阅读说明：本技术 宿主细菌特异性纳米粒子 (Host bacteria specific nanoparticles ) 是由 满仲翔一 安藤弘树 于 2019-12-05 设计创作，主要内容包括：本发明的课题在于提供安全性高、实用性和有用性优异的重组噬菌体。可提供通过将病毒粒子构成基因的一部分缺失的细菌噬菌体基因组存储于头部从而增殖能力被剥夺、仅能够感染一次的重组细菌噬菌体和其制备方法。另外,可提供通过将具有包装位点且编码目标基因的质粒存储于头部从而增殖能力被剥夺、仅能够感染一次的重组细菌噬菌体和其制备方法。(The present invention addresses the problem of providing a recombinant phage that is highly safe and has excellent utility and usefulness. A recombinant bacteriophage which can be infected only once because the proliferation ability is deprived by storing a bacteriophage genome in which a part of viral particle-constituting genes is deleted on the head, and a method for producing the same are provided. In addition, a recombinant bacteriophage which can be infected only once because the proliferation ability is deprived by storing a plasmid encoding a target gene having a packaging site in the head, and a method for producing the same can be provided.)

1. A method for preparing host bacterium-specific nanoparticles, comprising the following steps (1) and (2):

(1) preparing a recombinant vector to which a bacteriophage genome in which a part of a virus particle-constituting gene is deleted is ligated,

(2) a step of carrying out a packaging reaction in the coexistence of the recombinant vector and a plasmid encoding a deleted viral particle-constituting gene.

2. The method according to claim 1, wherein the bacteriophage genome is prepared as a plurality of fragments, and the recombinant vector is prepared by a seamless cloning method using the plurality of fragments and a linear vector.

3. The method according to claim 2, wherein the seamless cloning method is gap repair cloning or Jersen assembly using homologous recombination in yeast cells.

4. The method according to any one of claims 1 to 3, wherein the viral particle-constituting gene deleted is a head gene or a tail gene.

5. The method according to any one of claims 1 to 3, wherein the missing viral particle-constituting gene is a tail gene.

6. The production method according to any one of claims 1 to 5, wherein the bacteriophage genome is a genome of T7 bacteriophage.

7. The production method according to any one of claims 1 to 6, wherein the step (2) is composed of the following steps (2-1) and (2-2):

(2-1) a step of introducing the recombinant vector into a host bacterium holding a plasmid encoding the deleted viral particle-constituting gene,

(2-2) culturing the host bacterium after the introduction operation.

8. The method according to any one of claims 1 to 7, further comprising the step (3) of:

(3) a step of recovering the bacteriophage produced by the packaging reaction.

9. A host bacterium-specific nanoparticle comprising a recombinant bacteriophage having a head portion storing a bacteriophage genome in which a part of a virus particle-constituting gene is deleted and a tail portion, the recombinant bacteriophage having an infecting ability but no re-infecting ability to a host bacterium.

10. The host bacterium-specific nanoparticle according to claim 9, wherein the deleted virion constituent gene is a head gene or a tail gene.

11. The host bacterium-specific nanoparticle according to claim 9, wherein the deleted virion constituent gene is a tail gene.

12. The host bacteria-specific nanoparticle of any one of claims 9 to 11, wherein the bacteriophage genome is the genome of the T7 bacteriophage.

13. An antibacterial agent comprising the host bacterium-specific nanoparticle according to any one of claims 9 to 12 as an active ingredient.

14. A composition comprising the antibacterial agent of claim 13.

15. The composition according to claim 14, which is a pharmaceutical, disinfectant, cleanser or oral composition for bacterial infection.

16. A method for preparing host bacterium-specific nanoparticles, comprising the following steps (1) and (2):

(1) a step of preparing a recombinant vector to which a bacteriophage genome deleted in a packaging site is ligated,

(2) a step of performing a packaging reaction in the coexistence of the recombinant vector and a plasmid having a deleted packaging site and encoding a target gene.

17. The method according to claim 16, wherein the bacteriophage genome is prepared as a plurality of fragments, and the recombinant vector is prepared by a seamless cloning method using the plurality of fragments and a linear vector.

18. The method according to claim 17, wherein the seamless cloning method is a gap repair clone using homologous recombination in yeast cells.

19. The method according to any one of claims 16 to 18, wherein the bacteriophage genome is a genome of T7 bacteriophage.

20. The method according to any one of claims 16 to 19, wherein the target gene is one or more genes selected from the group consisting of a marker gene, a reporter gene, an enzyme gene, a gene for genome editing, a gene encoding an antimicrobial peptide, an antimicrobial gene, and a gene constituting a synthetic gene line.

21. The method according to any one of claims 16 to 20, wherein step (2) consists of the following steps (2-1) and (2-2):

(2-1) a step of introducing the recombinant vector into a host bacterium holding a plasmid having the deleted packaging site and encoding a target gene,

(2-2) culturing the host bacterium after the introduction operation, and then lysing the cultured host bacterium.

22. The method according to any one of claims 16 to 21, further comprising the step (3) of:

(3) a step of recovering the bacteriophage produced by the packaging reaction.

23. A host bacterium-specific nanoparticle consisting of a recombinant bacteriophage with a head and a tail, the head storing a plasmid having a packaging site and encoding a gene of interest, the recombinant bacteriophage being infectious to a host bacterium but not re-infectious.

24. The host bacteria-specific nanoparticle according to claim 23, wherein the bacteriophage genome is the genome of T7 bacteriophage.

25. The host bacterium-specific nanoparticle according to claim 23 or 24, wherein the target gene is one or more genes selected from a marker gene, a reporter gene, an enzyme gene, a gene for genome editing, a gene encoding an antimicrobial peptide, an antimicrobial gene, and a gene group constituting a synthetic gene line.

26. A transduction composition comprising the host bacterium-specific nanoparticle according to any one of claims 23 to 25 as an active ingredient.

Technical Field

The present invention relates to the use of bacteriophage (hereinafter, sometimes simply referred to as "phage" according to the conventional practice) which is a virus that infects bacteria. In particular, it relates to host bacteria-specific nanoparticles composed of recombinant phages and their uses. The present application claims priority based on japanese patent application No. 2018-244789, filed on 27/12/2018, the entire contents of which are incorporated by reference.

Background

Drug-resistant bacteria have spread throughout the world, and development of new antibacterial agents has been stagnated. Under such circumstances, a "phage therapy" has attracted attention (for example, see patent document 1 and non-patent documents 1 and 2 regarding phage therapy). Bacteriophages are natural enemy viruses that infect bacteria. Has a series of life cycles of (1) adhesion to bacteria, (2) injection of phage genome, (3) proliferation in bacteria, (4) lysis, (5) release of progeny phage, and (6) reinfection to bacteria. The use of bacteriophages for the treatment of bacterial infections is phage therapy. Since phages have extremely high host specificity, they can almost sterilize only target bacteria, rather than sterilizing indiscriminately as antimicrobial drugs do. Therefore, there is an advantage that treatment can be performed without disturbing the bacterial flora originally established.

Documents of the prior art

Patent document

Patent document 1: international publication No. 2016/071503 pamphlet

Non-patent document

Non-patent document 1: front Microbiol.2012; 3: 238.

non-patent document 2: curr Opin Investig drugs.2009Aug; 10(8): 766-74.

Disclosure of Invention

Bacteriophages are viruses that continue to proliferate as long as the target bacteria (host) are present. The possibility of obtaining variation during the propagation to cause unexpected side effects such as infectivity to humans, transfer of a harmful gene of a target bacterium (horizontal transmission), infection with a beneficial bacterium possessed by humans, and the like cannot be excluded. Accordingly, an object of the present invention is to provide a recombinant phage (host bacterium-specific nanoparticle) which is highly safe and excellent in practicality and usefulness. More specifically, it is an object to create a recombinant phage which is deprived of the ability to proliferate and can be infected only once. Recombinant phages showing the characteristic of being able to infect only once can of course be used in phage therapy, and are also useful as genetic engineering tools for genetic modification/genome editing and the like of target bacteria.

In order to solve the above problems, two strategies are established. As a strategy 1, the phage was modified so that the expression type was in a complete state and the genotype was in an incomplete state. Specifically, a phage genome in which a part of a virus particle-constituting gene (gene of a protein constituting a phage particle) is deleted (virus particle-constituting gene-deleted phage genome) is introduced into a host bacterium holding a plasmid encoding the deleted gene. In the host bacterium after the introduction operation, proteins other than the viral particle-constituting protein encoded by the deleted gene are expressed from the deleted phage genome, and the viral particle-constituting protein encoded by the deleted gene is expressed from a plasmid, and it is expected that a recombinant phage having a complete expression type and an incomplete genotype (a partial deletion of the viral particle-constituting gene) will be formed. As a result of performing a verification experiment using the tail gene as an example of the deleted gene, the resulting recombinant phage had its original function (lytic activity against the target bacterium), but could not be infected any more. On the other hand, as the 2 nd strategy, a phage genome (PAC site-deleted phage genome) in which a packaging site (PAC site) necessary for the packaging of phage, i.e., the storage of the phage genome to the head is deleted is introduced into a host bacterium having a PAC site on a plasmid. In the host bacterium after the introduction operation, various proteins required for phage formation are expressed from the PAC site-deleted phage genome, and a plasmid having the PAC site is packaged, and a recombinant phage that can be used as a carrier of the plasmid (i.e., a gene introduction tool) can be expected. The effectiveness of this strategy was verified, and the formation of recombinant phages exhibiting the expected properties was confirmed. Thus confirming its effectiveness for both strategies. It is worth noting that either strategy successfully performed 100% bio-masking.

The following invention is based on the above-described results.

[1] A method for preparing host bacterium-specific nanoparticles, comprising the following steps (1) and (2):

(1) preparing a recombinant vector to which a bacteriophage genome in which a part of a virus particle-constituting gene is deleted is ligated,

(2) a step of carrying out a packaging reaction in the coexistence of the recombinant vector and a plasmid encoding a deleted viral particle-constituting gene.

[2] The production method according to [1], wherein the bacteriophage genome is prepared as a plurality of fragments, and the recombinant vector is produced by a seamless cloning method using the plurality of fragments and a linear vector.

[3] The method according to [2], wherein the seamless cloning method is gap repair cloning or Gipson Assembly (Gibson Assembly) using homologous recombination in yeast cells.

[4] The production method according to any one of [1] to [3], wherein the viral particle-constituting gene deleted is a head gene or a tail gene.

[5] The production method according to any one of [1] to [3], wherein the viral particle-constituting gene deleted is a tail gene.

[6] The production method according to any one of [1] to [5], wherein the bacteriophage genome is a genome of T7 bacteriophage.

[7] The production method according to any one of [1] to [6], wherein the step (2) is composed of the following steps (2-1) and (2-2):

(2-1) a step of introducing the recombinant vector into a host bacterium holding a plasmid encoding the deleted viral particle-constituting gene,

(2-2) culturing the host bacterium after the introduction operation.

[8] The method according to any one of [1] to [7], further comprising the step (3) of:

(3) a step of recovering the bacteriophage produced by the packaging reaction.

[9] A host bacterium-specific nanoparticle comprising a recombinant bacteriophage having a head portion storing a bacteriophage genome in which a part of a virus particle-constituting gene is deleted and a tail portion, the recombinant bacteriophage having an infecting ability but no re-infecting ability to a host bacterium.

[10] The host bacterium-specific nanoparticle according to [9], wherein the deleted viral particle-constituting gene is a head gene or a tail gene.

[11] The host bacterium-specific nanoparticle according to [9], wherein the deleted viral particle-constituting gene is a tail gene.

[12] The host bacterium-specific nanoparticle according to any one of [9] to [11], wherein the bacteriophage genome is a genome of T7 bacteriophage.

[13] An antibacterial agent comprising the host bacterium-specific nanoparticle according to any one of [9] to [12] as an active ingredient.

[14] A composition comprising the antibacterial agent of [13 ].

[15] The composition according to [14], which is a pharmaceutical, disinfectant, cleanser or oral composition for bacterial infection.

[16] A method for preparing host bacterium-specific nanoparticles, comprising the following steps (1) and (2):

(1) a step of preparing a recombinant vector to which a bacteriophage genome deleted in a packaging site is ligated,

(2) a step of performing a packaging reaction in the coexistence of the recombinant vector and a plasmid having a deleted packaging site and encoding a target gene.

[17] The production method according to [16], wherein the bacteriophage genome is prepared as a plurality of fragments, and the recombinant vector is produced by a seamless cloning method using the plurality of fragments and a linear vector.

[18] The production method according to [17], wherein the seamless cloning method is a gap-repairing clone using homologous recombination in yeast cells.

[19] The production method according to any one of [16] to [18], wherein the bacteriophage genome is a genome of T7 bacteriophage.

[20] The production method according to any one of [16] to [19], wherein the target gene is one or more than two genes selected from the group consisting of a marker gene, a reporter gene, an enzyme gene, a gene for genome editing, a gene encoding an antimicrobial peptide, an antimicrobial gene, and a gene constituting a synthetic gene line.

[21] The method according to any one of [16] to [20], wherein the step (2) is constituted by the following steps (2-1) and (2-2):

(2-1) a step of introducing the recombinant vector into a host bacterium holding a plasmid having the deleted packaging site and encoding a target gene,

(2-2) culturing the host bacterium after the introduction operation, and then lysing the cultured host bacterium.

[22] The method according to any one of [16] to [21], further comprising the step (3) of:

(3) a step of recovering the bacteriophage produced by the packaging reaction.

[23] A host bacterium-specific nanoparticle consisting of a recombinant bacteriophage with a head and a tail, the head storing a plasmid having a packaging site and encoding a gene of interest, the recombinant bacteriophage being infectious to a host bacterium but not re-infectious.

[24] The host bacterium-specific nanoparticle according to [23], wherein the bacteriophage genome is a genome of T7 bacteriophage.

[25] The host bacterium-specific nanoparticle according to [23] or [24], wherein the target gene is one or more than two genes selected from a marker gene, a reporter gene, an enzyme gene, a gene for genome editing, a gene encoding an antimicrobial peptide, an antimicrobial gene, and a gene group constituting a synthetic gene line.

[26] A transduction composition comprising the host bacterium-specific nanoparticle according to any one of [23] to [25] as an active ingredient.

Drawings

FIG. 1 shows primers used for preparing DNA fragments for reconstructing a phage genome of a designer.

FIG. 2 shows the design and reconstruction of the designer phage genome (the genome of a phage with the virions constituting a gene deleted). The phage genome and the vector were amplified by PCR using primers designed in such a way that the tail gene was not amplified, and ligated in yeast.

FIG. 3 shows the structure of a vector for connecting a phage genome in which a viral particle constituting a gene is deleted.

FIG. 4 shows the design and reconstruction of the designer phage genome (the genome of the phage in which the virion constitutes a gene deletion). The phage genome and the vector were amplified by PCR using primers designed in such a manner that the head gene was not amplified, and ligated in yeast.

FIG. 5 is the mechanism of Gap repair (Gap-repairing). (1) The 5 '-end of the DNA fragment is cut off by the exonuclease complex, and the 3' -end is exposed. (2) If there is a homologous sequence at the exposed 3' end, a base pair is formed. (3) The DNA is elongated by a DNA polymerase. (4) The DNA is ligated by a ligase.

FIG. 6 shows the structure of a plasmid for cloning virus particle-constituting genes.

FIG. 7 shows the construction of a plasmid encoding a virus particle-constituting gene. The virus particle-constituting gene amplified by PCR is ligated with a plasmid vector.

FIG. 8 is the design and reconstruction of PAC site deleted phage genome. The phage genome and the vector were amplified by PCR using primers designed in such a way that the PAC site was not amplified, and ligated in yeast.

FIG. 9 is the structure of the phage.

FIG. 10 shows a process for preparing a phage with a deleted tail gene.

FIG. 11 shows the bactericidal effect of the tail gene-deleted phages. Coli was infected with a predetermined number of tail gene-deleted phages, and the number of viable bacteria was measured over time. LB: medium only (no tail gene deletion phage), MOI 1: infection with the same number of tail gene-deleted phages as e.coli, MOI 10: 10-fold of tail gene-deleted phages infected with e.coli, MOI 100: 100-fold deletion of tail genes from infected e.coli, MOI 1000: 1000-fold tail gene-deleted phage infected with E.coli

Figure 12 is plaque formation determination results. The samples after the killing measurement were further cultured to confirm the formation of plaque. WT: wild-type T7 phage (positive control), LB: medium only (negative control), MOI ═ 1: a sample infected with the same number of tail gene-deleted phages as those of escherichia coli was further cultured for 24 hours, and then the culture supernatant was mixed with escherichia coli, and the MOI was 10: a sample infected with 10 times as many tail gene-deleted phages of e.coli was further cultured for 24 hours, and then the culture supernatant was mixed with e.coli, and the MOI was 100: a sample infected with 100 times of the tail gene-deleted phage of escherichia coli was further cultured for 24 hours, and then the culture supernatant was mixed with escherichia coli, and MOI was 1000: and a substance obtained by further culturing a sample infected with 1000 times of a phage having a deleted tail gene of Escherichia coli for 24 hours and then mixing the culture supernatant with Escherichia coli.

FIG. 13 is a flow chart for the preparation of transduction nanoparticles.

Fig. 14 is transduction efficiency of transduction nanoparticles. Coli was infected with the packaged transduction nanoparticle with the PAC site maintenance plasmid inserted with the antibiotic resistance gene and lacZ gene encoding β -galactosidase (upper panel), and colony formation was observed (upper right panel). Compared to E.coli only (bottom left plate), transduction of nanoparticles only (bottom right plate). The upper left panel is the result of the plaque formation assay.

Figure 15 is a verification of biological shielding based on plaque formation assays. A solution of the phage lacking the head gene (upper panel of A) or the phage lacking the tail gene (lower panel of A) was added to E.coli, and the presence or absence of plaque (B) was observed.

FIG. 16 is a confirmation of the self-lysis phenomenon. If the head gene is deleted phage, autolysis occurs and plaque (BSP of the left panel) is not formed. When the head gene was expressed in E.coli, plaques (BSP of the right panel) were formed. BSP: bio-conjugated synthetic phase (host specific nanoparticles)

FIG. 17 shows confirmation of bactericidal action. (1) Escherichia coli BW25113, (2) BW25113+ phage T7(MOI ═ 10), (3) BW25113+ BSP Δ head (head gene-deficient phage) (MOI ═ 10), and (4) BW25113+ BSP Δ tail (tail gene-deficient phage) (MOI ═ 10).

Figure 18 is a verification of biological shielding based on plaque formation assay. Upper Left (LB): plates of host bacteria only, top right: plates with head gene-deleted phages added at MOI ═ 1, bottom left: plates with head gene-deleted phage added at MOI 10, bottom right: plates containing head gene-deleted phages added at MOI of 100.

Figure 19 is a validation of in vivo efficacy. Survival of mice vaccinated with salmonella LT2 was compared between the non-treatment group to which neither SP6 nor the head gene-deleted phage was administered, the SP 6-administered group (SP6), and the head gene-deleted phage-administered group (BSP).

Detailed Description

1. Host bacterium specific nano particle composed of virus particle constitutive gene deletion recombinant phage

The 1 st aspect of the present invention relates to a recombinant phage in which a part of a virus particle-constituting gene is deleted. Since the recombinant phage can be used as an active ingredient of an antibacterial agent (details will be described later) and has a size of nanometer order, the recombinant phage of this aspect may be referred to as "antibacterial nanoparticle of the present invention" for convenience of description.

The structure of a bacteriophage is generally roughly divided into a head and a tail. The head stores a phage genome. The tail is important for infecting host bacteria, and although its constitution differs depending on the kind of phage, it is composed of a tail fiber, a substrate, a spike, and the like. The phage genome constituting the antibacterial nanoparticle of the present invention lacks a part of the virus particle-constituting gene. That is, an incomplete phage genome in which a part of a virus particle-constituting gene is deleted is stored in the head. By this structural feature, the host bacterium has an ability to infect but not have an ability to re-infect. Thus, the antibacterial nanoparticles of the present invention show the expression pattern required for infecting host bacteria, and the genotype is incomplete and can be infected only 1 time.

The phage display host bacterial specificity. The host bacterium in the present invention is not particularly limited, and is not limited in shape (generally roughly classified into a sphere, a rod, and a helix), gram-staining property (gram-positive or gram-negative), aerobic property (aerobic property, anaerobic property, and facultative anaerobic property), pathogenic property, presence pattern, and the like. If exemplified by host bacteria, Escherichia coli (Escherichia coli), Shigella bacteria (Shigella) (s.dysenteriae, s.frexenri, s.sonnei, etc.), Salmonella bacteria (Salmonella) (s.typh, s.paratyphi-a, s.schottueller, s.typhimurium, s.enteridis, etc.), Enterobacter bacteria (Enterobacter) (e.aerogenes, e.cloacae, etc.), Klebsiella bacteria (Klebsiella) (k.pneumoniae, k.oxydycella, etc.), Proteus bacteria (Proteus) (p.mirabilis, p.vulgaricus, etc.), Yersinia bacteria (Yersinia) (y, y.enterobacter, etc.), rhodobacter bacteria (e.g., Pseudomonas, etc.), rhodobacter, Pseudomonas, etc. (Pseudomonas bacteria (e.g., Pseudomonas, etc.), rhodobacter, etc. (hydrogen bacillus, etc.), Pseudomonas bacteria (Pseudomonas, etc.), Pseudomonas bacteria (e.g., Pseudomonas sp.bacillus, Pseudomonas, etc. (hydrogen Examples of the bacterial strain include but are not limited to the genera consisting of terra terrestris (Francisella tularensis), Bacteroides (Bacteroides) (b.fragilis, b.melaninogenicus, etc.), Neisseria (Neisseria) (n.gonorrhoeae, n.menitididis, etc.), Staphylococcus (staphyloccocus) (s.aureus, s.epidermidis, s.saprophyticus, etc.), Streptococcus (Streptococcus) (s.pyelogenes, s.agalactiae, s.viridans, s.pyeloniaceae, etc.), Enterococcus (Enterococcus) (e.faecalis, e.faecium, e.avearium, etc.), Bacillus (Bacillus) (b.subtilis, b.antiferrococcus, b.ceylaecium, b.cebacillus, Clostridium) (Clostridium, strain, c.coli, c.c.c.), Clostridium (Clostridium, c.c.c.c.c.c.c.r.c.c.r.c.c.c.r.c.c.c.c.r.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.c.f Mooseri, r. tsutsutsugamushi, etc.), Chlamydia bacteria (Chlamydia) (c. trachomatis, c. psittaci, etc.), Listeria bacteria (Listeria) (l. monocytogenes, etc.).

The method for producing the antibacterial nanoparticles of the present invention will be described below.

< preparation method of antibacterial nanoparticles >

The production method of the present invention comprises the following steps (1) and (2).

(1) Preparing a recombinant vector to which a bacteriophage genome in which a part of a virus particle-constituting gene is deleted is ligated

(2) A step of carrying out a packaging reaction in the coexistence of the recombinant vector and a plasmid encoding the deleted viral particle-constituting gene

In the production method of the present invention, first, a recombinant vector to which a bacteriophage genome in which a part of a virus particle-constituting gene is deleted is ligated is prepared (step (1)). A bacteriophage genome in which a part of a virion-constituting gene is deleted, that is, a bacteriophage genome in which a complete set of virion-constituting genes is not present but is incomplete, will be referred to as "virion-constituting gene-deleted bacteriophage genome" in the present description.

The virus particle-constituting gene to be deleted (hereinafter referred to as "deleted virus particle gene") is not particularly limited as long as the structure required for infecting host bacteria is critically damaged and the ability of the phage to re-infect is lost. Therefore, it is sufficient to delete the head gene (a gene encoding a part or all of the head) or the tail gene (a gene encoding a part or all of the tail). The phage genome in which the viral particle-constituting gene is deleted can be prepared by genetic engineering methods. Therefore, in the genome of the virus particle-constituting gene-deleted phage, a part of the virus particle-constituting gene is deleted as a result of manual manipulation. As a specific example of the deleted virion gene, the sequence of the gene (gene10AB) encoding the head of T7 phage (formed by using gene10A in the genomic sequence registered in NCBI's GenBank as "DEFI NITION: Genome of bacteriophage T7. ACCESSION: V01146J 02518 X00411. VERSION: V01146.1" and gene 10B prepared by frame-shifting in translation of gene 10A) is shown in SEQ ID NO. 1, the sequence of the tail gene11 is shown in SEQ ID NO. 2 (sequence of "DEFINITION: Genome of bacteriophage T7. ACCESIO N: V01146J 518 X00411. VERSION: V01146.1" in NCBI's GenBank is shown in positions 24228 to 24818), the sequence of the tail gene12 is shown in SEQ ID NO. 3 (sequence of aforementioned fiber gene 24842 to 27226), and the sequence of the aforementioned fiber gene 36285 is shown in SEQ ID NO. 363417.

The phage from which the genome of the virus particle-constituting gene-deleted phage is derived is not particularly limited, and may be either a virulent phage or a temperate phage. Virulent phages proliferate within the host bacteria after infection, eventually causing their lysis and the death of the host bacteria (lytic cycle). Temperate phages proliferate through the lytic or lysogenic cycle. In the lysogenic cycle, the phage is encoded into the genomic DNA of the host bacterium. The phage in this state is called a prophage, and the host bacterium harboring the prophage is called a lysogen.

Examples of phages are Myoviridae (Myoviridae) phages (T4-like virus genus, P1-like virus genus, P2-like virus genus, Mu-like virus genus, SPO 1-like virus genus, phi H-like virus genus), Lepididae (Siphoridae) phages (Lambda-like virus genus, Gamma-like virus genus, T1-like virus genus, T5-like virus genus, c 2-like virus genus, L5-like virus genus, PsiM 1-like virus genus, phiC 31-like virus genus, N15-like virus genus), Brevibridae (Podoviridae) phages (T7-like virus genus, phi 29-like virus genus, P22-like virus genus, N4-like virus genus), layered bacteriophages (Tectiviridae) phages (Tectiviridae), overlaid family Corticaceae (Corticaviridae) phages (Fulviriviridae), genus Fulvirirus (Fulvirirus), genus Deltaviridae (Alliaceae) phages (Belviridae) phages (Belvirirus), genus Triviridae), genus (Belviriae) phages (Belviridae), genus, Rhabdiridae) phages (Belvirirus), genus Liporhinus), genus (Belvirises (Belvirirus), genus, Liporhinus), genus (Belvirirus (Belvirises (Belvirirus), and Myxoviridae) phages (Belviri) phages (Belviri), and Myxoviridae) phages (Belviri) phages, Filamentous phage (Inoviridae) phages (Inoviridae genus, Pectovirus genus, M13-like virus genus, fd-like virus genus), microphagaceae (Microviriae) phages (Microvirue genus, Spiromicprovirus genus, Bdellomicrovirus genus, Chlamydiamicrovirus genus), Rhabdoviridae (Leviviridae) phages (Levivivirus genus, Allolevivirus genus), Capillaviridae (Cystoviridae) phages (Cystovirus genus), Viridae (Ampullaviridae) phages, Bicaudavaviridae (Bicaudavaviridae) phages, Klebsviridae (Clavaviridae) phages, Geogreidae (Globuliridae) phages, and Trictaviridae (Guttiviridae) genus phages. The antibacterial nanoparticles of the present invention typically have a function of killing host bacteria by lysis. For this function, virulent phages (e.g., T-phage, SP6 phage, gh-1 phage, etc.) are preferred. The information on the genomic DNA of T7 phage, which is one of particularly preferred phages, is registered in public databases (for example, GenBank of NCBI, DEFINITION: Genome of bacteriophages T7. ACCESSION: V01146J 02518 X00411. VERSION: V01146.1), and can be used for designing/preparing the Genome of the virion-constituting gene-deleted phage of the present invention. The design and production of a genome of a phage with a deleted viral particle-constituting gene corresponding to a phage other than the T7 phage can be performed in the same manner by using known sequence information.

The genome of the phage lacking the viral particle-constituting gene can be prepared by a seamless cloning method. In the seamless cloning method, a target DNA sequence is prepared as a plurality of fragments, and the plurality of fragments are simultaneously cloned into a linear vector. Seamless Cloning methods such as Gap-repairing Cloning (Gap-repairing Cloning) (see, for example, Ando et al, Cell Systems: 1 (3); 2015), gepson Assembly (for example, a system and a Kit (Gibson Assembly Kit) available from New England Biolabs Japan), In-Fusion Cloning (for example, a system and a Kit (In-Fusion (registered trademark) HD Cloning Kit) available from Takara Bio corporation) and the like can be used) have been developed. In a preferred embodiment, the genome of the gene-deleted phage is reconstituted in yeast cells using nick-repair cloning. In this case, the virus particle-constituting gene-deleted phage genome is divided into, for example, 2 to 20 (preferably 2 to 8) fragments, and each fragment is amplified by a nucleic acid amplification reaction typified by the PCR (polymerase chain reaction) method. On the other hand, the vector having the yeast replication origin is introduced into the yeast cell together with the amplified DNA fragment in a linear form. The carrier can be linearized by a conventional method such as restriction enzyme treatment. In order to confirm success or failure of the clone in yeast, the vector is usually loaded with a selection marker gene such as an auxotrophic marker (e.g., URA3 gene, LEU2 gene), drug resistance marker (e.g., ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene) and the like. The details of the method for preparing a phage genome deficient in a viral particle-constituting gene cloned by gap repair are described in the section of examples described later. It should be noted that a gap repair clone can be used for the preparation (reconstruction) of a phage genome to which a modification has been applied (designer phage genome) (see, for example, U.S. Pat. No. 9,617,522).

In step (2) following step (1), a packaging reaction is carried out in the presence of the recombinant vector prepared in step (1), that is, the recombinant vector to which the reconstructed viral particle-constituting gene-deleted phage genome is ligated (hereinafter referred to as "deleted phage genome vector") and a plasmid encoding the deleted viral particle-constituting gene (that is, deleted viral particle gene). Various host bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus can be used for the packaging reaction. The host bacterium may not be the original host (host of phage derived from virus particle-constituting gene-deleted phage genome) as long as it is a bacterium in which phage formed by packaging is soluble. In the case of in vitro packaging using a host bacterium, a host bacterium holding a plasmid encoding a deleted virion gene is generally prepared in advance, and a deleted phage genome vector is introduced into the host bacterium. Plasmids encoding the deleted virion gene are usually loaded with a selectable marker gene (e.g., an auxotrophic marker or drug resistance marker) for confirming the presence of the gene, in addition to the origin of replication of the host bacterium. When the deleted virion gene does not contain a promoter, a promoter may be introduced when constructing the plasmid, or a plasmid vector having a promoter may be used. In the former case, for example, a primer used in the case of amplifying a missing virion gene by PCR is provided with a promoter sequence, and an amplification product in which the primer is located immediately upstream of the gene is obtained. Construction of a plasmid encoding a deleted virion gene can be carried out, for example, by assembling with Gepton, cloning using restriction enzymes and ligase, and the like. The plasmid may be introduced into the host bacterium by a conventional method such as a competent cell method or an electroporation method. On the other hand, the method for introducing the deleted phage genome vector into the host bacterium is not particularly limited, and electroporation may be used, for example.

When a deleted phage genome vector is introduced into a host bacterium holding a plasmid encoding a deleted virion gene as described above, in the host bacterium, proteins other than the virion constituent protein encoded by the deleted virion gene are expressed from the deleted phage genome vector, and the virion protein encoded by the deleted virion gene is expressed from the plasmid, thereby forming a recombinant phage having a complete expression pattern and an incomplete genotype (a part of the virion constituent gene is deleted). When the host bacteria are cultured under appropriate conditions, lysis occurs finally, and the recombinant phage is released. The released recombinant phage can be recovered by conventional methods. The host bacterium can be cultured using either a solid medium or a liquid medium, but from the viewpoint of efficiency of recovery of the recombinant phage, ease of recovery operation, and the like, it is preferable to use a liquid medium, that is, to use liquid culture. In the case of liquid culture, for example, after culturing to complete lysis, the culture solution is subjected to purification, sterilization, and other treatments as necessary to obtain a recombinant phage liquid. It is preferable that the remaining host bacteria are killed by chloroform treatment or the like in advance before the operation of recovering the culture solution.

< use of antibacterial nanoparticles >

The antibacterial nanoparticles of the present invention have a characteristic that they have an ability to infect host bacteria but have no ability to re-infect host bacteria due to their characteristic structure, and exhibit a host bacteria-specific bactericidal ability. Therefore, the compound is useful as an active ingredient of a bactericide having high safety and excellent specificity. The "antibacterial agent" is an agent having an action and an effect of inhibiting (inhibiting) the growth of bacteria or an action and an effect of killing (sterilizing) bacteria. The antibacterial agent of the present invention is used for various purposes in the form of a composition containing the same. Hereinafter, a pharmaceutical (therapeutic or prophylactic) agent, a disinfectant, a cleanser and an oral composition using the composition of the present invention will be described as typical applications.

(i) Pharmaceutical products

The pharmaceutical product of the present invention is used for the treatment or prevention of bacterial infection. The pharmaceutical product of the present invention can exhibit a therapeutic effect or a prophylactic effect on bacterial infection (these two effects are collectively referred to as "pharmaceutical effect"). The pharmaceutical effects herein include (1) prevention of bacterial infection, (2) prevention, inhibition, or delay of onset of bacterial infection, (3) alleviation (lightening) of symptoms characteristic of bacterial infection or accompanying symptoms, (4) prevention, inhibition, or delay of worsening of symptoms characteristic of bacterial infection or accompanying symptoms, and the like. Since the therapeutic effect and the prophylactic effect are partially overlapping concepts, it is sometimes difficult to clearly distinguish them from each other, and the practical significance of such a distinction is small.

The pharmaceutical preparations can be prepared by conventional methods. Preferably formulated in combination with a pharmaceutically acceptable vehicle. The "pharmaceutically acceptable medium" refers to a substance that does not substantially affect the efficacy of the active ingredient of the present invention (sterilization specific to the target bacterium) and provides advantages or benefits for administration, storage, and the like of the pharmaceutical product of the present invention. Examples of the "pharmaceutically acceptable medium" include deionized water, ultrapure water, physiological saline, phosphate buffered physiological saline (PBS), and a 5% dextrose aqueous solution. When formulated, the pharmaceutical composition may contain other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, physiological saline, and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, white sugar, etc. can be used. As the disintegrating agent, starch, carboxymethyl cellulose, calcium carbonate, or the like can be used. As the buffer, phosphate, citrate, acetate, or the like can be used. As the emulsifier, gum arabic, sodium alginate, tragacanth gum, and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate, or the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol, or the like can be used. As the stabilizer, propylene glycol, ascorbic acid, or the like can be used. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. As the preservative, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

The formulation for formulation is not particularly limited. Examples of the dosage form are tablets, powders, fine granules, capsules, syrups, injections, external preparations (ointments, creams, lotions, liquids, gels, cataplasms, plasters, tapes, aerosols, etc.) and suppositories. The pharmaceutical products are applied to subjects by oral administration or non-oral administration (local injection to affected parts, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. In addition, systemic administration and local administration are also applied depending on the subject. These administration routes are not mutually exclusive, and two or more kinds selected arbitrarily may be used in combination.

The pharmaceutical of the present invention contains an active ingredient in an amount necessary for obtaining a desired effect (i.e., a therapeutically or prophylactically effective amount). The amount of the active ingredient in the pharmaceutical of the present invention is usually set so that a desired dose can be achieved, for example, within a range of about 0.001% by weight to about 80% by weight, depending on the dosage form.

The dose of the drug of the present invention is set so that a desired effect can be obtained. In setting the therapeutically or prophylactically effective dose, the symptoms, the age, sex, body weight, and the like of the patient are generally considered. The appropriate dose can be set by those skilled in the art in consideration of these circumstances. In the case of the administration amount, the amount of the active ingredient (the amount of the antibacterial nanoparticles of the present invention) per day may be 10 for an adult (body weight of about 60kg)³pfu/mL～10¹¹pfu/mL, preferably 10⁷pfu/mL～10¹¹The dose was set in the form of pfu/mL. For example, 1 to several times a day, 1 time 2 days, or 1 time 3 days, etc. can be used as the administration schedule. In preparing the administration schedule, the condition of the patient, the duration of the effect of the active ingredient, and the like may be considered.

Treatment with another pharmaceutical, typically an antibacterial agent (e.g., a penicillin-based antibacterial agent, a cephem-based antibacterial agent, a carbapenem-based antibacterial agent, a penem-based antibacterial agent, a tetracycline-based antibacterial agent, a β -lactamase inhibitor, fosfomycin, vancomycin, an aminoglycoside-based antibacterial agent, or a macrolide-based antibacterial agent) can be performed in parallel with the treatment or prevention with the pharmaceutical of the present invention. The mechanism of action of the active ingredient of the present invention is different from that of conventional antibacterial agents generally used. Therefore, if the pharmaceutical product of the present invention is used in combination with a conventional antibacterial agent, it is expected that a combined action and effect will be exhibited, and the therapeutic effect will be increased.

As is clear from the above description, the present application also provides a method for treating or preventing various bacterial infections (so-called phage therapy) characterized by administering a therapeutically or prophylactically effective amount of a pharmaceutical containing the antibacterial nanoparticles of the present invention to a subject suffering from or at risk of suffering from a bacterial infection. The subject to be treated or prevented is typically a human, but may be mammals other than humans (e.g., monkeys, cows, pigs, horses, goats, sheep, dogs, cats, rabbits, etc.), birds (chickens, quails, turkeys, geese, ducks, ostriches, wild ducks, psiogos, asparagus, etc.), fish and shellfish, reptiles (lizards, snakes, iguana, chameleon, turtles, geckoes, etc.), amphibians (frogs, newts, salamanders, etc.), plants, etc.

(ii) Disinfectant and cleaning agent

The disinfectant or detergent of the present invention is used for disinfecting or cleaning living rooms (including sickrooms), kitchens, toilets, bathrooms, and the like, tableware, knives (knives, forks, spoons, and the like), kitchen utensils (knives, pots, mixers, microwave ovens, and the like), medical instruments, devices, and the like, and for disinfecting or cleaning hands, fingers, oral cavities, and the like, for example. The disinfectant or cleaning agent of the present invention is applied by, for example, applying, spraying, scattering, etc., in a liquid form (for example, spray, lotion), gel form, or solid form (for example, powder form). The disinfectant or bactericide of the present invention may be carried on or adhered to a carrier (for example, a sheet) made of natural fibers, synthetic fibers, or the like, and used as a product for wiping purposes or the like, an infection prevention mask, or the like.

Benzalkonium chloride and cetyl pyridinium chloride may also be addedAntibacterial or degerming components such as phenoxyethanol, isopropyl methylphenol, chlorhexidine gluconate and the like, pH regulators, surfactants, adsorbents, carriers and the like are added to the disinfectant or the cleaning agent of the present invention.

(iii) Oral composition

The oral composition of the present invention can be used for maintaining the oral hygiene state, improving the oral environment, and the like. In particular, it is expected to be applied to the prevention or treatment of periodontal diseases or diseases related thereto. According to the oral composition of the present invention, a specific bactericidal effect against a target bacterium can be expected. Typical target bacteria are bacteria causing periodontal disease, for example, actinobacillus actinomycetemcomitans (agregabacter actinomycetes), Prevotella (Prevotella intermedia), Treponema denticola (Treponema denticola), and the like. The oral composition of the present invention may be used as a dental disinfectant, and for example, may be used as a disinfectant after an implant operation or added to mouthwash (mouth wash), toothpaste, or the like.

The oral composition of the present invention is provided in the form of, for example, a dentifrice, a liquid dentifrice, a tooth gel, a mouthwash, a mouth wash, a candy, a troche, a chewing gum, or the like. The oral composition of the present invention may contain a base for oral use, an additive, and the like, which are generally used, in addition to the active ingredient (antibacterial nanoparticles) that is characteristic of the present invention. Examples of base agents for oral use are dental dibasic calcium phosphate, alumina, sorbitol solution. Examples of the additive include a binder, a wetting agent, a foaming agent, a surfactant, a solvent, a cosolvent, a preservative, a sweetener, and a colorant.

2. Host bacteria-specific nanoparticles for transduction

The 2 nd aspect of the present invention relates to a recombinant bacteriophage that can be utilized in transduction to a host bacterium. For ease of explanation, the recombinant bacteriophage of this aspect is sometimes referred to as a "transduction nanoparticle of the invention". In the transduction nanoparticle of the present invention, a plasmid having a packaging site (sometimes simply referred to as "PAC site") and encoding a target gene is stored in the head. By storing a specific plasmid in the head instead of the phage genome, the host bacterium can be infected with the plasmid but cannot be re-infected (can be infected only 1 time), and the plasmid functions as a means for transduction specific to the target bacterium. It should be noted that the PAC sites maintained by the plasmids stored in the head reflect the preparation process of the transduction nanoparticles of the present invention, and are characteristic to the present invention. The method for producing transduction nanoparticles of the present invention will be described below, but the same matters as in the first aspect are not described, and the above description is incorporated.

< preparation of transducing nanoparticles >

The production method of the present invention comprises the following steps (1) and (2).

(1) Preparation of recombinant vector ligated with bacteriophage genome deleted from packaging site

(2) A step of carrying out a packaging reaction in the coexistence of the recombinant vector and a plasmid having the packaging site deleted and encoding a target gene

In the production method of the present invention, first, a recombinant vector to which a bacteriophage genome deleted at the PAC site is ligated is prepared (step (1)). A bacteriophage genome with a PAC site deletion, i.e. an incomplete bacteriophage genome without a PAC site which itself is not packaged, is referred to as a "PAC site deletion bacteriophage genome". The PAC site is a sequence required for packaging of the phage genome when the phage is formed within the host bacterium. Thus, the PAC site-deleted phage genome is not packaged, generating phage that do not have a phage genome (with the phage genome deleted).

Typically, phage genomes with only PAC site deletions are used. However, phage genomes deleted in addition to PAC sites may also be used intentionally, or according to the necessity of recombination operations. In this case, if the portion to be additionally deleted is a gene required for phage formation (for example, a part of a virus particle-constituting gene), the gene may be previously encoded in a plasmid packaged by having a PAC site (see the description of step (2)), or another plasmid encoding the gene may be previously introduced into a host bacterium to be used for packaging. As an example of the PAC site, the sequence of the PAC site on the 5 'terminal side of the T7 phage is shown in sequence number 5, and the sequence of the PAC site on the 3' terminal side is shown in sequence number 6.

The PAC site deleted phage genome can be prepared by a seamless cloning method (preferably gap repair cloning) as in the case of aspect 1.

In step (2) following step (1), a packaging reaction is carried out in the coexistence of the recombinant vector prepared in step (1), that is, the recombinant vector to which the reconstructed PAC site-deleted phage genome is ligated (hereinafter, referred to as "PAC site-deleted phage genome vector") and a plasmid having a deleted PAC site and encoding a target gene (hereinafter, referred to as "PAC site-holding plasmid"). As in the case of claim 1, various host bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus can be used for the packaging reaction. The procedure for introducing a PAC site-deleted phage genome vector and a PAC site-maintained plasmid into a host bacterium is the same as in the case of the 1 st aspect.

Typically, a host bacterium holding a PAC site-retaining plasmid is prepared in advance, and a PAC site-deleted phage genome vector is introduced into the host bacterium to establish a state required for a packaging reaction (i.e., a condition under which the PAC site-deleted phage genome vector and the PAC site-retaining plasmid coexist). The PAC locus maintenance plasmid encodes a gene of interest in addition to the PAC locus. The target gene is a gene that is introduced into a target bacterium by the transduction nanoparticle of the present invention and is expressed in a target cell. Typically, 1 target gene is used, but 2 or more target genes may be encoded in the PAC locus maintenance plasmid. Various genes can be used as the target gene. Examples of the target gene include marker genes (drug resistance genes such as neomycin resistance gene (neo), kanamycin resistance gene (npt), hygromycin resistance gene (hph) and methotrexate resistance gene (dhfr), photoprotein genes such as β -galactosidase gene (lacZ), β -Glucuronidase (GUS) gene and luciferase gene (luc), fluorescent protein genes such as GFP gene, reporter genes (photoprotein genes such as luciferase gene (luc) and fluorescent protein genes such as GFP gene), (enzyme genes (ZFN (Zinc Finger Nuclease), TALENs (Transcription Ac activator-Like Effector Nuclease), gene for gene editing enzymes such as CRISPR-Cas9, α -amylase, β -amylase, glucan 1,4- α -glucosidase, glucose oxidase, glucose, Genes of sugar-related enzymes such as pullulanase and isoamylase, genes of protein-related enzymes such as aminopeptidase, dipeptidyl peptidase, carboxypeptidase, trypsin, chymotrypsin, papain, bromelain, pepsin and chymosin, genes of lipid-related enzymes such as lipase and phospholipase, genes of amino acid-related enzymes such as asparaginase and glutaminase, genes of plant tissue-disrupting enzymes such as cellulase, pectinase and hemicellulase, genes of endonuclease, exonuclease, DNA polymerase, helicase, DNA topoisomerase, RNA polymerase and adenylate deaminase, genes of enzymes for pharmaceuticals such as amylase, lipase, cellulase and galactosidase, glucose oxidase, mutase, peroxidase, glucose dehydrogenase, cholesterol esterase, cholesterol oxidase, Genes for diagnostic enzymes such as cholesterol dehydrogenase, lipoprotein lipase, glycerol kinase, L-alpha-glycerophosphate oxidase, lactase dehydrogenase, uricase, D-3-hydroxybutyrate dehydrogenase, bilirubin oxidase, glutaminase, and ascorbate oxidase), genes encoding antibacterial peptides (defensins, antibacterial peptides, dermatan, drosophila, etc.), antibacterial genes (lysozyme gene, endolysin gene, etc.), gene groups constituting synthetic gene lines (riboregulator, recombinase gene, fluorescent protein gene, etc.), genes that confer specific killing ability to target bacteria of bacteriophage (e.g., lysozyme gene of T7 bacteriophage), drugs, dietary supplements, and other substances (cytokines, hormones, neurotransmitters, fibrinogen, serum albumin, etc.), Lactoferrin, etc.), a gene that is beneficial to the survival or maintenance of a target cell, a gene that encodes a protein that enhances the function originally possessed by the target cell, and a gene that encodes a protein that is secreted from the target cell and acts on surrounding cells without acting on the target cell.

When a marker gene is used as a target gene, the target bacterium transduced with the nanoparticle can be detected using the expression of the gene as an index. Therefore, this method is useful for detection, tracking, and the like of target bacteria, and for a specific application, for example, it is conceivable to examine the contamination of food with food poisoning bacteria. In addition, as a method for confirming the introduction of the transduction nanoparticle of the present invention into a target bacterium, a marker gene can be used, and in this case, the marker gene and another gene (one or two or more genes for transduction into a target cell) can be usually used as a target gene.

When an enzyme gene is used as a target gene, a specific enzyme can be expressed and acted forcibly in a target bacterium, and the transduction nanoparticle of the present invention can be used, for example, for the production of a bacterium having a higher functionality or a new function. When a gene for genome editing in an enzyme gene is used as a target gene, the transduction nanoparticle of the present invention can be used for genome modification of a target bacterium. Examples of the use form of the enzyme gene as a target gene include alteration of drug-resistance of a drug-resistant bacterium (for example, disruption of a drug-resistant gene by genome editing and conversion to a drug-sensitive strain), improvement of productivity of industrial enzyme-producing bacteria, production of food and drink (particularly, fermented food and fermented drink), pharmaceutical products, or industrial products (chemical products and the like), and improvement of the ability (handling ability, production/production ability and the like) of bacteria used for production or bioremediation of bioenergy (for example, bioethanol).

The transduction nanoparticles of the present invention can be used as a target bacterium-specific antibacterial agent if a gene encoding an antibacterial peptide or a gene group that brings about target bacterium-specific killing ability of a bacteriophage is used as a target gene.

When the PAC site-deleted phage genome vector is introduced into the host bacterium holding the PAC site-retaining plasmid as described above, various proteins required for phage formation are expressed from the PAC site-deleted phage genome vector in the host bacterium, and the PAC site-retaining plasmid is packaged. That is, a recombinant phage having a PAC site-retaining plasmid stored at its head was formed. The resulting recombinant phage lacks the phage genome and, therefore, is generally not lysed. Therefore, the host bacterium is cultured under appropriate conditions to promote the formation of recombinant phage, and then lysed by chloroform treatment or the like to recover the recombinant phage. Culture conditions, recovery procedures, and the like are the same as those in the case of claim 1.

< use of transducing nanoparticles >

As described above, in the transduction nanoparticle of the present invention, a PAC site maintenance plasmid (having a PAC site and encoding a target gene) is stored in the head instead of the phage genome. This feature allows the host bacterium to have specific infectivity but no reinfection ability (which can be infected only 1 time), and thus the target bacterium-specific transduction device functions. Accordingly, the present invention also provides a transduction composition comprising the transduction nanoparticle as an active ingredient. As described above, since various target genes can be used, the transduction composition of the present invention can be used in a wide range of applications (for example, production of industrial enzymes, production of foods and beverages (particularly fermented foods and fermented beverages), pharmaceuticals, production of industrial products (chemicals, etc.), production of bioenergy (e.g., bioethanol), and bioremediation), and is extremely high in industrial value. Further, as described above, if a gene encoding an antibacterial peptide or a gene group having a target bacteria-specific killing ability of a bacteriophage is used as a target gene, the gene functions as an antibacterial agent specific to the target bacteria, and can be used as a pharmaceutical (therapeutic or prophylactic agent), a disinfectant, a cleanser, or an oral composition, as in the case of the 1 st aspect. In addition, the transduction nanoparticles of the present invention are useful as a tool for research (experiment).

Examples

In view of the potential usefulness of recombinant phages, the aim was to develop two "host bacteria-specific nanoparticles" that can only infect once.

< Material & operation >

1. Preparation and confirmation of phage (antibacterial nanoparticles) lacking viral particle-constituting genes

1-1. design and reconstruction of designer phage genomes (modified phage genomes)

The designer phage genome is reconstructed from PCR fragments or artificially synthesized DNA fragments. In this case, 20 to 40nt of homologous sequences to the ligated fragments are added to the 5 '-end and 3' -end of each fragment in advance. For example, when a fragment is amplified by PCR, primers are designed as shown in FIG. 1.

In this study, the tail gene or the head gene was deleted from the virus particle-constituting genes. When the tail gene was deleted, the phage genome in which the tail genes gene11, gene12, and gene17 of the T7 phage were deleted (tail gene-deleted phage genome) was reconstructed (sequence No. 7). Primers were designed in such a way that the tail genes (gene 11, gene12 and gene 17) were not amplified (FIG. 2), and the phage genome and the vector pTOW40836 (FIG. 3) were amplified using PCR. The vector is necessary for maintaining the phage genome in the yeast. The ends of the phage genome were made to have sequences homologous to the vector (FIG. 2). In this study, the phage genome was divided into 6 fragments (referred to as fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, and fragment 6 in order from the 5 '-end side to the 3' -end side) without containing the tail genes (gene 11, gene12, and gene 17) and amplified by PCR. The sequences of the primers used for amplification of the respective fragments are as follows.

(fragment 1)

Sequence of the forward primer: CTTGAAGACGAAAGGGCCTCGTGATACGCCTCTCACAGTGTACGGACCTAAAGTTCCCCC (Serial number 8)

Sequence of the reverse primer: ATTACGCGATGACAGTAGACAACCTTTCCG (Serial number 9)

(fragment 2)

Sequence of the forward primer: TGCAGCAATACCGGAAAGGTTGTCTACTGT (Serial number 10)

Sequence of the reverse primer: ATATGTCTCCTCATAGATGTGCCTATGTGG (Serial number 11)

(fragment 3)

Sequence of the forward primer: ACTTGTGACTCCACATAGGCACATCTATGA (Serial number 12)

Sequence of the reverse primer: AGGGAGAATATTTAAATAGTTCCTCCTTTCAGCAAAAAACCCCTC (Serial number 13)

(fragment 4)

Sequence of the forward primer: GAAAGGAGGAACTATTTAAATATTCTCCCTGTGGTGGCTCG (Serial number 14)

Sequence of the reverse primer: GAATAACCTGAGGGTCAATACCCTGCTTGT (Serial number 15)

(fragment 5)

Sequence of the forward primer: GACATGATGGACAAGCAGGGTATTGACCCT (Serial number 16)

Sequence of the reverse primer: CTTGTGATTTACCAATTGACCTCCTTAAAGTAAATCTAAGAGAC (Serial number 17)

(fragment 6)

Sequence of the forward primer: CTTTAAGGAGGTCAATTGGTAAATCACAAGGAAAGACGTGTAGTC (Serial number 18)

Sequence of the reverse primer: CATAATAGAAACGACACGAAATTACAAAATAGGGACACAGAGAGACACTCAAGGTAACAC (Serial number 19)

The sequences of primers used for amplification of the vector fragment are as follows.

Sequence of the forward primer: ATTTTGTAATTTCGTGTCGTTTCTATTATG (Serial number 20)

Sequence of the reverse primer: GGCGTATCACGAGGCCCTTTCGTCTTCAAG (Serial number 21)

The reconstruction of the head gene-deleted phage genome (head gene-deleted phage genome) (SEQ ID NO: 22) was also performed by the same method (FIG. 4). At this time, the phage genome was divided into 5 pieces so as not to contain the head gene (gene10AB), and 5 pieces (referred to as a piece 1 ', a piece 2 ', a piece 3 ', a piece 4 ', and a piece 5 ' in order from the 5 ' end side to the 3 ' end side) were amplified by PCR. The sequences of the primers used for amplification of the respective fragments are as follows.

(fragment 1')

Sequence of the forward primer: CTTGAAGACGAAAGGGCCTCGTGATACGCCTCTCACAGTGTACGGACCTAAAGTTCCCCC (Serial number 23)

Sequence of the reverse primer: ATTACGCGATGACAGTAGACAACCTTTCCG (Serial number 24)

(fragment 2')

Sequence of the forward primer: TGCAGCAATACCGGAAAGGTTGTCTACTGT (Serial number 25)

Sequence of the reverse primer: ATATGTCTCCTCATAGATGTGCCTATGTGG (Serial number 26)

(fragment 3')

Sequence of the forward primer: ACTTGTGACTCCACATAGGCACATCTATGA (Serial number 27)

Sequence of the reverse primer: GCCCCAAGGGGTTATGCTAGATTTCGAAGTCTATCAGAAGTTCGAATCGATTAC (Serial number 28)

(fragment 4')

Sequence of the forward primer: CTTCTGATAGACTTCGAAATCTAGCATAACCCCTTGGGGCCTCTAAACGG (Serial number 29)

Sequence of the reverse primer: GAATAACCTGAGGGTCAATACCCTGCTTGT (Serial number 30)

(fragment 5')

Sequence of the forward primer: GACATGATGGACAAGCAGGGTATTGACCCT (Serial number 31)

Sequence of the reverse primer: CATAATAGAAACGACACGAAATTACAAAATAGGGACACAGAGAGACACTCAAGGTAACAC (Serial number 32)

The same primer sets (SEQ ID NOS: 20 and 21) as in the case of the tail gene were used for amplification of the vector fragment.

1-2 preparation of Yeast competent cells

(1) Yeast were cultured in 5mL YPD medium at 30 ℃ for one-half hour.

(2) A total amount of yeast-evening culture medium was added to 45mL of YPD medium, and the mixture was cultured at 30 ℃ for 3 hours.

(3) Centrifugation was carried out at 8000g for 15 minutes at room temperature.

(4) The culture supernatant was discarded and suspended in 25mL of ultrapure water.

(5) Centrifugation was carried out at 8000g for 15 minutes at room temperature.

(6) The supernatant was discarded and suspended in 100mM LiAc 1 mL.

(7) Centrifugation was carried out at 12000g for 1 minute at room temperature.

(8) The supernatant was discarded and suspended in 100mM LiAc 400. mu.L. This was used as a competent cell.

1-3 transformation of Yeast

(1) Competent cells (50. mu.L) were centrifuged at 12000g for 1 minute at room temperature.

(2) The supernatant was discarded and the following reagents were added in order.

PEG3350(50％w/v)240μL

1M LiAc 36μL

ssDNA (obtained by leaving the ssDNA at 100 ℃ for 5 minutes or more and leaving the ssDNA on ice for 3 minutes or more) in an amount of 25. mu.L

50 μ L of DNA sample (all fragments required for the designer's genome. in the case of deletion of the tail gene, fragments 1 to 6 and vector DNA are combined; in the case of deletion of the head gene, fragments 1 ' to 5 ' and vector DNA are combined)

(3) The mixture was gently mixed by pipetting and allowed to stand at 30 ℃ for 30 minutes.

(4) The mixture was allowed to stand at 42 ℃ for 20 minutes.

(5) Centrifugation was carried out at 12000g for 1 minute at room temperature.

(6) The supernatant was discarded and suspended in 200. mu.L of LB liquid medium.

(7) Spread on selective agar medium.

(8) It was allowed to stand at 30 ℃ (colonies were visible in about 3 days).

The parent yeast strain used was auxotrophic and could not grow on selective media. When the designer genome is ligated to the vector in yeast, the nutrient source required for the parent strain can be supplied, and the strain can be grown on a selective medium. In the present method, the designer's genome is reconstructed by Gap-repairing cloning (Gap-repairing). Gap repair (Gap-repairing) is considered to be part of the DNA repair mechanism. The outline of the Gap repair (Gap-repairing) mechanism is shown in FIG. 5.

1-4 extraction of phage genome from Yeast

(1) The selection liquid medium was inoculated with yeast holding a phage genome and cultured at 30 ℃ for evening-out.

(2) 2mL of the culture broth was centrifuged at 12000g at room temperature for 2 minutes to collect the yeast.

(3) The culture supernatant was discarded, and the phage genome was extracted using YeaStar Genomic DNA Kit (Zymo Researh).

1-5 construction of plasmid encoding viral particle-constituting Gene and introduction of plasmid into Escherichia coli

After cloning the viral particle-constituting genes (tail genes or head genes) removed during phage genome reconstitution into plasmid vector pBR322 (fig. 6), the plasmid vector was introduced into escherichia coli, and escherichia coli having the viral particle-constituting genes retained on the plasmid was prepared. In the case where no promoter is present just upstream of the viral particle-constituting gene, the promoter sequence is added to the primer when cloning is performed on a plasmid vector by PCR (FIG. 7). The following describes the procedure (example) of the tail gene and the head gene.

< case of the Tail Gene (gene 11,12,17) >

(1) The tail genes gene11, gene12, and gene17 were amplified by stage 1 PCR, and the fragments were ligated and added with a promoter by stage 2 PCR (TAATACGACTCACTATAGGG: SEQ ID NO: 33). The sequences of the primers used in each PCR are shown below. The ends of the DNA fragments are provided with restriction enzyme sites in advance.

Stage 1 PCR: amplification of the Tail Gene

(fragment 1)

Sequence of the forward primer: CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGCGCTCATACGATATGAACG (Serial number 34)

Sequence of the reverse primer: CGTTAGCCATTGGTATATCTCCTTCTTTTAAATACCGGAACTTCTCCG (Serial number 35)

(fragment 2)

Sequence of the forward primer: CCGGTATTTAAAAGAAGGAGATATACCAATGGCTAACGTAATTAAAACCG (Serial number 36)

Sequence of the reverse primer: CCCGCGGATCCTTACTCGTTCTCCACCATGATTG (Serial number 37)

Stage 2 PCR: ligation of fragments and addition of promoters

Sequence of the forward primer: CCCGGAATTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAG (Serial number 38)

Sequence of the reverse primer: CCCGCGGATCCTTACTCGTTCTCCACCATGATTG (Serial number 39)

(2) The DNA fragment (tail gene) and the vector DNA were subjected to restriction enzyme treatment and ligated (5. mu.L in total).

(3) The reaction and E.coli chemically competent cells 45. mu.L were mixed and left on ice for 30 minutes.

(4) The mixture was allowed to stand at 42 ℃ for 1 minute.

(5) LB liquid medium (1 mL) was added thereto, and the mixture was allowed to stand at 37 ℃ for 30 minutes.

(6) Spread on agar medium with drug.

Coli that holds a plasmid can grow on an agar medium containing a drug because it obtains a drug (resistance) marker (drug resistance gene).

< case of head Gene (gene10AB) >

(1) The head gene (gene10AB) was amplified in a promoter-containing format. The sequences of the primers used in the PCR are shown below. The ends of the DNA fragments were previously provided with restriction enzyme sites.

Sequence of the forward primer: CCCGCGGATCCTTATTGCTCAGCGGTGGCAG (Serial number 40)

Sequence of the reverse primer: CCCGGAATTCTAATACGACTCACTATAGGGAGAC (Serial number 41)

(2) The DNA fragment (head gene) and the vector DNA were subjected to restriction enzyme treatment and ligated (5. mu.L in total).

(3) The reaction and E.coli chemically competent cells 45. mu.L were mixed and left on ice for 30 minutes.

(4) The mixture was allowed to stand at 42 ℃ for 1 minute.

(5) LB liquid medium (1 mL) was added thereto, and the mixture was allowed to stand at 37 ℃ for 30 minutes.

(6) Spread on agar medium with drug.

Coli that holds a plasmid can grow on an agar medium containing a drug because it obtains a drug (resistance) marker (drug resistance gene).

1-6 preparation of competent cells for electroporation

(1) Bacteria having a virus particle-constituting gene (tail gene or head gene) were inoculated into LB liquid medium containing the drug and cultured at 37 ℃ at first evening.

(2) The culture medium was added in an amount 1/100 times the volume of 20mL of the pharmaceutical-containing SOB broth, and the resulting mixture was cultured at 37 ℃ until the OD600 became 0.4.

(3) Centrifugation was carried out at 800g at 4 ℃ for 5 minutes.

(4) The culture supernatant was discarded and suspended in 10mL of cold 10% glycerol.

(5) Centrifugation was carried out at 800g at 4 ℃ for 5 minutes.

(6) The supernatant was discarded and suspended in 10mL of cold 10% glycerol.

(7) Centrifugation was carried out at 800g at 4 ℃ for 5 minutes.

(8) The supernatant was discarded and suspended in about 70 μ L of cold 10% glycerol. This was used as a competent cell for electroporation.

1-7 activation of phage with deletion of viral particle-constituting gene

(1) mu.L of the extracted virus particle-constituting gene-deleted phage genome (tail gene-deleted phage genome or head gene-deleted phage genome) and 20. mu.L of the electroporation competent cells were mixed and placed in a cuvette.

(2) Electroporation (2.5kV, 10. mu.F, 600. omega.).

(3) LB liquid medium (500. mu.L) was added to the cuvette and recovered.

(4) The mixture was mixed with soft agar medium and layered on LB agar medium.

(5) Standing at 37 ℃ until plaque (plaque) is formed.

1-8 confirmation of phage deficient in viral particle-constituting gene

(1) The plaques were picked up with toothpicks or wood chips and suspended in 100. mu.L of phage buffer.

(2) Bacteria expressing a virus particle-constituting gene (tail gene or head gene) or unexpressed bacteria were mixed with a soft agar medium and layered on an LB agar medium. To this, 2.5. mu.L of phage buffer (phase buffer) in which plaque was suspended was added dropwise and dried.

(3) Standing at 37 ℃ until plaque formation.

It was confirmed that plaque was formed in bacteria expressing a virus particle-constituting gene (tail gene or head gene), and that plaque was not formed in bacteria not expressing the virus particle-constituting gene.

1-9 recovery of phage lacking virus particle-constituting gene

(1) Bacteria expressing a virus particle-constituting gene (tail gene or head gene) were inoculated into LB liquid medium containing the drug, and then cultured at 37 ℃ at a low temperature.

(2) An amount of 1/100 cells of the bacterial culture was added to 50mL of LB liquid medium containing the drug.

(3) The culture was carried out at 37 ℃ until OD600 ═ 0.4.

(4) Phage buffer (phage buffer) in which phage was suspended was added and cultured until lysis was complete.

(5) Chloroform (1 mL) was added (to completely kill the remaining bacteria).

(6) Centrifugation was carried out at 12000g at 4 ℃ for 5 minutes. Thereby, a layer of a lysate and chloroform was formed.

(7) The lysate was filtered and sterilized with a 0.22 μm filter to prepare a phage solution.

1-10 killing assay (bactericidal assay)

(1) Bacteria were inoculated in LB liquid medium and cultured at 37 ℃ for evening-out.

(2) Prepare 5 bottles to be 1X 10⁶cfu/ml, a bacterial culture solution was added to an LB liquid medium, and a phage liquid was added thereto so as to have an MOI of 1, an MOI of 10, an MOI of 100, and an MOI of 1000.

(3) The culture was carried out at 37 ℃ and 1mL of the solution was sampled every 2 hours.

(4) The samples were centrifuged at 5000g for 5 minutes at 4 ℃.

(5) The culture supernatant was discarded and suspended in 1mL of PBS.

(6) Centrifugation was carried out at 5000g for 5 minutes at 4 ℃.

(7) The supernatant was discarded and suspended in 1mL of PBS. It was used as a stock solution.

(8) Stock solution was extracted from 10 with PBS^-1To 10^-710-fold stepwise dilution was performed, and 2.5. mu.L of each was added to LB agar medium and dried.

(9) Let stand at 37 ℃ for evening.

(10) The number of viable bacteria (number of colonies) was counted.

2. Preparation and validation of transduction nanoparticles

The same applies to the design and reconstruction of a phage genome by a designer, the preparation of a yeast competent cell, the transformation of a yeast, and the extraction of a phage genome from a yeast, as in the case of a virus particle gene-deleted phage. In the phage DNA, there is a sequence called a packaging (hereinafter PAC) site which is required for loading the head of the phage. Primers were designed to remove this sequence, and amplification was performed by PCR, while vector pTOW40836 (fig. 3) was amplified. In this study, the phage genome from which the PAC sites were removed was divided into 4 pieces, and 4 pieces (referred to as a piece I, a piece II, a piece III, and a piece IV in order from the 5 'end side to the 3' end side) were amplified by PCR. The sequences of the primers used for amplification of the respective fragments are as follows.

(fragment I)

Sequence of the forward primer: CTTGAAGACGAAAGGGCCTCGTGATACGCCGTCCATCCTAAAGCCAACACCTAAAGCC (Serial number 42)

Sequence of the reverse primer: ATTACGCGATGACAGTAGACAACCTTTCCG (Serial number 43)

(fragment II)

Sequence of the forward primer: TGCAGCAATACCGGAAAGGTTGTCTACTGT (Serial number 44)

Sequence of the reverse primer: ATATGTCTCCTCATAGATGTGCCTATGTGG (Serial number 45)

(fragment III)

Sequence of the forward primer: ACTTGTGACTCCACATAGGCACATCTATGA (Serial number 46)

Sequence of the reverse primer: GAATAACCTGAGGGTCAATACCCTGCTTGT (Serial number 47)

(fragment IV)

Sequence of the forward primer: GACATGATGGACAAGCAGGGTATTGACCCT (Serial number 48)

Sequence of the reverse primer: CATAATAGAAACGACACGAAATTACAAAATTGCATAAATCACCACTCAATGAAAGACG (Serial number 49)

The sequences of primers used for amplification of the vector fragment are as follows.

Sequence of the forward primer: ATTTTGTAATTTCGTGTCGTTTCTATTATG (Serial number 20)

Sequence of the reverse primer: GGCGTATCACGAGGCCCTTTCGTCTTCAAG (Serial number 21)

The phage genome DNA fragments (fragments I to IV) were mixed with the vector fragment, and the volume was adjusted to 50. mu.L with ultrapure water, and the obtained mixture was used as a DNA sample for yeast transformation. The yeast was transformed and the genome of the phage with the PAC site deleted (seq id No. 50) was reconstructed (fig. 8).

The plasmid having the PAC site was constructed in the same manner as in the case of the plasmid having the viral particle-constituting gene (the PAC site was cloned in plasmid pBR322 in place of the viral particle-constituting gene).

Competent cells for electroporation of bacteria retaining a plasmid having a PAC site were prepared in the same manner as in the case of a virus particle gene-deleted phage, and the transduction nanoparticles were activated by the following method.

(1) mu.L of the extracted PAC site-deleted phage genome and 20. mu.L of competent cells for electroporation were mixed and placed in a cuvette.

(2) Electroporation (2.5kV, 10. mu.F, 600. omega.).

(3) After electroporation, 1mL of LB liquid medium was added to a cuvette containing the PAC site-deleted phage genome and competent cells, and the cuvette was allowed to stand at 37 ℃ for 2 hours.

(4) To the mixture was added 100. mu.L of chloroform, and the mixture was gently tapped.

(5) Centrifugation was carried out at 12000g at 4 ℃ for 5 minutes. Thereby, a layer of the mixed solution and chloroform was formed.

(6) The mixture was filter-sterilized with a 0.22 μm filter.

(7) The recovered solution was used as transduction nanoparticle solution.

(8) The transduction nanoparticle solution and the bacterial culture solution were mixed and allowed to stand at 37 ℃ for 30 minutes.

(9) Spread on agar medium with drug.

Since the plasmid has a drug marker, the bacteria infected with the transduction nanoparticle are injected with the plasmid to obtain drug resistance. Bacteria infused with plasmids (i.e., bacteria that transduce nanoparticles function) can grow on agar media with the agent.

< development of phage having deleted Tail Gene >

1. Method of producing a composite material

The phage consists of a head where the genome is stored and a tail required for attachment to the bacterium (fig. 9). The bacteria were adhered to the surface of the bacteria via the foot (tail fiber) at the tip of the tail. The region other than the tail gene of the phage genome and a vector having homologous sequences to both ends of the phage genome were amplified by PCR, and these fragments were ligated in yeast to construct an artificial phage genome (SEQ ID NO: 7) (FIG. 10). In this experiment, although the artificial phage genome was constructed using the gap repair clone, the construction of the artificial phage genome was also successfully performed by the jeps assembly.

A phage genome was extracted from yeast, and the phage genome was introduced into E.coli harboring a plasmid encoding the tail gene (FIG. 10). The phage-constituting proteins other than the tail are expressed from the phage genome, and the tail is expressed from a plasmid, thereby forming a "phage having a tail as an expression type and no tail as a genotype" in E.coli. Finally, E.coli was lysed and the phage with the deleted tail gene was released (FIG. 10). The progeny phage produced from this phage, because they do not have a tail, cannot be re-infected.

2. Results (Sterilization effect of the phage with deletion of the Tail Gene)

1X 10 in each 1mL⁶Coli was prepared, and 1, 10, 100, and 1000 times (MOI 1, 10, 100, and 1000) the number of tail gene-deficient phages was added to the number of e.coli, and the number of live bacteria in e.coli was measured over time (killing measurement). Growth inhibition was observed by adding tail gene-deleted phage (MOI 1) to the same extent as escherichia coli, and the number of viable bacteria in escherichia coli was significantly reduced at MOI 10, 100, and 1000 (fig. 11). Further, each sample was directly cultured again to confirm whether or not a new phage was produced (plaque formation assay). When E.coli and phages were mixed with soft agar and continued to be cultured in the agar culture medium in the basal layer, as shown by "WT" in FIG. 12, transparent spots called lysoplaques (plaques) were formed. This is thatThe phage infects bacteria and makes them bacteriolytic. No plaque was observed even when each culture broth was mixed with E.coli. This strongly suggests that at least in the present experimental system, phages capable of reinfection may not be produced. The same experimental results were obtained by preparing a phage with a head gene deleted (see above for the preparation method).

< development of transducing nanoparticles >

1. Method of producing a composite material

The case where the phage genome is stored in the head is called packaging. There are sequences on the phage genome called Packaging (PAC) sites, which are recognized to store the phage genome at the head. By deleting the PAC site on the phage genome and having the PAC site on the plasmid, transduction nanoparticles capable of packaging arbitrary plasmids instead of the phage genome were developed. Similarly to the method for developing a phage lacking the tail gene described above, a phage genome lacking the PAC site (seq id No. 50) was constructed by amplifying the region other than the PAC site and the vector by PCR and ligating them in yeast. Next, a phage genome lacking the PAC site was extracted from the yeast, and the phage genome was introduced into E.coli holding a plasmid having the PAC site. Upon phage formation, the phage genome is not packaged because the PAC site is not present in the phage genome, alternatively, a plasmid with a PAC site is packaged (fig. 13). Even if the nanoparticles infect bacteria, only plasmids are introduced without bactericidal effect. In addition, since there is no phage genome, progeny phage are not formed.

2. Results (transduction efficiency of transduction nanoparticles)

The PAC site-maintaining plasmid was packaged in transduction nanoparticles by inserting an antibiotic resistance gene and lacZ gene encoding β -galactosidase into it. Beta-galactosidase is an enzyme that hydrolyzes beta-galactoside to produce galactose. When bacteria having this enzyme were grown on a medium containing a reagent called X-gal, the colonies turned blue. Coli having no lacZ gene and 10. mu.L of transduction nanoparticle were mixed and applied to a solution containing an antibodyBiotin and X-gal on agar medium (FIG. 14), and as a result, blue colonies were observed from the plates coated with E.coli infected with the transduction nanoparticles (upper right plate). Even if only E.coli (bottom left plate) or only 100. mu.L of transduction nanoparticles (bottom right plate) was spread on the agar medium, no colonies were formed, and therefore, it was considered that blue E.coli seen in the plate coated with E.coli infected with transduction nanoparticles acquired the antibiotic gene and lacZ gene through the transduction nanoparticles. In addition, plaque formation assay was performed considering the possibility of containing wild-type phage, but no plaque was formed (upper left panel). The transduction efficiency was about 4.2X 10 when calculated from the number of colonies⁵/L。

< summary >

Two "host bacteria-specific nanoparticles" were developed. One functions as a nanoparticle having bactericidal activity, and the other functions as a nanoparticle having transduction activity. The method should in principle be applicable to all phages. It is conceivable to use the recombinant vector in phage therapy, bacterial flora editing, as a tool for bacteria for which genetic methods have not been established, and the like. In addition, it is a single infection, with 100% successful bio-shielding at the laboratory level.

< verification of BioShielding >

The biological shielding of the virus particles constituting the gene-deleted phages (head gene-deleted phage and tail gene-deleted phage) was verified.

1. Plaque formation assay

Coli strain BW25113 was applied to an agar medium, and then a phage solution (containing a head gene-deleted phage (upper panel in FIG. 15A) or a tail gene-deleted phage (lower panel in FIG. 15A)) was added thereto to culture. Control phage solutions of wild-type phage were used. In contrast to the wild-type phages (left-hand (BW25113) T7 column in fig. 15B), no plaque formation was observed in the head gene-deleted phages (left-hand (BW25113) T7 Δ 10 column in fig. 15B) and tail gene-deleted phages (left-hand (BW25113) T7 Δ tail column in fig. 15B), and the masking was successfully performed. Similarly, when the E.coli strain expresses the head gene (the column of T7. DELTA.10 on the right side of FIG. 15B (BW25113/pBR-T7-g 10)) or the tail gene (the column of T7. DELTA.tail on the right side of FIG. 15B (BW25113/pBR-T7-g 11-12-17)), even the shield phage (head gene-deleted phage and tail gene-deleted phage) can form progeny phage and plaque.

2. Phenomenon of self-pyrolysis

Autolysis is a phenomenon in which when a large excess of phage is infected, the progeny phage, whether produced or not, causes death of the host bacteria, and is characterized by the absence of individual plaques. After the Escherichia coli strain BW25113 was applied to an agar medium, a phage solution (head gene-deleted phage) was added thereto and cultured. Control phage solutions of wild-type phage were used. For head gene-deleted phages, progeny phages could not be formed due to blocking, and plaques could not be formed (columns of BSP of the left panel of fig. 16). When the head gene is expressed, even the head gene-deleted phage can form a progeny phage and form a plaque (the BSP column of the right panel in fig. 16).

3. Bactericidal action of head gene deletion bacteriophage and tail gene deletion bacteriophage

A head gene-deficient phage or a tail gene-deficient phage was added to a solution of E.coli BW25113, and the mixture was cultured for 6 hours to evaluate the bactericidal activity. Like the wild-type phage having bactericidal activity (T7) (fig. 17 (2)), the head gene-deleted phage and the tail gene-deleted phage exhibited bactericidal activity ((3), (4) of fig. 17).

4. Proliferation property

After the E.coli strain BW25113 was applied to an agar medium, phage solutions (head gene-deficient phages) were added at respective concentrations. After further incubation for 24 hours, the formation of plaques was observed. No plaque was observed even at MOI of 100 (large excess of phage was added) (fig. 18). That is, the phage subjected to the masking measure (head gene-deleted phage) did not acquire the proliferative property, and only one infection was completed (i.e., the biological masking was successfully performed).

5. Validation of in vivo efficacy

The Salmonella LT2 is added in an amount of 0.8-1.4 × 10⁵After cfu is inoculated to mouse BJ6, SP6 or head gene deletion phage is added in an amount of 2.5-5.0 × 10⁷pfu was administered, and the survival rate was compared and evaluated. The control (non-treated group) was prepared in the case where neither SP6 nor the head gene-deleted phage was administered. The survival rate of the head gene-deleted phage-administered group (BSP) was significantly higher than that of the SP 6-administered group (SP6) compared to the non-treated group (fig. 19). On the other hand, no phage was detected in the blood and spleen of the mice in the group to which the head gene-deleted phage was administered, and 100% of the bioshielding was successfully performed.

Industrial applicability

The recombinant phage provided by the invention is deprived of the multiplication capacity and can only be infected once. This feature ensures high safety, and therefore, is suitable for various uses including clinical use. Among the recombinant phages of the present invention, phages that exhibit bactericidal activity by storing a phage genome (in which a part of a virus particle-constituting gene is deleted) on the head are expected to be used particularly in phage therapy. On the other hand, recombinant phages produced by storing a specific plasmid in the head instead of the phage genome are expected to be used and applied as a gene transfer tool in a wide range of applications such as alteration of drug sensitivity of drug-resistant bacteria, improvement in productivity of industrial enzyme-producing bacteria, production of foods and beverages (in particular, fermented foods and fermented beverages), pharmaceuticals and industrial products (chemicals and the like), production of bioenergy (e.g., bioethanol), and improvement in the ability (processing ability, production/production ability and the like) of bacteria used for bioremediation.

The present invention is not limited to the description of the embodiment and examples of the invention described above. Various modifications within the scope that can be easily conceived by those skilled in the art are also included in the present invention without departing from the description of the claims. The contents of the papers, patent publications, and the like disclosed in the present specification are incorporated herein by reference in their entirety.

Sequence Listing free text

Sequence number 7: description of artificial sequences: phage genome with deleted tail gene

Sequence number 8: description of artificial sequences: forward primer

Sequence number 9: description of artificial sequences: reverse primer

Sequence number 10: description of artificial sequences: forward primer

Sequence number 11: description of artificial sequences: reverse primer

Sequence number 12: description of artificial sequences: forward primer

Sequence number 13: description of artificial sequences: reverse primer

Sequence number 14: description of artificial sequences: forward primer

Sequence number 15: description of artificial sequences: reverse primer

Sequence number 16: description of artificial sequences: forward primer

Sequence number 17: description of artificial sequences: reverse primer

Sequence number 18: description of artificial sequences: forward primer

Sequence number 19: description of artificial sequences: reverse primer

Sequence number 20: description of artificial sequences: forward primer

Sequence number 21: description of artificial sequences: reverse primer

Sequence number 22: description of artificial sequences: head gene deleted phage genome

Sequence number 23: description of artificial sequences: forward primer

Sequence number 24: description of artificial sequences: reverse primer

Sequence number 25: description of artificial sequences: forward primer

Sequence number 26: description of artificial sequences: reverse primer

Sequence number 27: description of artificial sequences: forward primer

Sequence number 28: description of artificial sequences: reverse primer

Sequence number 29: description of artificial sequences: forward primer

Sequence number 30: description of artificial sequences: reverse primer

Sequence number 31: description of artificial sequences: forward primer

Sequence number 32: description of artificial sequences: reverse primer

Sequence number 33: description of artificial sequences: promoters

Sequence number 34: description of artificial sequences: forward primer

Sequence number 35: description of artificial sequences: reverse primer

Sequence number 36: description of artificial sequences: forward primer

Sequence number 37: description of artificial sequences: reverse primer

Sequence number 38: description of artificial sequences: forward primer

Sequence number 39: description of artificial sequences: reverse primer

Sequence number 40: description of artificial sequences: reverse primer

Sequence No. 41: description of artificial sequences: forward primer

Sequence number 42: description of artificial sequences: reverse primer

Sequence number 43: description of artificial sequences: forward primer

Sequence number 44: description of artificial sequences: reverse primer

Sequence number 45: description of artificial sequences: forward primer

Sequence number 46: description of artificial sequences: forward primer

Sequence number 47: description of artificial sequences: reverse primer

Sequence number 48: description of artificial sequences: forward primer

Sequence number 49: description of artificial sequences: and (3) a reverse primer.

Sequence listing

<110> eastern China sea national university institution of national university legal people

<120> host bacterium-specific nanoparticles

<130> GU18001P

<150> JP P2018-244789

<151> 2018-12-27

<160> 50

<170> PatentIn version 3.5

<210> 1

<211> 1197

<212> DNA

<213> bacteriophage T7

<400> 1

atggctagca tgactggtgg acagcaaatg ggtactaacc aaggtaaagg tgtagttgct 60

gctggagata aactggcgtt gttcttgaag gtatttggcg gtgaagtcct gactgcgttc 120

gctcgtacct ccgtgaccac ttctcgccac atggtacgtt ccatctccag cggtaaatcc 180

gctcagttcc ctgttctggg tcgcactcag gcagcgtatc tggctccggg cgagaacctc 240

gacgataaac gtaaggacat caaacacacc gagaaggtaa tcaccattga cggtctcctg 300

acggctgacg ttctgattta tgatattgag gacgcgatga accactacga cgttcgctct 360

gagtatacct ctcagttggg tgaatctctg gcgatggctg cggatggtgc ggttctggct 420

gagattgccg gtctgtgtaa cgtggaaagc aaatataatg agaacatcga gggcttaggt 480

actgctaccg taattgagac cactcagaac aaggccgcac ttaccgacca agttgcgctg 540

ggtaaggaga ttattgcggc tctgactaag gctcgtgcgg ctctgaccaa gaactatgtt 600

ccggctgctg accgtgtgtt ctactgtgac ccagatagct actctgcgat tctggcagca 660

ctgatgccga acgcagcaaa ctacgctgct ctgattgacc ctgagaaggg ttctatccgc 720

aacgttatgg gctttgaggt tgtagaagtt ccgcacctca ccgctggtgg tgctggtacc 780

gctcgtgagg gcactactgg tcagaagcac gtcttccctg ccaataaagg tgagggtaat 840

gtcaaggttg ctaaggacaa cgttatcggc ctgttcatgc accgctctgc ggtaggtact 900

gttaagctgc gtgacttggc tctggagcgc gctcgccgtg ctaacttcca agcggaccag 960

attatcgcta agtacgcaat gggccacggt ggtcttcgcc cagaagctgc tggtgcagtg 1020

gtttttcaaa gtggagtaat gctgggggtg gcctcaacgg tcgctgctag tcccgaagag 1080

gcgagtgtta cttcaacaga agaaacctta acgccagcac aggaggccgc acgcacccgc 1140

gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataa 1197

<210> 2

<211> 591

<212> DNA

<213> bacteriophage T7

<400> 2

atgcgctcat acgatatgaa cgttgagact gccgctgagt tatcagctgt gaacgacatt 60

ctggcgtcta tcggtgaacc tccggtatca acgctggaag gtgacgctaa cgcagatgca 120

gcgaacgctc ggcgtattct caacaagatt aaccgacaga ttcaatctcg tggatggacg 180

ttcaacattg aggaaggcat aacgctacta cctgatgttt actccaacct gattgtatac 240

agtgacgact atttatccct aatgtctact tccggtcaat ccatctacgt taaccgaggt 300

ggctatgtgt atgaccgaac gagtcaatca gaccgctttg actctggtat tactgtgaac 360

attattcgtc tccgcgacta cgatgagatg cctgagtgct tccgttactg gattgtcacc 420

aaggcttccc gtcagttcaa caaccgattc tttggggcac cggaagtaga gggtgtactc 480

caagaagagg aagatgaggc tagacgtctc tgcatggagt atgagatgga ctacggtggg 540

tacaatatgc tggatggaga tgcgttcact tctggtctac tgactcgcta a 591

<210> 3

<211> 2385

<212> DNA

<213> bacteriophage T7

<400> 3

atggcactca ttagccaatc aatcaagaac ttgaagggtg gtatcagcca acagcctgac 60

atccttcgtt atccagacca agggtcacgc caagttaacg gttggtcttc ggagaccgag 120

ggcctccaaa agcgtccacc tcttgttttc ttaaatacac ttggagacaa cggtgcgtta 180

ggtcaagctc cgtacatcca cctgattaac cgagatgagc acgaacagta ttacgctgtg 240

ttcactggta gcggaatccg agtgttcgac ctttctggta acgagaagca agttaggtat 300

cctaacggtt ccaactacat caagaccgct aatccacgta acgacctgcg aatggttact 360

gtagcagact atacgttcat cgttaaccgt aacgttgttg cacagaagaa cacaaagtct 420

gtcaacttac cgaattacaa ccctaatcaa gacggattga ttaacgttcg tggtggtcag 480

tatggtaggg aactaattgt acacattaac ggtaaagacg ttgcgaagta taagatacca 540

gatggtagtc aacctgaaca cgtaaacaat acggatgccc aatggttagc tgaagagtta 600

gccaagcaga tgcgcactaa cttgtctgat tggactgtaa atgtagggca agggttcatc 660

catgtgaccg cacctagtgg tcaacagatt gactccttca cgactaaaga tggctacgca 720

gaccagttga ttaaccctgt gacccactac gctcagtcgt tctctaagct gccacctaat 780

gctcctaacg gctacatggt gaaaatcgta ggggacgcct ctaagtctgc cgaccagtat 840

tacgttcggt atgacgctga gcggaaagtt tggactgaga ctttaggttg gaacactgag 900

gaccaagttc tatgggaaac catgccacac gctcttgtgc gagccgctga cggtaatttc 960

gacttcaagt ggcttgagtg gtctcctaag tcttgtggtg acgttgacac caacccttgg 1020

ccttcttttg ttggttcaag tattaacgat gtgttcttct tccgtaaccg cttaggattc 1080

cttagtgggg agaacatcat attgagtcgt acagccaaat acttcaactt ctaccctgcg 1140

tccattgcga accttagtga tgacgaccct atagacgtag ctgtgagtac caaccgaata 1200

gcaatcctta agtacgccgt tccgttctca gaagagttac tcatctggtc cgatgaagca 1260

caattcgtcc tgactgcctc gggtactctc acatctaagt cggttgagtt gaacctaacg 1320

acccagtttg acgtacagga ccgagcgaga ccttttggga ttgggcgtaa tgtctacttt 1380

gctagtccga ggtccagctt cacgtccatc cacaggtact acgctgtgca ggatgtcagt 1440

tccgttaaga atgctgagga cattacatca cacgttccta actacatccc taatggtgtg 1500

ttcagtattt gcggaagtgg tacggaaaac ttctgttcgg tactatctca cggggaccct 1560

agtaaaatct tcatgtacaa attcctgtac ctgaacgaag agttaaggca acagtcgtgg 1620

tctcattggg actttgggga aaacgtacag gttctagctt gtcagagtat cagctcagat 1680

atgtatgtga ttcttcgcaa tgagttcaat acgttcctag ctagaatctc tttcactaag 1740

aacgccattg acttacaggg agaaccctat cgtgccttta tggacatgaa gattcgatac 1800

acgattccta gtggaacata caacgatgac acattcacta cctctattca tattccaaca 1860

atttatggtg caaacttcgg gaggggcaaa atcactgtat tggagcctga tggtaagata 1920

accgtgtttg agcaacctac ggctgggtgg aatagcgacc cttggctgag actcagcggt 1980

aacttggagg gacgcatggt gtacattggg ttcaacatta acttcgtata tgagttctct 2040

aagttcctca tcaagcagac tgccgacgac gggtctacct ccacggaaga cattgggcgc 2100

ttacagttac gccgagcgtg ggttaactac gagaactctg gtacgtttga catttatgtt 2160

gagaaccaat cgtctaactg gaagtacaca atggctggtg cccgattagg ctctaacact 2220

ctgagggctg ggagactgaa cttagggacc ggacaatatc gattccctgt ggttggtaac 2280

gccaagttca acactgtata catcttgtca gatgagacta cccctctgaa catcattggg 2340

tgtggctggg aaggtaacta cttacggaga agttccggta tttaa 2385

<210> 4

<211> 1662

<212> DNA

<213> bacteriophage T7

<400> 4

atggctaacg taattaaaac cgttttgact taccagttag atggctccaa tcgtgatttt 60

aatatcccgt ttgagtatct agcccgtaag ttcgtagtgg taactcttat tggtgtagac 120

cgaaaggtcc ttacgattaa tacagactat cgctttgcta cacgtactac tatctctctg 180

acaaaggctt ggggtccagc cgatggctac acgaccatcg agttacgtcg agtaacctcc 240

actaccgacc gattggttga ctttacggat ggttcaatcc tccgcgcgta tgaccttaac 300

gtcgctcaga ttcaaacgat gcacgtagcg gaagaggccc gtgacctcac tacggatact 360

atcggtgtca ataacgatgg tcacttggat gctcgtggtc gtcgaattgt gaacctagcg 420

aacgccgtgg atgaccgcga tgctgttccg tttggtcaac taaagaccat gaaccagaac 480

tcatggcaag cacgtaatga agccttacag ttccgtaatg aggctgagac tttcagaaac 540

caagcggagg gctttaagaa cgagtccagt accaacgcta cgaacacaaa gcagtggcgc 600

gatgagacca agggtttccg agacgaagcc aagcggttca agaatacggc tggtcaatac 660

gctacatctg ctgggaactc tgcttccgct gcgcatcaat ctgaggtaaa cgctgagaac 720

tctgccacag catccgctaa ctctgctcat ttggcagaac agcaagcaga ccgtgcggaa 780

cgtgaggcag acaagctgga aaattacaat ggattggctg gtgcaattga taaggtagat 840

ggaaccaatg tgtactggaa aggaaatatt cacgctaacg ggcgccttta catgaccaca 900

aacggttttg actgtggcca gtatcaacag ttctttggtg gtgtcactaa tcgttactct 960

gtcatggagt ggggagatga gaacggatgg ctgatgtatg ttcaacgtag agagtggaca 1020

acagcgatag gcggtaacat ccagttagta gtaaacggac agatcatcac ccaaggtgga 1080

gccatgaccg gtcagctaaa attgcagaat gggcatgttc ttcaattaga gtccgcatcc 1140

gacaaggcgc actatattct atctaaagat ggtaacagga ataactggta cattggtaga 1200

gggtcagata acaacaatga ctgtaccttc cactcctatg tacatggtac gaccttaaca 1260

ctcaagcagg actatgcagt agttaacaaa cacttccacg taggtcaggc cgttgtggcc 1320

actgatggta atattcaagg tactaagtgg ggaggtaaat ggctggatgc ttacctacgt 1380

gacagcttcg ttgcgaagtc caaggcgtgg actcaggtgt ggtctggtag tgctggcggt 1440

ggggtaagtg tgactgtttc acaggatctc cgcttccgca atatctggat taagtgtgcc 1500

aacaactctt ggaacttctt ccgtactggc cccgatggaa tctacttcat agcctctgat 1560

ggtggatggt tacgattcca aatacactcc aacggtctcg gattcaagaa tattgcagac 1620

agtcgttcag tacctaatgc aatcatggtg gagaacgagt aa 1662

<210> 5

<211> 160

<212> DNA

<213> bacteriophage T7

<400> 5

tctcacagtg tacggaccta aagttccccc atagggggta cctaaagccc agccaatcac 60

ctaaagtcaa ccttcggttg accttgaggg ttccctaagg gttggggatg acccttgggt 120

ttgtctttgg gtgttacctt gagtgtctct ctgtgtccct 160

<210> 6

<211> 799

<212> DNA

<213> bacteriophage T7

<400> 6

ttaggactgc atagggatgc actatagacc acggatggtc agttctttaa gttactgaaa 60

agacacgata aattaatacg actcactata gggagaggag ggacgaaagg ttactatata 120

gatactgaat gaatacttat agagtgcata aagtatgcat aatggtgtac ctagagtgac 180

ctctaagaat ggtgattata ttgtattagt atcaccttaa cttaaggacc aacataaagg 240

gaggagactc atgttccgct tattgttgaa cctactgcgg catagagtca cctaccgatt 300

tcttgtggta ctttgtgctg cccttgggta cgcatctctt actggagacc tcagttcact 360

ggagtctgtc gtttgctcta tactcacttg tagcgattag ggtcttcctg accgactgat 420

ggctcaccga gggattcagc ggtatgattg catcacacca cttcatccct atagagtcaa 480

gtcctaaggt atacccataa agagcctcta atggtctatc ctaaggtcta tacctaaaga 540

taggccatcc tatcagtgtc acctaaagag ggtcttagag agggcctatg gagttcctat 600

agggtccttt aaaatatacc ataaaaatct gagtgactat ctcacagtgt acggacctaa 660

agttccccca tagggggtac ctaaagccca gccaatcacc taaagtcaac cttcggttga 720

ccttgagggt tccctaaggg ttggggatga cccttgggtt tgtctttggg tgttaccttg 780

agtgtctctc tgtgtccct 799

<210> 7

<211> 35276

<212> DNA

<213> Artificial sequence

<220>

<223> Tail Gene-deleted phage genome

<400> 7

tctcacagtg tacggaccta aagttccccc atagggggta cctaaagccc agccaatcac 60

ctaaagtcaa ccttcggttg accttgaggg ttccctaagg gttggggatg acccttgggt 120

ttgtctttgg gtgttacctt gagtgtctct ctgtgtccct atctgttaca gtctcctaaa 180

gtatcctcct aaagtcacct cctaacgtcc atcctaaagc caacacctaa agcctacacc 240

taaagaccca tcaagtcaac gcctatctta aagtttaaac ataaagacca gacctaaaga 300

ccagacctaa agacactaca taaagaccag acctaaagac gccttgttgt tagccataaa 360

gtgataacct ttaatcattg tctttattaa tacaactcac tataaggaga gacaacttaa 420

agagacttaa aagattaatt taaaatttat caaaaagagt attgacttaa agtctaacct 480

ataggatact tacagccatc gagagggaca cggcgaatag ccatcccaat cgacaccggg 540

gtcaaccgga taagtagaca gcctgataag tcgcacgaaa aacaggtatt gacaacatga 600

agtaacatgc agtaagatac aaatcgctag gtaacactag cagcgtcaac cgggcgcaca 660

gtgccttcta ggtgacttaa gcgcaccacg gcacataagg tgaaacaaaa cggttgacaa 720

catgaagtaa acacggtacg atgtaccaca tgaaacgaca gtgagtcacc acactgaaag 780

gtgatgcggt ctaacgaaac ctgacctaag acgctcttta acaatctggt aaatagctct 840

tgagtgcatg actagcggat aactcaaggg tatcgcaagg tgccctttat gatattcact 900

aataactgca cgaggtaaca caagatggct atgtctaaca tgacttacaa caacgttttc 960

gaccacgctt acgaaatgct gaaagaaaac atccgttatg atgacatccg tgacactgat 1020

gacctgcacg atgctattca catggctgcc gataatgcag ttccgcacta ctacgctgac 1080

atctttagcg taatggcaag tgagggcatt gaccttgagt tcgaagactc tggtctgatg 1140

cctgacacca aggacgtaat ccgcatcctg caagcgcgta tctatgagca attaacgatt 1200

gacctctggg aagacgcaga agacttgctc aatgaatact tggaggaagt cgaggagtac 1260

gaggaggatg aagagtaatg tctactacca acgtgcaata cggtctgacc gctcaaactg 1320

tacttttcta tagcgacatg gtgcgctgtg gctttaactg gtcactcgca atggcacagc 1380

tcaaagaact gtacgaaaac aacaaggcaa tagctttaga atctgctgag tgatagactc 1440

aaggtcgctc ctagcgagtg gcctttatga ttatcacttt acttatgagg gagtaatgta 1500

tatgcttact atcggtctac tcaccgctct aggtctagct gtaggtgcat cctttgggaa 1560

ggctttaggt gtagctgtag gttcctactt taccgcttgc atcatcatag gaatcatcaa 1620

aggggcacta cgcaaatgat gaagcactac gttatgccaa tccacacgtc caacggggca 1680

accgtatgta cacctgatgg gttcgcaatg aaacaacgaa tcgaacgcct taagcgtgaa 1740

ctccgcatta accgcaagat taacaagata ggttccggct atgacagaac gcactgatgg 1800

cttaaagaaa ggttatatgc ccaatggcac actatacgct gcaaatcggc gaatagtgag 1860

aacttggcga gagaacaacc tcgaacgccg caaggacaag agagggcggc gtggcataga 1920

cgaaaggaaa aggttaaagc caagaaactc gccgcacttg aacaggcact agccaacaca 1980

ctgaacgcta tctcataacg aacataaagg acacaatgca atgaacatta ccgacatcat 2040

gaacgctatc gacgcaatca aagcactgcc aatctgtgaa cttgacaagc gtcaaggtat 2100

gcttatcgac ttactggtcg agatggtcaa cagcgagacg tgtgatggcg agctaaccga 2160

actaaatcag gcacttgagc atcaagattg gtggactacc ttgaagtgtc tcacggctga 2220

cgcagggttc aagatgctcg gtaatggtca cttctcggct gcttatagtc acccgctgct 2280

acctaacaga gtgattaagg tgggctttaa gaaagaggat tcaggcgcag cctataccgc 2340

attctgccgc atgtatcagg gtcgtcctgg tatccctaac gtctacgatg tacagcgcca 2400

cgctggatgc tatacggtgg tacttgacgc acttaaggat tgcgagcgtt tcaacaatga 2460

tgcccattat aaatacgctg agattgcaag cgacatcatt gattgcaatt cggatgagca 2520

tgatgagtta actggatggg atggtgagtt tgttgaaact tgtaaactaa tccgcaagtt 2580

ctttgagggc atcgcctcat tcgacatgca tagcgggaac atcatgttct caaatggaga 2640

cgtaccatac atcaccgacc cggtatcatt ctcgcagaag aaagacggtg gcgcattcag 2700

catcgaccct gaggaactca tcaaggaagt cgaggaagtc gcacgacaga aagaaattga 2760

ccgcgctaag gcccgtaaag aacgtcacga ggggcgctta gaggcacgca gattcaaacg 2820

tcgcaaccgc aaggcacgta aagcacacaa agctaagcgc gaaagaatgc ttgctgcgtg 2880

gcgatgggct gaacgtcaag aacggcgtaa ccatgaggta gctgtagatg tactaggaag 2940

aaccaataac gctatgctct gggtcaacat gttctctggg gactttaagg cgcttgagga 3000

acgaatcgcg ctgcactggc gtaatgctga ccggatggct atcgctaatg gtcttacgct 3060

caacattgat aagcaacttg acgcaatgtt aatgggctga tagtcttatc ttacaggtca 3120

tctgcgggtg gcctgaatag gtacgattta ctaactggaa gaggcactaa atgaacacga 3180

ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg ttcaacactc 3240

tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag catgagtctt 3300

acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa gctggtgagg 3360

ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag atgattgcac 3420

gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg acagccttcc 3480

agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag accactctgg 3540

cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca atcggtcggg 3600

ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag cacttcaaga 3660

aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa gcatttatgc 3720

aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg tggtcttcgt 3780

ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc attgagtcaa 3840

ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac tctgagacta 3900

tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg ctggctggca 3960

tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc attactggtg 4020

gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac agtaagaaag 4080

cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt aacattgcgc 4140

aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta atcaccaagt 4200

ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc ccgatgaaac 4260

cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct gccgctgctg 4320

tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc atgcttgagc 4380

aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg gactggcgcg 4440

gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc aaaggactgc 4500

ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg aaaatccacg 4560

gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag ttcattgagg 4620

aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact tggtgggctg 4680

agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg gtacagcacc 4740

acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc tctggcatcc 4800

agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac ttgcttccta 4860

gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag attctacaag 4920

cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag aacactggtg 4980

aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg ctggcttacg 5040

gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg tccaaagagt 5100

tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat tccggcaagg 5160

gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg atttgggaat 5220

ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag tctgctgcta 5280

agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc aagcgttgcg 5340

ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag aagcctattc 5400

agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc attaacacca 5460

acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct aactttgtac 5520

acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag aagtacggaa 5580

tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac gctgcgaacc 5640

tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat gtactggctg 5700

atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa atgccagcac 5760

ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc gcgttcgcgt 5820

aacgccaaat caatacgact cactatagag ggacaaactc aaggtcattc gcaagagtgg 5880

cctttatgat tgaccttctt ccggttaata cgactcacta taggagaacc ttaaggttta 5940

actttaagac ccttaagtgt taattagaga tttaaattaa agaattacta agagaggact 6000

ttaagtatgc gtaacttcga aaagatgacc aaacgttcta accgtaatgc tcgtgacttc 6060

gaggcaacca aaggtcgcaa gttgaataag actaagcgtg accgctctca caagcgtagc 6120

tgggagggtc agtaagatgg gacgtttata tagtggtaat ctggcagcat tcaaggcagc 6180

aacaaacaag ctgttccagt tagacttagc ggtcatttat gatgactggt atgatgccta 6240

tacaagaaaa gattgcatac ggttacgtat tgaggacagg agtggaaacc tgattgatac 6300

tagcaccttc taccaccacg acgaggacgt tctgttcaat atgtgtactg attggttgaa 6360

ccatatgtat gaccagttga aggactggaa gtaatacgac tcagtatagg gacaatgctt 6420

aaggtcgctc tctaggagtg gccttagtca tttaaccaat aggagataaa cattatgatg 6480

aacattaaga ctaacccgtt taaagccgtg tctttcgtag agtctgccat taagaaggct 6540

ctggataacg ctgggtatct tatcgctgaa atcaagtacg atggtgtacg cgggaacatc 6600

tgcgtagaca atactgctaa cagttactgg ctctctcgtg tatctaaaac gattccggca 6660

ctggagcact taaacgggtt tgatgttcgc tggaagcgtc tactgaacga tgaccgttgc 6720

ttctacaaag atggctttat gcttgatggg gaactcatgg tcaagggcgt agactttaac 6780

acagggtccg gcctactgcg taccaaatgg actgacacga agaaccaaga gttccatgaa 6840

gagttattcg ttgaaccaat ccgtaagaaa gataaagttc cctttaagct gcacactgga 6900

caccttcaca taaaactgta cgctatcctc ccgctgcaca tcgtggagtc tggagaagac 6960

tgtgatgtca tgacgttgct catgcaggaa cacgttaaga acatgctgcc tctgctacag 7020

gaatacttcc ctgaaatcga atggcaagcg gctgaatctt acgaggtcta cgatatggta 7080

gaactacagc aactgtacga gcagaagcga gcagaaggcc atgagggtct cattgtgaaa 7140

gacccgatgt gtatctataa gcgcggtaag aaatctggct ggtggaaaat gaaacctgag 7200

aacgaagctg acggtatcat tcagggtctg gtatggggta caaaaggtct ggctaatgaa 7260

ggtaaagtga ttggttttga ggtgcttctt gagagtggtc gtttagttaa cgccacgaat 7320

atctctcgcg ccttaatgga tgagttcact gagacagtaa aagaggccac cctaagtcaa 7380

tggggattct ttagcccata cggtattggc gacaacgatg cttgtactat taacccttac 7440

gatggctggg cgtgtcaaat tagctacatg gaggaaacac ctgatggctc tttgcggcac 7500

ccatcgttcg taatgttccg tggcaccgag gacaaccctc aagagaaaat gtaatcacac 7560

tggctcacct tcgggtgggc ctttctgcgt ttataaggag acactttatg tttaagaagg 7620

ttggtaaatt ccttgcggct ttggcagcta tcctgacgct tgcgtatatt cttgcggtat 7680

accctcaagt agcactagta gtagttggcg cttgttactt agcggcagtg tgtgcttgcg 7740

tgtggagtat agttaactgg taatacgact cactaaagga ggtacacacc atgatgtact 7800

taatgccatt actcatcgtc attgtaggat gccttgcgct ccactgtagc gatgatgata 7860

tgccagatgg tcacgcttaa tacgactcac taaaggagac actatatgtt tcgacttcat 7920

tacaacaaaa gcgttaagaa tttcacggtt cgccgtgctg accgttcaat cgtatgtgcg 7980

agcgagcgcc gagctaagat acctcttatt ggtaacacag ttcctttggc accgagcgtc 8040

cacatcatta tcacccgtgg tgactttgag aaagcaatag acaagaaacg tccggttctt 8100

agtgtggcag tgacccgctt cccgttcgtc cgtctgttac tcaaacgaat caaggaggtg 8160

ttctgatggg actgttagat ggtgaagcct gggaaaaaga aaacccgcca gtacaagcaa 8220

ctgggtgtat agcttgctta gagaaagatg accgttatcc acacacctgt aacaaaggag 8280

ctaacgatat gaccgaacgt gaacaagaga tgatcattaa gttgatagac aataatgaag 8340

gtcgcccaga tgatttgaat ggctgcggta ttctctgctc caatgtccct tgccacctct 8400

gccccgcaaa taacgatcaa aagataacct taggtgaaat ccgagcgatg gacccacgta 8460

aaccacatct gaataaacct gaggtaactc ctacagatga ccagccttcc gctgagacaa 8520

tcgaaggtgt cactaagcct tcccactaca tgctgtttga cgacattgag gctatcgaag 8580

tgattgctcg ttcaatgacc gttgagcagt tcaagggata ctgcttcggt aacatcttaa 8640

agtacagact acgtgctggt aagaagtcag agttagcgta cttagagaaa gacctagcga 8700

aagcagactt ctataaagaa ctctttgaga aacataagga taaatgttat gcataacttc 8760

aagtcaaccc cacctgccga cagcctatct gatgacttca catcttgctc agagtggtgc 8820

cgaaagatgt gggaagagac attcgacgat gcgtacatca agctgtatga actttggaaa 8880

tcgagaggtc aatgactatg tcaaacgtaa atacaggttc acttagtgtg gacaataaga 8940

agttttgggc taccgtagag tcctcggagc attccttcga ggttccaatc tacgctgaga 9000

ccctagacga agctctggag ttagccgaat ggcaatacgt tccggctggc tttgaggtta 9060

ctcgtgtgcg tccttgtgta gcaccgaagt aatacgactc actattaggg aagactccct 9120

ctgagaaacc aaacgaaacc taaaggagat taacattatg gctaagaaga ttttcacctc 9180

tgcgctgggt accgctgaac cttacgctta catcgccaag ccggactacg gcaacgaaga 9240

gcgtggcttt gggaaccctc gtggtgtcta taaagttgac ctgactattc ccaacaaaga 9300

cccgcgctgc cagcgtatgg tcgatgaaat cgtgaagtgt cacgaagagg cttatgctgc 9360

tgccgttgag gaatacgaag ctaatccacc tgctgtagct cgtggtaaga aaccgctgaa 9420

accgtatgag ggtgacatgc cgttcttcga taacggtgac ggtacgacta cctttaagtt 9480

caaatgctac gcgtctttcc aagacaagaa gaccaaagag accaagcaca tcaatctggt 9540

tgtggttgac tcaaaaggta agaagatgga agacgttccg attatcggtg gtggctctaa 9600

gctgaaagtt aaatattctc tggttccata caagtggaac actgctgtag gtgcgagcgt 9660

taagctgcaa ctggaatccg tgatgctggt cgaactggct acctttggtg gcggtgaaga 9720

cgattgggct gacgaagttg aagagaacgg ctatgttgcc tctggttctg ccaaagcgag 9780

caaaccacgc gacgaagaaa gctgggacga agacgacgaa gagtccgagg aagcagacga 9840

agacggagac ttctaagtgg aactgcggga gaaaatcctt gagcgaatca aggtgacttc 9900

ctctgggtgt tgggagtggc agggcgctac gaacaataaa gggtacgggc aggtgtggtg 9960

cagcaatacc ggaaaggttg tctactgtca tcgcgtaatg tctaatgctc cgaaaggttc 10020

taccgtcctg cactcctgtg ataatccatt atgttgtaac cctgaacacc tatccatagg 10080

aactccaaaa gagaactcca ctgacatggt aaataagggt cgctcacaca aggggtataa 10140

actttcagac gaagacgtaa tggcaatcat ggagtccagc gagtccaatg tatccttagc 10200

tcgcacctat ggtgtctccc aacagactat ttgtgatata cgcaaaggga ggcgacatgg 10260

caggttacgg cgctaaagga atccgaaagg ttggagcgtt tcgctctggc ctagaggaca 10320

aggtttcaaa gcagttggaa tcaaaaggta ttaaattcga gtatgaagag tggaaagtgc 10380

cttatgtaat tccggcgagc aatcacactt acactccaga cttcttactt ccaaacggta 10440

tattcgttga gacaaagggt ctgtgggaaa gcgatgatag aaagaagcac ttattaatta 10500

gggagcagca ccccgagcta gacatccgta ttgtcttctc aagctcacgt actaagttat 10560

acaaaggttc tccaacgtct tatggagagt tctgcgaaaa gcatggtatt aagttcgctg 10620

ataaactgat acctgctgag tggataaagg aacccaagaa ggaggtcccc tttgatagat 10680

taaaaaggaa aggaggaaag aaataatggc tcgtgtacag tttaaacaac gtgaatctac 10740

tgacgcaatc tttgttcact gctcggctac caagccaagt cagaatgttg gtgtccgtga 10800

gattcgccag tggcacaaag agcagggttg gctcgatgtg ggataccact ttatcatcaa 10860

gcgagacggt actgtggagg caggacgaga tgagatggct gtaggctctc acgctaaggg 10920

ttacaaccac aactctatcg gcgtctgcct tgttggtggt atcgacgata aaggtaagtt 10980

cgacgctaac tttacgccag cccaaatgca atcccttcgc tcactgcttg tcacactgct 11040

ggctaagtac gaaggcgctg tgcttcgcgc ccatcatgag gtggcgccga aggcttgccc 11100

ttcgttcgac cttaagcgtt ggtgggagaa gaacgaactg gtcacttctg accgtggata 11160

attaattgaa ctcactaaag ggagaccaca gcggtttccc tttgttcgca ttggaggtca 11220

aataatgcgc aagtcttata aacaattcta taaggctccg aggaggcata tccaagtgtg 11280

ggaggcagcc aatgggccta taccaaaagg ttattatata gaccacattg acggcaatcc 11340

actcaacgac gccttagaca atctccgtct ggctctccca aaagaaaact catggaacat 11400

gaagactcca aagagcaata cctcaggact aaagggactg agttggagca aggaaaggga 11460

gatgtggaga ggcactgtaa cagctgaggg taaacagcat aactttcgta gtagagatct 11520

attggaagtc gttgcgtgga tttatagaac taggagggaa ttgcatggac aattcgcacg 11580

attccgatag tgtatttctt taccacattc cttgtgacaa ctgtgggagt agtgatggga 11640

actcgctgtt ctctgacgga cacacgttct gctacgtatg cgagaagtgg actgctggta 11700

atgaagacac taaagagagg gcttcaaaac ggaaaccctc aggaggtaaa ccaatgactt 11760

acaacgtgtg gaacttcggg gaatccaatg gacgctactc cgcgttaact gcgagaggaa 11820

tctccaagga aacctgtcag aaggctggct actggattgc caaagtagac ggtgtgatgt 11880

accaagtggc tgactatcgg gaccagaacg gcaacattgt gagtcagaag gttcgagata 11940

aagataagaa ctttaagacc actggtagtc acaagagtga cgctctgttc gggaagcact 12000

tgtggaatgg tggtaagaag attgtcgtta cagaaggtga aatcgacatg cttaccgtga 12060

tggaacttca agactgtaag tatcctgtag tgtcgttggg tcacggtgcc tctgccgcta 12120

agaagacatg cgctgccaac tacgaatact ttgaccagtt cgaacagatt atcttaatgt 12180

tcgatatgga cgaagcaggg cgcaaagcag tcgaagaggc tgcacaggtt ctacctgctg 12240

gtaaggtacg agtggcagtt cttccgtgta aggatgcaaa cgagtgtcac ctaaatggtc 12300

acgaccgtga aatcatggag caagtgtgga atgctggtcc ttggattcct gatggtgtgg 12360

tatcggctct ttcgttacgt gaacgaatcc gtgagcacct atcgtccgag gaatcagtag 12420

gtttactttt cagtggctgc actggtatca acgataagac cttaggtgcc cgtggtggtg 12480

aagtcattat ggtcacttcc ggttccggta tgggtaagtc aacgttcgtc cgtcaacaag 12540

ctctacaatg gggcacagcg atgggcaaga aggtaggctt agcgatgctt gaggagtccg 12600

ttgaggagac cgctgaggac cttataggtc tacacaaccg tgtccgactg agacaatccg 12660

actcactaaa gagagagatt attgagaacg gtaagttcga ccaatggttc gatgaactgt 12720

tcggcaacga tacgttccat ctatatgact cattcgccga ggctgagacg gatagactgc 12780

tcgctaagct ggcctacatg cgctcaggct tgggctgtga cgtaatcatt ctagaccaca 12840

tctcaatcgt cgtatccgct tctggtgaat ccgatgagcg taagatgatt gacaacctga 12900

tgaccaagct caaagggttc gctaagtcaa ctggggtggt gctggtcgta atttgtcacc 12960

ttaagaaccc agacaaaggt aaagcacatg aggaaggtcg ccccgtttct attactgacc 13020

tacgtggttc tggcgcacta cgccaactat ctgatactat tattgccctt gagcgtaatc 13080

agcaaggcga tatgcctaac cttgtcctcg ttcgtattct caagtgccgc tttactggtg 13140

atactggtat cgctggctac atggaataca acaaggaaac cggatggctt gaaccatcaa 13200

gttactcagg ggaagaagag tcacactcag agtcaacaga ctggtccaac gacactgact 13260

tctgacagga ttcttgatga ctttccagac gactacgaga agtttcgctg gagagtccca 13320

ttctaatacg actcactaaa ggagacacac catgttcaaa ctgattaaga agttaggcca 13380

actgctggtt cgtatgtaca acgtggaagc caagcgactg aacgatgagg ctcgtaaaga 13440

ggccacacag tcacgcgctc tggcgattcg ctccaacgaa ctggctgaca gtgcatccac 13500

taaagttacc gaggctgccc gtgtggcaaa ccaagctcaa cagctttcca aattctttga 13560

gtaatcaaac aggagaaacc attatgtcta acgtagctga aactatccgt ctatccgata 13620

cagctgacca gtggaaccgt cgagtccaca tcaacgttcg caacggtaag gcgactatgg 13680

tttaccgctg gaaggactct aagtcctcta agaatcacac tcagcgtatg acgttgacag 13740

atgagcaagc actgcgtctg gtcaatgcgc ttaccaaagc tgccgtgaca gcaattcatg 13800

aagctggtcg cgtcaatgaa gctatggcta tcctcgacaa gattgataac taagagtggt 13860

atcctcaagg tcgccaaagt ggtggccttc atgaatacta ttcgactcac tataggagat 13920

attaccatgc gtgaccctaa agttatccaa gcagaaatcg ctaaactgga agctgaactg 13980

gaggacgtta agtaccatga agctaagact cgctccgctg ttcacatctt gaagaactta 14040

ggctggactt ggacaagaca gactggctgg aagaaaccag aagttaccaa gctgagtcat 14100

aaggtgttcg ataaggacac tatgacccac atcaaggctg gtgattgggt taaggttgac 14160

atgggagttg ttggtggata cggctacgtc cgctcagtta gtggcaaata tgcacaagtg 14220

tcatacatca caggtgttac tccacgcggt gcaatcgttg ccgataagac caacatgatt 14280

cacacaggtt tcttgacagt tgtttcatat gaagagattg ttaagtcacg ataatcaata 14340

ggagaaatca atatgatcgt ttctgacatc gaagctaacg ccctcttaga gagcgtcact 14400

aagttccact gcggggttat ctacgactac tccaccgctg agtacgtaag ctaccgtccg 14460

agtgacttcg gtgcgtatct ggatgcgctg gaagccgagg ttgcacgagg cggtcttatt 14520

gtgttccaca acggtcacaa gtatgacgtt cctgcattga ccaaactggc aaagttgcaa 14580

ttgaaccgag agttccacct tcctcgtgag aactgtattg acacccttgt gttgtcacgt 14640

ttgattcatt ccaacctcaa ggacaccgat atgggtcttc tgcgttccgg caagttgccc 14700

ggaaaacgct ttgggtctca cgctttggag gcgtggggtt atcgcttagg cgagatgaag 14760

ggtgaataca aagacgactt taagcgtatg cttgaagagc agggtgaaga atacgttgac 14820

ggaatggagt ggtggaactt caacgaagag atgatggact ataacgttca ggacgttgtg 14880

gtaactaaag ctctccttga gaagctactc tctgacaaac attacttccc tcctgagatt 14940

gactttacgg acgtaggata cactacgttc tggtcagaat cccttgaggc cgttgacatt 15000

gaacatcgtg ctgcatggct gctcgctaaa caagagcgca acgggttccc gtttgacaca 15060

aaagcaatcg aagagttgta cgtagagtta gctgctcgcc gctctgagtt gctccgtaaa 15120

ttgaccgaaa cgttcggctc gtggtatcag cctaaaggtg gcactgagat gttctgccat 15180

ccgcgaacag gtaagccact acctaaatac cctcgcatta agacacctaa agttggtggt 15240

atctttaaga agcctaagaa caaggcacag cgagaaggcc gtgagccttg cgaacttgat 15300

acccgcgagt acgttgctgg tgctccttac accccagttg aacatgttgt gtttaaccct 15360

tcgtctcgtg accacattca gaagaaactc caagaggctg ggtgggtccc gaccaagtac 15420

accgataagg gtgctcctgt ggtggacgat gaggtactcg aaggagtacg tgtagatgac 15480

cctgagaagc aagccgctat cgacctcatt aaagagtact tgatgattca gaagcgaatc 15540

ggacagtctg ctgagggaga caaagcatgg cttcgttatg ttgctgagga tggtaagatt 15600

catggttctg ttaaccctaa tggagcagtt acgggtcgtg cgacccatgc gttcccaaac 15660

cttgcgcaaa ttccgggtgt acgttctcct tatggagagc agtgtcgcgc tgcttttggc 15720

gctgagcacc atttggatgg gataactggt aagccttggg ttcaggctgg catcgacgca 15780

tccggtcttg agctacgctg cttggctcac ttcatggctc gctttgataa cggcgagtac 15840

gctcacgaga ttcttaacgg cgacatccac actaagaacc agatagctgc tgaactacct 15900

acccgagata acgctaagac gttcatctat gggttcctct atggtgctgg tgatgagaag 15960

attggacaga ttgttggtgc tggtaaagag cgcggtaagg aactcaagaa gaaattcctt 16020

gagaacaccc ccgcgattgc agcactccgc gagtctatcc aacagacact tgtcgagtcc 16080

tctcaatggg tagctggtga gcaacaagtc aagtggaaac gccgctggat taaaggtctg 16140

gatggtcgta aggtacacgt tcgtagtcct cacgctgcct tgaataccct actgcaatct 16200

gctggtgctc tcatctgcaa actgtggatt atcaagaccg aagagatgct cgtagagaaa 16260

ggcttgaagc atggctggga tggggacttt gcgtacatgg catgggtaca tgatgaaatc 16320

caagtaggct gccgtaccga agagattgct caggtggtca ttgagaccgc acaagaagcg 16380

atgcgctggg ttggagacca ctggaacttc cggtgtcttc tggataccga aggtaagatg 16440

ggtcctaatt gggcgatttg ccactgatac aggaggctac tcatgaacga aagacactta 16500

acaggtgctg cttctgaaat gctagtagcc tacaaattta ccaaagctgg gtacactgtc 16560

tattacccta tgctgactca gagtaaagag gacttggttg tatgtaagga tggtaaattt 16620

agtaaggttc aggttaaaac agccacaacg gttcaaacca acacaggaga tgccaagcag 16680

gttaggctag gtggatgcgg taggtccgaa tataaggatg gagactttga cattcttgcg 16740

gttgtggttg acgaagatgt gcttattttc acatgggacg aagtaaaagg taagacatcc 16800

atgtgtgtcg gcaagagaaa caaaggcata aaactatagg agaaattatt atggctatga 16860

caaagaaatt taaagtgtcc ttcgacgtta ccgcaaagat gtcgtctgac gttcaggcaa 16920

tcttagagaa agatatgctg catctatgta agcaggtcgg ctcaggtgcg attgtcccca 16980

atggtaaaca gaaggaaatg attgtccagt tcctgacaca cggtatggaa ggattgatga 17040

cattcgtagt acgtacatca tttcgtgagg ccattaagga catgcacgaa gagtatgcag 17100

ataaggactc tttcaaacaa tctcctgcaa cagtacggga ggtgttctga tgtctgacta 17160

cctgaaagtg ctgcaagcaa tcaaaagttg ccctaagact ttccagtcca actatgtacg 17220

gaacaatgcg agcctcgtag cggaggccgc ttcccgtggt cacatctcgt gcctgactac 17280

tagtggacgt aacggtggcg cttgggaaat cactgcttcc ggtactcgct ttctgaaacg 17340

aatgggagga tgtgtctaat gtctcgtgac cttgtgacta ttccacgcga tgtgtggaac 17400

gatatacagg gctacatcga ctctctggaa cgtgagaacg atagccttaa gaatcaacta 17460

atggaagctg acgaatacgt agcggaacta gaggagaaac ttaatggcac ttcttgacct 17520

taaacaattc tatgagttac gtgaaggctg cgacgacaag ggtatccttg tgatggacgg 17580

cgactggctg gtcttccaag ctatgagtgc tgctgagttt gatgcctctt gggaggaaga 17640

gatttggcac cgatgctgtg accacgctaa ggcccgtcag attcttgagg attccattaa 17700

gtcctacgag acccgtaaga aggcttgggc aggtgctcca attgtccttg cgttcaccga 17760

tagtgttaac tggcgtaaag aactggttga cccgaactat aaggctaacc gtaaggccgt 17820

gaagaaacct gtagggtact ttgagttcct tgatgctctc tttgagcgcg aagagttcta 17880

ttgcatccgt gagcctatgc ttgagggtga tgacgttatg ggagttattg cttccaatcc 17940

gtctgccttc ggtgctcgta aggctgtaat catctcttgc gataaggact ttaagaccat 18000

ccctaactgt gacttcctgt ggtgtaccac tggtaacatc ctgactcaga ccgaagagtc 18060

cgctgactgg tggcacctct tccagaccat caagggtgac atcactgatg gttactcagg 18120

gattgctgga tggggtgata ccgccgagga cttcttgaat aacccgttca taaccgagcc 18180

taaaacgtct gtgcttaagt ccggtaagaa caaaggccaa gaggttacta aatgggttaa 18240

acgcgaccct gagcctcatg agacgctttg ggactgcatt aagtccattg gcgcgaaggc 18300

tggtatgacc gaagaggata ttatcaagca gggccaaatg gctcgaatcc tacggttcaa 18360

cgagtacaac tttattgaca aggagattta cctgtggaga ccgtagcgta tattggtctg 18420

ggtctttgtg ttctcggagt gtgcctcatt tcgtggggcc tttgggactt agccagaata 18480

atcaagtcgt tacacgacac taagtgataa actcaaggtc cctaaattaa tacgactcac 18540

tatagggaga taggggcctt tacgattatt actttaagat ttaactctaa gaggaatctt 18600

tattatgtta acacctatta accaattact taagaaccct aacgatattc cagatgtacc 18660

tcgtgcaacc gctgagtatc tacaggttcg attcaactat gcgtacctcg aagcgtctgg 18720

tcatatagga cttatgcgtg ctaatggttg tagtgaggcc cacatcttgg gtttcattca 18780

gggcctacag tatgcctcta acgtcattga cgagattgag ttacgcaagg aacaactaag 18840

agatgatggg gaggattgac actatgtgtt tctcaccgaa aattaaaact ccgaagatgg 18900

ataccaatca gattcgagcc gttgagccag cgcctctgac ccaagaagtg tcaagcgtgg 18960

agttcggtgg gtcttctgat gagacggata ccgagggcac cgaagtgtct ggacgcaaag 19020

gcctcaaggt cgaacgtgat gattccgtag cgaagtctaa agccagcggc aatggctccg 19080

ctcgtatgaa atcttccatc cgtaagtccg catttggagg taagaagtga tgtctgagtt 19140

cacatgtgtg gaggctaaga gtcgcttccg tgcaatccgg tggactgtgg aacaccttgg 19200

gttgcctaaa ggattcgaag gacactttgt gggctacagc ctctacgtag acgaagtgat 19260

ggacatgtct ggttgccgtg aagagtacat tctggactct accggaaaac atgtagcgta 19320

cttcgcgtgg tgcgtaagct gtgacattca ccacaaagga gacattctgg atgtaacgtc 19380

cgttgtcatt aatcctgagg cagactctaa gggcttacag cgattcctag cgaaacgctt 19440

taagtacctt gcggaactcc acgattgcga ttgggtgtct cgttgtaagc atgaaggcga 19500

gacaatgcgt gtatacttta aggaggtata agttatgggt aagaaagtta agaaggccgt 19560

gaagaaagtc accaagtccg ttaagaaagt cgttaaggaa ggggctcgtc cggttaaaca 19620

ggttgctggc ggtctagctg gtctggctgg tggtactggt gaagcacaga tggtggaagt 19680

accacaagct gccgcacaga ttgttgacgt acctgagaaa gaggtttcca ctgaggacga 19740

agcacagaca gaaagcggac gcaagaaagc tcgtgctggc ggtaagaaat ccttgagtgt 19800

agcccgtagc tccggtggcg gtatcaacat ttaatcagga ggttatcgtg gaagactgca 19860

ttgaatggac cggaggtgtc aactctaagg gttatggtcg taagtgggtt aatggtaaac 19920

ttgtgactcc acataggcac atctatgagg agacatatgg tccagttcca acaggaattg 19980

tggtgatgca tatctgcgat aaccctaggt gctataacat aaagcacctt acgcttggaa 20040

ctccaaagga taattccgag gacatggtta ccaaaggtag acaggctaaa ggagaggaac 20100

taagcaagaa acttacagag tcagacgttc tcgctatacg ctcttcaacc ttaagccacc 20160

gctccttagg agaactgtat ggagtcagtc aatcaaccat aacgcgaata ctacagcgta 20220

agacatggag acacatttaa tggctgagaa acgaacagga cttgcggagg atggcgcaaa 20280

gtctgtctat gagcgtttaa agaacgaccg tgctccctat gagacacgcg ctcagaattg 20340

cgctcaatat accatcccat cattgttccc taaggactcc gataacgcct ctacagatta 20400

tcaaactccg tggcaagccg tgggcgctcg tggtctgaac aatctagcct ctaagctcat 20460

gctggctcta ttccctatgc agacttggat gcgacttact atatctgaat atgaagcaaa 20520

gcagttactg agcgaccccg atggactcgc taaggtcgat gagggcctct cgatggtaga 20580

gcgtatcatc atgaactaca ttgagtctaa cagttaccgc gtgactctct ttgaggctct 20640

caaacagtta gtcgtagctg gtaacgtcct gctgtaccta ccggaaccgg aagggtcaaa 20700

ctataatccc atgaagctgt accgattgtc ttcttatgtg gtccaacgag acgcattcgg 20760

caacgttctg caaatggtga ctcgtgacca gatagctttt ggtgctctcc ctgaggacat 20820

ccgtaaggct gtagaaggtc aaggtggtga gaagaaagct gatgagacaa tcgacgtgta 20880

cactcacatc tatctggatg aggactcagg tgaatacctc cgatacgaag aggtcgaggg 20940

tatggaagtc caaggctccg atgggactta tcctaaagag gcttgcccat acatcccgat 21000

tcggatggtc agactagatg gtgaatccta cggtcgttcg tacattgagg aatacttagg 21060

tgacttacgg tcccttgaaa atctccaaga ggctatcgtc aagatgtcca tgattagctc 21120

taaggttatc ggcttagtga atcctgctgg tatcacccag ccacgccgac tgaccaaagc 21180

tcagactggt gacttcgtta ctggtcgtcc agaagacatc tcgttcctcc aactggagaa 21240

gcaagcagac tttactgtag ctaaagccgt aagtgacgct atcgaggctc gcctttcgtt 21300

tgcctttatg ttgaactctg cggttcagcg tacaggtgaa cgtgtgaccg ccgaagagat 21360

tcggtatgta gcttctgaac ttgaagatac tttaggtggt gtctactcta tcctttctca 21420

agaattacaa ttgcctctgg tacgagtgct cttgaagcaa ctacaagcca cgcaacagat 21480

tcctgagtta cctaaggaag ccgtagagcc aaccattagt acaggtctgg aagcaattgg 21540

tcgaggacaa gaccttgata agctggagcg gtgtgtcact gcgtgggctg cactggcacc 21600

tatgcgggac gaccctgata ttaaccttgc gatgattaag ttacgtattg ccaacgctat 21660

cggtattgac acttctggta ttctactcac cgaagaacag aagcaacaga agatggccca 21720

acagtctatg caaatgggta tggataatgg tgctgctgcg ctggctcaag gtatggctgc 21780

acaagctaca gcttcacctg aggctatggc tgctgccgct gattccgtag gtttacagcc 21840

gggaatttaa tacgactcac tatagggaga cctcatcttt gaaatgagcg atgacaagag 21900

gttggagtcc tcggtcttcc tgtagttcaa ctttaaggag acaataataa tggctgaatc 21960

taatgcagac gtatatgcat cttttggcgt gaactccgct gtgatgtctg gtggttccgt 22020

tgaggaacat gagcagaaca tgctggctct tgatgttgct gcccgtgatg gcgatgatgc 22080

aatcgagtta gcgtcagacg aagtggaaac agaacgtgac ctgtatgaca actctgaccc 22140

gttcggtcaa gaggatgacg aaggccgcat tcaggttcgt atcggtgatg gctctgagcc 22200

gaccgatgtg gacactggag aagaaggcgt tgagggcacc gaaggttccg aagagtttac 22260

cccactgggc gagactccag aagaactggt agctgcctct gagcaacttg gtgagcacga 22320

agagggcttc caagagatga ttaacattgc tgctgagcgt ggcatgagtg tcgagaccat 22380

tgaggctatc cagcgtgagt acgaggagaa cgaagagttg tccgccgagt cctacgctaa 22440

gctggctgaa attggctaca cgaaggcttt cattgactcg tatatccgtg gtcaagaagc 22500

tctggtggag cagtacgtaa acagtgtcat tgagtacgct ggtggtcgtg aacgttttga 22560

tgcactgtat aaccaccttg agacgcacaa ccctgaggct gcacagtcgc tggataatgc 22620

gttgaccaat cgtgacttag cgaccgttaa ggctatcatc aacttggctg gtgagtctcg 22680

cgctaaggcg ttcggtcgta agccaactcg tagtgtgact aatcgtgcta ttccggctaa 22740

acctcaggct accaagcgtg aaggctttgc ggaccgtagc gagatgatta aagctatgag 22800

tgaccctcgg tatcgcacag atgccaacta tcgtcgtcaa gtcgaacaga aagtaatcga 22860

ttcgaacttc tgatagactt cgaaattaat acgactcact atagggagac cacaacggtt 22920

tccctctaga aataattttg tttaacttta agaaggagat atacatatgg ctagcatgac 22980

tggtggacag caaatgggta ctaaccaagg taaaggtgta gttgctgctg gagataaact 23040

ggcgttgttc ttgaaggtat ttggcggtga agtcctgact gcgttcgctc gtacctccgt 23100

gaccacttct cgccacatgg tacgttccat ctccagcggt aaatccgctc agttccctgt 23160

tctgggtcgc actcaggcag cgtatctggc tccgggcgag aacctcgacg ataaacgtaa 23220

ggacatcaaa cacaccgaga aggtaatcac cattgacggt ctcctgacgg ctgacgttct 23280

gatttatgat attgaggacg cgatgaacca ctacgacgtt cgctctgagt atacctctca 23340

gttgggtgaa tctctggcga tggctgcgga tggtgcggtt ctggctgaga ttgccggtct 23400

gtgtaacgtg gaaagcaaat ataatgagaa catcgagggc ttaggtactg ctaccgtaat 23460

tgagaccact cagaacaagg ccgcacttac cgaccaagtt gcgctgggta aggagattat 23520

tgcggctctg actaaggctc gtgcggctct gaccaagaac tatgttccgg ctgctgaccg 23580

tgtgttctac tgtgacccag atagctactc tgcgattctg gcagcactga tgccgaacgc 23640

agcaaactac gctgctctga ttgaccctga gaagggttct atccgcaacg ttatgggctt 23700

tgaggttgta gaagttccgc acctcaccgc tggtggtgct ggtaccgctc gtgagggcac 23760

tactggtcag aagcacgtct tccctgccaa taaaggtgag ggtaatgtca aggttgctaa 23820

ggacaacgtt atcggcctgt tcatgcaccg ctctgcggta ggtactgtta agctgcgtga 23880

cttggctctg gagcgcgctc gccgtgctaa cttccaagcg gaccagatta tcgctaagta 23940

cgcaatgggc cacggtggtc ttcgcccaga agctgctggt gcagtggttt tcaaagtgga 24000

gtaatgctgg gggtggcctc aacggtcgct gctagtcccg aagaggcgag tgttacttca 24060

acagaagaaa ccttaacgcc agcacaggag gccgcacgca cccgcgctgc taacaaagcc 24120

cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 24180

gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactattta aatattctcc 24240

ctgtggtggc tcgaaattaa tacgactcac tatagggaga acaatacgac tacgggaggg 24300

ttttcttatg atgactataa gacctactaa aagtacagac tttgaggtat tcactccggc 24360

tcaccatgac attcttgaag ctaaggctgc tggtattgag ccgagtttcc ctgatgcttc 24420

cgagtgtgtc acgttgagcc tctatgggtt ccctctagct atcggtggta actgcgggga 24480

ccagtgctgg ttcgttacga gcgaccaagt gtggcgactt agtggaaagg ctaagcgaaa 24540

gttccgtaag ttaatcatgg agtatcgcga taagatgctt gagaagtatg atactctttg 24600

gaattacgta tgggtaggca atacgtccca cattcgtttc ctcaagacta tcggtgcggt 24660

attccatgaa gagtacacac gagatggtca atttcagtta tttacaatca cgaaaggagg 24720

ataaccatat gtgttgggca gccgcaatac ctatcgctat atctggcgct caggctatca 24780

gtggtcagaa cgctcaggcc aaaatgattg ccgctcagac cgctgctggt cgtcgtcaag 24840

ctatggaaat catgaggcag acgaacatcc agaatgctga cctatcgttg caagctcgaa 24900

gtaaacttga ggaagcgtcc gccgagttga cctcacagaa catgcagaag gtccaagcta 24960

ttgggtctat ccgagcggct atcggagaga gtatgcttga aggttcctca atggaccgca 25020

ttaagcgagt cacagaagga cagttcattc gggaagccaa tatggtaact gagaactatc 25080

gccgtgacta ccaagcaatc ttcgcacagc aacttggtgg tactcaaagt gctgcaagtc 25140

agattgacga aatctataag agcgaacaga aacagaagag taagctacag atggttctgg 25200

acccactggc tatcatgggg tcttccgctg cgagtgctta cgcatccggt gcgttcgact 25260

ctaagtccac aactaaggca cctattgttg ccgctaaagg aaccaagacg gggaggtaat 25320

gagctatgag taaaattgaa tctgcccttc aagcggcaca accgggactc tctcggttac 25380

gtggtggtgc tggaggtatg ggctatcgtg cagcaaccac tcaggccgaa cagccaaggt 25440

caagcctatt ggacaccatt ggtcggttcg ctaaggctgg tgccgatatg tataccgcta 25500

aggaacaacg agcacgagac ctagctgatg aacgctctaa cgagattatc cgtaagctga 25560

cccctgagca acgtcgagaa gctctcaaca acgggaccct tctgtatcag gatgacccat 25620

acgctatgga agcactccga gtcaagactg gtcgtaacgc tgcgtatctt gtggacgatg 25680

acgttatgca gaagataaaa gagggtgtct tccgtactcg cgaagagatg gaagagtatc 25740

gccatagtcg ccttcaagag ggcgctaagg tatacgctga gcagttcggc atcgaccctg 25800

aggacgttga ttatcagcgt ggtttcaacg gggacattac cgagcgtaac atctcgctgt 25860

atggtgcgca tgataacttc ttgagccagc aagctcagaa gggcgctatc atgaacagcc 25920

gagtggaact caacggtgtc cttcaagacc ctgatatgct gcgtcgtcca gactctgctg 25980

acttctttga gaagtatatc gacaacggtc tggttactgg cgcaatccca tctgatgctc 26040

aagccacaca gcttataagc caagcgttca gtgacgcttc tagccgtgct ggtggtgctg 26100

acttcctgat gcgagtcggt gacaagaagg taacacttaa cggagccact acgacttacc 26160

gagagttgat tggtgaggaa cagtggaacg ctctcatggt cacagcacaa cgttctcagt 26220

ttgagactga cgcgaagctg aacgagcagt atcgcttgaa gattaactct gcgctgaacc 26280

aagaggaccc aaggacagct tgggagatgc ttcaaggtat caaggctgaa ctagataagg 26340

tccaacctga tgagcagatg acaccacaac gtgagtggct aatctccgca caggaacaag 26400

ttcagaatca gatgaacgca tggacgaaag ctcaggccaa ggctctggac gattccatga 26460

agtcaatgaa caaacttgac gtaatcgaca agcaattcca gaagcgaatc aacggtgagt 26520

gggtctcaac ggattttaag gatatgccag tcaacgagaa cactggtgag ttcaagcata 26580

gcgatatggt taactacgcc aataagaagc tcgctgagat tgacagtatg gacattccag 26640

acggtgccaa ggatgctatg aagttgaagt accttcaagc ggactctaag gacggagcat 26700

tccgtacagc catcggaacc atggtcactg acgctggtca agagtggtct gccgctgtga 26760

ttaacggtaa gttaccagaa cgaaccccag ctatggatgc tctgcgcaga atccgcaatg 26820

ctgaccctca gttgattgct gcgctatacc cagaccaagc tgagctattc ctgacgatgg 26880

acatgatgga caagcagggt attgaccctc aggttattct tgatgccgac cgactgactg 26940

ttaagcggtc caaagagcaa cgctttgagg atgataaagc attcgagtct gcactgaatg 27000

catctaaggc tcctgagatt gcccgtatgc cagcgtcact gcgcgaatct gcacgtaaga 27060

tttatgactc cgttaagtat cgctcgggga acgaaagcat ggctatggag cagatgacca 27120

agttccttaa ggaatctacc tacacgttca ctggtgatga tgttgacggt gataccgttg 27180

gtgtgattcc taagaatatg atgcaggtta actctgaccc gaaatcatgg gagcaaggtc 27240

gggatattct ggaggaagca cgtaagggaa tcattgcgag caacccttgg ataaccaata 27300

agcaactgac catgtattct caaggtgact ccatttacct tatggacacc acaggtcaag 27360

tcagagtccg atacgacaaa gagttactct cgaaggtctg gagtgagaac cagaagaaac 27420

tcgaagagaa agctcgtgag aaggctctgg ctgatgtgaa caagcgagca cctatagttg 27480

ccgctacgaa ggcccgtgaa gctgctgcta aacgagtccg agagaaacgt aaacagactc 27540

ctaagttcat ctacggacgt aaggagtaac taaaggctac ataaggaggc cctaaatgga 27600

taagtacgat aagaacgtac caagtgatta tgatggtctg ttccaaaagg ctgctgatgc 27660

caacggggtc tcttatgacc ttttacgtaa agtcgcttgg acagaatcac gatttgtgcc 27720

tacagcaaaa tctaagactg gaccattagg catgatgcaa tttaccaagg caaccgctaa 27780

ggccctcggt ctgcgagtta ccgatggtcc agacgacgac cgactgaacc ctgagttagc 27840

tattaatgct gccgctaagc aacttgcagg tctggtaggg aagtttgatg gcgatgaact 27900

caaagctgcc cttgcgtaca accaaggcga gggacgcttg ggtaatccac aacttgaggc 27960

gtactctaag ggagacttcg catcaatctc tgaggaggga cgtaactaca tgcgtaacct 28020

tctggatgtt gctaagtcac ctatggctgg acagttggaa acttttggtg gcataacccc 28080

aaagggtaaa ggcattccgg ctgaggtagg attggctgga attggtcaca agcagaaagt 28140

aacacaggaa cttcctgagt ccacaagttt tgacgttaag ggtatcgaac aggaggctac 28200

ggcgaaacca ttcgccaagg acttttggga gacccacgga gaaacacttg acgagtacaa 28260

cagtcgttca accttcttcg gattcaaaaa tgctgccgaa gctgaactct ccaactcagt 28320

cgctgggatg gctttccgtg ctggtcgtct cgataatggt tttgatgtgt ttaaagacac 28380

cattacgccg actcgctgga actctcacat ctggactcca gaggagttag agaagattcg 28440

aacagaggtt aagaaccctg cgtacatcaa cgttgtaact ggtggttccc ctgagaacct 28500

cgatgacctc attaaattgg ctaacgagaa ctttgagaat gactcccgcg ctgccgaggc 28560

tggcctaggt gccaaactga gtgctggtat tattggtgct ggtgtggacc cgcttagcta 28620

tgttcctatg gtcggtgtca ctggtaaggg ctttaagtta atcaataagg ctcttgtagt 28680

tggtgccgaa agtgctgctc tgaacgttgc atccgaaggt ctccgtacct ccgtagctgg 28740

tggtgacgca gactatgcgg gtgctgcctt aggtggcttt gtgtttggcg caggcatgtc 28800

tgcaatcagt gacgctgtag ctgctggact gaaacgcagt aaaccagaag ctgagttcga 28860

caatgagttc atcggtccta tgatgcgatt ggaagcccgt gagacagcac gaaacgccaa 28920

ctctgcggac ctctctcgga tgaacactga gaacatgaag tttgaaggtg aacataatgg 28980

tgtcccttat gaggacttac caacagagag aggtgccgtg gtgttacatg atggctccgt 29040

tctaagtgca agcaacccaa tcaaccctaa gactctaaaa gagttctccg aggttgaccc 29100

tgagaaggct gcgcgaggaa tcaaactggc tgggttcacc gagattggct tgaagacctt 29160

ggggtctgac gatgctgaca tccgtagagt ggctatcgac ctcgttcgct ctcctactgg 29220

tatgcagtct ggtgcctcag gtaagttcgg tgcaacagct tctgacatcc atgagagact 29280

tcatggtact gaccagcgta cttataatga cttgtacaaa gcaatgtctg acgctatgaa 29340

agaccctgag ttctctactg gcggcgctaa gatgtcccgt gaagaaactc gatacactat 29400

ctaccgtaga gcggcactag ctattgagcg tccagaacta cagaaggcac tcactccgtc 29460

tgagagaatc gttatggaca tcattaagcg tcactttgac accaagcgtg aacttatgga 29520

aaacccagca atattcggta acacaaaggc tgtgagtatc ttccctgaga gtcgccacaa 29580

aggtacttac gttcctcacg tatatgaccg tcatgccaag gcgctgatga ttcaacgcta 29640

cggtgccgaa ggtttgcagg aagggattgc ccgctcatgg atgaacagct acgtctccag 29700

acctgaggtc aaggccagag tcgatgagat gcttaaggaa ttacacgggg tgaaggaagt 29760

aacaccagag atggtagaga agtacgctat ggataaggct tatggtatct cccactcaga 29820

ccagttcacc aacagttcca taatagaaga gaacattgag ggcttagtag gtatcgagaa 29880

taactcattc cttgaggcac gtaacttgtt tgattcggac ctatccatca ctatgccaga 29940

cggacagcaa ttctcagtga atgacctaag ggacttcgat atgttccgca tcatgccagc 30000

gtatgaccgc cgtgtcaatg gtgacatcgc catcatgggg tctactggta aaaccactaa 30060

ggaacttaag gatgagattt tggctctcaa agcgaaagct gagggagacg gtaagaagac 30120

tggcgaggta catgctttaa tggataccgt taagattctt actggtcgtg ctagacgcaa 30180

tcaggacact gtgtgggaaa cctcactgcg tgccatcaat gacctagggt tcttcgctaa 30240

gaacgcctac atgggtgctc agaacattac ggagattgct gggatgattg tcactggtaa 30300

cgttcgtgct ctagggcatg gtatcccaat tctgcgtgat acactctaca agtctaaacc 30360

agtttcagct aaggaactca aggaactcca tgcgtctctg ttcgggaagg aggtggacca 30420

gttgattcgg cctaaacgtg ctgacattgt gcagcgccta agggaagcaa ctgataccgg 30480

acctgccgtg gcgaacatcg tagggacctt gaagtattca acacaggaac tggctgctcg 30540

ctctccgtgg actaagctac tgaacggaac cactaactac cttctggatg ctgcgcgtca 30600

aggtatgctt ggggatgtta ttagtgccac cctaacaggt aagactaccc gctgggagaa 30660

agaaggcttc cttcgtggtg cctccgtaac tcctgagcag atggctggca tcaagtctct 30720

catcaaggaa catatggtac gcggtgagga cgggaagttt accgttaagg acaagcaagc 30780

gttctctatg gacccacggg ctatggactt atggagactg gctgacaagg tagctgatga 30840

ggcaatgctg cgtccacata aggtgtcctt acaggattcc catgcgttcg gagcactagg 30900

taagatggtt atgcagttta agtctttcac tatcaagtcc cttaactcta agttcctgcg 30960

aaccttctat gatggataca agaacaaccg agcgattgac gctgcgctga gcatcatcac 31020

ctctatgggt ctcgctggtg gtttctatgc tatggctgca cacgtcaaag catacgctct 31080

gcctaaggag aaacgtaagg agtacttgga gcgtgcactg gacccaacca tgattgccca 31140

cgctgcgtta tctcgtagtt ctcaattggg tgctcctttg gctatggttg acctagttgg 31200

tggtgtttta gggttcgagt cctccaagat ggctcgctct acgattctac ctaaggacac 31260

cgtgaaggaa cgtgacccaa acaaaccgta cacctctaga gaggtaatgg gcgctatggg 31320

ttcaaacctt ctggaacaga tgccttcggc tggctttgtg gctaacgtag gggctacctt 31380

aatgaatgct gctggcgtgg tcaactcacc taataaagca accgagcagg acttcatgac 31440

tggtcttatg aactccacaa aagagttagt accgaacgac ccattgactc aacagcttgt 31500

gttgaagatt tatgaggcga acggtgttaa cttgagggag cgtaggaaat aatacgactc 31560

actataggga gaggcgaaat aatcttctcc ctgtagtctc ttagatttac tttaaggagg 31620

tcaattggta aatcacaagg aaagacgtgt agtccacgga tggactctca aggaggtaca 31680

aggtgctatc attagacttt aacaacgaat tgattaaggc tgctccaatt gttgggacgg 31740

gtgtagcaga tgttagtgct cgactgttct ttgggttaag ccttaacgaa tggttctacg 31800

ttgctgctat cgcctacaca gtggttcaga ttggtgccaa ggtagtcgat aagatgattg 31860

actggaagaa agccaataag gagtgatatg tatggaaaag gataagagcc ttattacatt 31920

cttagagatg ttggacactg cgatggctca gcgtatgctt gcggaccttt cggaccatga 31980

gcgtcgctct ccgcaactct ataatgctat taacaaactg ttagaccgcc acaagttcca 32040

gattggtaag ttgcagccgg atgttcacat cttaggtggc cttgctggtg ctcttgaaga 32100

gtacaaagag aaagtcggtg ataacggtct tacggatgat gatatttaca cattacagtg 32160

atatactcaa ggccactaca gatagtggtc tttatggatg tcattgtcta tacgagatgc 32220

tcctacgtga aatctgaaag ttaacgggag gcattatgct agaattttta cgtaagctaa 32280

tcccttgggt tctcgctggg atgctattcg ggttaggatg gcatctaggg tcagactcaa 32340

tggacgctaa atggaaacag gaggtacaca atgagtacgt taagagagtt gaggctgcga 32400

agagcactca aagagcaatc gatgcggtat ctgctaagta tcaagaagac cttgccgcgc 32460

tggaagggag cactgatagg attatttctg atttgcgtag cgacaataag cggttgcgcg 32520

tcagagtcaa aactaccgga acctccgatg gtcagtgtgg attcgagcct gatggtcgag 32580

ccgaacttga cgaccgagat gctaaacgta ttctcgcagt gacccagaag ggtgacgcat 32640

ggattcgtgc gttacaggat actattcgtg aactgcaacg taagtaggaa atcaagtaag 32700

gaggcaatgt gtctactcaa tccaatcgta atgcgctcgt agtggcgcaa ctgaaaggag 32760

acttcgtggc gttcctattc gtcttatgga aggcgctaaa cctaccggtg cccactaagt 32820

gtcagattga catggctaag gtgctggcga atggagacaa caagaagttc atcttacagg 32880

ctttccgtgg tatcggtaag tcgttcatca catgtgcgtt cgttgtgtgg tccttatgga 32940

gagaccctca gttgaagata cttatcgtat cagcctctaa ggagcgtgca gacgctaact 33000

ccatctttat taagaacatc attgacctgc tgccattcct atctgagtta aagccaagac 33060

ccggacagcg tgactcggta atcagctttg atgtaggccc agccaatcct gaccactctc 33120

ctagtgtgaa atcagtaggt atcactggtc agttaactgg tagccgtgct gacattatca 33180

ttgcggatga cgttgagatt ccgtctaaca gcgcaactat gggtgcccgt gagaagctat 33240

ggactctggt tcaggagttc gctgcgttac ttaaaccgct gccttcctct cgcgttatct 33300

accttggtac acctcagaca gagatgactc tctataagga acttgaggat aaccgtgggt 33360

acacaaccat tatctggcct gctctgtacc caaggacacg tgaagagaac ctctattact 33420

cacagcgtct tgctcctatg ttacgcgctg agtacgatga gaaccctgag gcacttgctg 33480

ggactccaac agacccagtg cgctttgacc gtgatgacct gcgcgagcgt gagttggaat 33540

acggtaaggc tggctttacg ctacagttca tgcttaaccc taaccttagt gatgccgaga 33600

agtacccgct gaggcttcgt gacgctatcg tagcggcctt agacttagag aaggccccaa 33660

tgcattacca gtggcttccg aaccgtcaga acatcattga ggaccttcct aacgttggcc 33720

ttaagggtga tgacctgcat acgtaccacg attgttccaa caactcaggt cagtaccaac 33780

agaagattct ggtcattgac cctagtggtc gcggtaagga cgaaacaggt tacgctgtgc 33840

tgtacacact gaacggttac atctacctta tggaagctgg aggtttccgt gatggctact 33900

ccgataagac ccttgagtta ctcgctaaga aggcaaagca atggggagtc cagacggttg 33960

tctacgagag taacttcggt gacggtatgt tcggtaaggt attcagtcct atccttctta 34020

aacaccacaa ctgtgcgatg gaagagattc gtgcccgtgg tatgaaagag atgcgtattt 34080

gcgataccct tgagccagtc atgcagactc accgccttgt aattcgtgat gaggtcatta 34140

gggccgacta ccagtccgct cgtgacgtag acggtaagca tgacgttaag tactcgttgt 34200

tctaccagat gacccgtatc actcgtgaga aaggcgctct ggctcatgat gaccgattgg 34260

atgcccttgc gttaggcatt gagtatctcc gtgagtccat gcagttggat tccgttaagg 34320

tcgagggtga agtacttgct gacttccttg aggaacacat gatgcgtcct acggttgctg 34380

ctacgcatat cattgagatg tctgtgggag gagttgatgt gtactctgag gacgatgagg 34440

gttacggtac gtctttcatt gagtggtgat ttatgcatta ggactgcata gggatgcact 34500

atagaccacg gatggtcagt tctttaagtt actgaaaaga cacgataaat taatacgact 34560

cactataggg agaggaggga cgaaaggtta ctatatagat actgaatgaa tacttataga 34620

gtgcataaag tatgcataat ggtgtaccta gagtgacctc taagaatggt gattatattg 34680

tattagtatc accttaactt aaggaccaac ataaagggag gagactcatg ttccgcttat 34740

tgttgaacct actgcggcat agagtcacct accgatttct tgtggtactt tgtgctgccc 34800

ttgggtacgc atctcttact ggagacctca gttcactgga gtctgtcgtt tgctctatac 34860

tcacttgtag cgattagggt cttcctgacc gactgatggc tcaccgaggg attcagcggt 34920

atgattgcat cacaccactt catccctata gagtcaagtc ctaaggtata cccataaaga 34980

gcctctaatg gtctatccta aggtctatac ctaaagatag gccatcctat cagtgtcacc 35040

taaagagggt cttagagagg gcctatggag ttcctatagg gtcctttaaa atataccata 35100

aaaatctgag tgactatctc acagtgtacg gacctaaagt tcccccatag ggggtaccta 35160

aagcccagcc aatcacctaa agtcaacctt cggttgacct tgagggttcc ctaagggttg 35220

gggatgaccc ttgggtttgt ctttgggtgt taccttgagt gtctctctgt gtccct 35276

<210> 8

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 8

cttgaagacg aaagggcctc gtgatacgcc tctcacagtg tacggaccta aagttccccc 60

<210> 9

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 9

attacgcgat gacagtagac aacctttccg 30

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 10

tgcagcaata ccggaaaggt tgtctactgt 30

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 11

atatgtctcc tcatagatgt gcctatgtgg 30

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 12

acttgtgact ccacataggc acatctatga 30

<210> 13

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 13

agggagaata tttaaatagt tcctcctttc agcaaaaaac ccctc 45

<210> 14

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 14

gaaaggagga actatttaaa tattctccct gtggtggctc g 41

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 15

gaataacctg agggtcaata ccctgcttgt 30

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 16

gacatgatgg acaagcaggg tattgaccct 30

<210> 17

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 17

cttgtgattt accaattgac ctccttaaag taaatctaag agac 44

<210> 18

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 18

ctttaaggag gtcaattggt aaatcacaag gaaagacgtg tagtc 45

<210> 19

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 19

cataatagaa acgacacgaa attacaaaat agggacacag agagacactc aaggtaacac 60

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 20

attttgtaat ttcgtgtcgt ttctattatg 30

<210> 21

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 21

ggcgtatcac gaggcccttt cgtcttcaag 30

<210> 22

<211> 38661

<212> DNA

<213> Artificial sequence

<220>

<223> head Gene-deleted phage genome

<400> 22

tctcacagtg tacggaccta aagttccccc atagggggta cctaaagccc agccaatcac 60

ctaaagtcaa ccttcggttg accttgaggg ttccctaagg gttggggatg acccttgggt 120

ttgtctttgg gtgttacctt gagtgtctct ctgtgtccct atctgttaca gtctcctaaa 180

gtatcctcct aaagtcacct cctaacgtcc atcctaaagc caacacctaa agcctacacc 240

taaagaccca tcaagtcaac gcctatctta aagtttaaac ataaagacca gacctaaaga 300

ccagacctaa agacactaca taaagaccag acctaaagac gccttgttgt tagccataaa 360

gtgataacct ttaatcattg tctttattaa tacaactcac tataaggaga gacaacttaa 420

agagacttaa aagattaatt taaaatttat caaaaagagt attgacttaa agtctaacct 480

ataggatact tacagccatc gagagggaca cggcgaatag ccatcccaat cgacaccggg 540

gtcaaccgga taagtagaca gcctgataag tcgcacgaaa aacaggtatt gacaacatga 600

agtaacatgc agtaagatac aaatcgctag gtaacactag cagcgtcaac cgggcgcaca 660

gtgccttcta ggtgacttaa gcgcaccacg gcacataagg tgaaacaaaa cggttgacaa 720

catgaagtaa acacggtacg atgtaccaca tgaaacgaca gtgagtcacc acactgaaag 780

gtgatgcggt ctaacgaaac ctgacctaag acgctcttta acaatctggt aaatagctct 840

tgagtgcatg actagcggat aactcaaggg tatcgcaagg tgccctttat gatattcact 900

aataactgca cgaggtaaca caagatggct atgtctaaca tgacttacaa caacgttttc 960

gaccacgctt acgaaatgct gaaagaaaac atccgttatg atgacatccg tgacactgat 1020

gacctgcacg atgctattca catggctgcc gataatgcag ttccgcacta ctacgctgac 1080

atctttagcg taatggcaag tgagggcatt gaccttgagt tcgaagactc tggtctgatg 1140

cctgacacca aggacgtaat ccgcatcctg caagcgcgta tctatgagca attaacgatt 1200

gacctctggg aagacgcaga agacttgctc aatgaatact tggaggaagt cgaggagtac 1260

gaggaggatg aagagtaatg tctactacca acgtgcaata cggtctgacc gctcaaactg 1320

tacttttcta tagcgacatg gtgcgctgtg gctttaactg gtcactcgca atggcacagc 1380

tcaaagaact gtacgaaaac aacaaggcaa tagctttaga atctgctgag tgatagactc 1440

aaggtcgctc ctagcgagtg gcctttatga ttatcacttt acttatgagg gagtaatgta 1500

tatgcttact atcggtctac tcaccgctct aggtctagct gtaggtgcat cctttgggaa 1560

ggctttaggt gtagctgtag gttcctactt taccgcttgc atcatcatag gaatcatcaa 1620

aggggcacta cgcaaatgat gaagcactac gttatgccaa tccacacgtc caacggggca 1680

accgtatgta cacctgatgg gttcgcaatg aaacaacgaa tcgaacgcct taagcgtgaa 1740

ctccgcatta accgcaagat taacaagata ggttccggct atgacagaac gcactgatgg 1800

cttaaagaaa ggttatatgc ccaatggcac actatacgct gcaaatcggc gaatagtgag 1860

aacttggcga gagaacaacc tcgaacgccg caaggacaag agagggcggc gtggcataga 1920

cgaaaggaaa aggttaaagc caagaaactc gccgcacttg aacaggcact agccaacaca 1980

ctgaacgcta tctcataacg aacataaagg acacaatgca atgaacatta ccgacatcat 2040

gaacgctatc gacgcaatca aagcactgcc aatctgtgaa cttgacaagc gtcaaggtat 2100

gcttatcgac ttactggtcg agatggtcaa cagcgagacg tgtgatggcg agctaaccga 2160

actaaatcag gcacttgagc atcaagattg gtggactacc ttgaagtgtc tcacggctga 2220

cgcagggttc aagatgctcg gtaatggtca cttctcggct gcttatagtc acccgctgct 2280

acctaacaga gtgattaagg tgggctttaa gaaagaggat tcaggcgcag cctataccgc 2340

attctgccgc atgtatcagg gtcgtcctgg tatccctaac gtctacgatg tacagcgcca 2400

cgctggatgc tatacggtgg tacttgacgc acttaaggat tgcgagcgtt tcaacaatga 2460

tgcccattat aaatacgctg agattgcaag cgacatcatt gattgcaatt cggatgagca 2520

tgatgagtta actggatggg atggtgagtt tgttgaaact tgtaaactaa tccgcaagtt 2580

ctttgagggc atcgcctcat tcgacatgca tagcgggaac atcatgttct caaatggaga 2640

cgtaccatac atcaccgacc cggtatcatt ctcgcagaag aaagacggtg gcgcattcag 2700

catcgaccct gaggaactca tcaaggaagt cgaggaagtc gcacgacaga aagaaattga 2760

ccgcgctaag gcccgtaaag aacgtcacga ggggcgctta gaggcacgca gattcaaacg 2820

tcgcaaccgc aaggcacgta aagcacacaa agctaagcgc gaaagaatgc ttgctgcgtg 2880

gcgatgggct gaacgtcaag aacggcgtaa ccatgaggta gctgtagatg tactaggaag 2940

aaccaataac gctatgctct gggtcaacat gttctctggg gactttaagg cgcttgagga 3000

acgaatcgcg ctgcactggc gtaatgctga ccggatggct atcgctaatg gtcttacgct 3060

caacattgat aagcaacttg acgcaatgtt aatgggctga tagtcttatc ttacaggtca 3120

tctgcgggtg gcctgaatag gtacgattta ctaactggaa gaggcactaa atgaacacga 3180

ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg ttcaacactc 3240

tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag catgagtctt 3300

acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa gctggtgagg 3360

ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag atgattgcac 3420

gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg acagccttcc 3480

agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag accactctgg 3540

cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca atcggtcggg 3600

ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag cacttcaaga 3660

aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa gcatttatgc 3720

aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg tggtcttcgt 3780

ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc attgagtcaa 3840

ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac tctgagacta 3900

tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg ctggctggca 3960

tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc attactggtg 4020

gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac agtaagaaag 4080

cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt aacattgcgc 4140

aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta atcaccaagt 4200

ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc ccgatgaaac 4260

cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct gccgctgctg 4320

tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc atgcttgagc 4380

aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg gactggcgcg 4440

gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc aaaggactgc 4500

ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg aaaatccacg 4560

gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag ttcattgagg 4620

aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact tggtgggctg 4680

agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg gtacagcacc 4740

acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc tctggcatcc 4800

agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac ttgcttccta 4860

gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag attctacaag 4920

cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag aacactggtg 4980

aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg ctggcttacg 5040

gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg tccaaagagt 5100

tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat tccggcaagg 5160

gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg atttgggaat 5220

ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag tctgctgcta 5280

agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc aagcgttgcg 5340

ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag aagcctattc 5400

agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc attaacacca 5460

acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct aactttgtac 5520

acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag aagtacggaa 5580

tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac gctgcgaacc 5640

tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat gtactggctg 5700

atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa atgccagcac 5760

ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc gcgttcgcgt 5820

aacgccaaat caatacgact cactatagag ggacaaactc aaggtcattc gcaagagtgg 5880

cctttatgat tgaccttctt ccggttaata cgactcacta taggagaacc ttaaggttta 5940

actttaagac ccttaagtgt taattagaga tttaaattaa agaattacta agagaggact 6000

ttaagtatgc gtaacttcga aaagatgacc aaacgttcta accgtaatgc tcgtgacttc 6060

gaggcaacca aaggtcgcaa gttgaataag actaagcgtg accgctctca caagcgtagc 6120

tgggagggtc agtaagatgg gacgtttata tagtggtaat ctggcagcat tcaaggcagc 6180

aacaaacaag ctgttccagt tagacttagc ggtcatttat gatgactggt atgatgccta 6240

tacaagaaaa gattgcatac ggttacgtat tgaggacagg agtggaaacc tgattgatac 6300

tagcaccttc taccaccacg acgaggacgt tctgttcaat atgtgtactg attggttgaa 6360

ccatatgtat gaccagttga aggactggaa gtaatacgac tcagtatagg gacaatgctt 6420

aaggtcgctc tctaggagtg gccttagtca tttaaccaat aggagataaa cattatgatg 6480

aacattaaga ctaacccgtt taaagccgtg tctttcgtag agtctgccat taagaaggct 6540

ctggataacg ctgggtatct tatcgctgaa atcaagtacg atggtgtacg cgggaacatc 6600

tgcgtagaca atactgctaa cagttactgg ctctctcgtg tatctaaaac gattccggca 6660

ctggagcact taaacgggtt tgatgttcgc tggaagcgtc tactgaacga tgaccgttgc 6720

ttctacaaag atggctttat gcttgatggg gaactcatgg tcaagggcgt agactttaac 6780

acagggtccg gcctactgcg taccaaatgg actgacacga agaaccaaga gttccatgaa 6840

gagttattcg ttgaaccaat ccgtaagaaa gataaagttc cctttaagct gcacactgga 6900

caccttcaca taaaactgta cgctatcctc ccgctgcaca tcgtggagtc tggagaagac 6960

tgtgatgtca tgacgttgct catgcaggaa cacgttaaga acatgctgcc tctgctacag 7020

gaatacttcc ctgaaatcga atggcaagcg gctgaatctt acgaggtcta cgatatggta 7080

gaactacagc aactgtacga gcagaagcga gcagaaggcc atgagggtct cattgtgaaa 7140

gacccgatgt gtatctataa gcgcggtaag aaatctggct ggtggaaaat gaaacctgag 7200

aacgaagctg acggtatcat tcagggtctg gtatggggta caaaaggtct ggctaatgaa 7260

ggtaaagtga ttggttttga ggtgcttctt gagagtggtc gtttagttaa cgccacgaat 7320

atctctcgcg ccttaatgga tgagttcact gagacagtaa aagaggccac cctaagtcaa 7380

tggggattct ttagcccata cggtattggc gacaacgatg cttgtactat taacccttac 7440

gatggctggg cgtgtcaaat tagctacatg gaggaaacac ctgatggctc tttgcggcac 7500

ccatcgttcg taatgttccg tggcaccgag gacaaccctc aagagaaaat gtaatcacac 7560

tggctcacct tcgggtgggc ctttctgcgt ttataaggag acactttatg tttaagaagg 7620

ttggtaaatt ccttgcggct ttggcagcta tcctgacgct tgcgtatatt cttgcggtat 7680

accctcaagt agcactagta gtagttggcg cttgttactt agcggcagtg tgtgcttgcg 7740

tgtggagtat agttaactgg taatacgact cactaaagga ggtacacacc atgatgtact 7800

taatgccatt actcatcgtc attgtaggat gccttgcgct ccactgtagc gatgatgata 7860

tgccagatgg tcacgcttaa tacgactcac taaaggagac actatatgtt tcgacttcat 7920

tacaacaaaa gcgttaagaa tttcacggtt cgccgtgctg accgttcaat cgtatgtgcg 7980

agcgagcgcc gagctaagat acctcttatt ggtaacacag ttcctttggc accgagcgtc 8040

cacatcatta tcacccgtgg tgactttgag aaagcaatag acaagaaacg tccggttctt 8100

agtgtggcag tgacccgctt cccgttcgtc cgtctgttac tcaaacgaat caaggaggtg 8160

ttctgatggg actgttagat ggtgaagcct gggaaaaaga aaacccgcca gtacaagcaa 8220

ctgggtgtat agcttgctta gagaaagatg accgttatcc acacacctgt aacaaaggag 8280

ctaacgatat gaccgaacgt gaacaagaga tgatcattaa gttgatagac aataatgaag 8340

gtcgcccaga tgatttgaat ggctgcggta ttctctgctc caatgtccct tgccacctct 8400

gccccgcaaa taacgatcaa aagataacct taggtgaaat ccgagcgatg gacccacgta 8460

aaccacatct gaataaacct gaggtaactc ctacagatga ccagccttcc gctgagacaa 8520

tcgaaggtgt cactaagcct tcccactaca tgctgtttga cgacattgag gctatcgaag 8580

tgattgctcg ttcaatgacc gttgagcagt tcaagggata ctgcttcggt aacatcttaa 8640

agtacagact acgtgctggt aagaagtcag agttagcgta cttagagaaa gacctagcga 8700

aagcagactt ctataaagaa ctctttgaga aacataagga taaatgttat gcataacttc 8760

aagtcaaccc cacctgccga cagcctatct gatgacttca catcttgctc agagtggtgc 8820

cgaaagatgt gggaagagac attcgacgat gcgtacatca agctgtatga actttggaaa 8880

tcgagaggtc aatgactatg tcaaacgtaa atacaggttc acttagtgtg gacaataaga 8940

agttttgggc taccgtagag tcctcggagc attccttcga ggttccaatc tacgctgaga 9000

ccctagacga agctctggag ttagccgaat ggcaatacgt tccggctggc tttgaggtta 9060

ctcgtgtgcg tccttgtgta gcaccgaagt aatacgactc actattaggg aagactccct 9120

ctgagaaacc aaacgaaacc taaaggagat taacattatg gctaagaaga ttttcacctc 9180

tgcgctgggt accgctgaac cttacgctta catcgccaag ccggactacg gcaacgaaga 9240

gcgtggcttt gggaaccctc gtggtgtcta taaagttgac ctgactattc ccaacaaaga 9300

cccgcgctgc cagcgtatgg tcgatgaaat cgtgaagtgt cacgaagagg cttatgctgc 9360

tgccgttgag gaatacgaag ctaatccacc tgctgtagct cgtggtaaga aaccgctgaa 9420

accgtatgag ggtgacatgc cgttcttcga taacggtgac ggtacgacta cctttaagtt 9480

caaatgctac gcgtctttcc aagacaagaa gaccaaagag accaagcaca tcaatctggt 9540

tgtggttgac tcaaaaggta agaagatgga agacgttccg attatcggtg gtggctctaa 9600

gctgaaagtt aaatattctc tggttccata caagtggaac actgctgtag gtgcgagcgt 9660

taagctgcaa ctggaatccg tgatgctggt cgaactggct acctttggtg gcggtgaaga 9720

cgattgggct gacgaagttg aagagaacgg ctatgttgcc tctggttctg ccaaagcgag 9780

caaaccacgc gacgaagaaa gctgggacga agacgacgaa gagtccgagg aagcagacga 9840

agacggagac ttctaagtgg aactgcggga gaaaatcctt gagcgaatca aggtgacttc 9900

ctctgggtgt tgggagtggc agggcgctac gaacaataaa gggtacgggc aggtgtggtg 9960

cagcaatacc ggaaaggttg tctactgtca tcgcgtaatg tctaatgctc cgaaaggttc 10020

taccgtcctg cactcctgtg ataatccatt atgttgtaac cctgaacacc tatccatagg 10080

aactccaaaa gagaactcca ctgacatggt aaataagggt cgctcacaca aggggtataa 10140

actttcagac gaagacgtaa tggcaatcat ggagtccagc gagtccaatg tatccttagc 10200

tcgcacctat ggtgtctccc aacagactat ttgtgatata cgcaaaggga ggcgacatgg 10260

caggttacgg cgctaaagga atccgaaagg ttggagcgtt tcgctctggc ctagaggaca 10320

aggtttcaaa gcagttggaa tcaaaaggta ttaaattcga gtatgaagag tggaaagtgc 10380

cttatgtaat tccggcgagc aatcacactt acactccaga cttcttactt ccaaacggta 10440

tattcgttga gacaaagggt ctgtgggaaa gcgatgatag aaagaagcac ttattaatta 10500

gggagcagca ccccgagcta gacatccgta ttgtcttctc aagctcacgt actaagttat 10560

acaaaggttc tccaacgtct tatggagagt tctgcgaaaa gcatggtatt aagttcgctg 10620

ataaactgat acctgctgag tggataaagg aacccaagaa ggaggtcccc tttgatagat 10680

taaaaaggaa aggaggaaag aaataatggc tcgtgtacag tttaaacaac gtgaatctac 10740

tgacgcaatc tttgttcact gctcggctac caagccaagt cagaatgttg gtgtccgtga 10800

gattcgccag tggcacaaag agcagggttg gctcgatgtg ggataccact ttatcatcaa 10860

gcgagacggt actgtggagg caggacgaga tgagatggct gtaggctctc acgctaaggg 10920

ttacaaccac aactctatcg gcgtctgcct tgttggtggt atcgacgata aaggtaagtt 10980

cgacgctaac tttacgccag cccaaatgca atcccttcgc tcactgcttg tcacactgct 11040

ggctaagtac gaaggcgctg tgcttcgcgc ccatcatgag gtggcgccga aggcttgccc 11100

ttcgttcgac cttaagcgtt ggtgggagaa gaacgaactg gtcacttctg accgtggata 11160

attaattgaa ctcactaaag ggagaccaca gcggtttccc tttgttcgca ttggaggtca 11220

aataatgcgc aagtcttata aacaattcta taaggctccg aggaggcata tccaagtgtg 11280

ggaggcagcc aatgggccta taccaaaagg ttattatata gaccacattg acggcaatcc 11340

actcaacgac gccttagaca atctccgtct ggctctccca aaagaaaact catggaacat 11400

gaagactcca aagagcaata cctcaggact aaagggactg agttggagca aggaaaggga 11460

gatgtggaga ggcactgtaa cagctgaggg taaacagcat aactttcgta gtagagatct 11520

attggaagtc gttgcgtgga tttatagaac taggagggaa ttgcatggac aattcgcacg 11580

attccgatag tgtatttctt taccacattc cttgtgacaa ctgtgggagt agtgatggga 11640

actcgctgtt ctctgacgga cacacgttct gctacgtatg cgagaagtgg actgctggta 11700

atgaagacac taaagagagg gcttcaaaac ggaaaccctc aggaggtaaa ccaatgactt 11760

acaacgtgtg gaacttcggg gaatccaatg gacgctactc cgcgttaact gcgagaggaa 11820

tctccaagga aacctgtcag aaggctggct actggattgc caaagtagac ggtgtgatgt 11880

accaagtggc tgactatcgg gaccagaacg gcaacattgt gagtcagaag gttcgagata 11940

aagataagaa ctttaagacc actggtagtc acaagagtga cgctctgttc gggaagcact 12000

tgtggaatgg tggtaagaag attgtcgtta cagaaggtga aatcgacatg cttaccgtga 12060

tggaacttca agactgtaag tatcctgtag tgtcgttggg tcacggtgcc tctgccgcta 12120

agaagacatg cgctgccaac tacgaatact ttgaccagtt cgaacagatt atcttaatgt 12180

tcgatatgga cgaagcaggg cgcaaagcag tcgaagaggc tgcacaggtt ctacctgctg 12240

gtaaggtacg agtggcagtt cttccgtgta aggatgcaaa cgagtgtcac ctaaatggtc 12300

acgaccgtga aatcatggag caagtgtgga atgctggtcc ttggattcct gatggtgtgg 12360

tatcggctct ttcgttacgt gaacgaatcc gtgagcacct atcgtccgag gaatcagtag 12420

gtttactttt cagtggctgc actggtatca acgataagac cttaggtgcc cgtggtggtg 12480

aagtcattat ggtcacttcc ggttccggta tgggtaagtc aacgttcgtc cgtcaacaag 12540

ctctacaatg gggcacagcg atgggcaaga aggtaggctt agcgatgctt gaggagtccg 12600

ttgaggagac cgctgaggac cttataggtc tacacaaccg tgtccgactg agacaatccg 12660

actcactaaa gagagagatt attgagaacg gtaagttcga ccaatggttc gatgaactgt 12720

tcggcaacga tacgttccat ctatatgact cattcgccga ggctgagacg gatagactgc 12780

tcgctaagct ggcctacatg cgctcaggct tgggctgtga cgtaatcatt ctagaccaca 12840

tctcaatcgt cgtatccgct tctggtgaat ccgatgagcg taagatgatt gacaacctga 12900

tgaccaagct caaagggttc gctaagtcaa ctggggtggt gctggtcgta atttgtcacc 12960

ttaagaaccc agacaaaggt aaagcacatg aggaaggtcg ccccgtttct attactgacc 13020

tacgtggttc tggcgcacta cgccaactat ctgatactat tattgccctt gagcgtaatc 13080

agcaaggcga tatgcctaac cttgtcctcg ttcgtattct caagtgccgc tttactggtg 13140

atactggtat cgctggctac atggaataca acaaggaaac cggatggctt gaaccatcaa 13200

gttactcagg ggaagaagag tcacactcag agtcaacaga ctggtccaac gacactgact 13260

tctgacagga ttcttgatga ctttccagac gactacgaga agtttcgctg gagagtccca 13320

ttctaatacg actcactaaa ggagacacac catgttcaaa ctgattaaga agttaggcca 13380

actgctggtt cgtatgtaca acgtggaagc caagcgactg aacgatgagg ctcgtaaaga 13440

ggccacacag tcacgcgctc tggcgattcg ctccaacgaa ctggctgaca gtgcatccac 13500

taaagttacc gaggctgccc gtgtggcaaa ccaagctcaa cagctttcca aattctttga 13560

gtaatcaaac aggagaaacc attatgtcta acgtagctga aactatccgt ctatccgata 13620

cagctgacca gtggaaccgt cgagtccaca tcaacgttcg caacggtaag gcgactatgg 13680

tttaccgctg gaaggactct aagtcctcta agaatcacac tcagcgtatg acgttgacag 13740

atgagcaagc actgcgtctg gtcaatgcgc ttaccaaagc tgccgtgaca gcaattcatg 13800

aagctggtcg cgtcaatgaa gctatggcta tcctcgacaa gattgataac taagagtggt 13860

atcctcaagg tcgccaaagt ggtggccttc atgaatacta ttcgactcac tataggagat 13920

attaccatgc gtgaccctaa agttatccaa gcagaaatcg ctaaactgga agctgaactg 13980

gaggacgtta agtaccatga agctaagact cgctccgctg ttcacatctt gaagaactta 14040

ggctggactt ggacaagaca gactggctgg aagaaaccag aagttaccaa gctgagtcat 14100

aaggtgttcg ataaggacac tatgacccac atcaaggctg gtgattgggt taaggttgac 14160

atgggagttg ttggtggata cggctacgtc cgctcagtta gtggcaaata tgcacaagtg 14220

tcatacatca caggtgttac tccacgcggt gcaatcgttg ccgataagac caacatgatt 14280

cacacaggtt tcttgacagt tgtttcatat gaagagattg ttaagtcacg ataatcaata 14340

ggagaaatca atatgatcgt ttctgacatc gaagctaacg ccctcttaga gagcgtcact 14400

aagttccact gcggggttat ctacgactac tccaccgctg agtacgtaag ctaccgtccg 14460

agtgacttcg gtgcgtatct ggatgcgctg gaagccgagg ttgcacgagg cggtcttatt 14520

gtgttccaca acggtcacaa gtatgacgtt cctgcattga ccaaactggc aaagttgcaa 14580

ttgaaccgag agttccacct tcctcgtgag aactgtattg acacccttgt gttgtcacgt 14640

ttgattcatt ccaacctcaa ggacaccgat atgggtcttc tgcgttccgg caagttgccc 14700

ggaaaacgct ttgggtctca cgctttggag gcgtggggtt atcgcttagg cgagatgaag 14760

ggtgaataca aagacgactt taagcgtatg cttgaagagc agggtgaaga atacgttgac 14820

ggaatggagt ggtggaactt caacgaagag atgatggact ataacgttca ggacgttgtg 14880

gtaactaaag ctctccttga gaagctactc tctgacaaac attacttccc tcctgagatt 14940

gactttacgg acgtaggata cactacgttc tggtcagaat cccttgaggc cgttgacatt 15000

gaacatcgtg ctgcatggct gctcgctaaa caagagcgca acgggttccc gtttgacaca 15060

aaagcaatcg aagagttgta cgtagagtta gctgctcgcc gctctgagtt gctccgtaaa 15120

ttgaccgaaa cgttcggctc gtggtatcag cctaaaggtg gcactgagat gttctgccat 15180

ccgcgaacag gtaagccact acctaaatac cctcgcatta agacacctaa agttggtggt 15240

atctttaaga agcctaagaa caaggcacag cgagaaggcc gtgagccttg cgaacttgat 15300

acccgcgagt acgttgctgg tgctccttac accccagttg aacatgttgt gtttaaccct 15360

tcgtctcgtg accacattca gaagaaactc caagaggctg ggtgggtccc gaccaagtac 15420

accgataagg gtgctcctgt ggtggacgat gaggtactcg aaggagtacg tgtagatgac 15480

cctgagaagc aagccgctat cgacctcatt aaagagtact tgatgattca gaagcgaatc 15540

ggacagtctg ctgagggaga caaagcatgg cttcgttatg ttgctgagga tggtaagatt 15600

catggttctg ttaaccctaa tggagcagtt acgggtcgtg cgacccatgc gttcccaaac 15660

cttgcgcaaa ttccgggtgt acgttctcct tatggagagc agtgtcgcgc tgcttttggc 15720

gctgagcacc atttggatgg gataactggt aagccttggg ttcaggctgg catcgacgca 15780

tccggtcttg agctacgctg cttggctcac ttcatggctc gctttgataa cggcgagtac 15840

gctcacgaga ttcttaacgg cgacatccac actaagaacc agatagctgc tgaactacct 15900

acccgagata acgctaagac gttcatctat gggttcctct atggtgctgg tgatgagaag 15960

attggacaga ttgttggtgc tggtaaagag cgcggtaagg aactcaagaa gaaattcctt 16020

gagaacaccc ccgcgattgc agcactccgc gagtctatcc aacagacact tgtcgagtcc 16080

tctcaatggg tagctggtga gcaacaagtc aagtggaaac gccgctggat taaaggtctg 16140

gatggtcgta aggtacacgt tcgtagtcct cacgctgcct tgaataccct actgcaatct 16200

gctggtgctc tcatctgcaa actgtggatt atcaagaccg aagagatgct cgtagagaaa 16260

ggcttgaagc atggctggga tggggacttt gcgtacatgg catgggtaca tgatgaaatc 16320

caagtaggct gccgtaccga agagattgct caggtggtca ttgagaccgc acaagaagcg 16380

atgcgctggg ttggagacca ctggaacttc cggtgtcttc tggataccga aggtaagatg 16440

ggtcctaatt gggcgatttg ccactgatac aggaggctac tcatgaacga aagacactta 16500

acaggtgctg cttctgaaat gctagtagcc tacaaattta ccaaagctgg gtacactgtc 16560

tattacccta tgctgactca gagtaaagag gacttggttg tatgtaagga tggtaaattt 16620

agtaaggttc aggttaaaac agccacaacg gttcaaacca acacaggaga tgccaagcag 16680

gttaggctag gtggatgcgg taggtccgaa tataaggatg gagactttga cattcttgcg 16740

gttgtggttg acgaagatgt gcttattttc acatgggacg aagtaaaagg taagacatcc 16800

atgtgtgtcg gcaagagaaa caaaggcata aaactatagg agaaattatt atggctatga 16860

caaagaaatt taaagtgtcc ttcgacgtta ccgcaaagat gtcgtctgac gttcaggcaa 16920

tcttagagaa agatatgctg catctatgta agcaggtcgg ctcaggtgcg attgtcccca 16980

atggtaaaca gaaggaaatg attgtccagt tcctgacaca cggtatggaa ggattgatga 17040

cattcgtagt acgtacatca tttcgtgagg ccattaagga catgcacgaa gagtatgcag 17100

ataaggactc tttcaaacaa tctcctgcaa cagtacggga ggtgttctga tgtctgacta 17160

cctgaaagtg ctgcaagcaa tcaaaagttg ccctaagact ttccagtcca actatgtacg 17220

gaacaatgcg agcctcgtag cggaggccgc ttcccgtggt cacatctcgt gcctgactac 17280

tagtggacgt aacggtggcg cttgggaaat cactgcttcc ggtactcgct ttctgaaacg 17340

aatgggagga tgtgtctaat gtctcgtgac cttgtgacta ttccacgcga tgtgtggaac 17400

gatatacagg gctacatcga ctctctggaa cgtgagaacg atagccttaa gaatcaacta 17460

atggaagctg acgaatacgt agcggaacta gaggagaaac ttaatggcac ttcttgacct 17520

taaacaattc tatgagttac gtgaaggctg cgacgacaag ggtatccttg tgatggacgg 17580

cgactggctg gtcttccaag ctatgagtgc tgctgagttt gatgcctctt gggaggaaga 17640

gatttggcac cgatgctgtg accacgctaa ggcccgtcag attcttgagg attccattaa 17700

gtcctacgag acccgtaaga aggcttgggc aggtgctcca attgtccttg cgttcaccga 17760

tagtgttaac tggcgtaaag aactggttga cccgaactat aaggctaacc gtaaggccgt 17820

gaagaaacct gtagggtact ttgagttcct tgatgctctc tttgagcgcg aagagttcta 17880

ttgcatccgt gagcctatgc ttgagggtga tgacgttatg ggagttattg cttccaatcc 17940

gtctgccttc ggtgctcgta aggctgtaat catctcttgc gataaggact ttaagaccat 18000

ccctaactgt gacttcctgt ggtgtaccac tggtaacatc ctgactcaga ccgaagagtc 18060

cgctgactgg tggcacctct tccagaccat caagggtgac atcactgatg gttactcagg 18120

gattgctgga tggggtgata ccgccgagga cttcttgaat aacccgttca taaccgagcc 18180

taaaacgtct gtgcttaagt ccggtaagaa caaaggccaa gaggttacta aatgggttaa 18240

acgcgaccct gagcctcatg agacgctttg ggactgcatt aagtccattg gcgcgaaggc 18300

tggtatgacc gaagaggata ttatcaagca gggccaaatg gctcgaatcc tacggttcaa 18360

cgagtacaac tttattgaca aggagattta cctgtggaga ccgtagcgta tattggtctg 18420

ggtctttgtg ttctcggagt gtgcctcatt tcgtggggcc tttgggactt agccagaata 18480

atcaagtcgt tacacgacac taagtgataa actcaaggtc cctaaattaa tacgactcac 18540

tatagggaga taggggcctt tacgattatt actttaagat ttaactctaa gaggaatctt 18600

tattatgtta acacctatta accaattact taagaaccct aacgatattc cagatgtacc 18660

tcgtgcaacc gctgagtatc tacaggttcg attcaactat gcgtacctcg aagcgtctgg 18720

tcatatagga cttatgcgtg ctaatggttg tagtgaggcc cacatcttgg gtttcattca 18780

gggcctacag tatgcctcta acgtcattga cgagattgag ttacgcaagg aacaactaag 18840

agatgatggg gaggattgac actatgtgtt tctcaccgaa aattaaaact ccgaagatgg 18900

ataccaatca gattcgagcc gttgagccag cgcctctgac ccaagaagtg tcaagcgtgg 18960

agttcggtgg gtcttctgat gagacggata ccgagggcac cgaagtgtct ggacgcaaag 19020

gcctcaaggt cgaacgtgat gattccgtag cgaagtctaa agccagcggc aatggctccg 19080

ctcgtatgaa atcttccatc cgtaagtccg catttggagg taagaagtga tgtctgagtt 19140

cacatgtgtg gaggctaaga gtcgcttccg tgcaatccgg tggactgtgg aacaccttgg 19200

gttgcctaaa ggattcgaag gacactttgt gggctacagc ctctacgtag acgaagtgat 19260

ggacatgtct ggttgccgtg aagagtacat tctggactct accggaaaac atgtagcgta 19320

cttcgcgtgg tgcgtaagct gtgacattca ccacaaagga gacattctgg atgtaacgtc 19380

cgttgtcatt aatcctgagg cagactctaa gggcttacag cgattcctag cgaaacgctt 19440

taagtacctt gcggaactcc acgattgcga ttgggtgtct cgttgtaagc atgaaggcga 19500

gacaatgcgt gtatacttta aggaggtata agttatgggt aagaaagtta agaaggccgt 19560

gaagaaagtc accaagtccg ttaagaaagt cgttaaggaa ggggctcgtc cggttaaaca 19620

ggttgctggc ggtctagctg gtctggctgg tggtactggt gaagcacaga tggtggaagt 19680

accacaagct gccgcacaga ttgttgacgt acctgagaaa gaggtttcca ctgaggacga 19740

agcacagaca gaaagcggac gcaagaaagc tcgtgctggc ggtaagaaat ccttgagtgt 19800

agcccgtagc tccggtggcg gtatcaacat ttaatcagga ggttatcgtg gaagactgca 19860

ttgaatggac cggaggtgtc aactctaagg gttatggtcg taagtgggtt aatggtaaac 19920

ttgtgactcc acataggcac atctatgagg agacatatgg tccagttcca acaggaattg 19980

tggtgatgca tatctgcgat aaccctaggt gctataacat aaagcacctt acgcttggaa 20040

ctccaaagga taattccgag gacatggtta ccaaaggtag acaggctaaa ggagaggaac 20100

taagcaagaa acttacagag tcagacgttc tcgctatacg ctcttcaacc ttaagccacc 20160

gctccttagg agaactgtat ggagtcagtc aatcaaccat aacgcgaata ctacagcgta 20220

agacatggag acacatttaa tggctgagaa acgaacagga cttgcggagg atggcgcaaa 20280

gtctgtctat gagcgtttaa agaacgaccg tgctccctat gagacacgcg ctcagaattg 20340

cgctcaatat accatcccat cattgttccc taaggactcc gataacgcct ctacagatta 20400

tcaaactccg tggcaagccg tgggcgctcg tggtctgaac aatctagcct ctaagctcat 20460

gctggctcta ttccctatgc agacttggat gcgacttact atatctgaat atgaagcaaa 20520

gcagttactg agcgaccccg atggactcgc taaggtcgat gagggcctct cgatggtaga 20580

gcgtatcatc atgaactaca ttgagtctaa cagttaccgc gtgactctct ttgaggctct 20640

caaacagtta gtcgtagctg gtaacgtcct gctgtaccta ccggaaccgg aagggtcaaa 20700

ctataatccc atgaagctgt accgattgtc ttcttatgtg gtccaacgag acgcattcgg 20760

caacgttctg caaatggtga ctcgtgacca gatagctttt ggtgctctcc ctgaggacat 20820

ccgtaaggct gtagaaggtc aaggtggtga gaagaaagct gatgagacaa tcgacgtgta 20880

cactcacatc tatctggatg aggactcagg tgaatacctc cgatacgaag aggtcgaggg 20940

tatggaagtc caaggctccg atgggactta tcctaaagag gcttgcccat acatcccgat 21000

tcggatggtc agactagatg gtgaatccta cggtcgttcg tacattgagg aatacttagg 21060

tgacttacgg tcccttgaaa atctccaaga ggctatcgtc aagatgtcca tgattagctc 21120

taaggttatc ggcttagtga atcctgctgg tatcacccag ccacgccgac tgaccaaagc 21180

tcagactggt gacttcgtta ctggtcgtcc agaagacatc tcgttcctcc aactggagaa 21240

gcaagcagac tttactgtag ctaaagccgt aagtgacgct atcgaggctc gcctttcgtt 21300

tgcctttatg ttgaactctg cggttcagcg tacaggtgaa cgtgtgaccg ccgaagagat 21360

tcggtatgta gcttctgaac ttgaagatac tttaggtggt gtctactcta tcctttctca 21420

agaattacaa ttgcctctgg tacgagtgct cttgaagcaa ctacaagcca cgcaacagat 21480

tcctgagtta cctaaggaag ccgtagagcc aaccattagt acaggtctgg aagcaattgg 21540

tcgaggacaa gaccttgata agctggagcg gtgtgtcact gcgtgggctg cactggcacc 21600

tatgcgggac gaccctgata ttaaccttgc gatgattaag ttacgtattg ccaacgctat 21660

cggtattgac acttctggta ttctactcac cgaagaacag aagcaacaga agatggccca 21720

acagtctatg caaatgggta tggataatgg tgctgctgcg ctggctcaag gtatggctgc 21780

acaagctaca gcttcacctg aggctatggc tgctgccgct gattccgtag gtttacagcc 21840

gggaatttaa tacgactcac tatagggaga cctcatcttt gaaatgagcg atgacaagag 21900

gttggagtcc tcggtcttcc tgtagttcaa ctttaaggag acaataataa tggctgaatc 21960

taatgcagac gtatatgcat cttttggcgt gaactccgct gtgatgtctg gtggttccgt 22020

tgaggaacat gagcagaaca tgctggctct tgatgttgct gcccgtgatg gcgatgatgc 22080

aatcgagtta gcgtcagacg aagtggaaac agaacgtgac ctgtatgaca actctgaccc 22140

gttcggtcaa gaggatgacg aaggccgcat tcaggttcgt atcggtgatg gctctgagcc 22200

gaccgatgtg gacactggag aagaaggcgt tgagggcacc gaaggttccg aagagtttac 22260

cccactgggc gagactccag aagaactggt agctgcctct gagcaacttg gtgagcacga 22320

agagggcttc caagagatga ttaacattgc tgctgagcgt ggcatgagtg tcgagaccat 22380

tgaggctatc cagcgtgagt acgaggagaa cgaagagttg tccgccgagt cctacgctaa 22440

gctggctgaa attggctaca cgaaggcttt cattgactcg tatatccgtg gtcaagaagc 22500

tctggtggag cagtacgtaa acagtgtcat tgagtacgct ggtggtcgtg aacgttttga 22560

tgcactgtat aaccaccttg agacgcacaa ccctgaggct gcacagtcgc tggataatgc 22620

gttgaccaat cgtgacttag cgaccgttaa ggctatcatc aacttggctg gtgagtctcg 22680

cgctaaggcg ttcggtcgta agccaactcg tagtgtgact aatcgtgcta ttccggctaa 22740

acctcaggct accaagcgtg aaggctttgc ggaccgtagc gagatgatta aagctatgag 22800

tgaccctcgg tatcgcacag atgccaacta tcgtcgtcaa gtcgaacaga aagtaatcga 22860

ttcgaacttc tgatagactt cgaaatctag cataacccct tggggcctct aaacgggtct 22920

tgaggggttt tttgctgaaa ggaggaacta tatgcgctca tacgatatga acgttgagac 22980

tgccgctgag ttatcagctg tgaacgacat tctggcgtct atcggtgaac ctccggtatc 23040

aacgctggaa ggtgacgcta acgcagatgc agcgaacgct cggcgtattc tcaacaagat 23100

taaccgacag attcaatctc gtggatggac gttcaacatt gaggaaggca taacgctact 23160

acctgatgtt tactccaacc tgattgtata cagtgacgac tatttatccc taatgtctac 23220

ttccggtcaa tccatctacg ttaaccgagg tggctatgtg tatgaccgaa cgagtcaatc 23280

agaccgcttt gactctggta ttactgtgaa cattattcgt ctccgcgact acgatgagat 23340

gcctgagtgc ttccgttact ggattgtcac caaggcttcc cgtcagttca acaaccgatt 23400

ctttggggca ccggaagtag agggtgtact ccaagaagag gaagatgagg ctagacgtct 23460

ctgcatggag tatgagatgg actacggtgg gtacaatatg ctggatggag atgcgttcac 23520

ttctggtcta ctgactcgct aacattaata aataaggagg ctctaatggc actcattagc 23580

caatcaatca agaacttgaa gggtggtatc agccaacagc ctgacatcct tcgttatcca 23640

gaccaagggt cacgccaagt taacggttgg tcttcggaga ccgagggcct ccaaaagcgt 23700

ccacctcttg ttttcttaaa tacacttgga gacaacggtg cgttaggtca agctccgtac 23760

atccacctga ttaaccgaga tgagcacgaa cagtattacg ctgtgttcac tggtagcgga 23820

atccgagtgt tcgacctttc tggtaacgag aagcaagtta ggtatcctaa cggttccaac 23880

tacatcaaga ccgctaatcc acgtaacgac ctgcgaatgg ttactgtagc agactatacg 23940

ttcatcgtta accgtaacgt tgttgcacag aagaacacaa agtctgtcaa cttaccgaat 24000

tacaacccta atcaagacgg attgattaac gttcgtggtg gtcagtatgg tagggaacta 24060

attgtacaca ttaacggtaa agacgttgcg aagtataaga taccagatgg tagtcaacct 24120

gaacacgtaa acaatacgga tgcccaatgg ttagctgaag agttagccaa gcagatgcgc 24180

actaacttgt ctgattggac tgtaaatgta gggcaagggt tcatccatgt gaccgcacct 24240

agtggtcaac agattgactc cttcacgact aaagatggct acgcagacca gttgattaac 24300

cctgtgaccc actacgctca gtcgttctct aagctgccac ctaatgctcc taacggctac 24360

atggtgaaaa tcgtagggga cgcctctaag tctgccgacc agtattacgt tcggtatgac 24420

gctgagcgga aagtttggac tgagacttta ggttggaaca ctgaggacca agttctatgg 24480

gaaaccatgc cacacgctct tgtgcgagcc gctgacggta atttcgactt caagtggctt 24540

gagtggtctc ctaagtcttg tggtgacgtt gacaccaacc cttggccttc ttttgttggt 24600

tcaagtatta acgatgtgtt cttcttccgt aaccgcttag gattccttag tggggagaac 24660

atcatattga gtcgtacagc caaatacttc aacttctacc ctgcgtccat tgcgaacctt 24720

agtgatgacg accctataga cgtagctgtg agtaccaacc gaatagcaat ccttaagtac 24780

gccgttccgt tctcagaaga gttactcatc tggtccgatg aagcacaatt cgtcctgact 24840

gcctcgggta ctctcacatc taagtcggtt gagttgaacc taacgaccca gtttgacgta 24900

caggaccgag cgagaccttt tgggattggg cgtaatgtct actttgctag tccgaggtcc 24960

agcttcacgt ccatccacag gtactacgct gtgcaggatg tcagttccgt taagaatgct 25020

gaggacatta catcacacgt tcctaactac atccctaatg gtgtgttcag tatttgcgga 25080

agtggtacgg aaaacttctg ttcggtacta tctcacgggg accctagtaa aatcttcatg 25140

tacaaattcc tgtacctgaa cgaagagtta aggcaacagt cgtggtctca ttgggacttt 25200

ggggaaaacg tacaggttct agcttgtcag agtatcagct cagatatgta tgtgattctt 25260

cgcaatgagt tcaatacgtt cctagctaga atctctttca ctaagaacgc cattgactta 25320

cagggagaac cctatcgtgc ctttatggac atgaagattc gatacacgat tcctagtgga 25380

acatacaacg atgacacatt cactacctct attcatattc caacaattta tggtgcaaac 25440

ttcgggaggg gcaaaatcac tgtattggag cctgatggta agataaccgt gtttgagcaa 25500

cctacggctg ggtggaatag cgacccttgg ctgagactca gcggtaactt ggagggacgc 25560

atggtgtaca ttgggttcaa cattaacttc gtatatgagt tctctaagtt cctcatcaag 25620

cagactgccg acgacgggtc tacctccacg gaagacattg ggcgcttaca gttacgccga 25680

gcgtgggtta actacgagaa ctctggtacg tttgacattt atgttgagaa ccaatcgtct 25740

aactggaagt acacaatggc tggtgcccga ttaggctcta acactctgag ggctgggaga 25800

ctgaacttag ggaccggaca atatcgattc cctgtggttg gtaacgccaa gttcaacact 25860

gtatacatct tgtcagatga gactacccct ctgaacatca ttgggtgtgg ctgggaaggt 25920

aactacttac ggagaagttc cggtatttaa ttaaatattc tccctgtggt ggctcgaaat 25980

taatacgact cactataggg agaacaatac gactacggga gggttttctt atgatgacta 26040

taagacctac taaaagtaca gactttgagg tattcactcc ggctcaccat gacattcttg 26100

aagctaaggc tgctggtatt gagccgagtt tccctgatgc ttccgagtgt gtcacgttga 26160

gcctctatgg gttccctcta gctatcggtg gtaactgcgg ggaccagtgc tggttcgtta 26220

cgagcgacca agtgtggcga cttagtggaa aggctaagcg aaagttccgt aagttaatca 26280

tggagtatcg cgataagatg cttgagaagt atgatactct ttggaattac gtatgggtag 26340

gcaatacgtc ccacattcgt ttcctcaaga ctatcggtgc ggtattccat gaagagtaca 26400

cacgagatgg tcaatttcag ttatttacaa tcacgaaagg aggataacca tatgtgttgg 26460

gcagccgcaa tacctatcgc tatatctggc gctcaggcta tcagtggtca gaacgctcag 26520

gccaaaatga ttgccgctca gaccgctgct ggtcgtcgtc aagctatgga aatcatgagg 26580

cagacgaaca tccagaatgc tgacctatcg ttgcaagctc gaagtaaact tgaggaagcg 26640

tccgccgagt tgacctcaca gaacatgcag aaggtccaag ctattgggtc tatccgagcg 26700

gctatcggag agagtatgct tgaaggttcc tcaatggacc gcattaagcg agtcacagaa 26760

ggacagttca ttcgggaagc caatatggta actgagaact atcgccgtga ctaccaagca 26820

atcttcgcac agcaacttgg tggtactcaa agtgctgcaa gtcagattga cgaaatctat 26880

aagagcgaac agaaacagaa gagtaagcta cagatggttc tggacccact ggctatcatg 26940

gggtcttccg ctgcgagtgc ttacgcatcc ggtgcgttcg actctaagtc cacaactaag 27000

gcacctattg ttgccgctaa aggaaccaag acggggaggt aatgagctat gagtaaaatt 27060

gaatctgccc ttcaagcggc acaaccggga ctctctcggt tacgtggtgg tgctggaggt 27120

atgggctatc gtgcagcaac cactcaggcc gaacagccaa ggtcaagcct attggacacc 27180

attggtcggt tcgctaaggc tggtgccgat atgtataccg ctaaggaaca acgagcacga 27240

gacctagctg atgaacgctc taacgagatt atccgtaagc tgacccctga gcaacgtcga 27300

gaagctctca acaacgggac ccttctgtat caggatgacc catacgctat ggaagcactc 27360

cgagtcaaga ctggtcgtaa cgctgcgtat cttgtggacg atgacgttat gcagaagata 27420

aaagagggtg tcttccgtac tcgcgaagag atggaagagt atcgccatag tcgccttcaa 27480

gagggcgcta aggtatacgc tgagcagttc ggcatcgacc ctgaggacgt tgattatcag 27540

cgtggtttca acggggacat taccgagcgt aacatctcgc tgtatggtgc gcatgataac 27600

ttcttgagcc agcaagctca gaagggcgct atcatgaaca gccgagtgga actcaacggt 27660

gtccttcaag accctgatat gctgcgtcgt ccagactctg ctgacttctt tgagaagtat 27720

atcgacaacg gtctggttac tggcgcaatc ccatctgatg ctcaagccac acagcttata 27780

agccaagcgt tcagtgacgc ttctagccgt gctggtggtg ctgacttcct gatgcgagtc 27840

ggtgacaaga aggtaacact taacggagcc actacgactt accgagagtt gattggtgag 27900

gaacagtgga acgctctcat ggtcacagca caacgttctc agtttgagac tgacgcgaag 27960

ctgaacgagc agtatcgctt gaagattaac tctgcgctga accaagagga cccaaggaca 28020

gcttgggaga tgcttcaagg tatcaaggct gaactagata aggtccaacc tgatgagcag 28080

atgacaccac aacgtgagtg gctaatctcc gcacaggaac aagttcagaa tcagatgaac 28140

gcatggacga aagctcaggc caaggctctg gacgattcca tgaagtcaat gaacaaactt 28200

gacgtaatcg acaagcaatt ccagaagcga atcaacggtg agtgggtctc aacggatttt 28260

aaggatatgc cagtcaacga gaacactggt gagttcaagc atagcgatat ggttaactac 28320

gccaataaga agctcgctga gattgacagt atggacattc cagacggtgc caaggatgct 28380

atgaagttga agtaccttca agcggactct aaggacggag cattccgtac agccatcgga 28440

accatggtca ctgacgctgg tcaagagtgg tctgccgctg tgattaacgg taagttacca 28500

gaacgaaccc cagctatgga tgctctgcgc agaatccgca atgctgaccc tcagttgatt 28560

gctgcgctat acccagacca agctgagcta ttcctgacga tggacatgat ggacaagcag 28620

ggtattgacc ctcaggttat tcttgatgcc gaccgactga ctgttaagcg gtccaaagag 28680

caacgctttg aggatgataa agcattcgag tctgcactga atgcatctaa ggctcctgag 28740

attgcccgta tgccagcgtc actgcgcgaa tctgcacgta agatttatga ctccgttaag 28800

tatcgctcgg ggaacgaaag catggctatg gagcagatga ccaagttcct taaggaatct 28860

acctacacgt tcactggtga tgatgttgac ggtgataccg ttggtgtgat tcctaagaat 28920

atgatgcagg ttaactctga cccgaaatca tgggagcaag gtcgggatat tctggaggaa 28980

gcacgtaagg gaatcattgc gagcaaccct tggataacca ataagcaact gaccatgtat 29040

tctcaaggtg actccattta ccttatggac accacaggtc aagtcagagt ccgatacgac 29100

aaagagttac tctcgaaggt ctggagtgag aaccagaaga aactcgaaga gaaagctcgt 29160

gagaaggctc tggctgatgt gaacaagcga gcacctatag ttgccgctac gaaggcccgt 29220

gaagctgctg ctaaacgagt ccgagagaaa cgtaaacaga ctcctaagtt catctacgga 29280

cgtaaggagt aactaaaggc tacataagga ggccctaaat ggataagtac gataagaacg 29340

taccaagtga ttatgatggt ctgttccaaa aggctgctga tgccaacggg gtctcttatg 29400

accttttacg taaagtcgct tggacagaat cacgatttgt gcctacagca aaatctaaga 29460

ctggaccatt aggcatgatg caatttacca aggcaaccgc taaggccctc ggtctgcgag 29520

ttaccgatgg tccagacgac gaccgactga accctgagtt agctattaat gctgccgcta 29580

agcaacttgc aggtctggta gggaagtttg atggcgatga actcaaagct gcccttgcgt 29640

acaaccaagg cgagggacgc ttgggtaatc cacaacttga ggcgtactct aagggagact 29700

tcgcatcaat ctctgaggag ggacgtaact acatgcgtaa ccttctggat gttgctaagt 29760

cacctatggc tggacagttg gaaacttttg gtggcataac cccaaagggt aaaggcattc 29820

cggctgaggt aggattggct ggaattggtc acaagcagaa agtaacacag gaacttcctg 29880

agtccacaag ttttgacgtt aagggtatcg aacaggaggc tacggcgaaa ccattcgcca 29940

aggacttttg ggagacccac ggagaaacac ttgacgagta caacagtcgt tcaaccttct 30000

tcggattcaa aaatgctgcc gaagctgaac tctccaactc agtcgctggg atggctttcc 30060

gtgctggtcg tctcgataat ggttttgatg tgtttaaaga caccattacg ccgactcgct 30120

ggaactctca catctggact ccagaggagt tagagaagat tcgaacagag gttaagaacc 30180

ctgcgtacat caacgttgta actggtggtt cccctgagaa cctcgatgac ctcattaaat 30240

tggctaacga gaactttgag aatgactccc gcgctgccga ggctggccta ggtgccaaac 30300

tgagtgctgg tattattggt gctggtgtgg acccgcttag ctatgttcct atggtcggtg 30360

tcactggtaa gggctttaag ttaatcaata aggctcttgt agttggtgcc gaaagtgctg 30420

ctctgaacgt tgcatccgaa ggtctccgta cctccgtagc tggtggtgac gcagactatg 30480

cgggtgctgc cttaggtggc tttgtgtttg gcgcaggcat gtctgcaatc agtgacgctg 30540

tagctgctgg actgaaacgc agtaaaccag aagctgagtt cgacaatgag ttcatcggtc 30600

ctatgatgcg attggaagcc cgtgagacag cacgaaacgc caactctgcg gacctctctc 30660

ggatgaacac tgagaacatg aagtttgaag gtgaacataa tggtgtccct tatgaggact 30720

taccaacaga gagaggtgcc gtggtgttac atgatggctc cgttctaagt gcaagcaacc 30780

caatcaaccc taagactcta aaagagttct ccgaggttga ccctgagaag gctgcgcgag 30840

gaatcaaact ggctgggttc accgagattg gcttgaagac cttggggtct gacgatgctg 30900

acatccgtag agtggctatc gacctcgttc gctctcctac tggtatgcag tctggtgcct 30960

caggtaagtt cggtgcaaca gcttctgaca tccatgagag acttcatggt actgaccagc 31020

gtacttataa tgacttgtac aaagcaatgt ctgacgctat gaaagaccct gagttctcta 31080

ctggcggcgc taagatgtcc cgtgaagaaa ctcgatacac tatctaccgt agagcggcac 31140

tagctattga gcgtccagaa ctacagaagg cactcactcc gtctgagaga atcgttatgg 31200

acatcattaa gcgtcacttt gacaccaagc gtgaacttat ggaaaaccca gcaatattcg 31260

gtaacacaaa ggctgtgagt atcttccctg agagtcgcca caaaggtact tacgttcctc 31320

acgtatatga ccgtcatgcc aaggcgctga tgattcaacg ctacggtgcc gaaggtttgc 31380

aggaagggat tgcccgctca tggatgaaca gctacgtctc cagacctgag gtcaaggcca 31440

gagtcgatga gatgcttaag gaattacacg gggtgaagga agtaacacca gagatggtag 31500

agaagtacgc tatggataag gcttatggta tctcccactc agaccagttc accaacagtt 31560

ccataataga agagaacatt gagggcttag taggtatcga gaataactca ttccttgagg 31620

cacgtaactt gtttgattcg gacctatcca tcactatgcc agacggacag caattctcag 31680

tgaatgacct aagggacttc gatatgttcc gcatcatgcc agcgtatgac cgccgtgtca 31740

atggtgacat cgccatcatg gggtctactg gtaaaaccac taaggaactt aaggatgaga 31800

ttttggctct caaagcgaaa gctgagggag acggtaagaa gactggcgag gtacatgctt 31860

taatggatac cgttaagatt cttactggtc gtgctagacg caatcaggac actgtgtggg 31920

aaacctcact gcgtgccatc aatgacctag ggttcttcgc taagaacgcc tacatgggtg 31980

ctcagaacat tacggagatt gctgggatga ttgtcactgg taacgttcgt gctctagggc 32040

atggtatccc aattctgcgt gatacactct acaagtctaa accagtttca gctaaggaac 32100

tcaaggaact ccatgcgtct ctgttcggga aggaggtgga ccagttgatt cggcctaaac 32160

gtgctgacat tgtgcagcgc ctaagggaag caactgatac cggacctgcc gtggcgaaca 32220

tcgtagggac cttgaagtat tcaacacagg aactggctgc tcgctctccg tggactaagc 32280

tactgaacgg aaccactaac taccttctgg atgctgcgcg tcaaggtatg cttggggatg 32340

ttattagtgc caccctaaca ggtaagacta cccgctggga gaaagaaggc ttccttcgtg 32400

gtgcctccgt aactcctgag cagatggctg gcatcaagtc tctcatcaag gaacatatgg 32460

tacgcggtga ggacgggaag tttaccgtta aggacaagca agcgttctct atggacccac 32520

gggctatgga cttatggaga ctggctgaca aggtagctga tgaggcaatg ctgcgtccac 32580

ataaggtgtc cttacaggat tcccatgcgt tcggagcact aggtaagatg gttatgcagt 32640

ttaagtcttt cactatcaag tcccttaact ctaagttcct gcgaaccttc tatgatggat 32700

acaagaacaa ccgagcgatt gacgctgcgc tgagcatcat cacctctatg ggtctcgctg 32760

gtggtttcta tgctatggct gcacacgtca aagcatacgc tctgcctaag gagaaacgta 32820

aggagtactt ggagcgtgca ctggacccaa ccatgattgc ccacgctgcg ttatctcgta 32880

gttctcaatt gggtgctcct ttggctatgg ttgacctagt tggtggtgtt ttagggttcg 32940

agtcctccaa gatggctcgc tctacgattc tacctaagga caccgtgaag gaacgtgacc 33000

caaacaaacc gtacacctct agagaggtaa tgggcgctat gggttcaaac cttctggaac 33060

agatgccttc ggctggcttt gtggctaacg taggggctac cttaatgaat gctgctggcg 33120

tggtcaactc acctaataaa gcaaccgagc aggacttcat gactggtctt atgaactcca 33180

caaaagagtt agtaccgaac gacccattga ctcaacagct tgtgttgaag atttatgagg 33240

cgaacggtgt taacttgagg gagcgtagga aataatacga ctcactatag ggagaggcga 33300

aataatcttc tccctgtagt ctcttagatt tactttaagg aggtcaaatg gctaacgtaa 33360

ttaaaaccgt tttgacttac cagttagatg gctccaatcg tgattttaat atcccgtttg 33420

agtatctagc ccgtaagttc gtagtggtaa ctcttattgg tgtagaccga aaggtcctta 33480

cgattaatac agactatcgc tttgctacac gtactactat ctctctgaca aaggcttggg 33540

gtccagccga tggctacacg accatcgagt tacgtcgagt aacctccact accgaccgat 33600

tggttgactt tacggatggt tcaatcctcc gcgcgtatga ccttaacgtc gctcagattc 33660

aaacgatgca cgtagcggaa gaggcccgtg acctcactac ggatactatc ggtgtcaata 33720

acgatggtca cttggatgct cgtggtcgtc gaattgtgaa cctagcgaac gccgtggatg 33780

accgcgatgc tgttccgttt ggtcaactaa agaccatgaa ccagaactca tggcaagcac 33840

gtaatgaagc cttacagttc cgtaatgagg ctgagacttt cagaaaccaa gcggagggct 33900

ttaagaacga gtccagtacc aacgctacga acacaaagca gtggcgcgat gagaccaagg 33960

gtttccgaga cgaagccaag cggttcaaga atacggctgg tcaatacgct acatctgctg 34020

ggaactctgc ttccgctgcg catcaatctg aggtaaacgc tgagaactct gccacagcat 34080

ccgctaactc tgctcatttg gcagaacagc aagcagaccg tgcggaacgt gaggcagaca 34140

agctggaaaa ttacaatgga ttggctggtg caattgataa ggtagatgga accaatgtgt 34200

actggaaagg aaatattcac gctaacgggc gcctttacat gaccacaaac ggttttgact 34260

gtggccagta tcaacagttc tttggtggtg tcactaatcg ttactctgtc atggagtggg 34320

gagatgagaa cggatggctg atgtatgttc aacgtagaga gtggacaaca gcgataggcg 34380

gtaacatcca gttagtagta aacggacaga tcatcaccca aggtggagcc atgaccggtc 34440

agctaaaatt gcagaatggg catgttcttc aattagagtc cgcatccgac aaggcgcact 34500

atattctatc taaagatggt aacaggaata actggtacat tggtagaggg tcagataaca 34560

acaatgactg taccttccac tcctatgtac atggtacgac cttaacactc aagcaggact 34620

atgcagtagt taacaaacac ttccacgtag gtcaggccgt tgtggccact gatggtaata 34680

ttcaaggtac taagtgggga ggtaaatggc tggatgctta cctacgtgac agcttcgttg 34740

cgaagtccaa ggcgtggact caggtgtggt ctggtagtgc tggcggtggg gtaagtgtga 34800

ctgtttcaca ggatctccgc ttccgcaata tctggattaa gtgtgccaac aactcttgga 34860

acttcttccg tactggcccc gatggaatct acttcatagc ctctgatggt ggatggttac 34920

gattccaaat acactccaac ggtctcggat tcaagaatat tgcagacagt cgttcagtac 34980

ctaatgcaat catggtggag aacgagtaat tggtaaatca caaggaaaga cgtgtagtcc 35040

acggatggac tctcaaggag gtacaaggtg ctatcattag actttaacaa cgaattgatt 35100

aaggctgctc caattgttgg gacgggtgta gcagatgtta gtgctcgact gttctttggg 35160

ttaagcctta acgaatggtt ctacgttgct gctatcgcct acacagtggt tcagattggt 35220

gccaaggtag tcgataagat gattgactgg aagaaagcca ataaggagtg atatgtatgg 35280

aaaaggataa gagccttatt acattcttag agatgttgga cactgcgatg gctcagcgta 35340

tgcttgcgga cctttcggac catgagcgtc gctctccgca actctataat gctattaaca 35400

aactgttaga ccgccacaag ttccagattg gtaagttgca gccggatgtt cacatcttag 35460

gtggccttgc tggtgctctt gaagagtaca aagagaaagt cggtgataac ggtcttacgg 35520

atgatgatat ttacacatta cagtgatata ctcaaggcca ctacagatag tggtctttat 35580

ggatgtcatt gtctatacga gatgctccta cgtgaaatct gaaagttaac gggaggcatt 35640

atgctagaat ttttacgtaa gctaatccct tgggttctcg ctgggatgct attcgggtta 35700

ggatggcatc tagggtcaga ctcaatggac gctaaatgga aacaggaggt acacaatgag 35760

tacgttaaga gagttgaggc tgcgaagagc actcaaagag caatcgatgc ggtatctgct 35820

aagtatcaag aagaccttgc cgcgctggaa gggagcactg ataggattat ttctgatttg 35880

cgtagcgaca ataagcggtt gcgcgtcaga gtcaaaacta ccggaacctc cgatggtcag 35940

tgtggattcg agcctgatgg tcgagccgaa cttgacgacc gagatgctaa acgtattctc 36000

gcagtgaccc agaagggtga cgcatggatt cgtgcgttac aggatactat tcgtgaactg 36060

caacgtaagt aggaaatcaa gtaaggaggc aatgtgtcta ctcaatccaa tcgtaatgcg 36120

ctcgtagtgg cgcaactgaa aggagacttc gtggcgttcc tattcgtctt atggaaggcg 36180

ctaaacctac cggtgcccac taagtgtcag attgacatgg ctaaggtgct ggcgaatgga 36240

gacaacaaga agttcatctt acaggctttc cgtggtatcg gtaagtcgtt catcacatgt 36300

gcgttcgttg tgtggtcctt atggagagac cctcagttga agatacttat cgtatcagcc 36360

tctaaggagc gtgcagacgc taactccatc tttattaaga acatcattga cctgctgcca 36420

ttcctatctg agttaaagcc aagacccgga cagcgtgact cggtaatcag ctttgatgta 36480

ggcccagcca atcctgacca ctctcctagt gtgaaatcag taggtatcac tggtcagtta 36540

actggtagcc gtgctgacat tatcattgcg gatgacgttg agattccgtc taacagcgca 36600

actatgggtg cccgtgagaa gctatggact ctggttcagg agttcgctgc gttacttaaa 36660

ccgctgcctt cctctcgcgt tatctacctt ggtacacctc agacagagat gactctctat 36720

aaggaacttg aggataaccg tgggtacaca accattatct ggcctgctct gtacccaagg 36780

acacgtgaag agaacctcta ttactcacag cgtcttgctc ctatgttacg cgctgagtac 36840

gatgagaacc ctgaggcact tgctgggact ccaacagacc cagtgcgctt tgaccgtgat 36900

gacctgcgcg agcgtgagtt ggaatacggt aaggctggct ttacgctaca gttcatgctt 36960

aaccctaacc ttagtgatgc cgagaagtac ccgctgaggc ttcgtgacgc tatcgtagcg 37020

gccttagact tagagaaggc cccaatgcat taccagtggc ttccgaaccg tcagaacatc 37080

attgaggacc ttcctaacgt tggccttaag ggtgatgacc tgcatacgta ccacgattgt 37140

tccaacaact caggtcagta ccaacagaag attctggtca ttgaccctag tggtcgcggt 37200

aaggacgaaa caggttacgc tgtgctgtac acactgaacg gttacatcta ccttatggaa 37260

gctggaggtt tccgtgatgg ctactccgat aagacccttg agttactcgc taagaaggca 37320

aagcaatggg gagtccagac ggttgtctac gagagtaact tcggtgacgg tatgttcggt 37380

aaggtattca gtcctatcct tcttaaacac cacaactgtg cgatggaaga gattcgtgcc 37440

cgtggtatga aagagatgcg tatttgcgat acccttgagc cagtcatgca gactcaccgc 37500

cttgtaattc gtgatgaggt cattagggcc gactaccagt ccgctcgtga cgtagacggt 37560

aagcatgacg ttaagtactc gttgttctac cagatgaccc gtatcactcg tgagaaaggc 37620

gctctggctc atgatgaccg attggatgcc cttgcgttag gcattgagta tctccgtgag 37680

tccatgcagt tggattccgt taaggtcgag ggtgaagtac ttgctgactt ccttgaggaa 37740

cacatgatgc gtcctacggt tgctgctacg catatcattg agatgtctgt gggaggagtt 37800

gatgtgtact ctgaggacga tgagggttac ggtacgtctt tcattgagtg gtgatttatg 37860

cattaggact gcatagggat gcactataga ccacggatgg tcagttcttt aagttactga 37920

aaagacacga taaattaata cgactcacta tagggagagg agggacgaaa ggttactata 37980

tagatactga atgaatactt atagagtgca taaagtatgc ataatggtgt acctagagtg 38040

acctctaaga atggtgatta tattgtatta gtatcacctt aacttaagga ccaacataaa 38100

gggaggagac tcatgttccg cttattgttg aacctactgc ggcatagagt cacctaccga 38160

tttcttgtgg tactttgtgc tgcccttggg tacgcatctc ttactggaga cctcagttca 38220

ctggagtctg tcgtttgctc tatactcact tgtagcgatt agggtcttcc tgaccgactg 38280

atggctcacc gagggattca gcggtatgat tgcatcacac cacttcatcc ctatagagtc 38340

aagtcctaag gtatacccat aaagagcctc taatggtcta tcctaaggtc tatacctaaa 38400

gataggccat cctatcagtg tcacctaaag agggtcttag agagggccta tggagttcct 38460

atagggtcct ttaaaatata ccataaaaat ctgagtgact atctcacagt gtacggacct 38520

aaagttcccc catagggggt acctaaagcc cagccaatca cctaaagtca accttcggtt 38580

gaccttgagg gttccctaag ggttggggat gacccttggg tttgtctttg ggtgttacct 38640

tgagtgtctc tctgtgtccc t 38661

<210> 23

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 23

cttgaagacg aaagggcctc gtgatacgcc tctcacagtg tacggaccta aagttccccc 60

<210> 24

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 24

attacgcgat gacagtagac aacctttccg 30

<210> 25

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 25

tgcagcaata ccggaaaggt tgtctactgt 30

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 26

atatgtctcc tcatagatgt gcctatgtgg 30

<210> 27

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 27

acttgtgact ccacataggc acatctatga 30

<210> 28

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 28

gccccaaggg gttatgctag atttcgaagt ctatcagaag ttcgaatcga ttac 54

<210> 29

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 29

cttctgatag acttcgaaat ctagcataac cccttggggc ctctaaacgg 50

<210> 30

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 30

gaataacctg agggtcaata ccctgcttgt 30

<210> 31

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 31

gacatgatgg acaagcaggg tattgaccct 30

<210> 32

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 32

cataatagaa acgacacgaa attacaaaat agggacacag agagacactc aaggtaacac 60

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> promoter

<400> 33

taatacgact cactataggg 20

<210> 34

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 34

ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga tataccaatg 60

cgctcatacg atatgaacg 79

<210> 35

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 35

cgttagccat tggtatatct ccttctttta aataccggaa cttctccg 48

<210> 36

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 36

ccggtattta aaagaaggag atataccaat ggctaacgta attaaaaccg 50

<210> 37

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 37

cccgcggatc cttactcgtt ctccaccatg attg 34

<210> 38

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 38

cccggaattc gaaattaata cgactcacta tagggagacc acaacggttt ccctctag 58

<210> 39

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 39

cccgcggatc cttactcgtt ctccaccatg attg 34

<210> 40

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 40

cccgcggatc cttattgctc agcggtggca g 31

<210> 41

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 41

cccggaattc taatacgact cactataggg agac 34

<210> 42

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 42

cttgaagacg aaagggcctc gtgatacgcc gtccatccta aagccaacac ctaaagcc 58

<210> 43

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 43

attacgcgat gacagtagac aacctttccg 30

<210> 44

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 44

tgcagcaata ccggaaaggt tgtctactgt 30

<210> 45

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 45

atatgtctcc tcatagatgt gcctatgtgg 30

<210> 46

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 46

acttgtgact ccacataggc acatctatga 30

<210> 47

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 47

gaataacctg agggtcaata ccctgcttgt 30

<210> 48

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 48

gacatgatgg acaagcaggg tattgaccct 30

<210> 49

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer

<400> 49

cataatagaa acgacacgaa attacaaaat tgcataaatc accactcaat gaaagacg 58

<210> 50

<211> 38932

<212> DNA

<213> Artificial sequence

<220>

<223> PAC site deleted phage genome

<400> 50

gtccatccta aagccaacac ctaaagccta cacctaaaga cccatcaagt caacgcctat 60

cttaaagttt aaacataaag accagaccta aagaccagac ctaaagacac tacataaaga 120

ccagacctaa agacgccttg ttgttagcca taaagtgata acctttaatc attgtcttta 180

ttaatacaac tcactataag gagagacaac ttaaagagac ttaaaagatt aatttaaaat 240

ttatcaaaaa gagtattgac ttaaagtcta acctatagga tacttacagc catcgagagg 300

gacacggcga atagccatcc caatcgacac cggggtcaac cggataagta gacagcctga 360

taagtcgcac gaaaaacagg tattgacaac atgaagtaac atgcagtaag atacaaatcg 420

ctaggtaaca ctagcagcgt caaccgggcg cacagtgcct tctaggtgac ttaagcgcac 480

cacggcacat aaggtgaaac aaaacggttg acaacatgaa gtaaacacgg tacgatgtac 540

cacatgaaac gacagtgagt caccacactg aaaggtgatg cggtctaacg aaacctgacc 600

taagacgctc tttaacaatc tggtaaatag ctcttgagtg catgactagc ggataactca 660

agggtatcgc aaggtgccct ttatgatatt cactaataac tgcacgaggt aacacaagat 720

ggctatgtct aacatgactt acaacaacgt tttcgaccac gcttacgaaa tgctgaaaga 780

aaacatccgt tatgatgaca tccgtgacac tgatgacctg cacgatgcta ttcacatggc 840

tgccgataat gcagttccgc actactacgc tgacatcttt agcgtaatgg caagtgaggg 900

cattgacctt gagttcgaag actctggtct gatgcctgac accaaggacg taatccgcat 960

cctgcaagcg cgtatctatg agcaattaac gattgacctc tgggaagacg cagaagactt 1020

gctcaatgaa tacttggagg aagtcgagga gtacgaggag gatgaagagt aatgtctact 1080

accaacgtgc aatacggtct gaccgctcaa actgtacttt tctatagcga catggtgcgc 1140

tgtggcttta actggtcact cgcaatggca cagctcaaag aactgtacga aaacaacaag 1200

gcaatagctt tagaatctgc tgagtgatag actcaaggtc gctcctagcg agtggccttt 1260

atgattatca ctttacttat gagggagtaa tgtatatgct tactatcggt ctactcaccg 1320

ctctaggtct agctgtaggt gcatcctttg ggaaggcttt aggtgtagct gtaggttcct 1380

actttaccgc ttgcatcatc ataggaatca tcaaaggggc actacgcaaa tgatgaagca 1440

ctacgttatg ccaatccaca cgtccaacgg ggcaaccgta tgtacacctg atgggttcgc 1500

aatgaaacaa cgaatcgaac gccttaagcg tgaactccgc attaaccgca agattaacaa 1560

gataggttcc ggctatgaca gaacgcactg atggcttaaa gaaaggttat atgcccaatg 1620

gcacactata cgctgcaaat cggcgaatag tgagaacttg gcgagagaac aacctcgaac 1680

gccgcaagga caagagaggg cggcgtggca tagacgaaag gaaaaggtta aagccaagaa 1740

actcgccgca cttgaacagg cactagccaa cacactgaac gctatctcat aacgaacata 1800

aaggacacaa tgcaatgaac attaccgaca tcatgaacgc tatcgacgca atcaaagcac 1860

tgccaatctg tgaacttgac aagcgtcaag gtatgcttat cgacttactg gtcgagatgg 1920

tcaacagcga gacgtgtgat ggcgagctaa ccgaactaaa tcaggcactt gagcatcaag 1980

attggtggac taccttgaag tgtctcacgg ctgacgcagg gttcaagatg ctcggtaatg 2040

gtcacttctc ggctgcttat agtcacccgc tgctacctaa cagagtgatt aaggtgggct 2100

ttaagaaaga ggattcaggc gcagcctata ccgcattctg ccgcatgtat cagggtcgtc 2160

ctggtatccc taacgtctac gatgtacagc gccacgctgg atgctatacg gtggtacttg 2220

acgcacttaa ggattgcgag cgtttcaaca atgatgccca ttataaatac gctgagattg 2280

caagcgacat cattgattgc aattcggatg agcatgatga gttaactgga tgggatggtg 2340

agtttgttga aacttgtaaa ctaatccgca agttctttga gggcatcgcc tcattcgaca 2400

tgcatagcgg gaacatcatg ttctcaaatg gagacgtacc atacatcacc gacccggtat 2460

cattctcgca gaagaaagac ggtggcgcat tcagcatcga ccctgaggaa ctcatcaagg 2520

aagtcgagga agtcgcacga cagaaagaaa ttgaccgcgc taaggcccgt aaagaacgtc 2580

acgaggggcg cttagaggca cgcagattca aacgtcgcaa ccgcaaggca cgtaaagcac 2640

acaaagctaa gcgcgaaaga atgcttgctg cgtggcgatg ggctgaacgt caagaacggc 2700

gtaaccatga ggtagctgta gatgtactag gaagaaccaa taacgctatg ctctgggtca 2760

acatgttctc tggggacttt aaggcgcttg aggaacgaat cgcgctgcac tggcgtaatg 2820

ctgaccggat ggctatcgct aatggtctta cgctcaacat tgataagcaa cttgacgcaa 2880

tgttaatggg ctgatagtct tatcttacag gtcatctgcg ggtggcctga ataggtacga 2940

tttactaact ggaagaggca ctaaatgaac acgattaaca tcgctaagaa cgacttctct 3000

gacatcgaac tggctgctat cccgttcaac actctggctg accattacgg tgagcgttta 3060

gctcgcgaac agttggccct tgagcatgag tcttacgaga tgggtgaagc acgcttccgc 3120

aagatgtttg agcgtcaact taaagctggt gaggttgcgg ataacgctgc cgccaagcct 3180

ctcatcacta ccctactccc taagatgatt gcacgcatca acgactggtt tgaggaagtg 3240

aaagctaagc gcggcaagcg cccgacagcc ttccagttcc tgcaagaaat caagccggaa 3300

gccgtagcgt acatcaccat taagaccact ctggcttgcc taaccagtgc tgacaataca 3360

accgttcagg ctgtagcaag cgcaatcggt cgggccattg aggacgaggc tcgcttcggt 3420

cgtatccgtg accttgaagc taagcacttc aagaaaaacg ttgaggaaca actcaacaag 3480

cgcgtagggc acgtctacaa gaaagcattt atgcaagttg tcgaggctga catgctctct 3540

aagggtctac tcggtggcga ggcgtggtct tcgtggcata aggaagactc tattcatgta 3600

ggagtacgct gcatcgagat gctcattgag tcaaccggaa tggttagctt acaccgccaa 3660

aatgctggcg tagtaggtca agactctgag actatcgaac tcgcacctga atacgctgag 3720

gctatcgcaa cccgtgcagg tgcgctggct ggcatctctc cgatgttcca accttgcgta 3780

gttcctccta agccgtggac tggcattact ggtggtggct attgggctaa cggtcgtcgt 3840

cctctggcgc tggtgcgtac tcacagtaag aaagcactga tgcgctacga agacgtttac 3900

atgcctgagg tgtacaaagc gattaacatt gcgcaaaaca ccgcatggaa aatcaacaag 3960

aaagtcctag cggtcgccaa cgtaatcacc aagtggaagc attgtccggt cgaggacatc 4020

cctgcgattg agcgtgaaga actcccgatg aaaccggaag acatcgacat gaatcctgag 4080

gctctcaccg cgtggaaacg tgctgccgct gctgtgtacc gcaaggacaa ggctcgcaag 4140

tctcgccgta tcagccttga gttcatgctt gagcaagcca ataagtttgc taaccataag 4200

gccatctggt tcccttacaa catggactgg cgcggtcgtg tttacgctgt gtcaatgttc 4260

aacccgcaag gtaacgatat gaccaaagga ctgcttacgc tggcgaaagg taaaccaatc 4320

ggtaaggaag gttactactg gctgaaaatc cacggtgcaa actgtgcggg tgtcgataag 4380

gttccgttcc ctgagcgcat caagttcatt gaggaaaacc acgagaacat catggcttgc 4440

gctaagtctc cactggagaa cacttggtgg gctgagcaag attctccgtt ctgcttcctt 4500

gcgttctgct ttgagtacgc tggggtacag caccacggcc tgagctataa ctgctccctt 4560

ccgctggcgt ttgacgggtc ttgctctggc atccagcact tctccgcgat gctccgagat 4620

gaggtaggtg gtcgcgcggt taacttgctt cctagtgaaa ccgttcagga catctacggg 4680

attgttgcta agaaagtcaa cgagattcta caagcagacg caatcaatgg gaccgataac 4740

gaagtagtta ccgtgaccga tgagaacact ggtgaaatct ctgagaaagt caagctgggc 4800

actaaggcac tggctggtca atggctggct tacggtgtta ctcgcagtgt gactaagcgt 4860

tcagtcatga cgctggctta cgggtccaaa gagttcggct tccgtcaaca agtgctggaa 4920

gataccattc agccagctat tgattccggc aagggtctga tgttcactca gccgaatcag 4980

gctgctggat acatggctaa gctgatttgg gaatctgtga gcgtgacggt ggtagctgcg 5040

gttgaagcaa tgaactggct taagtctgct gctaagctgc tggctgctga ggtcaaagat 5100

aagaagactg gagagattct tcgcaagcgt tgcgctgtgc attgggtaac tcctgatggt 5160

ttccctgtgt ggcaggaata caagaagcct attcagacgc gcttgaacct gatgttcctc 5220

ggtcagttcc gcttacagcc taccattaac accaacaaag atagcgagat tgatgcacac 5280

aaacaggagt ctggtatcgc tcctaacttt gtacacagcc aagacggtag ccaccttcgt 5340

aagactgtag tgtgggcaca cgagaagtac ggaatcgaat cttttgcact gattcacgac 5400

tccttcggta ccattccggc tgacgctgcg aacctgttca aagcagtgcg cgaaactatg 5460

gttgacacat atgagtcttg tgatgtactg gctgatttct acgaccagtt cgctgaccag 5520

ttgcacgagt ctcaattgga caaaatgcca gcacttccgg ctaaaggtaa cttgaacctc 5580

cgtgacatct tagagtcgga cttcgcgttc gcgtaacgcc aaatcaatac gactcactat 5640

agagggacaa actcaaggtc attcgcaaga gtggccttta tgattgacct tcttccggtt 5700

aatacgactc actataggag aaccttaagg tttaacttta agacccttaa gtgttaatta 5760

gagatttaaa ttaaagaatt actaagagag gactttaagt atgcgtaact tcgaaaagat 5820

gaccaaacgt tctaaccgta atgctcgtga cttcgaggca accaaaggtc gcaagttgaa 5880

taagactaag cgtgaccgct ctcacaagcg tagctgggag ggtcagtaag atgggacgtt 5940

tatatagtgg taatctggca gcattcaagg cagcaacaaa caagctgttc cagttagact 6000

tagcggtcat ttatgatgac tggtatgatg cctatacaag aaaagattgc atacggttac 6060

gtattgagga caggagtgga aacctgattg atactagcac cttctaccac cacgacgagg 6120

acgttctgtt caatatgtgt actgattggt tgaaccatat gtatgaccag ttgaaggact 6180

ggaagtaata cgactcagta tagggacaat gcttaaggtc gctctctagg agtggcctta 6240

gtcatttaac caataggaga taaacattat gatgaacatt aagactaacc cgtttaaagc 6300

cgtgtctttc gtagagtctg ccattaagaa ggctctggat aacgctgggt atcttatcgc 6360

tgaaatcaag tacgatggtg tacgcgggaa catctgcgta gacaatactg ctaacagtta 6420

ctggctctct cgtgtatcta aaacgattcc ggcactggag cacttaaacg ggtttgatgt 6480

tcgctggaag cgtctactga acgatgaccg ttgcttctac aaagatggct ttatgcttga 6540

tggggaactc atggtcaagg gcgtagactt taacacaggg tccggcctac tgcgtaccaa 6600

atggactgac acgaagaacc aagagttcca tgaagagtta ttcgttgaac caatccgtaa 6660

gaaagataaa gttcccttta agctgcacac tggacacctt cacataaaac tgtacgctat 6720

cctcccgctg cacatcgtgg agtctggaga agactgtgat gtcatgacgt tgctcatgca 6780

ggaacacgtt aagaacatgc tgcctctgct acaggaatac ttccctgaaa tcgaatggca 6840

agcggctgaa tcttacgagg tctacgatat ggtagaacta cagcaactgt acgagcagaa 6900

gcgagcagaa ggccatgagg gtctcattgt gaaagacccg atgtgtatct ataagcgcgg 6960

taagaaatct ggctggtgga aaatgaaacc tgagaacgaa gctgacggta tcattcaggg 7020

tctggtatgg ggtacaaaag gtctggctaa tgaaggtaaa gtgattggtt ttgaggtgct 7080

tcttgagagt ggtcgtttag ttaacgccac gaatatctct cgcgccttaa tggatgagtt 7140

cactgagaca gtaaaagagg ccaccctaag tcaatgggga ttctttagcc catacggtat 7200

tggcgacaac gatgcttgta ctattaaccc ttacgatggc tgggcgtgtc aaattagcta 7260

catggaggaa acacctgatg gctctttgcg gcacccatcg ttcgtaatgt tccgtggcac 7320

cgaggacaac cctcaagaga aaatgtaatc acactggctc accttcgggt gggcctttct 7380

gcgtttataa ggagacactt tatgtttaag aaggttggta aattccttgc ggctttggca 7440

gctatcctga cgcttgcgta tattcttgcg gtataccctc aagtagcact agtagtagtt 7500

ggcgcttgtt acttagcggc agtgtgtgct tgcgtgtgga gtatagttaa ctggtaatac 7560

gactcactaa aggaggtaca caccatgatg tacttaatgc cattactcat cgtcattgta 7620

ggatgccttg cgctccactg tagcgatgat gatatgccag atggtcacgc ttaatacgac 7680

tcactaaagg agacactata tgtttcgact tcattacaac aaaagcgtta agaatttcac 7740

ggttcgccgt gctgaccgtt caatcgtatg tgcgagcgag cgccgagcta agatacctct 7800

tattggtaac acagttcctt tggcaccgag cgtccacatc attatcaccc gtggtgactt 7860

tgagaaagca atagacaaga aacgtccggt tcttagtgtg gcagtgaccc gcttcccgtt 7920

cgtccgtctg ttactcaaac gaatcaagga ggtgttctga tgggactgtt agatggtgaa 7980

gcctgggaaa aagaaaaccc gccagtacaa gcaactgggt gtatagcttg cttagagaaa 8040

gatgaccgtt atccacacac ctgtaacaaa ggagctaacg atatgaccga acgtgaacaa 8100

gagatgatca ttaagttgat agacaataat gaaggtcgcc cagatgattt gaatggctgc 8160

ggtattctct gctccaatgt cccttgccac ctctgccccg caaataacga tcaaaagata 8220

accttaggtg aaatccgagc gatggaccca cgtaaaccac atctgaataa acctgaggta 8280

actcctacag atgaccagcc ttccgctgag acaatcgaag gtgtcactaa gccttcccac 8340

tacatgctgt ttgacgacat tgaggctatc gaagtgattg ctcgttcaat gaccgttgag 8400

cagttcaagg gatactgctt cggtaacatc ttaaagtaca gactacgtgc tggtaagaag 8460

tcagagttag cgtacttaga gaaagaccta gcgaaagcag acttctataa agaactcttt 8520

gagaaacata aggataaatg ttatgcataa cttcaagtca accccacctg ccgacagcct 8580

atctgatgac ttcacatctt gctcagagtg gtgccgaaag atgtgggaag agacattcga 8640

cgatgcgtac atcaagctgt atgaactttg gaaatcgaga ggtcaatgac tatgtcaaac 8700

gtaaatacag gttcacttag tgtggacaat aagaagtttt gggctaccgt agagtcctcg 8760

gagcattcct tcgaggttcc aatctacgct gagaccctag acgaagctct ggagttagcc 8820

gaatggcaat acgttccggc tggctttgag gttactcgtg tgcgtccttg tgtagcaccg 8880

aagtaatacg actcactatt agggaagact ccctctgaga aaccaaacga aacctaaagg 8940

agattaacat tatggctaag aagattttca cctctgcgct gggtaccgct gaaccttacg 9000

cttacatcgc caagccggac tacggcaacg aagagcgtgg ctttgggaac cctcgtggtg 9060

tctataaagt tgacctgact attcccaaca aagacccgcg ctgccagcgt atggtcgatg 9120

aaatcgtgaa gtgtcacgaa gaggcttatg ctgctgccgt tgaggaatac gaagctaatc 9180

cacctgctgt agctcgtggt aagaaaccgc tgaaaccgta tgagggtgac atgccgttct 9240

tcgataacgg tgacggtacg actaccttta agttcaaatg ctacgcgtct ttccaagaca 9300

agaagaccaa agagaccaag cacatcaatc tggttgtggt tgactcaaaa ggtaagaaga 9360

tggaagacgt tccgattatc ggtggtggct ctaagctgaa agttaaatat tctctggttc 9420

catacaagtg gaacactgct gtaggtgcga gcgttaagct gcaactggaa tccgtgatgc 9480

tggtcgaact ggctaccttt ggtggcggtg aagacgattg ggctgacgaa gttgaagaga 9540

acggctatgt tgcctctggt tctgccaaag cgagcaaacc acgcgacgaa gaaagctggg 9600

acgaagacga cgaagagtcc gaggaagcag acgaagacgg agacttctaa gtggaactgc 9660

gggagaaaat ccttgagcga atcaaggtga cttcctctgg gtgttgggag tggcagggcg 9720

ctacgaacaa taaagggtac gggcaggtgt ggtgcagcaa taccggaaag gttgtctact 9780

gtcatcgcgt aatgtctaat gctccgaaag gttctaccgt cctgcactcc tgtgataatc 9840

cattatgttg taaccctgaa cacctatcca taggaactcc aaaagagaac tccactgaca 9900

tggtaaataa gggtcgctca cacaaggggt ataaactttc agacgaagac gtaatggcaa 9960

tcatggagtc cagcgagtcc aatgtatcct tagctcgcac ctatggtgtc tcccaacaga 10020

ctatttgtga tatacgcaaa gggaggcgac atggcaggtt acggcgctaa aggaatccga 10080

aaggttggag cgtttcgctc tggcctagag gacaaggttt caaagcagtt ggaatcaaaa 10140

ggtattaaat tcgagtatga agagtggaaa gtgccttatg taattccggc gagcaatcac 10200

acttacactc cagacttctt acttccaaac ggtatattcg ttgagacaaa gggtctgtgg 10260

gaaagcgatg atagaaagaa gcacttatta attagggagc agcaccccga gctagacatc 10320

cgtattgtct tctcaagctc acgtactaag ttatacaaag gttctccaac gtcttatgga 10380

gagttctgcg aaaagcatgg tattaagttc gctgataaac tgatacctgc tgagtggata 10440

aaggaaccca agaaggaggt cccctttgat agattaaaaa ggaaaggagg aaagaaataa 10500

tggctcgtgt acagtttaaa caacgtgaat ctactgacgc aatctttgtt cactgctcgg 10560

ctaccaagcc aagtcagaat gttggtgtcc gtgagattcg ccagtggcac aaagagcagg 10620

gttggctcga tgtgggatac cactttatca tcaagcgaga cggtactgtg gaggcaggac 10680

gagatgagat ggctgtaggc tctcacgcta agggttacaa ccacaactct atcggcgtct 10740

gccttgttgg tggtatcgac gataaaggta agttcgacgc taactttacg ccagcccaaa 10800

tgcaatccct tcgctcactg cttgtcacac tgctggctaa gtacgaaggc gctgtgcttc 10860

gcgcccatca tgaggtggcg ccgaaggctt gcccttcgtt cgaccttaag cgttggtggg 10920

agaagaacga actggtcact tctgaccgtg gataattaat tgaactcact aaagggagac 10980

cacagcggtt tccctttgtt cgcattggag gtcaaataat gcgcaagtct tataaacaat 11040

tctataaggc tccgaggagg catatccaag tgtgggaggc agccaatggg cctataccaa 11100

aaggttatta tatagaccac attgacggca atccactcaa cgacgcctta gacaatctcc 11160

gtctggctct cccaaaagaa aactcatgga acatgaagac tccaaagagc aatacctcag 11220

gactaaaggg actgagttgg agcaaggaaa gggagatgtg gagaggcact gtaacagctg 11280

agggtaaaca gcataacttt cgtagtagag atctattgga agtcgttgcg tggatttata 11340

gaactaggag ggaattgcat ggacaattcg cacgattccg atagtgtatt tctttaccac 11400

attccttgtg acaactgtgg gagtagtgat gggaactcgc tgttctctga cggacacacg 11460

ttctgctacg tatgcgagaa gtggactgct ggtaatgaag acactaaaga gagggcttca 11520

aaacggaaac cctcaggagg taaaccaatg acttacaacg tgtggaactt cggggaatcc 11580

aatggacgct actccgcgtt aactgcgaga ggaatctcca aggaaacctg tcagaaggct 11640

ggctactgga ttgccaaagt agacggtgtg atgtaccaag tggctgacta tcgggaccag 11700

aacggcaaca ttgtgagtca gaaggttcga gataaagata agaactttaa gaccactggt 11760

agtcacaaga gtgacgctct gttcgggaag cacttgtgga atggtggtaa gaagattgtc 11820

gttacagaag gtgaaatcga catgcttacc gtgatggaac ttcaagactg taagtatcct 11880

gtagtgtcgt tgggtcacgg tgcctctgcc gctaagaaga catgcgctgc caactacgaa 11940

tactttgacc agttcgaaca gattatctta atgttcgata tggacgaagc agggcgcaaa 12000

gcagtcgaag aggctgcaca ggttctacct gctggtaagg tacgagtggc agttcttccg 12060

tgtaaggatg caaacgagtg tcacctaaat ggtcacgacc gtgaaatcat ggagcaagtg 12120

tggaatgctg gtccttggat tcctgatggt gtggtatcgg ctctttcgtt acgtgaacga 12180

atccgtgagc acctatcgtc cgaggaatca gtaggtttac ttttcagtgg ctgcactggt 12240

atcaacgata agaccttagg tgcccgtggt ggtgaagtca ttatggtcac ttccggttcc 12300

ggtatgggta agtcaacgtt cgtccgtcaa caagctctac aatggggcac agcgatgggc 12360

aagaaggtag gcttagcgat gcttgaggag tccgttgagg agaccgctga ggaccttata 12420

ggtctacaca accgtgtccg actgagacaa tccgactcac taaagagaga gattattgag 12480

aacggtaagt tcgaccaatg gttcgatgaa ctgttcggca acgatacgtt ccatctatat 12540

gactcattcg ccgaggctga gacggataga ctgctcgcta agctggccta catgcgctca 12600

ggcttgggct gtgacgtaat cattctagac cacatctcaa tcgtcgtatc cgcttctggt 12660

gaatccgatg agcgtaagat gattgacaac ctgatgacca agctcaaagg gttcgctaag 12720

tcaactgggg tggtgctggt cgtaatttgt caccttaaga acccagacaa aggtaaagca 12780

catgaggaag gtcgccccgt ttctattact gacctacgtg gttctggcgc actacgccaa 12840

ctatctgata ctattattgc ccttgagcgt aatcagcaag gcgatatgcc taaccttgtc 12900

ctcgttcgta ttctcaagtg ccgctttact ggtgatactg gtatcgctgg ctacatggaa 12960

tacaacaagg aaaccggatg gcttgaacca tcaagttact caggggaaga agagtcacac 13020

tcagagtcaa cagactggtc caacgacact gacttctgac aggattcttg atgactttcc 13080

agacgactac gagaagtttc gctggagagt cccattctaa tacgactcac taaaggagac 13140

acaccatgtt caaactgatt aagaagttag gccaactgct ggttcgtatg tacaacgtgg 13200

aagccaagcg actgaacgat gaggctcgta aagaggccac acagtcacgc gctctggcga 13260

ttcgctccaa cgaactggct gacagtgcat ccactaaagt taccgaggct gcccgtgtgg 13320

caaaccaagc tcaacagctt tccaaattct ttgagtaatc aaacaggaga aaccattatg 13380

tctaacgtag ctgaaactat ccgtctatcc gatacagctg accagtggaa ccgtcgagtc 13440

cacatcaacg ttcgcaacgg taaggcgact atggtttacc gctggaagga ctctaagtcc 13500

tctaagaatc acactcagcg tatgacgttg acagatgagc aagcactgcg tctggtcaat 13560

gcgcttacca aagctgccgt gacagcaatt catgaagctg gtcgcgtcaa tgaagctatg 13620

gctatcctcg acaagattga taactaagag tggtatcctc aaggtcgcca aagtggtggc 13680

cttcatgaat actattcgac tcactatagg agatattacc atgcgtgacc ctaaagttat 13740

ccaagcagaa atcgctaaac tggaagctga actggaggac gttaagtacc atgaagctaa 13800

gactcgctcc gctgttcaca tcttgaagaa cttaggctgg acttggacaa gacagactgg 13860

ctggaagaaa ccagaagtta ccaagctgag tcataaggtg ttcgataagg acactatgac 13920

ccacatcaag gctggtgatt gggttaaggt tgacatggga gttgttggtg gatacggcta 13980

cgtccgctca gttagtggca aatatgcaca agtgtcatac atcacaggtg ttactccacg 14040

cggtgcaatc gttgccgata agaccaacat gattcacaca ggtttcttga cagttgtttc 14100

atatgaagag attgttaagt cacgataatc aataggagaa atcaatatga tcgtttctga 14160

catcgaagct aacgccctct tagagagcgt cactaagttc cactgcgggg ttatctacga 14220

ctactccacc gctgagtacg taagctaccg tccgagtgac ttcggtgcgt atctggatgc 14280

gctggaagcc gaggttgcac gaggcggtct tattgtgttc cacaacggtc acaagtatga 14340

cgttcctgca ttgaccaaac tggcaaagtt gcaattgaac cgagagttcc accttcctcg 14400

tgagaactgt attgacaccc ttgtgttgtc acgtttgatt cattccaacc tcaaggacac 14460

cgatatgggt cttctgcgtt ccggcaagtt gcccggaaaa cgctttgggt ctcacgcttt 14520

ggaggcgtgg ggttatcgct taggcgagat gaagggtgaa tacaaagacg actttaagcg 14580

tatgcttgaa gagcagggtg aagaatacgt tgacggaatg gagtggtgga acttcaacga 14640

agagatgatg gactataacg ttcaggacgt tgtggtaact aaagctctcc ttgagaagct 14700

actctctgac aaacattact tccctcctga gattgacttt acggacgtag gatacactac 14760

gttctggtca gaatcccttg aggccgttga cattgaacat cgtgctgcat ggctgctcgc 14820

taaacaagag cgcaacgggt tcccgtttga cacaaaagca atcgaagagt tgtacgtaga 14880

gttagctgct cgccgctctg agttgctccg taaattgacc gaaacgttcg gctcgtggta 14940

tcagcctaaa ggtggcactg agatgttctg ccatccgcga acaggtaagc cactacctaa 15000

ataccctcgc attaagacac ctaaagttgg tggtatcttt aagaagccta agaacaaggc 15060

acagcgagaa ggccgtgagc cttgcgaact tgatacccgc gagtacgttg ctggtgctcc 15120

ttacacccca gttgaacatg ttgtgtttaa cccttcgtct cgtgaccaca ttcagaagaa 15180

actccaagag gctgggtggg tcccgaccaa gtacaccgat aagggtgctc ctgtggtgga 15240

cgatgaggta ctcgaaggag tacgtgtaga tgaccctgag aagcaagccg ctatcgacct 15300

cattaaagag tacttgatga ttcagaagcg aatcggacag tctgctgagg gagacaaagc 15360

atggcttcgt tatgttgctg aggatggtaa gattcatggt tctgttaacc ctaatggagc 15420

agttacgggt cgtgcgaccc atgcgttccc aaaccttgcg caaattccgg gtgtacgttc 15480

tccttatgga gagcagtgtc gcgctgcttt tggcgctgag caccatttgg atgggataac 15540

tggtaagcct tgggttcagg ctggcatcga cgcatccggt cttgagctac gctgcttggc 15600

tcacttcatg gctcgctttg ataacggcga gtacgctcac gagattctta acggcgacat 15660

ccacactaag aaccagatag ctgctgaact acctacccga gataacgcta agacgttcat 15720

ctatgggttc ctctatggtg ctggtgatga gaagattgga cagattgttg gtgctggtaa 15780

agagcgcggt aaggaactca agaagaaatt ccttgagaac acccccgcga ttgcagcact 15840

ccgcgagtct atccaacaga cacttgtcga gtcctctcaa tgggtagctg gtgagcaaca 15900

agtcaagtgg aaacgccgct ggattaaagg tctggatggt cgtaaggtac acgttcgtag 15960

tcctcacgct gccttgaata ccctactgca atctgctggt gctctcatct gcaaactgtg 16020

gattatcaag accgaagaga tgctcgtaga gaaaggcttg aagcatggct gggatgggga 16080

ctttgcgtac atggcatggg tacatgatga aatccaagta ggctgccgta ccgaagagat 16140

tgctcaggtg gtcattgaga ccgcacaaga agcgatgcgc tgggttggag accactggaa 16200

cttccggtgt cttctggata ccgaaggtaa gatgggtcct aattgggcga tttgccactg 16260

atacaggagg ctactcatga acgaaagaca cttaacaggt gctgcttctg aaatgctagt 16320

agcctacaaa tttaccaaag ctgggtacac tgtctattac cctatgctga ctcagagtaa 16380

agaggacttg gttgtatgta aggatggtaa atttagtaag gttcaggtta aaacagccac 16440

aacggttcaa accaacacag gagatgccaa gcaggttagg ctaggtggat gcggtaggtc 16500

cgaatataag gatggagact ttgacattct tgcggttgtg gttgacgaag atgtgcttat 16560

tttcacatgg gacgaagtaa aaggtaagac atccatgtgt gtcggcaaga gaaacaaagg 16620

cataaaacta taggagaaat tattatggct atgacaaaga aatttaaagt gtccttcgac 16680

gttaccgcaa agatgtcgtc tgacgttcag gcaatcttag agaaagatat gctgcatcta 16740

tgtaagcagg tcggctcagg tgcgattgtc cccaatggta aacagaagga aatgattgtc 16800

cagttcctga cacacggtat ggaaggattg atgacattcg tagtacgtac atcatttcgt 16860

gaggccatta aggacatgca cgaagagtat gcagataagg actctttcaa acaatctcct 16920

gcaacagtac gggaggtgtt ctgatgtctg actacctgaa agtgctgcaa gcaatcaaaa 16980

gttgccctaa gactttccag tccaactatg tacggaacaa tgcgagcctc gtagcggagg 17040

ccgcttcccg tggtcacatc tcgtgcctga ctactagtgg acgtaacggt ggcgcttggg 17100

aaatcactgc ttccggtact cgctttctga aacgaatggg aggatgtgtc taatgtctcg 17160

tgaccttgtg actattccac gcgatgtgtg gaacgatata cagggctaca tcgactctct 17220

ggaacgtgag aacgatagcc ttaagaatca actaatggaa gctgacgaat acgtagcgga 17280

actagaggag aaacttaatg gcacttcttg accttaaaca attctatgag ttacgtgaag 17340

gctgcgacga caagggtatc cttgtgatgg acggcgactg gctggtcttc caagctatga 17400

gtgctgctga gtttgatgcc tcttgggagg aagagatttg gcaccgatgc tgtgaccacg 17460

ctaaggcccg tcagattctt gaggattcca ttaagtccta cgagacccgt aagaaggctt 17520

gggcaggtgc tccaattgtc cttgcgttca ccgatagtgt taactggcgt aaagaactgg 17580

ttgacccgaa ctataaggct aaccgtaagg ccgtgaagaa acctgtaggg tactttgagt 17640

tccttgatgc tctctttgag cgcgaagagt tctattgcat ccgtgagcct atgcttgagg 17700

gtgatgacgt tatgggagtt attgcttcca atccgtctgc cttcggtgct cgtaaggctg 17760

taatcatctc ttgcgataag gactttaaga ccatccctaa ctgtgacttc ctgtggtgta 17820

ccactggtaa catcctgact cagaccgaag agtccgctga ctggtggcac ctcttccaga 17880

ccatcaaggg tgacatcact gatggttact cagggattgc tggatggggt gataccgccg 17940

aggacttctt gaataacccg ttcataaccg agcctaaaac gtctgtgctt aagtccggta 18000

agaacaaagg ccaagaggtt actaaatggg ttaaacgcga ccctgagcct catgagacgc 18060

tttgggactg cattaagtcc attggcgcga aggctggtat gaccgaagag gatattatca 18120

agcagggcca aatggctcga atcctacggt tcaacgagta caactttatt gacaaggaga 18180

tttacctgtg gagaccgtag cgtatattgg tctgggtctt tgtgttctcg gagtgtgcct 18240

catttcgtgg ggcctttggg acttagccag aataatcaag tcgttacacg acactaagtg 18300

ataaactcaa ggtccctaaa ttaatacgac tcactatagg gagatagggg cctttacgat 18360

tattacttta agatttaact ctaagaggaa tctttattat gttaacacct attaaccaat 18420

tacttaagaa ccctaacgat attccagatg tacctcgtgc aaccgctgag tatctacagg 18480

ttcgattcaa ctatgcgtac ctcgaagcgt ctggtcatat aggacttatg cgtgctaatg 18540

gttgtagtga ggcccacatc ttgggtttca ttcagggcct acagtatgcc tctaacgtca 18600

ttgacgagat tgagttacgc aaggaacaac taagagatga tggggaggat tgacactatg 18660

tgtttctcac cgaaaattaa aactccgaag atggatacca atcagattcg agccgttgag 18720

ccagcgcctc tgacccaaga agtgtcaagc gtggagttcg gtgggtcttc tgatgagacg 18780

gataccgagg gcaccgaagt gtctggacgc aaaggcctca aggtcgaacg tgatgattcc 18840

gtagcgaagt ctaaagccag cggcaatggc tccgctcgta tgaaatcttc catccgtaag 18900

tccgcatttg gaggtaagaa gtgatgtctg agttcacatg tgtggaggct aagagtcgct 18960

tccgtgcaat ccggtggact gtggaacacc ttgggttgcc taaaggattc gaaggacact 19020

ttgtgggcta cagcctctac gtagacgaag tgatggacat gtctggttgc cgtgaagagt 19080

acattctgga ctctaccgga aaacatgtag cgtacttcgc gtggtgcgta agctgtgaca 19140

ttcaccacaa aggagacatt ctggatgtaa cgtccgttgt cattaatcct gaggcagact 19200

ctaagggctt acagcgattc ctagcgaaac gctttaagta ccttgcggaa ctccacgatt 19260

gcgattgggt gtctcgttgt aagcatgaag gcgagacaat gcgtgtatac tttaaggagg 19320

tataagttat gggtaagaaa gttaagaagg ccgtgaagaa agtcaccaag tccgttaaga 19380

aagtcgttaa ggaaggggct cgtccggtta aacaggttgc tggcggtcta gctggtctgg 19440

ctggtggtac tggtgaagca cagatggtgg aagtaccaca agctgccgca cagattgttg 19500

acgtacctga gaaagaggtt tccactgagg acgaagcaca gacagaaagc ggacgcaaga 19560

aagctcgtgc tggcggtaag aaatccttga gtgtagcccg tagctccggt ggcggtatca 19620

acatttaatc aggaggttat cgtggaagac tgcattgaat ggaccggagg tgtcaactct 19680

aagggttatg gtcgtaagtg ggttaatggt aaacttgtga ctccacatag gcacatctat 19740

gaggagacat atggtccagt tccaacagga attgtggtga tgcatatctg cgataaccct 19800

aggtgctata acataaagca ccttacgctt ggaactccaa aggataattc cgaggacatg 19860

gttaccaaag gtagacaggc taaaggagag gaactaagca agaaacttac agagtcagac 19920

gttctcgcta tacgctcttc aaccttaagc caccgctcct taggagaact gtatggagtc 19980

agtcaatcaa ccataacgcg aatactacag cgtaagacat ggagacacat ttaatggctg 20040

agaaacgaac aggacttgcg gaggatggcg caaagtctgt ctatgagcgt ttaaagaacg 20100

accgtgctcc ctatgagaca cgcgctcaga attgcgctca atataccatc ccatcattgt 20160

tccctaagga ctccgataac gcctctacag attatcaaac tccgtggcaa gccgtgggcg 20220

ctcgtggtct gaacaatcta gcctctaagc tcatgctggc tctattccct atgcagactt 20280

ggatgcgact tactatatct gaatatgaag caaagcagtt actgagcgac cccgatggac 20340

tcgctaaggt cgatgagggc ctctcgatgg tagagcgtat catcatgaac tacattgagt 20400

ctaacagtta ccgcgtgact ctctttgagg ctctcaaaca gttagtcgta gctggtaacg 20460

tcctgctgta cctaccggaa ccggaagggt caaactataa tcccatgaag ctgtaccgat 20520

tgtcttctta tgtggtccaa cgagacgcat tcggcaacgt tctgcaaatg gtgactcgtg 20580

accagatagc ttttggtgct ctccctgagg acatccgtaa ggctgtagaa ggtcaaggtg 20640

gtgagaagaa agctgatgag acaatcgacg tgtacactca catctatctg gatgaggact 20700

caggtgaata cctccgatac gaagaggtcg agggtatgga agtccaaggc tccgatggga 20760

cttatcctaa agaggcttgc ccatacatcc cgattcggat ggtcagacta gatggtgaat 20820

cctacggtcg ttcgtacatt gaggaatact taggtgactt acggtccctt gaaaatctcc 20880

aagaggctat cgtcaagatg tccatgatta gctctaaggt tatcggctta gtgaatcctg 20940

ctggtatcac ccagccacgc cgactgacca aagctcagac tggtgacttc gttactggtc 21000

gtccagaaga catctcgttc ctccaactgg agaagcaagc agactttact gtagctaaag 21060

ccgtaagtga cgctatcgag gctcgccttt cgtttgcctt tatgttgaac tctgcggttc 21120

agcgtacagg tgaacgtgtg accgccgaag agattcggta tgtagcttct gaacttgaag 21180

atactttagg tggtgtctac tctatccttt ctcaagaatt acaattgcct ctggtacgag 21240

tgctcttgaa gcaactacaa gccacgcaac agattcctga gttacctaag gaagccgtag 21300

agccaaccat tagtacaggt ctggaagcaa ttggtcgagg acaagacctt gataagctgg 21360

agcggtgtgt cactgcgtgg gctgcactgg cacctatgcg ggacgaccct gatattaacc 21420

ttgcgatgat taagttacgt attgccaacg ctatcggtat tgacacttct ggtattctac 21480

tcaccgaaga acagaagcaa cagaagatgg cccaacagtc tatgcaaatg ggtatggata 21540

atggtgctgc tgcgctggct caaggtatgg ctgcacaagc tacagcttca cctgaggcta 21600

tggctgctgc cgctgattcc gtaggtttac agccgggaat ttaatacgac tcactatagg 21660

gagacctcat ctttgaaatg agcgatgaca agaggttgga gtcctcggtc ttcctgtagt 21720

tcaactttaa ggagacaata ataatggctg aatctaatgc agacgtatat gcatcttttg 21780

gcgtgaactc cgctgtgatg tctggtggtt ccgttgagga acatgagcag aacatgctgg 21840

ctcttgatgt tgctgcccgt gatggcgatg atgcaatcga gttagcgtca gacgaagtgg 21900

aaacagaacg tgacctgtat gacaactctg acccgttcgg tcaagaggat gacgaaggcc 21960

gcattcaggt tcgtatcggt gatggctctg agccgaccga tgtggacact ggagaagaag 22020

gcgttgaggg caccgaaggt tccgaagagt ttaccccact gggcgagact ccagaagaac 22080

tggtagctgc ctctgagcaa cttggtgagc acgaagaggg cttccaagag atgattaaca 22140

ttgctgctga gcgtggcatg agtgtcgaga ccattgaggc tatccagcgt gagtacgagg 22200

agaacgaaga gttgtccgcc gagtcctacg ctaagctggc tgaaattggc tacacgaagg 22260

ctttcattga ctcgtatatc cgtggtcaag aagctctggt ggagcagtac gtaaacagtg 22320

tcattgagta cgctggtggt cgtgaacgtt ttgatgcact gtataaccac cttgagacgc 22380

acaaccctga ggctgcacag tcgctggata atgcgttgac caatcgtgac ttagcgaccg 22440

ttaaggctat catcaacttg gctggtgagt ctcgcgctaa ggcgttcggt cgtaagccaa 22500

ctcgtagtgt gactaatcgt gctattccgg ctaaacctca ggctaccaag cgtgaaggct 22560

ttgcggaccg tagcgagatg attaaagcta tgagtgaccc tcggtatcgc acagatgcca 22620

actatcgtcg tcaagtcgaa cagaaagtaa tcgattcgaa cttctgatag acttcgaaat 22680

taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac 22740

tttaagaagg agatatacat atggctagca tgactggtgg acagcaaatg ggtactaacc 22800

aaggtaaagg tgtagttgct gctggagata aactggcgtt gttcttgaag gtatttggcg 22860

gtgaagtcct gactgcgttc gctcgtacct ccgtgaccac ttctcgccac atggtacgtt 22920

ccatctccag cggtaaatcc gctcagttcc ctgttctggg tcgcactcag gcagcgtatc 22980

tggctccggg cgagaacctc gacgataaac gtaaggacat caaacacacc gagaaggtaa 23040

tcaccattga cggtctcctg acggctgacg ttctgattta tgatattgag gacgcgatga 23100

accactacga cgttcgctct gagtatacct ctcagttggg tgaatctctg gcgatggctg 23160

cggatggtgc ggttctggct gagattgccg gtctgtgtaa cgtggaaagc aaatataatg 23220

agaacatcga gggcttaggt actgctaccg taattgagac cactcagaac aaggccgcac 23280

ttaccgacca agttgcgctg ggtaaggaga ttattgcggc tctgactaag gctcgtgcgg 23340

ctctgaccaa gaactatgtt ccggctgctg accgtgtgtt ctactgtgac ccagatagct 23400

actctgcgat tctggcagca ctgatgccga acgcagcaaa ctacgctgct ctgattgacc 23460

ctgagaaggg ttctatccgc aacgttatgg gctttgaggt tgtagaagtt ccgcacctca 23520

ccgctggtgg tgctggtacc gctcgtgagg gcactactgg tcagaagcac gtcttccctg 23580

ccaataaagg tgagggtaat gtcaaggttg ctaaggacaa cgttatcggc ctgttcatgc 23640

accgctctgc ggtaggtact gttaagctgc gtgacttggc tctggagcgc gctcgccgtg 23700

ctaacttcca agcggaccag attatcgcta agtacgcaat gggccacggt ggtcttcgcc 23760

cagaagctgc tggtgcagtg gttttcaaag tggagtaatg ctgggggtgg cctcaacggt 23820

cgctgctagt cccgaagagg cgagtgttac ttcaacagaa gaaaccttaa cgccagcaca 23880

ggaggccgca cgcacccgcg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 23940

caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 24000

tttgctgaaa ggaggaacta tatgcgctca tacgatatga acgttgagac tgccgctgag 24060

ttatcagctg tgaacgacat tctggcgtct atcggtgaac ctccggtatc aacgctggaa 24120

ggtgacgcta acgcagatgc agcgaacgct cggcgtattc tcaacaagat taaccgacag 24180

attcaatctc gtggatggac gttcaacatt gaggaaggca taacgctact acctgatgtt 24240

tactccaacc tgattgtata cagtgacgac tatttatccc taatgtctac ttccggtcaa 24300

tccatctacg ttaaccgagg tggctatgtg tatgaccgaa cgagtcaatc agaccgcttt 24360

gactctggta ttactgtgaa cattattcgt ctccgcgact acgatgagat gcctgagtgc 24420

ttccgttact ggattgtcac caaggcttcc cgtcagttca acaaccgatt ctttggggca 24480

ccggaagtag agggtgtact ccaagaagag gaagatgagg ctagacgtct ctgcatggag 24540

tatgagatgg actacggtgg gtacaatatg ctggatggag atgcgttcac ttctggtcta 24600

ctgactcgct aacattaata aataaggagg ctctaatggc actcattagc caatcaatca 24660

agaacttgaa gggtggtatc agccaacagc ctgacatcct tcgttatcca gaccaagggt 24720

cacgccaagt taacggttgg tcttcggaga ccgagggcct ccaaaagcgt ccacctcttg 24780

ttttcttaaa tacacttgga gacaacggtg cgttaggtca agctccgtac atccacctga 24840

ttaaccgaga tgagcacgaa cagtattacg ctgtgttcac tggtagcgga atccgagtgt 24900

tcgacctttc tggtaacgag aagcaagtta ggtatcctaa cggttccaac tacatcaaga 24960

ccgctaatcc acgtaacgac ctgcgaatgg ttactgtagc agactatacg ttcatcgtta 25020

accgtaacgt tgttgcacag aagaacacaa agtctgtcaa cttaccgaat tacaacccta 25080

atcaagacgg attgattaac gttcgtggtg gtcagtatgg tagggaacta attgtacaca 25140

ttaacggtaa agacgttgcg aagtataaga taccagatgg tagtcaacct gaacacgtaa 25200

acaatacgga tgcccaatgg ttagctgaag agttagccaa gcagatgcgc actaacttgt 25260

ctgattggac tgtaaatgta gggcaagggt tcatccatgt gaccgcacct agtggtcaac 25320

agattgactc cttcacgact aaagatggct acgcagacca gttgattaac cctgtgaccc 25380

actacgctca gtcgttctct aagctgccac ctaatgctcc taacggctac atggtgaaaa 25440

tcgtagggga cgcctctaag tctgccgacc agtattacgt tcggtatgac gctgagcgga 25500

aagtttggac tgagacttta ggttggaaca ctgaggacca agttctatgg gaaaccatgc 25560

cacacgctct tgtgcgagcc gctgacggta atttcgactt caagtggctt gagtggtctc 25620

ctaagtcttg tggtgacgtt gacaccaacc cttggccttc ttttgttggt tcaagtatta 25680

acgatgtgtt cttcttccgt aaccgcttag gattccttag tggggagaac atcatattga 25740

gtcgtacagc caaatacttc aacttctacc ctgcgtccat tgcgaacctt agtgatgacg 25800

accctataga cgtagctgtg agtaccaacc gaatagcaat ccttaagtac gccgttccgt 25860

tctcagaaga gttactcatc tggtccgatg aagcacaatt cgtcctgact gcctcgggta 25920

ctctcacatc taagtcggtt gagttgaacc taacgaccca gtttgacgta caggaccgag 25980

cgagaccttt tgggattggg cgtaatgtct actttgctag tccgaggtcc agcttcacgt 26040

ccatccacag gtactacgct gtgcaggatg tcagttccgt taagaatgct gaggacatta 26100

catcacacgt tcctaactac atccctaatg gtgtgttcag tatttgcgga agtggtacgg 26160

aaaacttctg ttcggtacta tctcacgggg accctagtaa aatcttcatg tacaaattcc 26220

tgtacctgaa cgaagagtta aggcaacagt cgtggtctca ttgggacttt ggggaaaacg 26280

tacaggttct agcttgtcag agtatcagct cagatatgta tgtgattctt cgcaatgagt 26340

tcaatacgtt cctagctaga atctctttca ctaagaacgc cattgactta cagggagaac 26400

cctatcgtgc ctttatggac atgaagattc gatacacgat tcctagtgga acatacaacg 26460

atgacacatt cactacctct attcatattc caacaattta tggtgcaaac ttcgggaggg 26520

gcaaaatcac tgtattggag cctgatggta agataaccgt gtttgagcaa cctacggctg 26580

ggtggaatag cgacccttgg ctgagactca gcggtaactt ggagggacgc atggtgtaca 26640

ttgggttcaa cattaacttc gtatatgagt tctctaagtt cctcatcaag cagactgccg 26700

acgacgggtc tacctccacg gaagacattg ggcgcttaca gttacgccga gcgtgggtta 26760

actacgagaa ctctggtacg tttgacattt atgttgagaa ccaatcgtct aactggaagt 26820

acacaatggc tggtgcccga ttaggctcta acactctgag ggctgggaga ctgaacttag 26880

ggaccggaca atatcgattc cctgtggttg gtaacgccaa gttcaacact gtatacatct 26940

tgtcagatga gactacccct ctgaacatca ttgggtgtgg ctgggaaggt aactacttac 27000

ggagaagttc cggtatttaa ttaaatattc tccctgtggt ggctcgaaat taatacgact 27060

cactataggg agaacaatac gactacggga gggttttctt atgatgacta taagacctac 27120

taaaagtaca gactttgagg tattcactcc ggctcaccat gacattcttg aagctaaggc 27180

tgctggtatt gagccgagtt tccctgatgc ttccgagtgt gtcacgttga gcctctatgg 27240

gttccctcta gctatcggtg gtaactgcgg ggaccagtgc tggttcgtta cgagcgacca 27300

agtgtggcga cttagtggaa aggctaagcg aaagttccgt aagttaatca tggagtatcg 27360

cgataagatg cttgagaagt atgatactct ttggaattac gtatgggtag gcaatacgtc 27420

ccacattcgt ttcctcaaga ctatcggtgc ggtattccat gaagagtaca cacgagatgg 27480

tcaatttcag ttatttacaa tcacgaaagg aggataacca tatgtgttgg gcagccgcaa 27540

tacctatcgc tatatctggc gctcaggcta tcagtggtca gaacgctcag gccaaaatga 27600

ttgccgctca gaccgctgct ggtcgtcgtc aagctatgga aatcatgagg cagacgaaca 27660

tccagaatgc tgacctatcg ttgcaagctc gaagtaaact tgaggaagcg tccgccgagt 27720

tgacctcaca gaacatgcag aaggtccaag ctattgggtc tatccgagcg gctatcggag 27780

agagtatgct tgaaggttcc tcaatggacc gcattaagcg agtcacagaa ggacagttca 27840

ttcgggaagc caatatggta actgagaact atcgccgtga ctaccaagca atcttcgcac 27900

agcaacttgg tggtactcaa agtgctgcaa gtcagattga cgaaatctat aagagcgaac 27960

agaaacagaa gagtaagcta cagatggttc tggacccact ggctatcatg gggtcttccg 28020

ctgcgagtgc ttacgcatcc ggtgcgttcg actctaagtc cacaactaag gcacctattg 28080

ttgccgctaa aggaaccaag acggggaggt aatgagctat gagtaaaatt gaatctgccc 28140

ttcaagcggc acaaccggga ctctctcggt tacgtggtgg tgctggaggt atgggctatc 28200

gtgcagcaac cactcaggcc gaacagccaa ggtcaagcct attggacacc attggtcggt 28260

tcgctaaggc tggtgccgat atgtataccg ctaaggaaca acgagcacga gacctagctg 28320

atgaacgctc taacgagatt atccgtaagc tgacccctga gcaacgtcga gaagctctca 28380

acaacgggac ccttctgtat caggatgacc catacgctat ggaagcactc cgagtcaaga 28440

ctggtcgtaa cgctgcgtat cttgtggacg atgacgttat gcagaagata aaagagggtg 28500

tcttccgtac tcgcgaagag atggaagagt atcgccatag tcgccttcaa gagggcgcta 28560

aggtatacgc tgagcagttc ggcatcgacc ctgaggacgt tgattatcag cgtggtttca 28620

acggggacat taccgagcgt aacatctcgc tgtatggtgc gcatgataac ttcttgagcc 28680

agcaagctca gaagggcgct atcatgaaca gccgagtgga actcaacggt gtccttcaag 28740

accctgatat gctgcgtcgt ccagactctg ctgacttctt tgagaagtat atcgacaacg 28800

gtctggttac tggcgcaatc ccatctgatg ctcaagccac acagcttata agccaagcgt 28860

tcagtgacgc ttctagccgt gctggtggtg ctgacttcct gatgcgagtc ggtgacaaga 28920

aggtaacact taacggagcc actacgactt accgagagtt gattggtgag gaacagtgga 28980

acgctctcat ggtcacagca caacgttctc agtttgagac tgacgcgaag ctgaacgagc 29040

agtatcgctt gaagattaac tctgcgctga accaagagga cccaaggaca gcttgggaga 29100

tgcttcaagg tatcaaggct gaactagata aggtccaacc tgatgagcag atgacaccac 29160

aacgtgagtg gctaatctcc gcacaggaac aagttcagaa tcagatgaac gcatggacga 29220

aagctcaggc caaggctctg gacgattcca tgaagtcaat gaacaaactt gacgtaatcg 29280

acaagcaatt ccagaagcga atcaacggtg agtgggtctc aacggatttt aaggatatgc 29340

cagtcaacga gaacactggt gagttcaagc atagcgatat ggttaactac gccaataaga 29400

agctcgctga gattgacagt atggacattc cagacggtgc caaggatgct atgaagttga 29460

agtaccttca agcggactct aaggacggag cattccgtac agccatcgga accatggtca 29520

ctgacgctgg tcaagagtgg tctgccgctg tgattaacgg taagttacca gaacgaaccc 29580

cagctatgga tgctctgcgc agaatccgca atgctgaccc tcagttgatt gctgcgctat 29640

acccagacca agctgagcta ttcctgacga tggacatgat ggacaagcag ggtattgacc 29700

ctcaggttat tcttgatgcc gaccgactga ctgttaagcg gtccaaagag caacgctttg 29760

aggatgataa agcattcgag tctgcactga atgcatctaa ggctcctgag attgcccgta 29820

tgccagcgtc actgcgcgaa tctgcacgta agatttatga ctccgttaag tatcgctcgg 29880

ggaacgaaag catggctatg gagcagatga ccaagttcct taaggaatct acctacacgt 29940

tcactggtga tgatgttgac ggtgataccg ttggtgtgat tcctaagaat atgatgcagg 30000

ttaactctga cccgaaatca tgggagcaag gtcgggatat tctggaggaa gcacgtaagg 30060

gaatcattgc gagcaaccct tggataacca ataagcaact gaccatgtat tctcaaggtg 30120

actccattta ccttatggac accacaggtc aagtcagagt ccgatacgac aaagagttac 30180

tctcgaaggt ctggagtgag aaccagaaga aactcgaaga gaaagctcgt gagaaggctc 30240

tggctgatgt gaacaagcga gcacctatag ttgccgctac gaaggcccgt gaagctgctg 30300

ctaaacgagt ccgagagaaa cgtaaacaga ctcctaagtt catctacgga cgtaaggagt 30360

aactaaaggc tacataagga ggccctaaat ggataagtac gataagaacg taccaagtga 30420

ttatgatggt ctgttccaaa aggctgctga tgccaacggg gtctcttatg accttttacg 30480

taaagtcgct tggacagaat cacgatttgt gcctacagca aaatctaaga ctggaccatt 30540

aggcatgatg caatttacca aggcaaccgc taaggccctc ggtctgcgag ttaccgatgg 30600

tccagacgac gaccgactga accctgagtt agctattaat gctgccgcta agcaacttgc 30660

aggtctggta gggaagtttg atggcgatga actcaaagct gcccttgcgt acaaccaagg 30720

cgagggacgc ttgggtaatc cacaacttga ggcgtactct aagggagact tcgcatcaat 30780

ctctgaggag ggacgtaact acatgcgtaa ccttctggat gttgctaagt cacctatggc 30840

tggacagttg gaaacttttg gtggcataac cccaaagggt aaaggcattc cggctgaggt 30900

aggattggct ggaattggtc acaagcagaa agtaacacag gaacttcctg agtccacaag 30960

ttttgacgtt aagggtatcg aacaggaggc tacggcgaaa ccattcgcca aggacttttg 31020

ggagacccac ggagaaacac ttgacgagta caacagtcgt tcaaccttct tcggattcaa 31080

aaatgctgcc gaagctgaac tctccaactc agtcgctggg atggctttcc gtgctggtcg 31140

tctcgataat ggttttgatg tgtttaaaga caccattacg ccgactcgct ggaactctca 31200

catctggact ccagaggagt tagagaagat tcgaacagag gttaagaacc ctgcgtacat 31260

caacgttgta actggtggtt cccctgagaa cctcgatgac ctcattaaat tggctaacga 31320

gaactttgag aatgactccc gcgctgccga ggctggccta ggtgccaaac tgagtgctgg 31380

tattattggt gctggtgtgg acccgcttag ctatgttcct atggtcggtg tcactggtaa 31440

gggctttaag ttaatcaata aggctcttgt agttggtgcc gaaagtgctg ctctgaacgt 31500

tgcatccgaa ggtctccgta cctccgtagc tggtggtgac gcagactatg cgggtgctgc 31560

cttaggtggc tttgtgtttg gcgcaggcat gtctgcaatc agtgacgctg tagctgctgg 31620

actgaaacgc agtaaaccag aagctgagtt cgacaatgag ttcatcggtc ctatgatgcg 31680

attggaagcc cgtgagacag cacgaaacgc caactctgcg gacctctctc ggatgaacac 31740

tgagaacatg aagtttgaag gtgaacataa tggtgtccct tatgaggact taccaacaga 31800

gagaggtgcc gtggtgttac atgatggctc cgttctaagt gcaagcaacc caatcaaccc 31860

taagactcta aaagagttct ccgaggttga ccctgagaag gctgcgcgag gaatcaaact 31920

ggctgggttc accgagattg gcttgaagac cttggggtct gacgatgctg acatccgtag 31980

agtggctatc gacctcgttc gctctcctac tggtatgcag tctggtgcct caggtaagtt 32040

cggtgcaaca gcttctgaca tccatgagag acttcatggt actgaccagc gtacttataa 32100

tgacttgtac aaagcaatgt ctgacgctat gaaagaccct gagttctcta ctggcggcgc 32160

taagatgtcc cgtgaagaaa ctcgatacac tatctaccgt agagcggcac tagctattga 32220

gcgtccagaa ctacagaagg cactcactcc gtctgagaga atcgttatgg acatcattaa 32280

gcgtcacttt gacaccaagc gtgaacttat ggaaaaccca gcaatattcg gtaacacaaa 32340

ggctgtgagt atcttccctg agagtcgcca caaaggtact tacgttcctc acgtatatga 32400

ccgtcatgcc aaggcgctga tgattcaacg ctacggtgcc gaaggtttgc aggaagggat 32460

tgcccgctca tggatgaaca gctacgtctc cagacctgag gtcaaggcca gagtcgatga 32520

gatgcttaag gaattacacg gggtgaagga agtaacacca gagatggtag agaagtacgc 32580

tatggataag gcttatggta tctcccactc agaccagttc accaacagtt ccataataga 32640

agagaacatt gagggcttag taggtatcga gaataactca ttccttgagg cacgtaactt 32700

gtttgattcg gacctatcca tcactatgcc agacggacag caattctcag tgaatgacct 32760

aagggacttc gatatgttcc gcatcatgcc agcgtatgac cgccgtgtca atggtgacat 32820

cgccatcatg gggtctactg gtaaaaccac taaggaactt aaggatgaga ttttggctct 32880

caaagcgaaa gctgagggag acggtaagaa gactggcgag gtacatgctt taatggatac 32940

cgttaagatt cttactggtc gtgctagacg caatcaggac actgtgtggg aaacctcact 33000

gcgtgccatc aatgacctag ggttcttcgc taagaacgcc tacatgggtg ctcagaacat 33060

tacggagatt gctgggatga ttgtcactgg taacgttcgt gctctagggc atggtatccc 33120

aattctgcgt gatacactct acaagtctaa accagtttca gctaaggaac tcaaggaact 33180

ccatgcgtct ctgttcggga aggaggtgga ccagttgatt cggcctaaac gtgctgacat 33240

tgtgcagcgc ctaagggaag caactgatac cggacctgcc gtggcgaaca tcgtagggac 33300

cttgaagtat tcaacacagg aactggctgc tcgctctccg tggactaagc tactgaacgg 33360

aaccactaac taccttctgg atgctgcgcg tcaaggtatg cttggggatg ttattagtgc 33420

caccctaaca ggtaagacta cccgctggga gaaagaaggc ttccttcgtg gtgcctccgt 33480

aactcctgag cagatggctg gcatcaagtc tctcatcaag gaacatatgg tacgcggtga 33540

ggacgggaag tttaccgtta aggacaagca agcgttctct atggacccac gggctatgga 33600

cttatggaga ctggctgaca aggtagctga tgaggcaatg ctgcgtccac ataaggtgtc 33660

cttacaggat tcccatgcgt tcggagcact aggtaagatg gttatgcagt ttaagtcttt 33720

cactatcaag tcccttaact ctaagttcct gcgaaccttc tatgatggat acaagaacaa 33780

ccgagcgatt gacgctgcgc tgagcatcat cacctctatg ggtctcgctg gtggtttcta 33840

tgctatggct gcacacgtca aagcatacgc tctgcctaag gagaaacgta aggagtactt 33900

ggagcgtgca ctggacccaa ccatgattgc ccacgctgcg ttatctcgta gttctcaatt 33960

gggtgctcct ttggctatgg ttgacctagt tggtggtgtt ttagggttcg agtcctccaa 34020

gatggctcgc tctacgattc tacctaagga caccgtgaag gaacgtgacc caaacaaacc 34080

gtacacctct agagaggtaa tgggcgctat gggttcaaac cttctggaac agatgccttc 34140

ggctggcttt gtggctaacg taggggctac cttaatgaat gctgctggcg tggtcaactc 34200

acctaataaa gcaaccgagc aggacttcat gactggtctt atgaactcca caaaagagtt 34260

agtaccgaac gacccattga ctcaacagct tgtgttgaag atttatgagg cgaacggtgt 34320

taacttgagg gagcgtagga aataatacga ctcactatag ggagaggcga aataatcttc 34380

tccctgtagt ctcttagatt tactttaagg aggtcaaatg gctaacgtaa ttaaaaccgt 34440

tttgacttac cagttagatg gctccaatcg tgattttaat atcccgtttg agtatctagc 34500

ccgtaagttc gtagtggtaa ctcttattgg tgtagaccga aaggtcctta cgattaatac 34560

agactatcgc tttgctacac gtactactat ctctctgaca aaggcttggg gtccagccga 34620

tggctacacg accatcgagt tacgtcgagt aacctccact accgaccgat tggttgactt 34680

tacggatggt tcaatcctcc gcgcgtatga ccttaacgtc gctcagattc aaacgatgca 34740

cgtagcggaa gaggcccgtg acctcactac ggatactatc ggtgtcaata acgatggtca 34800

cttggatgct cgtggtcgtc gaattgtgaa cctagcgaac gccgtggatg accgcgatgc 34860

tgttccgttt ggtcaactaa agaccatgaa ccagaactca tggcaagcac gtaatgaagc 34920

cttacagttc cgtaatgagg ctgagacttt cagaaaccaa gcggagggct ttaagaacga 34980

gtccagtacc aacgctacga acacaaagca gtggcgcgat gagaccaagg gtttccgaga 35040

cgaagccaag cggttcaaga atacggctgg tcaatacgct acatctgctg ggaactctgc 35100

ttccgctgcg catcaatctg aggtaaacgc tgagaactct gccacagcat ccgctaactc 35160

tgctcatttg gcagaacagc aagcagaccg tgcggaacgt gaggcagaca agctggaaaa 35220

ttacaatgga ttggctggtg caattgataa ggtagatgga accaatgtgt actggaaagg 35280

aaatattcac gctaacgggc gcctttacat gaccacaaac ggttttgact gtggccagta 35340

tcaacagttc tttggtggtg tcactaatcg ttactctgtc atggagtggg gagatgagaa 35400

cggatggctg atgtatgttc aacgtagaga gtggacaaca gcgataggcg gtaacatcca 35460

gttagtagta aacggacaga tcatcaccca aggtggagcc atgaccggtc agctaaaatt 35520

gcagaatggg catgttcttc aattagagtc cgcatccgac aaggcgcact atattctatc 35580

taaagatggt aacaggaata actggtacat tggtagaggg tcagataaca acaatgactg 35640

taccttccac tcctatgtac atggtacgac cttaacactc aagcaggact atgcagtagt 35700

taacaaacac ttccacgtag gtcaggccgt tgtggccact gatggtaata ttcaaggtac 35760

taagtgggga ggtaaatggc tggatgctta cctacgtgac agcttcgttg cgaagtccaa 35820

ggcgtggact caggtgtggt ctggtagtgc tggcggtggg gtaagtgtga ctgtttcaca 35880

ggatctccgc ttccgcaata tctggattaa gtgtgccaac aactcttgga acttcttccg 35940

tactggcccc gatggaatct acttcatagc ctctgatggt ggatggttac gattccaaat 36000

acactccaac ggtctcggat tcaagaatat tgcagacagt cgttcagtac ctaatgcaat 36060

catggtggag aacgagtaat tggtaaatca caaggaaaga cgtgtagtcc acggatggac 36120

tctcaaggag gtacaaggtg ctatcattag actttaacaa cgaattgatt aaggctgctc 36180

caattgttgg gacgggtgta gcagatgtta gtgctcgact gttctttggg ttaagcctta 36240

acgaatggtt ctacgttgct gctatcgcct acacagtggt tcagattggt gccaaggtag 36300

tcgataagat gattgactgg aagaaagcca ataaggagtg atatgtatgg aaaaggataa 36360

gagccttatt acattcttag agatgttgga cactgcgatg gctcagcgta tgcttgcgga 36420

cctttcggac catgagcgtc gctctccgca actctataat gctattaaca aactgttaga 36480

ccgccacaag ttccagattg gtaagttgca gccggatgtt cacatcttag gtggccttgc 36540

tggtgctctt gaagagtaca aagagaaagt cggtgataac ggtcttacgg atgatgatat 36600

ttacacatta cagtgatata ctcaaggcca ctacagatag tggtctttat ggatgtcatt 36660

gtctatacga gatgctccta cgtgaaatct gaaagttaac gggaggcatt atgctagaat 36720

ttttacgtaa gctaatccct tgggttctcg ctgggatgct attcgggtta ggatggcatc 36780

tagggtcaga ctcaatggac gctaaatgga aacaggaggt acacaatgag tacgttaaga 36840

gagttgaggc tgcgaagagc actcaaagag caatcgatgc ggtatctgct aagtatcaag 36900

aagaccttgc cgcgctggaa gggagcactg ataggattat ttctgatttg cgtagcgaca 36960

ataagcggtt gcgcgtcaga gtcaaaacta ccggaacctc cgatggtcag tgtggattcg 37020

agcctgatgg tcgagccgaa cttgacgacc gagatgctaa acgtattctc gcagtgaccc 37080

agaagggtga cgcatggatt cgtgcgttac aggatactat tcgtgaactg caacgtaagt 37140

aggaaatcaa gtaaggaggc aatgtgtcta ctcaatccaa tcgtaatgcg ctcgtagtgg 37200

cgcaactgaa aggagacttc gtggcgttcc tattcgtctt atggaaggcg ctaaacctac 37260

cggtgcccac taagtgtcag attgacatgg ctaaggtgct ggcgaatgga gacaacaaga 37320

agttcatctt acaggctttc cgtggtatcg gtaagtcgtt catcacatgt gcgttcgttg 37380

tgtggtcctt atggagagac cctcagttga agatacttat cgtatcagcc tctaaggagc 37440

gtgcagacgc taactccatc tttattaaga acatcattga cctgctgcca ttcctatctg 37500

agttaaagcc aagacccgga cagcgtgact cggtaatcag ctttgatgta ggcccagcca 37560

atcctgacca ctctcctagt gtgaaatcag taggtatcac tggtcagtta actggtagcc 37620

gtgctgacat tatcattgcg gatgacgttg agattccgtc taacagcgca actatgggtg 37680

cccgtgagaa gctatggact ctggttcagg agttcgctgc gttacttaaa ccgctgcctt 37740

cctctcgcgt tatctacctt ggtacacctc agacagagat gactctctat aaggaacttg 37800

aggataaccg tgggtacaca accattatct ggcctgctct gtacccaagg acacgtgaag 37860

agaacctcta ttactcacag cgtcttgctc ctatgttacg cgctgagtac gatgagaacc 37920

ctgaggcact tgctgggact ccaacagacc cagtgcgctt tgaccgtgat gacctgcgcg 37980

agcgtgagtt ggaatacggt aaggctggct ttacgctaca gttcatgctt aaccctaacc 38040

ttagtgatgc cgagaagtac ccgctgaggc ttcgtgacgc tatcgtagcg gccttagact 38100

tagagaaggc cccaatgcat taccagtggc ttccgaaccg tcagaacatc attgaggacc 38160

ttcctaacgt tggccttaag ggtgatgacc tgcatacgta ccacgattgt tccaacaact 38220

caggtcagta ccaacagaag attctggtca ttgaccctag tggtcgcggt aaggacgaaa 38280

caggttacgc tgtgctgtac acactgaacg gttacatcta ccttatggaa gctggaggtt 38340

tccgtgatgg ctactccgat aagacccttg agttactcgc taagaaggca aagcaatggg 38400

gagtccagac ggttgtctac gagagtaact tcggtgacgg tatgttcggt aaggtattca 38460

gtcctatcct tcttaaacac cacaactgtg cgatggaaga gattcgtgcc cgtggtatga 38520

aagagatgcg tatttgcgat acccttgagc cagtcatgca gactcaccgc cttgtaattc 38580

gtgatgaggt cattagggcc gactaccagt ccgctcgtga cgtagacggt aagcatgacg 38640

ttaagtactc gttgttctac cagatgaccc gtatcactcg tgagaaaggc gctctggctc 38700

atgatgaccg attggatgcc cttgcgttag gcattgagta tctccgtgag tccatgcagt 38760

tggattccgt taaggtcgag ggtgaagtac ttgctgactt ccttgaggaa cacatgatgc 38820

gtcctacggt tgctgctacg catatcattg agatgtctgt gggaggagtt gatgtgtact 38880

ctgaggacga tgagggttac ggtacgtctt tcattgagtg gtgatttatg ca 38932