagggtat 5280

gcaaacacat gtccccctcg tccacatcca ggaaggtgat tggcttgtaa gtgtaggcca 5340

cgtgaccggg ggtcccggcc gggggggtat aaaagggtgc gggtccctgc tcgtcctcac 5400

tgtcttccgg atcgctgtcc aggagcgcca gctgttgggg taggtattcc ctctcgaagg 5460

cgggcatgac ctcggcactc aggttgtcag tttctagaaa cgaggaggat ttgatattga 5520

cggtgccggc ggagatgcct ttcaagagcc cctcgtccat ctggtcagaa aagacgatct 5580

ttttgttgtc gagcttggtg gcgaaggagc cgtagagggc gttggagagg agcttggcga 5640

tggagcgcat ggtctggttt ttttccttgt cggcgcgctc cttggcggcg atgttgagct 5700

gcacgtactc gcgcgccacg cacttccatt cggggaagac ggtggtcagc tcgtcgggca 5760

cgattctgac ctgccagccc cgattatgca gggtgatgag gtccacactg gtggccacct 5820

cgccgcgcag gggctcatta gtccagcaga ggcgtccgcc cttgcgcgag cagaaggggg 5880

gcagggggtc cagcatgacc tcgtcggggg ggtcggcatc gatggtgaag atgccgggca 5940

ggaggtcggg gtcaaagtag ctgatggaag tggccagatc gtccagggca gcttgccatt 6000

cgcgcacggc cagcgcgcgc tcgtagggac tgaggggcgt gccccagggc atgggatggg 6060

taagcgcgga ggcgtacatg ccgcagatgt cgtagacgta gaggggctcc tcgaggatgc 6120

cgatgtaggt ggggtagcag cgccccccgc ggatgctggc gcgcacgtag tcatacagct 6180

cgtgcgaggg ggcgaggagc cccgggccca ggttggtgcg actgggcttt tcggcgcggt 6240

agacgatctg gcggaaaatg gcatgcgagt tggaggagat ggtgggcctt tggaagatgt 6300

tgaagtgggc gtggggcagt ccgaccgagt cgcggatgaa gtgggcgtag gagtcttgca 6360

gcttggcgac gagctcggcg gtgactagga cgtccagagc gcagtagtcg agggtctcct 6420

ggatgatgtc atacttgagc tgtccctttt gtttccacag ctcgcggttg agaaggaact 6480

cttcgcggtc cttccagtac tcttcgaggg ggaacccgtc ctgatctgca cggtaagagc 6540

ctagcatgta gaactggttg acggccttgt aggcgcagca gcccttctcc acggggaggg 6600

cgtaggcctg ggcggccttg cgcagggagg tgtgcgtgag ggcgaaagtg tccctgacca 6660

tgaccttgag gaactggtgc ttgaagtcga tatcgtcgca gcccccctgc tcccagagct 6720

ggaagtccgt gcgcttcttg taggcggggt tgggcaaagc gaaagtaaca tcgttgaaga 6780

ggatcttgcc cgcgcggggc ataaagttgc gagtgatgcg gaaaggttgg ggcacctcgg 6840

cccggttgtt gatgacctgg gcggcgagca cgatctcgtc gaagccgttg atgttgtggc 6900

ccacgatgta gagttccacg aatcgcggac ggcccttgac gtggggcagt ttcttgagct 6960

cctcgtaggt gagctcgtcg gggtcgctga gcccgtgctg ctcgagcgcc cagtcggcga 7020

gatgggggtt ggcgcggagg aaggaagtcc agagatccac ggccagggcg gtttgcagac 7080

ggtcccggta ctgacggaac tgctgcccga cggccatttt ttcgggggtg acgcagtaga 7140

aggtgcgggg gtccccgtgc cagcgatccc atttgagctg gagggcgaga tcgagggcga 7200

gctcgacgag ccggtcgtcc ccggagagtt tcatgaccag catgaagggg acgagctgct 7260

tgccgaagga ccccatccag gtgtaggttt ccacatcgta ggtgaggaag agcctttcgg 7320

tgcgaggatg cgagccgatg gggaagaact ggatctcctg ccaccaattg gaggaatggc 7380

tgttgatgtg atggaagtag aaatgccgac ggcgcgccga acactcgtgc ttgtgtttat 7440

acaagcggcc acagtgctcg caacgctgca cgggatgcac gtgctgcacg agctgtacct 7500

gagttccttt gacgaggaat ttcagtggga agtggagtcg tggcgcctgc atctcgtgct 7560

gtactacgtc gtggtggtcg gcctggccct cttctgcctc gatggtggtc atgctgacga 7620

gcccgcgcgg gaggcaggtc cagacctcgg cgcgagcggg tcggagagcg aggacgaggg 7680

cgcgcaggcc ggagctgtcc agggtcctga gacgctgcgg agtcaggtca gtgggcagcg 7740

gcggcgcgcg gttgacttgc aggagttttt ccagggcgcg cgggaggtcc agatggtact 7800

tgatctccac cgcgccattg gtggcgacgt cgatggcttg cagggtcccg tgcccctggg 7860

gtgtgaccac cgtcccccgt ttcttcttgg gcggctgggg cgacgggggc ggtgcctctt 7920

ccatggttag aagcggcggc gaggacgcgc gccgggcggc aggggcggct cggggcccgg 7980

aggcaggggc ggcaggggca cgtcggcgcc gcgcgcgggt aggttctggt actgcgcccg 8040

gagaagactg gcgtgagcga cgacgcgacg gttgacgtcc tggatctgac gcctctgggt 8100

gaaggccacg ggacccgtga gtttgaacct gaaagagagt tcgacagaat caatctcggt 8160

atcgttgacg gcggcctgcc gcaggatctc ttgcacgtcg cccgagttgt cctggtaggc 8220

gatctcggtc atgaactgct cgatctcctc ctcttgaagg tctccgcggc cggcgcgctc 8280

cacggtggcc gcgaggtcgt tggagatgcg gcccatgagc tgcgagaagg cgttcatgcc 8340

cgcctcgttc cagacgcggc tgtagaccac gacgccctcg ggatcgccgg cgcgcatgac 8400

cacctgggcg aggttgagct ccacgtggcg cgtgaagacc gcgtagttgc agaggcgctg 8460

gtagaggtag ttgagcgtgg tggcgatgtg ctcggtgacg aagaaataca tgatccagcg 8520

gcggagcggc atctcgctga cgtcgcccag cgcctccaaa cgttccatgg cctcgtaaaa 8580

gtccacggcg aagttgaaaa actgggagtt gcgcgccgag acggtcaact cctcctccag 8640

aagacggatg agctcggcga tggtggcgcg cacctcgcgc tcgaaggccc ccgggagttc 8700

ctccacttcc tcttcttcct cctccactaa catctcttct acttcctcct caggcggcag 8760

tggtggcggg ggagggggcc tgcgtcgccg gcggcgcacg ggcagacggt cgatgaagcg 8820

ctcgatggtc tcgccgcgcc ggcgtcgcat ggtctcggtg acggcgcgcc cgtcctcgcg 8880

gggccgcagc gtgaagacgc cgccgcgcat ctccaggtgg ccgggggggt ccccgttggg 8940

cagggagagg gcgctgacga tgcatcttat caattgcccc gtagggactc cgcgcaagga 9000

cctgagcgtc tcgagatcca cgggatctga aaaccgctga acgaaggctt cgagccagtc 9060

gcagtcgcaa ggtaggctga gcacggtttc ttctggcggg tcatgttggt tgggagcggg 9120

gcgggcgatg ctgctggtga tgaagttgaa ataggcggtt ctgagacggc ggatggtggc 9180

gaggagcacc aggtctttgg gcccggcttg ctggatgcgc agacggtcgg ccatgcccca 9240

ggcgtggtcc tgacacctgg ccaggtcctt gtagtagtcc tgcatgagcc gctccacggg 9300

cacctcctcc tcgcccgcgc ggccgtgcat gcgcgtgagc ccgaagccgc gctggggctg 9360

gacgagcgcc aggtcggcga cgacgcgctc ggcgaggatg gcttgctgga tctgggtgag 9420

ggtggtctgg aagtcatcaa agtcgacgaa gcggtggtag gctccggtgt tgatggtgta 9480

ggagcagttg gccatgacgg accagttgac ggtctggtgg cccggacgca cgagctcgtg 9540

gtacttgagg cgcgagtagg cgcgcgtgtc gaagatgtag tcgttgcagg tgcgcaccag 9600

gtactggtag ccgatgagga agtgcggcgg cggctggcgg tagagcggcc atcgctcggt 9660

ggcgggggcg ccgggcgcga ggtcctcgag catggtgcgg tggtagccgt agatgtacct 9720

ggacatccag gtgatgccgg cggcggtggt ggaggcgcgc gggaactcgc ggacgcggtt 9780

ccagatgttg cgcagcggca ggaagtagtt catggtgggc acggtctggc ccgtgaggcg 9840

cgcgcagtcg tggatgctct atacgggcaa aaacgaaagc ggtcagcggc tcgactccgt 9900

ggcctggagg ctaagcgaac gggttgggct gcgcgtgtac cccggttcga atctcgaatc 9960

aggctggagc cgcagctaac gtggtattgg cactcccgtc tcgacccaag cctgcaccaa 10020

ccctccagga tacggaggcg ggtcgttttg caactttttt ttggaggccg gatgagacta 10080

gtaagcgcgg aaagcggccg accgcgatgg ctcgctgccg tagtctggag aagaatcgcc 10140

agggttgcgt tgcggtgtgc cccggttcga ggccggccgg attccgcggc taacgagggc 10200

gtggctgccc cgtcgtttcc aagaccccat agccagccga cttctccagt tacggagcga 10260

gcccctcttt tgttttgttt gtttttgcca gatgcatccc gtactgcggc agatgcgccc 10320

ccaccaccct ccaccgcaac aacagccccc tccacagccg gcgcttctgc ccccgcccca 10380

gcagcaactt ccagccacga ccgccgcggc cgccgtgagc ggggctggac agagttatga 10440

tcaccagctg gccttggaag agggcgaggg gctggcgcgc ctgggggcgt cgtcgccgga 10500

gcggcacccg cgcgtgcaga tgaaaaggga cgctcgcgag gcctacgtgc ccaagcagaa 10560

cctgttcaga gacaggagcg gcgaggagcc cgaggagatg cgcgcggccc ggttccacgc 10620

ggggcgggag ctgcggcgcg gcctggaccg aaagagggtg ctgagggacg aggatttcga 10680

ggcggacgag ctgacgggga tcagccccgc gcgcgcgcac gtggccgcgg ccaacctggt 10740

cacggcgtac gagcagaccg tgaaggagga gagcaacttc caaaaatcct tcaacaacca 10800

cgtgcgcacc ctgatcgcgc gcgaggaggt gaccctgggc ctgatgcacc tgtgggacct 10860

gctggaggcc atcgtgcaga accccaccag caagccgctg acggcgcagc tgttcctggt 10920

ggtgcagcat agtcgggaca acgaagcgtt cagggaggcg ctgctgaata tcaccgagcc 10980

cgagggccgc tggctcctgg acctggtgaa cattctgcag agcatcgtgg tgcaggagcg 11040

cgggctgccg ctgtccgaga agctggcggc catcaacttc tcggtgctga gtttgggcaa 11100

gtactacgct aggaagatct acaagacccc gtacgtgccc atagacaagg aggtgaagat 11160

cgacgggttt tacatgcgca tgaccctgaa agtgctgacc ctgagcgacg atctgggggt 11220

gtaccgcaac gacaggatgc accgtgcggt gagcgccagc aggcggcgcg agctgagcga 11280

ccaggagctg atgcatagtc tgcagcgggc cctgaccggg gccgggaccg agggggagag 11340

ctactttgac atgggcgcgg acctgcactg gcagcccagc cgccgggcct tggaggcggc 11400

ggcaggaccc tacgtagaag aggtggacga tgaggtggac gaggagggcg agtacctgga 11460

agactgatgg cgcgaccgta tttttgctag atgcaacaac aacagccacc tcctgatccc 11520

gcgatgcggg cggcgctgca gagccagccg tccggcatta actcctcgga cgattggacc 11580

caggccatgc aacgcatcat ggcgctgacg acccgcaacc ccgaagcctt tagacagcag 11640

ccccaggcca accggctctc ggccatcctg gaggccgtgg tgccctcgcg ctccaacccc 11700

acgcacgaga aggtcctggc catcgtgaac gcgctggtgg agaacaaggc catccgcggc 11760

gacgaggccg gcctggtgta caacgcgctg ctggagcgcg tggcccgcta caacagcacc 11820

aacgtgcaga ccaacctgga ccgcatggtg accgacgtgc gcgaggccgt ggcccagcgc 11880

gagcggttcc accgcgagtc caacctggga tccatggtgg cgctgaacgc cttcctcagc 11940

acccagcccg ccaacgtgcc ccggggccag gaggactaca ccaacttcat cagcgccctg 12000

cgcctgatgg tgaccgaggt gccccagagc gaggtgtacc agtccgggcc ggactacttc 12060

ttccagacca gtcgccaggg cttgcagacc gtgaacctga gccaggcttt caagaacttg 12120

cagggcctgt ggggcgtgca ggccccggtc ggggaccgcg cgacggtgtc gagcctgctg 12180

acgccgaact cgcgcctgct gctgctgctg gtggccccct tcacggacag cggcagcatc 12240

aaccgcaact cgtacctggg ctacctgatt aacctgtacc gcgaggccat cggccaggcg 12300

cacgtggacg agcagaccta ccaggagatc acccacgtga gccgcgccct gggccaggac 12360

gacccgggca acctggaagc caccctgaac tttttgctga ccaaccggtc gcagaagatc 12420

ccgccccagt acgcgctcag caccgaggag gagcgcatcc tgcgttacgt gcagcagagc 12480

gtgggcctgt tcctgatgca ggagggggcc acccccagcg ccgcgctcga catgaccgcg 12540

cgcaacatgg agcccagcat gtacgccagc aaccgcccgt tcatcaataa actgatggac 12600

tacttgcatc gggcggccgc catgaactct gactatttca ccaacgccat cctgaatccc 12660

cactggctcc cgccgccggg gttctacacg ggcgagtacg acatgcccga ccccaatgac 12720

gggttcctgt gggacgatgt ggacagcagc gtgttctccc cccgaccggg tgctaacgag 12780

cgccccttgt ggaagaagga aggcagcgac cgacgcccgt cctcggcgct gtccggccgc 12840

gagggtgctg ccgcggcggt gcccgaggcc gccagtcctt tcccgagctt gcccttctcg 12900

ctgaacagta tccgcagcag cgagctgggc aggatcacgc gcccgcgctt gctgggcgaa 12960

gaggagtact tgaatgactc gctgttgaga cccgagcggg agaagaactt ccccaataac 13020

gggatagaaa gcctggtgga caagatgagc cgctggaaga cgtatgcgca ggagcacagg 13080

gacgatcccc gggcgtcgca gggggccacg agccggggca gcgccgcccg taaacgccgg 13140

tggcacgaca ggcagcgggg acagatgtgg gacgatgagg actccgccga cgacagcagc 13200

gtgttggact tgggtgggag tggtaacccg ttcgctcacc tgcgcccccg tatcgggcgc 13260

atgatgtaag agaaaccgaa aataaatgat actcaccaag gccatggcga ccagcgtgcg 13320

ttcgtttctt ctctgttgtt gttgtatcta gtatgatgag gcgtgcgtac ccggagggtc 13380

ctcctccctc gtacgagagc gtgatgcagc aggcgatggc ggcggcggcg atgcagcccc 13440

cgctggaggc tccttacgtg cccccgcggt acctggcgcc tacggagggg cggaacagca 13500

ttcgttactc ggagctggca cccttgtacg ataccacccg gttgtacctg gtggacaaca 13560

agtcggcgga catcgcctcg ctgaactacc agaacgacca cagcaacttc ctgaccaccg 13620

tggtgcagaa caatgacttc acccccacgg aggccagcac ccagaccatc aactttgacg 13680

agcgctcgcg gtggggcggc cagctgaaaa ccatcatgca caccaacatg cccaacgtga 13740

acgagttcat gtacagcaac aagttcaagg cgcgggtgat ggtctcccgc aagaccccca 13800

atggggtgac agtgacagag gattatgatg gtagtcagga tgagctgaag tatgaatggg 13860

tggaatttga gctgcccgaa ggcaacttct cggtgaccat gaccatcgac ctgatgaaca 13920

acgccatcat cgacaattac ttggcggtgg ggcggcagaa cggggtgctg gagagcgaca 13980

tcggcgtgaa gttcgacact aggaacttca ggctgggctg ggaccccgtg accgagctgg 14040

tcatgcccgg ggtgtacacc aacgaggctt tccatcccga tattgtcttg ctgcccggct 14100

gcggggtgga cttcaccgag agccgcctca gcaacctgct gggcattcgc aagaggcagc 14160

ccttccagga aggcttccag atcatgtacg aggatctgga ggggggcaac atccccgcgc 14220

tcctggatgt cgacgcctat gagaaaagca aggaggatgc agcagctgaa gcaactgcag 14280

ccgtagctac cgcctctacc gaggtcaggg gcgataattt tgcaagcgcc gcagcagtgg 14340

cagcggccga ggcggctgaa accgaaagta agatagtcat tcagccggtg gagaaggata 14400

gcaagaacag gagctacaac gtactaccgg acaagataaa caccgcctac cgcagctggt 14460

acctagccta caactatggc gaccccgaga agggcgtgcg ctcctggacg ctgctcacca 14520

cctcggacgt cacctgcggc gtggagcaag tctactggtc gctgcccgac atgatgcaag 14580

acccggtcac cttccgctcc acgcgtcaag ttagcaacta cccggtggtg ggcgccgagc 14640

tcctgcccgt ctactccaag agcttcttca acgagcaggc cgtctactcg cagcagctgc 14700

gcgccttcac ctcgcttacg cacgtcttca accgcttccc cgagaaccag atcctcgtcc 14760

gcccgcccgc gcccaccatt accaccgtca gtgaaaacgt tcctgctctc acagatcacg 14820

ggaccctgcc gctgcgcagc agtatccggg gagtccagcg cgtgaccgtt actgacgcca 14880

gacgccgcac ctgcccctac gtctacaagg ccctgggcat agtcgcgccg cgcgtcctct 14940

cgagccgcac cttctaaatg tccattctca tctcgcccag taataacacc ggttggggcc 15000

tgcgcgcgcc cagcaagatg tacggaggcg ctcgccaacg ctccacgcaa caccccgtgc 15060

gcgtgcgcgg gcacttccgc gctccctggg gcgccctcaa gggccgcgtg cggtcgcgca 15120

ccaccgtcga cgacgtgatc gaccaggtgg tggccgacgc gcgcaactac acccccgccg 15180

ccgcgcccgt ctccaccgtg gacgccgtca tcgacagcgt ggtggcggac gcgcgccggt 15240

acgcccgcgc caagagccgg cggcggcgca tcgcccggcg gcaccggagc acccccgcca 15300

tgcgcgcggc gcgagccttg ctgcgcaggg ccaggcgcac gggacgcagg gccatgctca 15360

gggcggccag acgcgcggct tcaggcgcca gcgccggcag gacccggaga cgcgcggcca 15420

cggcggcggc agcggccatc gccagcatgt cccgcccgcg gcgagggaac gtgtactggg 15480

tgcgcgacgc cgccaccggt gtgcgcgtgc ccgtgcgcac ccgcccccct cgcacttgaa 15540

gatgttcact tcgcgatgtt gatgtgtccc agcggcgagg aggatgtcca agcgcaaatt 15600

caaggaagag atgctccagg tcatcgcgcc tgagatctac ggccctgcgg tggtgaagga 15660

ggaaagaaag ccccgcaaaa tcaagcgggt caaaaaggac aaaaaggaag aagaaagtga 15720

tgtggacgga ttggtggagt ttgtgcgcga gttcgccccc cggcggcgcg tgcagtggcg 15780

cgggcggaag gtgcaaccgg tgctgagacc cggcaccacc gtggtcttca cgcccggcga 15840

gcgctccggc accgcttcca agcgctccta cgacgaggtg tacggggatg atgatattct 15900

ggagcaggcg gccgagcgcc tgggcgagtt tgcttacggc aagcgcagcc gttccgcacc 15960

gaaggaagag gcggtgtcca tcccgctgga ccacggcaac cccacgccga gcctcaagcc 16020

cgtgaccttg cagcaggtgc tgccgaccgc ggcgccgcgc cgggggttca agcgcgaggg 16080

cgaggatctg taccccacca tgcagctgat ggtgcccaag cgccagaagc tggaagacgt 16140

gctggagacc atgaaggtgg acccggacgt gcagcccgag gtcaaggtgc ggcccatcaa 16200

gcaggtggcc ccgggcctgg gcgtgcagac cgtggacatc aagattccca cggagcccat 16260

ggaaacgcag accgagccca tgatcaagcc cagcaccagc accatggagg tgcagacgga 16320

tccctggatg ccatcggctc ctagtcgaag accccggcgc aagtacggcg cggccagcct 16380

gctgatgccc aactacgcgc tgcatccttc catcatcccc acgccgggct accgcggcac 16440

gcgcttctac cgcggtcata ccagcagccg ccgccgcaag accaccactc gccgccgccg 16500

tcgccgcacc gccgctgcaa ccacccctgc cgccctggtg cggagagtgt accgccgcgg 16560

ccgcgcacct ctgaccctgc cgcgcgcgcg ctaccacccg agcatcgcca tttaaacttt 16620

cgccagcttt gcagatcaat ggccctcaca tgccgccttc gcgttcccat tacgggctac 16680

cgaggaagaa aaccgcgccg tagaaggctg gcggggaacg ggatgcgtcg ccaccaccac 16740

cggcggcggc gcgccatcag caagcggttg gggggaggct tcctgcccgc gctgatcccc 16800

atcatcgccg cggcgatcgg ggcgatcccc ggcattgctt ccgtggcggt gcaggcctct 16860

cagcgccact gagacacact tggaaacatc ttgtaataaa cccatggact ctgacgctcc 16920

tggtcctgtg atgtgttttc gtagacagat ggaagacatc aatttttcgt ccctggctcc 16980

gcgacacggc acgcggccgt tcatgggcac ctggagcgac atcggcacca gccaactgaa 17040

cgggggcgcc ttcaattgga gcagtctctg gagcgggctt aagaatttcg ggtccacgct 17100

taaaacctat ggcagcaagg cgtggaacag caccacaggg caggcgctga gggataagct 17160

gaaagagcag aacttccagc agaaggtggt cgatgggctc gcctcgggca tcaacggggt 17220

ggtggacctg gccaaccagg ccgtgcagcg gcagatcaac agccgcctgg acccggtgcc 17280

gcccgccggc tccgtggaga tgccgcaggt ggaggaggag ctgcctcccc tggacaagcg 17340

gggcgagaag cgaccccgcc ccgatgcgga ggagacgctg ctgacgcaca cggacgagcc 17400

gcccccgtac gaggaggcgg tgaaactggg tctgcccacc acgcggccca tcgcgcccct 17460

ggccaccggg gtgctgaaac ccgaaaagcc cgcgaccctg gacttgcctc ctccccagcc 17520

ttcccgcccc tctacagtgg ctaagcccct gccgccggtg gccgtggccc gcgcgcgacc 17580

cgggggcacc gcccgccctc atgcgaactg gcagagcact ctgaacagca tcgtgggtct 17640

gggagtgcag agtgtgaagc gccgccgctg ctattaaacc taccgtagcg cttaacttgc 17700

ttgtctgtgt gtgtatgtat tatgtcgccg ccgccgctgt ccaccagaag gaggagtgaa 17760

gaggcgcgtc gccgagttgc aagatggcca ccccatcgat gctgccccag tgggcgtaca 17820

tgcacatcgc cggacaggac gcttcggagt acctgagtcc gggtctggtg cagtttgccc 17880

gcgccacaga cacctacttc agtctgggga acaagtttag gaaccccacg gtggcgccca 17940

cgcacgatgt gaccaccgac cgcagccagc ggctgacgct gcgcttcgtg cccgtggacc 18000

gcgaggacaa cacctactcg tacaaagtgc gctacacgct ggccgtgggc gacaaccgcg 18060

tgctggacat ggccagcacc tactttgaca tccgcggcgt gctggatcgg ggccctagct 18120

tcaaacccta ctccggcacc gcctacaaca gtctggcccc caagggagca cccaacactt 18180

gtcagtggac atataaagcc gatggtgaaa ctgccacaga aaaaacctat acatatggaa 18240

atgcacccgt gcagggcatt aacatcacaa aagatggtat tcaacttgga actgacaccg 18300

atgatcagcc aatctacgca gataaaacct atcagcctga acctcaagtg ggtgatgctg 18360

aatggcatga catcactggt actgatgaaa agtatggagg cagagctctt aagcctgata 18420

ccaaaatgaa gccttgttat ggttcttttg ccaagcctac taataaagaa ggaggtcagg 18480

caaatgtgaa aacaggaaca ggcactacta aagaatatga catagacatg gctttctttg 18540

acaacagaag tgcggctgct gctggcctag ctccagaaat tgttttgtat actgaaaatg 18600

tggatttgga aactccagat acccatattg tatacaaagc aggcacagat gacagcagct 18660

cttctattaa tttgggtcag caagccatgc ccaacagacc taactacatt ggtttcagag 18720

acaactttat cgggctcatg tactacaaca gcactggcaa tatgggggtg ctggccggtc 18780

aggcttctca gctgaatgct gtggttgact tgcaagacag aaacaccgag ctgtcctacc 18840

agctcttgct tgactctctg ggtgacagaa cccggtattt cagtatgtgg aatcaggcgg 18900

tggacagcta tgatcctgat gtgcgcatta ttgaaaatca tggtgtggag gatgaacttc 18960

ccaactattg tttccctctg gatgctgttg gcagaacaga tacttatcag ggaattaagg 19020

ctaatggaac tgatcaaacc acatggacca aagatgacag tgtcaatgat gctaatgaga 19080

taggcaaggg taatccattc gccatggaaa tcaacatcca agccaacctg tggaggaact 19140

tcctctacgc caacgtggcc ctgtacctgc ccgactctta caagtacacg ccggccaatg 19200

ttaccctgcc caccaacacc aacacctacg attacatgaa cggccgggtg gtggcgccct 19260

cgctggtgga ctcctacatc aacatcgggg cgcgctggtc gctggatccc atggacaacg 19320

tgaacccctt caaccaccac cgcaatgcgg ggctgcgcta ccgctccatg ctcctgggca 19380

acgggcgcta cgtgcccttc cacatccagg tgccccagaa atttttcgcc atcaagagcc 19440

tcctgctcct gcccgggtcc tacacctacg agtggaactt ccgcaaggac gtcaacatga 19500

tcctgcagag ctccctcggc aacgacctgc gcacggacgg ggcctccatc tccttcacca 19560

gcatcaacct ctacgccacc ttcttcccca tggcgcacaa cacggcctcc acgctcgagg 19620

ccatgctgcg caacgacacc aacgaccagt ccttcaacga ctacctctcg gcggccaaca 19680

tgctctaccc catcccggcc aacgccacca acgtgcccat ctccatcccc tcgcgcaact 19740

gggccgcctt ccgcggctgg tccttcacgc gtctcaagac caaggagacg ccctcgctgg 19800

gctccgggtt cgacccctac ttcgtctact cgggctccat cccctacctc gacggcacct 19860

tctacctcaa ccacaccttc aagaaggtct ccatcacctt cgactcctcc gtcagctggc 19920

ccggcaacga ccggctcctg acgcccaacg agttcgaaat caagcgcacc gtcgacggcg 19980

agggctacaa cgtggcccag tgcaacatga ccaaggactg gttcctggtc cagatgctgg 20040

cccactacaa catcggctac cagggcttct acgtgcccga gggctacaag gaccgcatgt 20100

actccttctt ccgcaacttc cagcccatga gccgccaggt ggtggacgag gtcaactaca 20160

aggactacca ggccgtcacc ctggcctacc agcacaacaa ctcgggcttc gtcggctacc 20220

tcgcgcccac catgcgccag ggccagccct accccgccaa ctacccctac ccgctcatcg 20280

gcaagagcgc cgtcaccagc gtcacccaga aaaagttcct ctgcgacagg gtcatgtggc 20340

gcatcccctt ctccagcaac ttcatgtcca tgggcgcgct caccgacctc ggccagaaca 20400

tgctctatgc caactccgcc cacgcgctag acatgaattt cgaagtcgac cccatggatg 20460

agtccaccct tctctatgtt gtcttcgaag tcttcgacgt cgtccgagtg caccagcccc 20520

accgcggcgt catcgaggcc gtctacctgc gcaccccctt ctcggccggt aacgccacca 20580

cctaagctct tgcttcttgc aagccatggc cgcgggctcc ggcgagcagg agctcagggc 20640

catcatccgc gacctgggct gcgggcccta cttcctgggc accttcgata agcgcttccc 20700

gggattcatg gccccgcaca agctggcctg cgccatcgtc aacacggccg gccgcgagac 20760

cgggggcgag cactggctgg ccttcgcctg gaacccgcgc tcgaacacct gctacctctt 20820

cgaccccttc gggttctcgg acgagcgcct caagcagatc taccagttcg agtacgaggg 20880

cctgctgcgc cgcagcgccc tggccaccga ggaccgctgc gtcaccctgg aaaagtccac 20940

ccagaccgtg cagggtccgc gctcggccgc ctgcgggctc ttctgctgca tgttcctgca 21000

cgccttcgtg cactggcccg accgccccat ggacaagaac cccaccatga acttgctgac 21060

gggggtgccc aacggcatgc tccagtcgcc ccaggtggaa cccaccctgc gccgcaacca 21120

ggaggcgctc taccgcttcc tcaactccca ctccgcctac tttcgctccc accgcgcgcg 21180

catcgagaag gccaccgcct tcgaccgcat gaatcaagac atgtaaaccg tgtgtgtatg 21240

ttaaatgtct ttaataaaca gcactttcat gttacacatg catctgagat gatttattta 21300

gaaatcgaaa gggttctgcc gggtctcggc atggcccgcg ggcagggaca cgttgcggaa 21360

ctggtacttg gccagccact tgaactcggg gatcagcagt ttgggcagcg gggtgtcggg 21420

gaaggagtcg gtccacagct tccgcgtcag ttgcagggcg cccagcaggt cgggcgcgga 21480

gatcttgaaa tcgcagttgg gacccgcgtt ctgcgcgcgg gagttgcggt acacggggtt 21540

gcagcactgg aacaccatca gggccgggtg cttcacgctc gccagcaccg tcgcgtcggt 21600

gatgctctcc acgtcgaggt cctcggcgtt ggccatcccg aagggggtca tcttgcaggt 21660

ctgccttccc atggtgggca cgcacccggg cttgtggttg caatcgcagt gcagggggat 21720

cagcatcatc tgggcctggt cggcgttcat ccccgggtac atggccttca tgaaagcctc 21780

caattgcctg aacgcctgct gggccttggc tccctcggtg aagaagaccc cgcaggactt 21840

gctagagaac tggttggtgg cgcacccggc gtcgtgcacg cagcagcgcg cgtcgttgtt 21900

ggccagctgc accacgctgc gcccccagcg gttctgggtg atcttggccc ggtcggggtt 21960

ctccttcagc gcgcgctgcc cgttctcgct cgccacatcc atctcgatca tgtgctcctt 22020

ctggatcatg gtggtcccgt gcaggcaccg cagcttgccc tcggcctcgg tgcacccgtg 22080

cagccacagc gcgcacccgg tgcactccca gttcttgtgg gcgatctggg aatgcgcgtg 22140

cacgaagccc tgcaggaagc ggcccatcat ggtggtcagg gtcttgttgc tagtgaaggt 22200

cagcggaatg ccgcggtgct cctcgttgat gtacaggtgg cagatgcggc ggtacacctc 22260

gccctgctcg ggcatcagct ggaagttggc tttcaggtcg gtctccacgc ggtagcggtc 22320

catcagcata gtcatgattt ccataccctt ctcccaggcc gagacgatgg gcaggctcat 22380

agggttcttc accatcatct tagcgctagc agccgcggcc agggggtcgc tctcgtccag 22440

ggtctcaaag ctccgcttgc cgtccttctc ggtgatccgc accggggggt agctgaagcc 22500

cacggccgcc agctcctcct cggcctgtct ttcgtcctcg ctgtcctggc tgacgtcctg 22560

caggaccaca tgcttggtct tgcggggttt cttcttgggc ggcagcggcg gcggagatgt 22620

tggagatggc gagggggagc gcgagttctc gctcaccact actatctctt cctcttcttg 22680

gtccgaggcc acgcggcggt aggtatgtct cttcgggggc agaggcggag gcgacgggct 22740

ctcgccgccg cgacttggcg gatggctggc agagcccctt ccgcgttcgg gggtgcgctc 22800

ccggcggcgc tctgactgac ttcctccgcg gccggccatt gtgttctcct agggaggaac 22860

aacaagcatg gagactcagc catcgccaac ctcgccatct gcccccaccg ccgacgagaa 22920

gcagcagcag cagaatgaaa gcttaaccgc cccgccgccc agccccgcca cctccgacgc 22980

ggccgtccca gacatgcaag agatggagga atccatcgag attgacctgg gctatgtgac 23040

gcccgcggag cacgaggagg agctggcagt gcgcttttca caagaagaga tacaccaaga 23100

acagccagag caggaagcag agaatgagca gagtcaggct gggctcgagc atgacggcga 23160

ctacctccac ctgagcgggg gggaggacgc gctcatcaag catctggccc ggcaggccac 23220

catcgtcaag gatgcgctgc tcgaccgcac cgaggtgccc ctcagcgtgg aggagctcag 23280

ccgcgcctac gagttgaacc tcttctcgcc gcgcgtgccc cccaagcgcc agcccaatgg 23340

cacctgcgag cccaacccgc gcctcaactt ctacccggtc ttcgcggtgc ccgaggccct 23400

ggccacctac cacatctttt tcaagaacca aaagatcccc gtctcctgcc gcgccaaccg 23460

cacccgcgcc gacgcccttt tcaacctggg tcccggcgcc cgcctacctg atatcgcctc 23520

cttggaagag gttcccaaga tcttcgaggg tctgggcagc gacgagactc gggccgcgaa 23580

cgctctgcaa ggagaaggag gagagcatga gcaccacagc gccctggtcg agttggaagg 23640

cgacaacgcg cggctggcgg tgctcaaacg cacggtcgag ctgacccatt tcgcctaccc 23700

ggctctgaac ctgcccccca aagtcatgag cgcggtcatg gaccaggtgc tcatcaagcg 23760

cgcgtcgccc atctccgagg acgagggcat gcaagactcc gaggagggca agcccgtggt 23820

cagcgacgag cagctggccc ggtggctggg tcctaatgct agtccccaga gtttggaaga 23880

gcggcgcaaa ctcatgatgg ccgtggtcct ggtgaccgtg gagctggagt gcctgcgccg 23940

cttcttcgcc gacgcggaga ccctgcgcaa ggtcgaggag aacctgcact acctcttcag 24000

gcacgggttc gtgcgccagg cctgcaagat ctccaacgtg gagctgacca acctggtctc 24060

ctacatgggc atcttgcacg agaaccgcct ggggcagaac gtgctgcaca ccaccctgcg 24120

cggggaggcc cggcgcgact acatccgcga ctgcgtctac ctctacctct gccacacctg 24180

gcagacgggc atgggcgtgt ggcagcagtg tctggaggag cagaacctga aagagctctg 24240

caagctcctg cagaagaacc tcaagggtct gtggaccggg ttcgacgagc gcaccaccgc 24300

ctcggacctg gccgacctca ttttccccga gcgcctcagg ctgacgctgc gcaacggcct 24360

gcccgacttt atgagccaaa gcatgttgca aaactttcgc tctttcatcc tcgaacgctc 24420

cggaatcctg cccgccacct gctccgcgct gccctcggac ttcgtgccgc tgaccttccg 24480

cgagtgcccc ccgccgctgt ggagccactg ctacctgctg cgcctggcca actacctggc 24540

ctaccactcg gacgtgatcg aggacgtcag cggcgagggc ctgctcgagt gccactgccg 24600

ctgcaacctc tgcacgccgc accgctccct ggcctgcaac ccccagctgc tgagcgagac 24660

ccagatcatc ggcaccttcg agttgcaagg gcccagcgaa ggcgagggtt cagccgccaa 24720

ggggggtctg aaactcaccc cggggctgtg gacctcggcc tacttgcgca agttcgtgcc 24780

cgaggactac catcccttcg agatcaggtt ctacgaggac caatcccatc cgcccaaggc 24840

cgagctgtcg gcctgcgtca tcacccaggg ggcgatcctg gcccaattgc aagccatcca 24900

gaaatcccgc caagaattct tgctgaaaaa gggccgcggg gtctacctcg acccccagac 24960

cggtgaggag ctcaaccccg gcttccccca ggatgccccg aggaaacaag aagctgaaag 25020

tggagctgcc gcccgtggag gatttggagg aagactggga gaacagcagt caggcagagg 25080

aggaggagat ggaggaagac tgggacagca ctcaggcaga ggaggacagc ctgcaagaca 25140

gtctggagga agacgaggag gaggcagagg aggaggtgga agaagcagcc gccgccagac 25200

cgtcgtcctc ggcgggggag aaagcaagca gcacggatac catctccgct ccgggtcggg 25260

gtcccgctcg accacacagt agatgggacg agaccggacg attcccgaac cccaccaccc 25320

agaccggtaa gaaggagcgg cagggataca agtcctggcg ggggcacaaa aacgccatcg 25380

tctcctgctt gcaggcctgc gggggcaaca tctccttcac ccggcgctac ctgctcttcc 25440

accgcggggt gaactttccc cgcaacatct tgcattacta ccgtcacctc cacagcccct 25500

actacttcca agaagaggca gcagcagcag aaaaagacca gcagaaaacc agcagctaga 25560

aaatccacag cggcggcagc aggtggactg aggatcgcgg cgaacgagcc ggcgcaaacc 25620

cgggagctga ggaaccggat ctttcccacc ctctatgcca tcttccagca gagtcggggg 25680

caggagcagg aactgaaagt caagaaccgt tctctgcgct cgctcacccg cagttgtctg 25740

tatcacaaga gcgaagacca acttcagcgc actctcgagg acgccgaggc tctcttcaac 25800

aagtactgcg cgctcactct taaagagtag cccgcgcccg cccagtcgca gaaaaaggcg 25860

ggaattacgt cacctgtgcc cttcgcccta gccgcctcca cccatcatca tgagcaaaga 25920

gattcccacg ccttacatgt ggagctacca gccccagatg ggcctggccg ccggtgccgc 25980

ccaggactac tccacccgca tgaattggct cagcgccggg cccgcgatga tctcacgggt 26040

gaatgacatc cgcgcccacc gaaaccagat actcctagaa cagtcagcgc tcaccgccac 26100

gccccgcaat cacctcaatc cgcgtaattg gcccgccgcc ctggtgtacc aggaaattcc 26160

ccagcccacg accgtactac ttccgcgaga cgcccaggcc gaagtccagc tgactaactc 26220

aggtgtccag ctggcgggcg gcgccaccct gtgtcgtcac cgccccgctc agggtataaa 26280

gcggctggtg atccggggca gaggcacaca gctcaacgac gaggtggtga gctcttcgct 26340

gggtctgcga cctgacggag tcttccaact cgccggatcg gggagatctt ccttcacgcc 26400

tcgtcaggcc gtcctgactt tggagagttc gtcctcgcag ccccgctcgg gtggcatcgg 26460

cactctccag ttcgtggagg agttcactcc ctcggtctac ttcaacccct tctccggctc 26520

ccccggccac tacccggacg agttcatccc gaacttcgac gccatcagcg agtcggtgga 26580

cggctacgat tgaatgtccc atggtggcgc agctgaccta gctcggcttc gacacctgga 26640

ccactgccgc cgcttccgct gcttcgctcg ggatctcgcc gagtttgcct actttgagct 26700

gcccgaggag caccctcagg gcccggccca cggagtgcgg atcgtcgtcg aagggggcct 26760

cgactcccac ctgcttcgga tcttcagcca gcgtccgatc ctggtcgagc gcgagcaagg 26820

acagaccctt ctgactctgt actgcatctg caaccacccc ggcctgcatg aaagtctttg 26880

ttgtctgctg tgtactgagt ataataaaag ctgagatcag cgactactcc ggacttccgt 26940

gtgttcctga atccatcaac cagtctttgt tcttcaccgg gaacgagacc gagctccagc 27000

tccagtgtaa gccccacaag aagtacctca cctggctgtt ccagggctcc ccgatcgccg 27060

ttgtcaacca ctgcgacaac gactatttaa atccacaata catgcccata ttagactatg 27120

aggccgagcc acagcgaccc atgctccccg ctattagtta cttcaatcta accggcggag 27180

atgactgacc cactggccaa caacaacgtc aacgaccttc tcctggacat ggacggccgc 27240

gcctcggagc agcgactcgc ccaacttcgc attcgccagc agcaggagag agccgtcaag 27300

gagctgcagg atgcggtggc catccaccag tgcaagagag gcatcttctg cctggtgaaa 27360

caggccaaga tctcctacga ggtcactcca aacgaccatc gcctctccta cgagctcctg 27420

cagcagcgcc agaagttcac ctgcctggtc ggagtcaacc ccatcgtcat cacccagcag 27480

tctggcgata ccaaggggtg catccactgc tcctgcgact cccccgactg cgtccacact 27540

ctgatcaaga ccctctgcgg cctccgcgac ctcctcccca tgaactaatc acccccttat 27600

ccagtgaaat aaagatcata ttgatgatga ttttacagaa ataaaaaata atcatttgat 27660

ttgaaataaa gatacaatca tattgatgat ttgagtttaa caaaaaaata aagaatcact 27720

tacttgaaat ctgataccag gtctctgtcc atgttttctg ccaacaccac ttcactcccc 27780

tcttcccagc tctggtactg caggccccgg cgggctgcaa acttcctcca cacgctgaag 27840

gggatgtcaa attcctcctg tccctcaatc ttcattttat cttctatcag atgtccaaaa 27900

agcgcgtccg ggtggatgat gacttcgacc ccgtctaccc ctacgatgca gacaacgcac 27960

cgaccgtgcc cttcatcaac ccccccttcg tctcttcaga tggattccaa gagaagcccc 28020

tgggggtgtt gtccctgcga ctggccgacc ccgtcaccac caagaacggg gaaatcaccc 28080

tcaagctggg agagggggtg gacctcgatt cctcgggaaa actcatctcc aacacggcca 28140

ccaaggccgc cgcccctctc agtttttcca acaacaccat ttcccttaac atggatcacc 28200

ccttttacac taaagatgga aaattatcct tacaagtttc tccaccatta aatatactga 28260

gaacaagcat tctaaacaca ctagctttag gttttggatc aggtttagga ctccgtggct 28320

ctgccttggc agtacagtta gtctctccac ttacatttga tactgatgga aacataaagc 28380

ttaccttaga cagaggtttg catgttacaa caggagatgc aattgaaagc aacataagct 28440

gggctaaagg tttaaaattt gaagatggag ccatagcaac caacattgga aatgggttag 28500

agtttggaag cagtagtaca gaaacaggtg ttgatgatgc ttacccaatc caagttaaac 28560

ttggatctgg ccttagcttt gacagtacag gagccataat ggctggtaac aaagaagacg 28620

ataaactcac tttgtggaca acacctgatc catcaccaaa ctgtcaaata ctcgcagaaa 28680

atgatgcaaa actaacactt tgcttgacta aatgtggtag tcaaatactg gccactgtgt 28740

cagtcttagt tgtaggaagt ggaaacctaa accccattac tggcaccgta agcagtgctc 28800

aggtgtttct acgttttgat gcaaacggtg ttcttttaac agaacattct acactaaaaa 28860

aatactgggg gtataggcag ggagatagca tagatggcac tccatatacc aatgctgtag 28920

gattcatgcc caatttaaaa gcttatccaa agtcacaaag ttctactact aaaaataata 28980

tagtagggca agtatacatg aatggagatg tttcaaaacc tatgcttctc actataaccc 29040

tcaatggtac tgatgacagc aacagtacat attcaatgtc attttcatac acctggacta 29100

atggaagcta tgttggagca acatttgggg ctaactctta taccttctca tacatcgccc 29160

aagaatgaac actgtatccc accctgcatg ccaacccttc ccaccccact ctgtggaaca 29220

aactctgaaa cacaaaataa aataaagttc aagtgtttta ttgattcaac agttaactta 29280

caggattcga gcagttattt ttcctccacc ctcccaggac atggaataca ccaccctctc 29340

cccccgcaca gccttgaaca tctgaatgcc attggtgatg gacatgcttt tggtctccac 29400

gttccacaca gtttcagagc gagccagtct cgggtcggtc agggagatga aaccctccgg 29460

gcactcccgc atctgcacct cacagctcaa cagctgagga ttgtcctcgg tggtcgggat 29520

cacggttatc tggaagaagc agaagagcgg cggtgggaat catagtccgc gaacgggatc 29580

ggccggtggt gtcgcatcag gccccgcagc agtcgctgcc gccgccgctc cgtcaagctg 29640

ctgctcaggg ggtccgggtc cagggactcc ctcagcatga tgcccacggc cctcagcatc 29700

agtcgtctgg tgcggcgggc gcagcagcgc atgcggatct cgctcaggtc gctgcagtac 29760

gtgcaacaca gaaccaccag gttgttcaac agtccatagt tcaacacgct ccagccgaaa 29820

ctcatcgcgg gaaggatgct acccacgtgg ccgtcgtacc agatcctcag gtaaatcaag 29880

tggtgccccc tccagaacac gctgcccacg tacatgatct ccttgggcat gtggcggttc 29940

accacctccc ggtaccacat caccctctgg ttgaacatgc agccccggat gatcctgcgg 30000

aaccacaggg ccagcaccgc cccgcccgcc atgcagcgaa gagaccccgg gtcccggcaa 30060

tggcaatgga ggacccaccg ctcgtacccg tggatcatct gggagctgaa caagtctatg 30120

ttggcacagc acaggcatat gctcatgcat ctcttcagca ctctcaactc ctcgggggtc 30180

aaaaccatat cccagggcac ggggaactct tgcaggacag cgaaccccgc agaacagggc 30240

aatcctcgca cagaacttac attgtgcatg gacagggtat cgcaatcagg cagcaccggg 30300

tgatcctcca ccagagaagc gcgggtctcg gtctcctcac agcgtggtaa gggggccggc 30360

cgatacgggt gatggcggga cgcggctgat cgtgttcgcg accgtgtcat gatgcagttg 30420

ctttcggaca ttttcgtact tgctgtagca gaacctggtc cgggcgctgc acaccgatcg 30480

ccggcggcgg tctcggcgct tggaacgctc ggtgttgaaa ttgtaaaaca gccactctct 30540

cagaccgtgc agcagatcta gggcctcagg agtgatgaag atcccatcat gcctgatggc 30600

tctgatcaca tcgaccaccg tggaatgggc cagacccagc cagatgatgc aattttgttg 30660

ggtttcggtg acggcggggg agggaagaac aggaagaacc atgattaact tttaatccaa 30720

acggtctcgg agtacttcaa aatgaagatc gcggagatgg cacctctcgc ccccgctgtg 30780

ttggtggaaa ataacagcca ggtcaaaggt gatacggttc tcgagatgtt ccacggtggc 30840

ttccagcaaa gcctccacgc gcacatccag aaacaagaca atagcgaaag cgggagggtt 30900

ctctaattcc tcaatcatca tgttacactc ctgcaccatc cccagataat tttcattttt 30960

ccagccttga atgattcgaa ctagttcgtg aggtaaatcc aagccagcca tgataaagag 31020

ctcgcgcaga gcgccctcca ccggcattct taagcacacc ctcataattc caagatattc 31080

tgctcctggt tcacctgcag cagattgaca agcggaatat caaaatctct gccgcgatcc 31140

ctgagctcct ccctcagcaa taactgtaag tactctttca tatcctctcc gaaattttta 31200

gccataggac caccaggaat aagattaggg caagccacag tacagataaa ccgaagtcct 31260

ccccagtgag cattgccaaa tgcaagactg ctataagcat gctggctaga cccggtgata 31320

tcttccagat aactggacag aaaatcgccc aggcaatttt taagaaaatc aacaaaagaa 31380

aaatcctcca ggtggacgtt tagagcctcg ggaacaacga tgaagtaaat gcaagcggtg 31440

cgttccagca tggttagtta gctgatctgt agaaaaaaca aaaatgaaca ttaaaccatg 31500

ctagcctggc gaacaggtgg gtaaatcgtt ctctccagca ccaggcaggc cacggggtct 31560

ccggcgcgac cctcgtaaaa attgtcgcta tgattgaaaa ccatcacaga gagacgttcc 31620

cggtggccgg cgtgaatgat tcgacaagat gaatacaccc ccggaacatt ggcgtccgcg 31680

agtgaaaaaa agcgcccgag gaagcaataa ggcactacaa tgctcagtct caagtccagc 31740

aaagcgatgc catgcggatg aagcacaaaa ttctcaggtg cgtacaaaat gtaattactc 31800

ccctcctgca caggcagcaa agcccccgat ccctccaggt acacatacaa agcctcagcg 31860

tccatgttaa cagcttaccg agcagcagca cacaacaggc gcaagagtca gagaaaggct 31920

gagctctaac ctgtccaccc gctctctgct caatatatag cccagatcta cactgacgta 31980

aaggccaaag tctaaaaata cccgccaaat aatcacacac gcccagcaca cgcccagaaa 32040

ccggtgacac actcaaaaaa atacgcgcac ttcctcaaac gcccaaaact gccgtcattt 32100

ccgggttccc acgctacgtc atcaaaacac gactttcaaa ttccgtcgac cgttaaaaac 32160

gtcacccgcc ccgcccctaa cggtcgcccg tctctcagcc aatcagcgcc ccgcatcccc 32220

aaattcaaac acctcatttg catattaacg cgcacaaaaa gtttgaggta tattattgat 32280

gatggttaat taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 32340

gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 32400

aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 32460

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 32520

tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 32580

ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 32640

tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 32700

agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 32760

cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 32820

tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 32880

gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 32940

cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 33000

atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 33060

agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 33120

gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 33180

ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 33240

cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 33300

ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 33360

atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 33420

gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 33480

tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 33540

gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 33600

gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 33660

tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 33720

ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 33780

tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 33840

ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 33900

ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 33960

tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 34020

ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 34080

tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 34140

tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 34200

actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 34260

cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcttt 34320

gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 34380

tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 34440

ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 34500

ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 34560

cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 34620

cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 34680

gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 34740

attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 34800

ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 34860

gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 34920

attacgccaa gcttgcatgc ctgcaggttt aaacttaatt aaccatcttc aataatatac 34980

ctcaaacttt ttgtgcgcgt taatatgcaa atgaggcgtt tgaatttggg gaggaagggc 35040

ggtgattg 35048

<210> 2

<211> 1426

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1426)

<223> CMV-EGFP nucleotide sequence

<400> 2

cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120

cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180

tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag 240

tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300

ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360

acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420

gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480

tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540

cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600

ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660

aagctggcta gcgtttaaac gggcccgccc gggcttataa gccaccatgg tgagcaaggg 720

cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 780

ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct 840

gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct 900

gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt 960

caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg 1020

caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 1080

gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 1140

ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa 1200

cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca 1260

gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 1320

gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 1380

gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaa 1426

<210> 3

<211> 2728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(2728)

<223> AdHu5E4 nucleotide sequence

<400> 3

tcacagaacc ctagtattca acctgccacc tccctcccaa cacacagagt acacagtcct 60

ttctccccgg ctggccttaa aaagcatcat atcatgggta acagacatat tcttaggtgt 120

tatattccac acggtttcct gtcgagccaa acgctcatca gtgatattaa taaactcccc 180

gggcagctca cttaagttca tgtcgctgtc cagctgctga gccacaggct gctgtccaac 240

ttgcggttgc ttaacgggcg gcgaaggaga agtccacgcc tacatggggg tagagtcata 300

atcgtgcatc aggatagggc ggtggtgctg cagcagcgcg cgaataaact gctgccgccg 360

ccgctccgtc ctgcaggaat acaacatggc agtggtctcc tcagcgatga ttcgcaccgc 420

ccgcagcata aggcgccttg tcctccgggc acagcagcgc accctgatct cacttaaatc 480

agcacagtaa ctgcagcaca gcaccacaat attgttcaaa atcccacagt gcaaggcgct 540

gtatccaaag ctcatggcgg ggaccacaga acccacgtgg ccatcatacc acaagcgcag 600

gtagattaag tggcgacccc tcataaacac gctggacata aacattacct cttttggcat 660

gttgtaattc accacctccc ggtaccatat aaacctctga ttaaacatgg cgccatccac 720

caccatccta aaccagctgg ccaaaacctg cccgccggct atacactgca gggaaccggg 780

actggaacaa tgacagtgga gagcccagga ctcgtaacca tggatcatca tgctcgtcat 840

gatatcaatg ttggcacaac acaggcacac gtgcatacac ttcctcagga ttacaagctc 900

ctcccgcgtt agaaccatat cccagggaac aacccattcc tgaatcagcg taaatcccac 960

actgcaggga agacctcgca cgtaactcac gttgtgcatt gtcaaagtgt tacattcggg 1020

cagcagcgga tgatcctcca gtatggtagc gcgggtttct gtctcaaaag gaggtagacg 1080

atccctactg tacggagtgc gccgagacaa ccgagatcgt gttggtcgta gtgtcatgcc 1140

aaatggaacg ccggacgtag tcatatttcc tgaagcaaaa ccaggtgcgg gcgtgacaaa 1200

cagatctgcg tctccggtct cgccgcttag atcgctctgt gtagtagttg tagtatatcc 1260

actctctcaa agcatccagg cgccccctgg cttcgggttc tatgtaaact ccttcatgcg 1320

ccgctgccct gataacatcc accaccgcag aataagccac acccagccaa cctacacatt 1380

cgttctgcga gtcacacacg ggaggagcgg gaagagctgg aagaaccatg tttttttttt 1440

tattccaaaa gattatccaa aacctcaaaa tgaagatcta ttaagtgaac gcgctcccct 1500

ccggtggcgt ggtcaaactc tacagccaaa gaacagataa tggcatttgt aagatgttgc 1560

acaatggctt ccaaaaggca aacggccctc acgtccaagt ggacgtaaag gctaaaccct 1620

tcagggtgaa tctcctctat aaacattcca gcaccttcaa ccatgcccaa ataattctca 1680

tctcgccacc ttctcaatat atctctaagc aaatcccgaa tattaagtcc ggccattgta 1740

aaaatctgct ccagagcgcc ctccaccttc agcctcaagc agcgaatcat gattgcaaaa 1800

attcaggttc ctcacagacc tgtataagat tcaaaagcgg aacattaaca aaaataccgc 1860

gatcccgtag gtcccttcgc agggccagct gaacataatc gtgcaggtct gcacggacca 1920

gcgcggccac ttccccgcca ggaaccatga caaaagaacc cacactgatt atgacacgca 1980

tactcggagc tatgctaacc agcgtagccc cgatgtaagc ttgttgcatg ggcggcgata 2040

taaaatgcaa ggtgctgctc aaaaaatcag gcaaagcctc gcgcaaaaaa gaaagcacat 2100

cgtagtcatg ctcatgcaga taaaggcagg taagctccgg aaccaccaca gaaaaagaca 2160

ccatttttct ctcaaacatg tctgcgggtt tctgcataaa cacaaaataa aataacaaaa 2220

aaacatttaa acattagaag cctgtcttac aacaggaaaa acaaccctta taagcataag 2280

acggactacg gccatgccgg cgtgaccgta aaaaaactgg tcaccgtgat taaaaagcac 2340

caccgacagc tcctcggtca tgtccggagt cataatgtaa gactcggtaa acacatcagg 2400

ttgattcaca tcggtcagtg ctaaaaagcg accgaaatag cccgggggaa tacatacccg 2460

caggcgtaga gacaacatta cagcccccat aggaggtata acaaaattaa taggagagaa 2520

aaacacataa acacctgaaa aaccctcctg cctaggcaaa atagcaccct cccgctccag 2580

aacaacatac agcgcttcca cagcggcagc cataacagtc agccttacca gtaaaaaaga 2640

aaacctatta aaaaaacacc actcgacacg gcaccagctc aatcagtcac agtgtaaaaa 2700

agggccaagt gcagagcgag tatatata 2728

<210> 4

<211> 1164

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1164)

<223> AdHu5E 4ORF 6/7 nucleotide sequence

<400> 4

tcacagaacc ctagtattca acctgccacc tccctcccaa cacacagagt acacagtcct 60

ttctccccgg ctggccttaa aaagcatcat atcatgggta acagacatat tcttaggtgt 120

tatattccac acggtttcct gtcgagccaa acgctcatca gtgatattaa taaactcccc 180

gggcagctca cttaagttca tgtcgctgtc cagctgctga gccacaggct gctgtccaac 240

ttgcggttgc ttaacgggcg gcgaaggaga agtccacgcc tacatggggg tagagtcata 300

atcgtgcatc aggatagggc ggtggtgctg cagcagcgcg cgaataaact gctgccgccg 360

ccgctccgtc ctgcaggaat acaacatggc agtggtctcc tcagcgatga ttcgcaccgc 420

ccgcagcata aggcgccttg tcctccgggc acagcagcgc accctgatct cacttaaatc 480

agcacagtaa ctgcagcaca gcaccacaat attgttcaaa atcccacagt gcaaggcgct 540

gtatccaaag ctcatggcgg ggaccacaga acccacgtgg ccatcatacc acaagcgcag 600

gtagattaag tggcgacccc tcataaacac gctggacata aacattacct cttttggcat 660

gttgtaattc accacctccc ggtaccatat aaacctctga ttaaacatgg cgccatccac 720

caccatccta aaccagctgg ccaaaacctg cccgccggct atacactgca gggaaccggg 780

actggaacaa tgacagtgga gagcccagga ctcgtaacca tggatcatca tgctcgtcat 840

gatatcaatg ttggcacaac acaggcacac gtgcatacac ttcctcagga ttacaagctc 900

ctcccgcgtt agaaccatat cccagggaac aacccattcc tgaatcagcg taaatcccac 960

actgcaggga agacctcgca cgtaactcac gttgtgcatt gtcaaagtgt tacattcggg 1020

cagcagcgga tgatcctcca gtatggtagc gcgggtttct gtctcaaaag gaggtagacg 1080

atccctactg tacggagtgc gccgagacaa ccgagatcgt gttggtcgta gtgtcatgcc 1140

aaatggaacg ccggacgtag tcat 1164

<210> 5

<211> 1511

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1511)

<223> AdHu5E 4ORF 3-6 nucleotide sequence

<400> 5

ctacatgggg gtagagtcat aatcgtgcat caggataggg cggtggtgct gcagcagcgc 60

gcgaataaac tgctgccgcc gccgctccgt cctgcaggaa tacaacatgg cagtggtctc 120

ctcagcgatg attcgcaccg cccgcagcat aaggcgcctt gtcctccggg cacagcagcg 180

caccctgatc tcacttaaat cagcacagta actgcagcac agcaccacaa tattgttcaa 240

aatcccacag tgcaaggcgc tgtatccaaa gctcatggcg gggaccacag aacccacgtg 300

gccatcatac cacaagcgca ggtagattaa gtggcgaccc ctcataaaca cgctggacat 360

aaacattacc tcttttggca tgttgtaatt caccacctcc cggtaccata taaacctctg 420

attaaacatg gcgccatcca ccaccatcct aaaccagctg gccaaaacct gcccgccggc 480

tatacactgc agggaaccgg gactggaaca atgacagtgg agagcccagg actcgtaacc 540

atggatcatc atgctcgtca tgatatcaat gttggcacaa cacaggcaca cgtgcataca 600

cttcctcagg attacaagct cctcccgcgt tagaaccata tcccagggaa caacccattc 660

ctgaatcagc gtaaatccca cactgcaggg aagacctcgc acgtaactca cgttgtgcat 720

tgtcaaagtg ttacattcgg gcagcagcgg atgatcctcc agtatggtag cgcgggtttc 780

tgtctcaaaa ggaggtagac gatccctact gtacggagtg cgccgagaca accgagatcg 840

tgttggtcgt agtgtcatgc caaatggaac gccggacgta gtcatatttc ctgaagcaaa 900

accaggtgcg ggcgtgacaa acagatctgc gtctccggtc tcgccgctta gatcgctctg 960

tgtagtagtt gtagtatatc cactctctca aagcatccag gcgccccctg gcttcgggtt 1020

ctatgtaaac tccttcatgc gccgctgccc tgataacatc caccaccgca gaataagcca 1080

cacccagcca acctacacat tcgttctgcg agtcacacac gggaggagcg ggaagagctg 1140

gaagaaccat gttttttttt ttattccaaa agattatcca aaacctcaaa atgaagatct 1200

attaagtgaa cgcgctcccc tccggtggcg tggtcaaact ctacagccaa agaacagata 1260

atggcatttg taagatgttg cacaatggct tccaaaaggc aaacggccct cacgtccaag 1320

tggacgtaaa ggctaaaccc ttcagggtga atctcctcta taaacattcc agcaccttca 1380

accatgccca aataattctc atctcgccac cttctcaata tatctctaag caaatcccga 1440

atattaagtc cggccattgt aaaaatctgc tccagagcgc cctccacctt cagcctcaag 1500

cagcgaatca t 1511