CN112011520B - Temperate phage VneM1 for regulating coral flora and application thereof - Google Patents

Abstract

The invention discloses a temperate bacteriophage VneM1 for regulating and controlling coral flora and application thereof. BacteriophageVibrio neocaledonicus bacteriophageVneM1, accession number: GDMCC NO. 61215-B1. The invention relates to coral symbiotic bacteriaVibrio neocaledonicusIsolation of a novel phage, from SCSIO 43009Vibrio neocaledonicus bacteriophageVneM1, the phage VneM1 has a cracking effect on coral symbiotic vibrios, the composition of coral symbiotic microorganisms is regulated and controlled, and hydranth infection experiments prove that coral albinism caused by temperature rise can be delayed. The bacteriophage VneM1 is a temperate bacteriophage and is normally present as a prophage integrated into the genome of the lysogen SCSIO 43009. The lysogenic-lytic conversion can occur spontaneously to produce phage, and environmental factors, such as SOS stress, can promote the lysogenic-lytic conversion.

Description

Temperate phage VneM1 for regulating coral flora and application thereof

The invention belongs to the field of marine organisms, and particularly relates to a temperate bacteriophage for regulating and controlling coral intestinal flora and application thereof.

Background

The coral reef is a marine ecosystem which is composed of marine animals and plants such as coral, fish, algae and the like, microorganisms and the like and has rich varieties. The coral reef ecosystem has extremely high productivity, has important economic value and also has important function in the processes of biogeochemical circulation and the like. However, the productivity of the coral reef ecosystem has declined dramatically during the past 30 years under the combined environmental and artificial pressures such as global warming. The current situation of coral reefs in China is particularly severe, and the number of reef-building corals forming the submerged reefs in continents and Hainan islands is reduced by 80%. It is reported that one of the important causes of its sharp decline is the outbreak of coral disease. Among them, more than half of coral diseases are closely related to symbiotic microorganisms. Coral tissues are rich in microorganisms both in their internal and surface mucus, and coral and these associated microorganisms together form a coral hologen (coral hologen). The community composition and structure of coral symbiotic microorganisms are important factors influencing the overall health of coral symbiosis. In the environmentUnder the pressure, the coral symbiosis population evolves in various modes such as microbial genome recombination and mutation, acquisition or loss of functional microbial groups, horizontal gene transfer and the like, and a microbial group beneficial to the coral symbiosis population is selected, so that the coral can better resist the environmental pressure. Meanwhile, the microorganisms in the coral symbiotic population can maintain the balance of the symbiotic population as a result of the direct dynamic action of the symbiotic microorganisms and environmental factors through various means, such as photosynthesis, nitrogen fixation, nutrition supply, disease resistance, and the like. However, coral commensal microorganisms may also directly cause coral disease. Pathogenic bacteria of coral condition under the environmental pressure of increased temperature, acidification, etcVibrio coralliilyticus、Vibrio shiloiAnd the abundance in the community is greatly increased, and the overall health of coral symbiosis is threatened. Reports show that the bacteriophage in intestinal tracts of human beings and animals can regulate and control the composition of intestinal flora by cracking and lysogenic conversion of intestinal bacteria, thereby influencing the intestinal health. Researches show that similar to the situation in intestinal tracts of human beings and animals, a large amount of temperate phages exist in coral symbiotic population, but few reports exist on whether the coral symbiotic microbial flora can be regulated. Therefore, the identified bacteriophage with the regulation and control function on the coral flora plays an important role in balancing coral symbiotic microbiome under environmental pressure by using the bacteriophage and maintaining the composition of the coral flora.

In human and animal symbiosis populations, bacteriophages are core members of the human and animal gut flora and potential regulators, affecting human and animal health by regulating gut microflora composition. The research of using a bacterium-limiting mouse model discovers that the lysis effect of the bacteriophage not only directly regulates and controls the population of sensitive bacteria, but also generates cascade effect on other bacterial populations through the interaction between bacteria, thereby influencing the population of other bacteriophage non-sensitive bacteria planted in intestinal tracts. Moreover, changes in the flora caused by bacteriophages directly affect the gut metabolome and thus the health of the animal host. Researchers have also been working on finding effective treatments by insightfully understanding the relationship between bacteriophage and gut flora for human diseases such as type II diabetes, asthma and obesity. In coral symbiotic totality, bacteriophagiaThe body plays an important role in the health of coral. The phage may help coral protect against disease by lysing pathogenic bacteria, such as by specifically killing coral pathogenic bacteria with the BA3 phageThalassomonas loyanaThe spreading of pathogen in coral can be effectively controlled, however, because the phage infection has species specificity, the BA3 phage can not act on coral diseases caused by other coral pathogens. Macro-microbiome studies have found that some corals inherently possess beneficial bacteriophages that enhance the resistance of corals to disease, but these bacteriophages have not been identified. In the coral symbiotic totality, some pathogenic vibrios, e.g.Vibrio coralliilyticus、Vibrio shiloi、Vibrio carchariaeUnder the environmental pressure, the enrichment is obviously increased, and the flora balance is broken, thereby causing coral diseases. Therefore, the screening of the phage with the cracking effect on the coral pathogenic vibrios plays an important role in balancing coral intestinal flora and preventing and controlling coral diseases by using the phage.

Disclosure of Invention

The first purpose of the invention is to provide a bacteriophage which can crack coral symbiotic vibrio, regulate and control coral symbiotic microbial flora and enhance the high-temperature tolerance of corallinaVibrio neocaledonicus bacteriophageVneM1, deposited at the guangdong province collection of microorganisms at 28 months 9 of 2020 with accession number: GDMCC number 61215-B1.

It is a second object of the present invention to provide a bacteriophageVibrio neocaledonicus bacteriophageApplication of VneM1 in preparation of a preparation for cracking coral symbiotic vibrios, a preparation for regulating coral symbiotic microbial flora and/or a preparation for enhancing high-temperature tolerance of coral.

The third purpose of the invention is to provide a preparation for cracking coral symbiotic vibrio, a preparation for regulating coral symbiotic microbial flora and/or a preparation for enhancing coral high-temperature tolerance, which contains bacteriophageVibrio neocaledonicus bacteriophageVneM1 as active ingredient.

The coral commensal vibrio is preferably Vibrio parahaemolyticusV neocaledonicus、V. fortis、V. chagasiiOrV. coralliilyticus。

The coral is preferably a tufted helmet-shaped coral.

The invention relates to coral symbiotic bacteriaVibrio neocaledonicusIsolation of a novel phage, from SCSIO 43009Vibrio neocaledonicus bacteriophageVneM1, the phage VneM1 has a cracking effect on coral symbiotic vibrios, the composition of coral symbiotic microorganisms is regulated and controlled, and hydranth infection experiments prove that coral albinism caused by temperature rise can be delayed. The bacteriophage VneM1 is a temperate bacteriophage and is normally present as a prophage integrated into the genome of the lysogen SCSIO 43009. The lysogenic-lytic conversion can occur spontaneously to produce phage, and environmental factors, such as SOS stress, can promote the lysogenic-lytic conversion.

In conclusion, the invention finds that the regulation and control effect of VneM1 on coral intestinal flora is pioneering in the field, and provides an alternative phage for treating coral diseases caused by Vibrio corallinus by using the phage.

BacteriophageVibrio neocaledonicus bacteriophageVneM1, deposited at the guangdong province collection of microorganisms (GDMCC) at 28 month 9, 2020, address: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61215-B1.

Drawings

A in FIG. 1 is a sequence analysis of the phage VneM1, and B in FIG. 1 is a transmission electron microscope observation of the morphology of the phage VneM 1.

FIG. 2A shows that prophage VneM1 can undergo autolysis of the host bacterium mediated by lysogenic-lytic conversion, FIG. 2B shows that plaques are produced (the indicator bacterium is SCSIO 43009), and FIG. 2C shows that phage VneM1 can undergo lysogenic-lytic conversion spontaneously during the cultivation of lysogenic bacterium SCSIO 43009 under the observation of a fluorescence microscope. The fluorescence intensity indicates the proliferation of the phage. Wherein SCSIO 43009 represents a host bacterium containing VneM1 prophage. + MMC indicates that addition of 1 mM mitomycin C induced SOS stress in the cells. Δ VneM1 represents a mutant in SCSIO 43009 that knocks out VneM1 prophage. PC denotes the field of view under white light; GFP represents the field under fluorescence; merge means that two fields of view overlap.

FIG. 3 is the lysis of various coral commensals by bacteriophage VneM 1.

FIG. 4 is a single hydranth invasion model used for detecting the influence and whitening rate of sea water injection, SCSIO 43009 bacterial liquid without VneM1 prophage, SCSIO 43009 bacterial liquid integrated with VneM1 and VneM1 phage injection on the health of the tufted coral under high temperature stress conditions (32 ℃).

FIG. 5 is the result of diversity analysis of coral symbiotic microorganisms after 7 days of treatment of sea water-injected coral samples with bacteriophage VneM1 under high temperature stress conditions (32 ℃).

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

a,Vibrio neocaledonicusExtraction of SCSIO 43009 genomic DNA:

coral symbiotic bacteria preserved at-80 deg.CVibrio neocaledonicusSCSIO 43009 (SCSIO 43009 for short), streaking 2216E culture medium flat plate, and standing overnight in an incubator at 30 ℃; picking single colony in a 250 ml triangular flask containing 25 ml fresh 2216E culture medium, culturing in an incubator at 25 ℃ for 12 hours under 220 rpm shaking; transferred at 1:100 into a 250 ml Erlenmeyer flask containing 25 ml of fresh 2216E medium, and shake-cultured at 220 rpm for 5-6 hours (to log phase); centrifuging at 12,000 rpm for 1 min, collecting thallus, and removing supernatant; total DNA extraction was performed using a bacterial genomic DNA extraction kit (TIANGEN, BEIJING) to obtain SCSIO 43009 genomic DNA, and whole genome sequencing and annotation analysis were performed on SCSIO 43009.

II, separation and identification of phage VneM1

The VneM1 phage was isolated and purified by the double-layer plate method: firstly, preparing semisolid R-top culture medium (2216E culture medium containing 0.75% of agar) and R-bottom culture medium (2216E culture medium containing 1.5% of agar) respectively; culturing the SCSIO 43009 in 2216E culture medium overnight, adding 1 microgram/ml of mitomycin into the SCSIO 43009 bacterial solution cultured overnight, and mixing with the semi-solid R-top culture medium in a volume ratio of 1:3And laid on a poured R-bottom medium, and incubated in an incubator at 30 ℃ overnight until plaques are observed. Next, a single plaque was gently picked with toothpick on a plate where a large number of plaques were formed and dissolved in 100. mu.l of SM buffer (100 mM NaCl, 8 mM MgSO 2)₄50 mM Tris-HCl); mixing 2 mul of the culture solution with the SCSIO 43009 bacterial solution cultured overnight, and incubating overnight in an incubator at 30 ℃ until plaques can be observed; repeating the purification steps for three times, and dissolving the plaques formed on the plate into the SM buffer to finally obtain a large amount of pure phage storage solution for later use.

Extracting total DNA of the phage: a single colony of SCSIO 43009 was picked up in 25 ml of 2216E medium and shake-cultured overnight at 30 ℃. Inoculating overnight bacteria liquid into 1L 2216E culture medium according to the proportion of 1:100, and shaking-culturing for 12 h at 30 ℃. Adding 1 ml of phage storage solution into 1L of bacterial liquid, and continuing shaking culture for 6 h. The phage were precipitated with PEG8000 as follows: cooling the incubated bacterial liquid to room temperature, adding pancreatic DNase I and RNase until the final concentration is 1 mug/ml, and incubating for 30 min at room temperature; adding 29.2 g of solid NaCl into each 500 ml of culture, stirring to dissolve the NaCl, and then carrying out ice bath for 1 h; centrifuging at 4 deg.C for 10 min at 11000 g to remove cell debris; taking the supernatant, adding PEG8000 to a final concentration of 10% (m/V), and slowly stirring until the supernatant is completely dissolved; the dissolved solution was allowed to stand at 4 ℃ and precipitated overnight. Centrifuging at 4 deg.C for 10 min at 11000 g to recover precipitated phage, removing supernatant, and removing residual liquid to obtain phage precipitate. The bacteriophage after precipitation has high purity, and is directly extracted by using a bacterial genome DNA extraction kit (TIANGEN, BEIJING) to obtain bacteriophage genome DNA for whole genome sequencing of VneM1 bacteriophage.

Next, we performed total DNA sequencing and transmission electron microscopy on the phages, respectively. Sequencing results show that the VneM1 phage genome is double-stranded circular DNA, the nucleotide sequence of the VneM1 phage genome is shown as SEQ ID No.1, the size of the VneM phage genome is about 35kb, 48 ORFs are coded, 22 functional ORFs including integrase, regulatory genes, replication proteins, structural proteins and the like of the phage are found through annotation, and the rest ORFs are hypothetical proteins or unknown functional proteins (Table 1 and FIG. 1A). Determination of the phage VneM1In-situ analysis, it was found that the DNA of VneM1 integrated into the 5' -end of the DsbA family thioredoxin-encoding gene on the SCSIO 43009 chromosome, and that the integration of VneM1 did not affect the sequences of the flanking genes due to the consensus sequence of the integration sites at both ends of the VneM1 prophage. Meanwhile, the transmission electron microscope detection result shows that the phage VneM1 is a typical tailed phage, has an icosahedral head and a flexible tail, belongs to the myotailphage family (Myoviridae) (fig. 1B), and the phage VneM1 is named as: bacteriophageVibrio neocaledonicus bacteriophageVneM1, deposited at the guangdong province collection of microorganisms (GDMCC) at 28 month 9 2020, address: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61215-B1.

TABLE 1 functional annotation of genes contained in bacteriophage VneM1

Thirdly, prophage VneM1 can be converted into phage by lysogenic-lysis

The construction method of the original phage VneM1 knockout strain in SCSIO 43097 is as follows:

the plasmid pMBLcas9 (the construction method is shown in Wang et al,Journal of Antimicrobial Chemotherapy2019, 74(9), 2559-2565) for the prophage VneM1 knockout. We selected the prophage-encoded integrase gene as the target for the sgRNA. Firstly, gene fragments of upstream and downstream homologous arms of prophage and sgRNA are obtained by PCR amplification by using primers (table 2); purification of homology arms and sgRNA fragments, pMBLcas9AhdI, enzyme digestion, namely, carrying out multi-fragment recombination by adopting a Clonexpress MultiS kit (Vazyme), introducing the recombinant fragments into escherichia coli WM3064 competent cells by a chemical conversion method, and sequencing to verify the recombinant plasmids. Subsequent transfer of the recombinant plasmid to S by means of conjugative transferCSIO 43009. Briefly, WM3064 containing recombinant plasmid was used as donor, and SCSIO 43009 was used as recipient to perform conjugation transfer experiment, and the donor and recipient were cultured to OD₆₀₀At 1.0, cells were collected by centrifugation at 6000 rpm for 2 minutes, washed 2 times with each medium, resuspended in 50. mu.L of LB after removing the supernatant, and spotted onto an LB plate containing 0.3 mM DAP after mixing the two cells. After overnight incubation, the lawn was streaked out using an inoculating loop to 2216E plates containing chloramphenicol (15. mu.g/mL). And (3) determining a correct knockout strain by using wF/wR primer PCR (polymerase chain reaction) of the zygote obtained on the resistant plate, thereby realizing the knockout of VneM1 prophage and obtaining a prophage knockout mutant strain delta VneM 1.

It was found that the cells spontaneously lysed when SCSIO 43009 was cultured overnight in 2216E medium, whereas the VneM1 prophage mutant was knocked out and no autolysis occurred. The double-layer plate detection confirms that the VneM1 prophage integrated on the SCSIO 43009 is caused by the self-lysis of the host bacterium SCSIO 43009 due to the generation of phage particles through lysogenic lysis conversion. Addition of 1 μ g/ml mitomycin to 2216E medium promoted the development of autolysis with a 1000-fold increase in titer of released phage particles (FIGS. 2A and 2B), suggesting that SOS stress in bacteria would activate lysogenic-lytic conversion of VneM1 prophage. The main capsid protein MCP encoded by the phage is an essential factor for phage assembly, the expression amount of the phage is low when the phage is integrated on a gene in a prophage form, green fluorescence can not be observed by a fluorescence microscope, and the fluorescence can be observed only when the phage is highly expressed in the processes of massive replication and assembly, so that the phage formation and release processes can be indicated.

The study then carried out green fluorescence labeling of the Peg902 Major Capsid Protein (MCP) of the VneM1 phage using the suicide plasmid plp12 (Luo et al, PLoS ONE 2015, 10 (12): e 0144465) in the following experimental procedure: first, design primers willgfpInsertion of coding region of Gene into MCP-encoding GenemcpBefore the stop codon of (3), MCP and GFP proteins were expressed as a fusion (Table 2); obtained by PCR amplification using genomic DNA of VneM1 phage as templatemcpUpstream and downstream homology arms andgfpthe fragments were purified separately, and then subjected to multi-fragment recombination with a Clonexpress MultiS kit (Vazyme) and a linear plp12 plasmid obtained by reverse PCR amplification, and the recombinant fragments were introduced into E.coli WM3064 competent cells by a chemical transformation method, and the recombinant plasmids were verified by sequencing. Taking WM3064 containing recombinant plasmid as donor bacteria, taking SCSIO 43009 as acceptor bacteria to perform conjugation transfer experiment, and culturing the donor bacteria and the acceptor bacteria to OD respectively₆₀₀At 1.0, cells were collected by centrifugation at 6000 rpm for 2 minutes, washed 2 times with each medium, resuspended in 50. mu.L of LB after removing the supernatant, and spotted onto an LB plate containing 0.3 mM DAP after mixing the two cells. After overnight incubation, the lawn was streaked using an inoculating loop to a 2216E plate containing 15. mu.g/mL chloramphenicol. And (3) determining the correct single-exchange strain by using MCP-dF/wR and MCP-wF/dR primers through PCR (polymerase chain reaction) on the monoclonal obtained on the resistant plate, inoculating the single-exchange strain into 25 ml 2216E culture medium containing 1% arabinose, carrying out shake flask culture at 30 ℃ for 12 h, then scribing the 2216E plate, and carrying out incubator culture at 30 ℃ until a single colony grows out. Single colonies were picked and streaked onto 2216E plates containing 15. mu.g/mL chloramphenicol and 2216E non-resistant plates, after incubation at 30 ℃ single colonies were picked that did not grow on 2216E plates containing 15. mu.g/mL chloramphenicol but on 2216E plates, verified and sequenced with MCP-wF/-wR and MCP-dF/-dR primers, and the correct strain was constructed and named 43009 MCP:: gfp.

Culturing strain 43009 MCP in 2216E liquid culture medium overnight, taking a small amount of thallus to perform fluorescence microscope observation, and finding that 43009 MCP shows that part of cells can emit green fluorescence during the culture process, and some cells have strong fluorescence and are full of the whole cells; the presence of a large number of fluorescent particles extracellularly in the fluorescent field (see FIG. 2C) indicates that VneM1 is a temperate phage that undergoes a lysogenic-lytic transition during culture, replicates and expands within the host cell, and is released extracellularly.

TABLE 2 primers used in the present invention

Fourthly, bacteriophage VneM1 cracks various coral vibrio symbiosis

The lysis effect of the phage VneM1 on coral symbiotic bacteria is detected by a double-layer plate method, firstly, a semisolid R-top culture medium (2216E culture medium containing 0.75% of agar) and an R-bottom culture medium (2216E culture medium containing 1.0% of agar) are respectively prepared; secondly, taking symbiotic vibrios separated from various coral intestinal tract digestive circulation cavities as test bacteria, performing overnight culture by using 2216E culture medium, mixing the obtained bacterial liquid with semisolid R-top culture medium in a volume ratio of 1:3, paving the mixture on poured R-bottom culture medium, then dropping a gradient diluted VneM1 phage storage solution on the poured R-top culture medium, performing overnight incubation in an incubator at 30 ℃, observing the generation condition of plaques and recording and taking photos. The detection of the double-layer plate method shows that the phage VneM1 can crack various coral symbiotic vibrios including those fromV. fortis、V. chagasiiCoral pathogenicityV. coralliilyticusMultiple strains of the arc species (FIG. 3). These results show that the bacteriophage VneM1 has a lysis effect on coral vibrio symbiosis and a potential function of regulating coral intestinal flora.

Fifthly, the phage VneM1 can regulate and control coral symbiotic microbial flora and enhance the high-temperature tolerance of the cluster helmet-shaped coral

The influence of VneM1 phage on coral intestinal flora composition and coral health is detected by a single hydranth infection model in coral, and the adopted experimental coral is south sea hermatypic coral Bunge helmet-shaped coral. Firstly, selecting mature and healthy single cups of non-marginal parts of coral groups, fixing the single cups of coral on a ceramic substrate by using glue, and temporarily culturing for 14 days in a coral circulating culture system to eliminate physiological damage to the coral in the separation process. Subsequently, the experimental group and the control group were set up, each group containing 10 hydranth coral, in a beaker containing 1.8L of seawater, and in a circulating heating system. 20. mu.l of seawater and bacterial suspension of SCSIO 43009 (10. mu.l) without VneM1 prophage⁸CFU/ml), VneM 1-integrated SCSIO 43009 bacterial liquid (10)⁸CFU/ml) and VneM1 phage (10)⁷CFU/ml) the sea water suspension is dropped on the surface of the hydranth of coral until the sea water is fully absorbed. Culturing at 30 deg.C for 12 hr, heating to 32 deg.C, and changing water 2 times per dayAnd (5) observing the whitening condition of the coral by photographing, and counting the whitening rate.

Detecting the influence of the phage VneM1 on the health of the coral under a high-temperature stress condition (32 ℃) by using a single hydranth infection model, wherein the result shows that the whitening rate of a control group only injected with seawater is 20% at 3 days after the temperature is raised, and the whitening rate reaches 100% at 5 days; the whitening rate of coral injected with SCSIO 43009 bacterial liquid without VneM1 at the 4 th day is 10%, and the whitening rate reaches 100% at the 6 th day; the coral whitening phenomenon of the coral injected with the SCSIO 43009 bacterial solution integrated with VneM1 and the coral whitening phenomenon of the phage VneM1 are delayed obviously, wherein the coral whitening delaying effect of the phage suspension injected with VneM1 is the most obvious, the whitening phenomenon starts at the 6 th day, and the whitening rate is 10% (figure 4). And then, selecting coral individuals subjected to temperature rise culture for 7 days before experiments, injecting seawater and phage, grinding the hydranth living body parts, performing freeze preservation, and sending to a company for gene amplicon high-throughput sequencing to determine the influence of lysogenic phage VneM1 on the structure and diversity of the clustered coralline bacteria. Through 16s rRNA amplicon high-throughput sequencing analysis, the coral injected with phage has richer microbial diversity and smaller vibrio increase ratio than the control group coral injected with seawater under high-temperature stress conditions (32 ℃) compared with healthy corals before experiments (FIG. 5). The above results show that: the VneM1 phage can regulate and control the microbial flora of the fasciola corals and enhance the high-temperature tolerance of the fasciola corals.

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> temperate phage VneM1 for regulating coral flora and application thereof

<141> 2020-10-21

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35529

<212> DNA

<213> bacteriophage (Vibrio neocaledonicus bacteriophage VnegM 1)

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catgtagctt aaaccacaag tcaactaact cagacaaacg gcggtgatcg ggtttgtcac 1680

caagccaggg cttgtcgtta acttcgcgca tggtgaattg ttcaaaggaa actgcttcac 1740

cttttgtggc aaagcgtttg cgaatgcgct taccggctcg gccctgtggg tagcactcac 1800

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gcaaaattta agagagaaaa gtagtcgttt ctaactgcat gtttgtcgga ttctaagttg 1920

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ggattaattt cagcacttat atatgaattt atgtaggtca tatcgttaat atctgaattt 2040

gttttaatgt tacgagatag ttgatacaaa atatgattta cagaaacttt tcctgaagag 2100

tgtttggtta attggaaatg cggaacaata ctagactgat ttattctatc tttactatga 2160

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taacgatgac gaagaataga gagaataact agaggcagca ctctataaga tttcttttgt 2280

gtgcatgcta cttcaatatc attcagtatt ttttttactt cacgtaatga tgtaatgcca 2340

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cttctatcaa aaaaacgact caaatagcta gttgaatcaa acccttcacc atatacagct 2520

ctaattgaat gttgtaactg ctcggtatcc gtagccagca caaaaacaaa gttttttgta 2580

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agttcatcaa tcaggacata gataggagta gatttatcat tgtgaacctt tatatatgca 2700

tcacttaatt caacaagagt ctgtttaaag tcattaatgc cttctacctt ttgcttttgt 2760

tcaagcatgg aggagaaagt gtctttaagt gaatcggtta aatcgctagt ttctagacca 2820

agatctttta gtccatctga tatgtctgaa aacagtgaat catcagcttc cattccccct 2880

tggcgtttta cgaagccaag gattaacttc catgaagtat ttttggcaaa atgtttgcca 2940

ccttccatta gtttgtttag taggtctttg ttctctgtaa tcgcactcat cagctgtgaa 3000

tgaaaagcct caagtaacgc taacatagcg tcatgagtaa aatcagactt ccatgcatca 3060

aagtaaacaa cagggtgttt ttctttcagc tcgttatacc agcactggag gaaagtagtt 3120

ttacccgctc cccattcagc attgaggtta agaacgtaac catcttcagc tttttcgttg 3180

aggaagtcgg ttagatgttc ggcataagac ctacgatcga tgactggaaa tttctcgaaa 3240

attatgtctg atgggacttc atggatgtta gacatagtac ggcctttaat gtgttacgca 3300

tgcagctttc tatagctcgg tataaatcgc cactggttcg attatgattt cgttgtgctc 3360

caagccatag gtaaatttgc tgacacctat catcgaagac gaaatagctc tcattttaga 3420

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aggatcatca gagcacttgt ggctgatgac accaagtaca acaaactctc tttgtgtcag 3540

ccttgagtac tttttgataa ttgaatttaa aggctctctt agatggtctc ggtctagata 3600

cgatgagaaa agagtatcgt gcaataactg atgaaattga agagcaccat tttggccaaa 3660

ttcaagcaac tcaataagag actcctgaaa caacttaggc tgtcttaagt tggattcaag 3720

cgcttgtttt tcgatgttat ggtttttgaa aaactcttta gacaccgcag agtctttgtc 3780

tttaacgttt gaacttaaat gcttttgctc aagtattgtt aactcagttt gcatctggga 3840

gatgattata ttctcaccaa tgcgattaaa gtttcgatag atatttagta cttcttgcga 3900

atccgcgacc agcatttgag ccttgatttt gacaaaggaa taatcggaaa ttgaggagag 3960

gatatcctct gtagaggcca gtgaatttat gtcaagaagc ttgctttctt cttctaatcg 4020

tttttgaaac aggttgaatg agtgatcgtg aaggaatttc tttgttgttg aagaggatgt 4080

ttctctaata acatccgcaa tgatttttcc agaggctagt ttaccttttt gtgtgtgctg 4140

atcggtgtgt tcaacatgat gttcgttgag gacgtattcg gtaacgcctt cgaagagttg 4200

agaggagaaa gaatagagtt ttggttcatc caaatagata aagtttttaa tcattttcct 4260

agcctagttt tatctttatg ccgttttagc gcagcagata agcgctcttt tttagattcg 4320

ttaaaagcgt cgatagagta tttgcgtttg gtatcgataa atgtccaaac ggaaaatgtg 4380

acagttaata ttaccgcgat gatgccgatt gcattgataa ggatactcaa catgtaactt 4440

ctccacctgt ttgctcgtgc tgttcagatt ttgcttttga actgagtcta aagaaaatga 4500

caaagcagag tataaatgaa caagcggcga gaaccataga cgagttggtt aaatgttcga 4560

cattgatacc tttaacaccc gattcgagca tcaataataa acacgtcagc gctccgtaaa 4620

aaaagattgc gacaatcgac ccggcgttac tcattgtttt ccaggtttga tcgcctgaca 4680

tatgttccaa ttcattgaaa atggatatgt gtagtacaaa acctacagcg ataaagtctg 4740

ccgcgttgag tggcttaact gtttccataa acgaacaaaa gaacaaacgt aaccctattg 4800

gtattagccc aatgattacg gtataccaaa gccatttcgt taacttcgac acctcagatg 4860

atttgctttt agtcacgata ttcccttata gttttgcgtc accgtactat cgattagttg 4920

aatcaaatag ccaacctact cagtaacttt ctttcgatct tcctgctttt gctgcgcaac 4980

accaataagg gcctgcatac gttgttcacg ttcatgcctg ttgatgtacc actctttaat 5040

gagaatttct aaaaggttga ttaacagagc tgcttcttct ggctcaacat caactatcac 5100

gttaatgtct ttttccatat gagcgccgat attcccgata ccacggacac cgtcaatggc 5160

tgcccatgta agagggtcaa ttttgtcttt aatactcttg atttcatcta ctagacgcgc 5220

aggtttaact tcccaaaaat ctcgcaagat tccctgtaaa catctgcgtg ccaatgttgc 5280

cgaagacttc gggctaaggt ctctgattaa gcatgcttct tcatagtcgg agagaattgc 5340

ttttggaatg taatctggaa aagttttcgc tgttgaattt ggctgaagct gccaagagcg 5400

tagtacggca tctggctctt ggtctatgtg tgcatatttt ttaaccaaaa ttgcagaaat 5460

cgaaatttcg cagcaattgg ggtttggaca agtaatcacc tcagtttcaa tcatgagcgc 5520

caccttgaat ttattaccgt ggtcaaaact gtggcgacca atactcaagt gatttccatc 5580

aatagttgca ttgtgattgc agtatgggca ttgccatgaa tgagccattt caaactccta 5640

accaatttaa ttattaaact ttctttaaag tcaccgccac acggccaatt accttaatat 5700

ctttttccga ctatttcatt gtcatttcac caggtgtcag ttggtgattc cagtcaatgt 5760

gtcataaaaa tcaccttcca ctgttagggt cttaccacca agtgcaagct gtttgtcgaa 5820

gtctttcgat gaaacaagaa caaggttata gctaagcgtt agctttttag cttctttatc 5880

gttttcgatg acttctacat caaaacgagt cttgtgctta tttgagataa cgaaatattg 5940

accatccatg tttgcagtgg cctcaaggtc taagttatct ctcatgttgt tggtcagctg 6000

attaccattt aaaagcgctg acagagattt ggtgttgaaa gtgtattgga tgcagctttg 6060

ctcgtcgcta gtgcagttat agatggtggc tgctggttga ttgtttttag tgaaaccatc 6120

accaactaat atcactttca tatcgtcttg cgcgtaactg aatgcagcac acagcccaag 6180

aactaagcca attacatatt tcattatctt tccctagcca atttttatta tttaagtttt 6240

gtttctaaca ctatcttgcc gattacctcg atatcaccgt cctgaacttc aatcgtttgt 6300

ccgtcgaaag cgatggcgag tttgccgggt aggcgttgaa cctggttgat ggagtatcgg 6360

ccgtcgatgc caatgaggta attaccattc attgcgtcgt tctctttctt atcgaccaat 6420

accacacttt ggttggtttc aatttcaatc agatcggcat tctctaaacc aaagccatta 6480

atgcggcgaa ctggatacgg cacttcgcct gtatctagca gctttccatt agttaagcag 6540

aagcttttga tgatgacaga gttagcctgc agatttatcg aacgttccga actaggataa 6600

ttagccgatc ctttttgagt ttcagagttt ctatcctctg gtctcaaagc aagttcatct 6660

agagggattc cctttgctaa atgaagccta accattaact catgcgatgt tctgtcatgt 6720

gcattccatg tagtaaatgt agattttggt actcccaaga tctcacttag ctcttctagg 6780

ttgcgacaat ttaagacctt tttgaggtta tcagtgaatt ctcgaccttt ctgatactcg 6840

tacggcaata ttttttcgtt ctcattcatg ctcaaataag acctagctct aaaaaatccc 6900

atttaaatgt tgattggtgt caaataagac cttgcaacta tgtcaaatgt gatcaataat 6960

tcctgtgtac ctgattagtg cgaataatcg tgagtacagt gaaatgttat taaatcaacg 7020

aaataggata ccaccatgtt gtcatacaac ccagtattac ccgtgccatt tgtgacgttt 7080

gaggaatact ctcgcatcac tggtctcaag cttgaaacga ttcgagacta cgtgcgtaaa 7140

ggtcgcatca tcatccaaga aaaaagagcc ccaaaagaaa agccgctggt aaacctcatt 7200

gcaatgaatg agatagccgc tcgtgaagcg ctggcaaaac taggatgagc tatgcgtttt 7260

tcctctctga ttccaaccaa aacttactgc ccgctttggc ttcatttatt cgcctgggca 7320

gtcatttgca ttccaacgtt gctataagga ttgtcaaaca tggacgcaaa catcgccatg 7380

tgcagattac gtgaggccaa acaaaacgcg tttgacgagg cgtgctgcgc atttgcaacc 7440

aatcacaaca tggcaggatt agccagaaag atggacatgg gcgaaaccat gctgcgcaac 7500

aagctcaacc cagagcagcc gcacaagctt tacgccattg agttagcctg gttgtgttac 7560

cactccggcg actactcaat ccataacgtt ctttatagca acttgggcac cgtgaccgtg 7620

gcactgccac aggaatcaga acagaaaaac ttcattgagc gcacgttgct caacaacgcg 7680

ctaagcggcg agctttctgg cgatgcaatg caaatgtgca ccgcagagcg cctgccgcgt 7740

tcaaccaaaa acaagacctt agccaaagcg catgccgcac tcggcaacct tgttttgatg 7800

atttccgatt tagagaaccg cacgactggc gcaacaccta tgttgtctat ggggctggat 7860

ttcttcgcca acggtgcgcc acttccgggt ttagcctaag gagcaataat gagtcagtta 7920

gctattcagc aagaacaatt acaacaagca cccaccgcca gcgagagcat tgctgcatgc 7980

aaagcgcttt tcaatggttc cgctacacgc ggcaagttga ggaaggtgtt caacgagttg 8040

ccagataaaa gtcgcggttt ggttcttatc gctggcggta tgtcaccaaa agattaccaa 8100

cgtgagtttg agtcgttcaa cgatttagag ctgcaaaaaa tccgttcagg aatgcagtac 8160

ctcaaagaaa tgatcgtcgg ctttgacaac acgctcggtg atgttcgtcg cctcaagcac 8220

taccaattca gtaataccca ttaatcacca agccaacctt tgcccccgct gtatccatgg 8280

gggccttttt ttcgtctaag cgtaggagca tagagatgaa taccaatcaa aagttaaccg 8340

aagcaagaaa cctcatagac caagtcctaa accgcaatgt cggcacgatt gggcatgcgg 8400

atttaccagc agaagcattg gctcaaaagc aaaagctatt tagcggccag agcaccaacc 8460

gtgaagtgtt agacattgtt aacgccatta atgcgctggc tatcagtaat caggatgtaa 8520

ttgaaattca caccggcttt gtagccaacg taaattggtt cgagacaatt gcttatgtga 8580

aaggatgtca gtacgagccg ggtgcaaata ggttgtttca gaagcgagtt cgactcgatg 8640

aagaagatgc cggcactcag ttattagcaa tcgaaagcga gataacagaa cttgtcattg 8700

aagcccgtga agaagctgaa gcaaaagcgg aggtggaagc atgagtatcc tgactatcta 8760

tcgcaaagac ttagagttcg gccttcgtca tgaaggcttc acttcacgca aaatcgagca 8820

gtttattcgc gtattcaatc cggtggaatc aagccagggc aacatgctgg agctggattc 8880

tactcgagca atgttggtga acgtgaacgg cactaagcaa ggtttgtgtc tggaagactt 8940

catcaccgca tggtgggttt actgggtagt ggtattcaac caatccacag acaacgcaac 9000

gcatcatcag gccattggcg caatccgcgc tcttttcttt atctctgcat gcacgaaaag 9060

cacaagccaa aacaccacaa tgcaaacgtg gtggcgagac tgcgaacctc tgcatggata 9120

cccaacgttg gaggctatct gatatggcat cactaactca gttacttaat gaaatcggtg 9180

acgagaacgt aacggttcaa gcacttcacc agtgtatgga ctcagctcaa tacaacaaag 9240

ggcgaaccac aatcaaattc aaaaccgatg gtcttggcgc tatcgaacta gcggatgggg 9300

aaaagaccgc tcttatcgtt tgggttgatg cggacaaata caacaatgca ctagcgaacg 9360

ccaaaacaat gaagaaatcg agcattgtgg aatcactacg tgaagctatc caacaggccg 9420

aagaactagg tttagtaaga actgaagatg gcacggtcat caccggagcg attgaaacac 9480

ctgatggcat tgttttggtt aaggagtaaa gagcaaatgg aatacgcagc aatcatgctt 9540

tgtccagatg gcggcgttgt tcgccatgaa gacactcaag aagttgccaa tgtaatggtt 9600

ggcgacttcg aatcactaga ccaggcaatc gagcaggcat gcatttctct tagttgtact 9660

cacctaacca aaggtgtgtt gagcaaagga aacggtaaag gcggctttat gttggtgacc 9720

actcaggaac tggaggcggt atgaaataca agaagattac ttccctctct gtagatgaag 9780

acaaaatcca agtctttgaa ggaagaacgg cgacattcga aaaatgcgcg gttgcttact 9840

ttgctggacc agaaggttgg ggtgtaacca tgaatattca gattgaagag ttggaaagct 9900

tcgttaatga gccagattgg caaaagcggt ttgttgaatt tgcgaaagaa aaattaggga 9960

tggtagcagc atgagatcag attacatcta cgaactagac ggtcataaag gtcaatccgc 10020

gatcgcgaaa gcgtttggcg ttccactggg tactttgaaa tcacgcctac gtaacggcaa 10080

aaccatccgt gaagcggttc attttgttga tggccgtgag aacaattgcg gcgttgctac 10140

tcacgagtgg aagggcatta aaggtatcga caatatcgct aaagcgattg gtaccacgca 10200

caccacaatc tacaagcatt tgaaagcagg ttgcactctg gatgaagcgg tcgcaaaagt 10260

tcagaaaagc cataagcgag cagagcaagt tcgcaaactt cgcgctaaat caatagccaa 10320

accaattgaa caggttggca tcaagaaacc aacggacatt agcccgcttt ggcgtgttgc 10380

tttaggtttt ggtggataaa aacaatcgtg tcgaagtacc taagcgaacc aacagaaatc 10440

gaactattag acttagataa gttcggtttt gaccatgact acaaacgtgc tgcttctctg 10500

gcttgtcaaa gttggggaag tttgcacgtt tttccgcaag cgccaaaaac actagcggat 10560

gcttgctttg gtgcaagacg ttttgatgag tcgcaagaac ctgacgacct gagcgtactc 10620

gaacgtaaac tattcgaagc aaacccagat gattttgagt gggcaaaaga caaaatccat 10680

ggcctgccag actatttaac caagtacttt gtgactcgat acatttcagt tttcgaaaag 10740

aaaggtcgta gagaagcaaa catctttttg cgtgagcgca tggggccagc agctgatcgc 10800

gccgttatgg tattacgcaa atacaaaaag ctaccaacaa cccaaaaggt tgctttgctc 10860

agtgaagaat tagataacac tgaccaaagt gactttacgt ctgcacaccc agagatagca 10920

aaacctcaac ttcgctttga tttcgacaag gcggagaaaa accgcaagcc agtgaaaagc 10980

cgtatcttgg cagagctaga acaagatgaa ctcaaagaaa tggcgttcaa gattggcaaa 11040

atcatgaatg ctcgtttcca aattatctct tctaagttgg caagcattac tgaagctgag 11100

ctggaaaaag acgatacgtt ctgtcctgtc gttgaaggtt accatcagtt agcggctttc 11160

acttctctgt ttggcatcaa gccgccttgt aaatacaaga agcaaaatga gctttccgct 11220

ctgcaagaca tctcgcgcat gattagcgaa aagtggtggc ttggtcgttt ggttaaagct 11280

cgcaaaatta tgcgtgagca cctagccatt gcaatggggc aagtatcttc aaaagcttct 11340

gcttatgcgt cttgggattg cgttcgtgag caccaggagc aacaaaagcg caactgggaa 11400

tacatcaagc agtgtgaact ctttgacgaa gaaaccgaag ataaagccga cctagccgaa 11460

atggttctga aaagcgtgtc taatcctgcg attcgccgtc atgaactgat ggtgcgttgt 11520

cgtggttgtg aagacatcgg caatgaattg ggtttgcaag gcttgttcct tactttgacc 11580

acgccctcga agtaccacaa ctcctacaag aaaggcggtt tcattgagca ttggaatggt 11640

gcgagccctc gagaggcgca agcgtactta aacaatgctt ggcagcgtat ccgcgctaag 11700

ttaggtcgtg aagaaatccg ctggtttggc gtacgtgttg ctgagccaca tcatgacggc 11760

acaccacact ggcatttgct gatctgggta aaaccagaag aagtggcgca ggtgcgtgac 11820

atctttattt cgtacgctac ccaagaagac cgcgcagaac tgcacccgca atacgaaaag 11880

gaaaagcaaa agccatttcg taagtgtgct tatgttggtc ctatggatta ccgcccacgt 11940

tgtgactttg gttacatcga cccagagaaa ggcacagcaa caggctacat cgcgaaatac 12000

atttctaaga acatcgacgg ctttgctatg gacgatgaag tgtccgacga aacaggcaaa 12060

tcagtcaaag acatggcgaa gaacgtcagc gcatggaaaa gccgctgggc gattcgccag 12120

tttcaatttt ttggtggtgc gccggttacg acttaccgag agctgcgccg cttcgcgagc 12180

caaaacaaaa aagcctttat ggagtacctc ttcatgcaag tgcgagagga tttgcttgct 12240

atgtacatta agctacagcg tgacctagtt ggacctatca agcctagtaa gctgatttct 12300

aataaagagt taataaaagt aataggggat agtttccaag caagtttaaa cactgatgat 12360

tcaagcatca cagaaacttt aaaagccgca gatcatggta attggcaagg ctacataatg 12420

gggcagggtg gaccattcgt taagcgcgaa gatttgctga tcactaactc atatcaagta 12480

cttcctttcg catcacctca cggtgaagac gtgcgcaagg tggaaggttt ccaaacaccg 12540

gaagcgatcg tgaaaactcg cactaaggtc tggacgattc agaagaaatc aaaagttatc 12600

gcagaagcag aagcgatcac ccaagggagc gcagcgaccg ctattggagc ctctggctcc 12660

tctcggagtt ctgtcaataa ctgtacggag catcgcgagg tacaggtcag cgatcagcta 12720

acccgaattt tagacccggt gaatagtcgg gcgaataacc cgccaaatat tgatgaaacg 12780

gcactggccg cactgctaaa aggcagttca attcgcatcg acgatgcaac cagtattcaa 12840

atccgccctg cggaggtaga cgaacacggg aatatacgtc cggcacagct ggtcgaggta 12900

agccgtgcgc ctgcagatga tcttaaatgg atggatttcg aaggttggga caaggtattc 12960

gcccaaccag aacagcaaga gtatcaacaa ccagacctgt cgttcttccc tgatggggac 13020

gactggccgt tggtttagat taaatcaact agccagtaag ctagtagttt atggattttt 13080

tgattgagaa ttcggagtaa ctgaagcttg catttttgag cactgaacta aaaacttatc 13140

cagagagtct gctgttgatt tcgcttgagt actagaaaat gattcgtcta agttgtagtc 13200

agcttttttt cgcaaagttt tataaatttc taagaacgtt tttagccgtc ttaggtcatt 13260

cttatcatac ggctcatttt gctttgcatc ataagatact aagtagtgct taagtgattc 13320

gtgacacccg acaccttgaa taggtaccgg gtcgcaagtc aatatttcta aaacaccgtg 13380

atacatacca tagtaacttc tactgattat gtttctgaac tcaatttcag ttgtagcact 13440

agacaatgac tgtttgacta aagtattaaa attttgagcg ttaaccgaca tttcgtaacc 13500

tcgttgcaag agtcggtgta tcatcaccga ctagttcgaa gtgtgttata catttaatat 13560

cgtcaaggtc agcctcaact aattcgcctg acaaatccca gttcaaatcg aacagttgtt 13620

caggttcgca ctcaacgtaa atcgttagcg ttgcgtgtcc tgactctggt ggtatatcta 13680

ataaagtgcc gttaagcaca acgcccttgt gagtttctgc aactcttgca gcgattaaag 13740

ataagtcggt aattgtgttt gatgaaacgt caaaccttgc ttcaatatca cgcataatca 13800

tgcgtctaac tcccgtacct ttttgaattc gcgtcaacaa ctcgttttgt tgagttcttg 13860

atagccactc tactaatttg gcatctagac caaagtcatt gaacaaaaag ttagtactta 13920

caatgttctc tacatcatat tcatctgcac attctagtgg catacgcttc actatttcgt 13980

aagctacagt aggcataccg agatagagca ttgtccagat taagttggaa gatacaaaag 14040

attcatgcgc gtatgaacga gatatactca tagctttatc ttttacttca tccaatctat 14100

cagcggcaca atatacgaga acttcaatta aagaagattc ttcaatactt ggaagattta 14160

aagcttctct cttatacgtg gcaaactcaa tatcgttcag cttttcatcg ctaaagataa 14220

ctgcgcgtaa ttcatcgagt aattcgttgg ttcgtgattt tgcaacagat tcagtcaagg 14280

ttgagcccca agttaagaac taaaaagtaa atataaatat aatttttaag agacgaaact 14340

ataccccagt tgaaatcttc aaacaatgtg cacattgtca aaaattaacc aatatgcgac 14400

aacattcatc aatatgcgcc atgtgtcaat ggctattata gtccactccc accaaaacta 14460

ttcacaattc taaaagtgta ttttctgtga gtttgatcac gtttctaaga ccttgcgcga 14520

tctggaacct aagttagctt ggtttggtaa acaacgcatt tgcccattta ggaaatgctt 14580

taccgcctgt tttaatgatg tagcagcgtg atttctatta actcgaagta aaaaaggaaa 14640

attcttaatg agtgaattat gtctaaaaac tactgtagaa acctgtatgg atatacagta 14700

ttatttccga attcagtggt tagggggtgc tatgttgttg aaaaatgaga atgaattaca 14760

acaagcgtta gacatcatta ttgatggaat agcagtgtca aatgcaaaac cggaaatgat 14820

agcaatgggc gtaaagatga tgaacatggt gctggcgaac taccagggag aaatcgacga 14880

gaagaagaga aaagccattc agtcgataat ccaaatggct gtggaagtag atagtccggt 14940

attttcttta taatatcgca agctgttgct tgagtgattg ccgctgttct ggcggcaacg 15000

ctttcaccag attaaaagcc agttggcttg tcgtttttgc cgatggactc aacgtatggc 15060

tataagacaa attcatcaca aatgtgtgtc cacattctgg gtcactgcag ctgcaataca 15120

aatctgaatg actgttggtt aagcggttgg atttttggat tcggcttttg ctaccacact 15180

ccgggcacaa cactctcata taaaatacct agcttattga ctgacttgat aatcatacgc 15240

caaaaggctg gttttttata cagcttggag aggggtttac gccgtatctc catgtattaa 15300

attaaaatta agtcgcaaat gcttaggtac ctctgggtct gagttcacct catccataat 15360

cagctcacat acaggaatga tctcatcctt agcgtattca ctgcctatct tcgttgggtc 15420

gcctaaactg gtcgtgccct gcggaataat gccagcttta cccaccggga agcggtggcc 15480

gacaagaatg tcctgcgcgg taatattttt gatgcgctca aactcatctt tggtggcgat 15540

atcaccaacc ggaatcagct gaatcccttt ctctttgccg ttcggaatat tcacaaacat 15600

actgcggaag ttacccacgc ctttagagct ggcgatcttc tgcttcaaca tttcttcatc 15660

ttcttcactc aagtttgggt cggtggcgta gaagataaag cccatatgtg cgccgttctt 15720

gtaatagcgg cgtcggaaca aggtcgcgtc tttatttagc aagctgctct gaatgctgcc 15780

taagtaatcc gccaaaccat agatttgctg ctgcgggtca tactgaggca agaaaatcac 15840

atcttccttt ttatattcgc gttgctggtt gtcgcgttcc agaatgacaa agttgccgtt 15900

tttgcgtcgg cgaaggtaca tgccaggtaa tggatgcaag cgaaccacac gtttgaaacc 15960

atcacggatt tttagaaatg cagcatcgcc aaaggtgaag taatcacggc aaaaagcttg 16020

aatatgtcgg cggcgggttg cgcctccact ttggaagcgt ccggctacat aattagcacg 16080

agctatcaac agtgaaccat gataggcgtt ggcacgagca atatccgcta agccactgcg 16140

tgaaatcgga ggttcccaat agttatcagc atcgttgtag aacaaatctg aataggtggt 16200

catccaactg tttgagtcga tggactctgg tgaggagtcg atgtgataga ccgactctgg 16260

cgcgtgttct tcttgtttga ctaaagttgt ggtttgttcg gtcatgctgc agtggcccag 16320

gttgatttag ttggtgttga gtgatctaac ggttcgttag atacggcgtg agaaatcgcc 16380

cagaatgcat cggcgtgtcc ggtcgtttca ctgcgttcgg ctttaaaggt catggcgtta 16440

ccgctggcgg ttgggacgcg cttaatcgcc ataaacgcca tggcaatgtc tttatgttcg 16500

gcatcgaact gcaggcgttt ggcttctact acatcaatca tcttcatgac cagacggttc 16560

ttgttttcgt tgctgtagtg gatggcgtga gcttcacgtg ggtatttctt agaaattaag 16620

tcccaaacac cgccgccaat gcccgtggtg tcgaccccga tgtaagtcac tttgtagcgc 16680

tgaaatactt tgtctatttc cgcaacgtga tattggaagt taagcccttt ccaatagtgc 16740

ttctcgagca cacggaattt ttcacccgcg acgacaggcg gagcgacaac caccaagcag 16800

gcattgtctc gggttcggct tgggtcgtaa cccaaccaaa cttcgcgatt ggcaaaaggt 16860

cgtttgttat ttggtttgta gtcctgccag tgagcagcat ccaccatgcc tttttcgagg 16920

tcagagaatt tgaatacaga caacgaaccg tcgacaaaca cacacataaa caggtttttg 16980

aagtcatcat cactgtattc gtcgcgcagt tcgtcaatgt caaacaggtc acaaccaccg 17040

tttgccgcat cttcaatggt gacaacataa cgccactgtt tgtcggggca gagtcgcccg 17100

ccgtctcgga attcatcaaa agtcgggaat tcgacgtggg cacgagagtc tttacctttg 17160

cgccactggt caccagtcca gaacgggtaa gcctggtgca ttttcgacga tggcgtagaa 17220

aagtaagttt tgcgccactt cttatgcgtt gccattgccg aagccagttt gttcagctcg 17280

tcgaacttag gtatccagaa atattcatcg acatacacgt ggccgtggta actctgtgcg 17340

gttttactgt tcgtggacaa aaagcgcagt tcggcgccat tggaaagaat aatcgggttg 17400

ccggttaact cgatgtctaa gaactcttta ccaatcgcaa tgatgtagct gcggaaaacc 17460

tcagcctgtg ctcgtgatgc agaaagaaag atttggttgt caccagtaag aatcgcatct 17520

tctaaggctt caccactgaa atagtaggta gcgcctatct ggcgagattt cagaatatta 17580

cgaatacgct gtttgatgtt attgcgcatc gtgtgctggt attcgaaaag ggattcgtgc 17640

cagcctttaa agtcatcttc tgtcaggtgc tcaatgctgt tcttcttacg gctacttttg 17700

cggttaccgt tactgctgct tttagaagag tcaccattac tctttggtga gctgtctggc 17760

gaaaggtgtc gctctgcttt ggcttttgca tcagcgtgag cttttaacag cttaacgtgg 17820

tgatcgataa gcttgtccat ttccttgagc tgctgatcgg ttttctcatc cttatctatc 17880

agcaccgcca aacggcggtt aatcatttcc tcaacagaaa gttcattcag caacaaagcc 17940

cagccgaatt tctccgccca ggtataaagg atgcgctcac tattaaggtt cagttgcgcc 18000

gcaatttctt tcggaggaac accgcgtaaa tagagctttt tcgcggcttc tcttatttca 18060

tctgaatatg ccatagctgc atcatacgcg ccgaaaactt gcagatgacc tagataaatt 18120

cctgaaaatt ctagttttga ttaaatccga attggcagga attgaagtgg ctgaaagccg 18180

ttattcaaag gcgtattgtt tgctcacacc gatatgtgaa tgacaagttt gagtaccaaa 18240

atgccaaaga ttagtgactg gaaaatcatt gctactgagg ggccaaccgt agacgggcgc 18300

aaaattaccc gtgattggat tgagcaaatg gcggcaagct atgacccgaa agagtacacc 18360

gcgctaatct ggccagaaca ccgtcgattt tatggctacg gtgaaaactg gggcaatgtt 18420

gctgagctta aagctgaaga agaagacggc aaattacgtc tgttcgcgaa gcttgagcca 18480

aacgaataca tgcttgaagc caaccgcaaa aagcaaaagc tgttcacatc aatcgagcca 18540

aatcccgatt acaagggcga agggcgttgc tatttaatgg gcctagctgc gactgactcc 18600

ccagcctcca caggtacctc gctccttcaa ttctctcgtc aatctggcga aaccaccgaa 18660

ttagagtgca gtgcattgga agaagtcgat ttttccgagt gcttcacgcg caaagaccgc 18720

tttttcgcgg cattcaatga atttttctct tctggcgatg aagtgccaga aactccatca 18780

atagacgagg ataccgaagt gactgaagag caacttaaag cagcactgaa agaacagttt 18840

gctgcattca aaggcgagtt caagcaagaa ctgaaagaag agttcaactt gcaaggcgaa 18900

ccagaaacac cagagacaga agagcagggc acaactgttg agcagttctc tgccactctg 18960

gacgaaaagc taaaaccgtt aactgagaaa gtgaacggtc tcgaaaccaa attcgctgaa 19020

ctttcacaag aagtaccagg ccaagagccg aacccgtcgg gtaccagtga gaagttctca 19080

tctgaggaga ttttttaatg ctgaatgcac tatctaccac ctacctacaa gagttttgtg 19140

cagcgacgtt agcagcggca aatgctccgc agggtactca gtcatttaac ctgacgccac 19200

caatggaaac caaacttcgc caggcgatca tggagtcgga tgctttcctc ggcatggtct 19260

cattgctgcc agttcagcaa attaaaggtc aggttgttga tgtgggtgat gatggcctgt 19320

ctacaggtcg taaagacagt ggtcgcttta gcgttgaagt tggtcaaagt ggtaatactt 19380

acgagctgac taagaccgat tctggcgcac atatcttgtg ggaaaccatg actcagtggg 19440

caaactcagg ctcaaaaggt cagtggttga ccatgatgaa aaacgccatc tctcgccgtt 19500

tcgcattaga catgttgcgc gttggcttta atggtacgtc tatcgcagct aacacagacc 19560

cagtcgctaa cccgcttggt caggatgtta acaaaggttg gttaaccatc gccaaagaga 19620

agaaagcgag ccaagttctt gcatctgctc aactcgaccc aacaggcgca accgcagatt 19680

cttacaaaaa cctagattcg ctggttcaag acctgattaa tacaacgatt gcaccagagc 19740

accgccaaga ccctgatcta gtggttctcg tcggttccaa cctggtcgca gcagaacagc 19800

atcgtttact ggaatcagca aacacgccaa ccgaacacaa agctgcgcag cagttagcca 19860

aaaccattgc gggtaaacag gcctatacac cgcctttctt ccctgccgat gcggtgtggg 19920

taactaacac taagaacctg caaattctta cccaagaagg tacgcagtgg cgcaagcaga 19980

agaatgacga agacgagctt cgcttcaagc aaaaccatat ccgtatggaa ggttatgcaa 20040

tcggcaacct gaacaagttc gctgcgattg aagcggtaac cgttgttgag cctgcagctt 20100

aaggagtgac gcatggttag cccattagca aaacagcgtc gacagctcat tgaaaagcag 20160

gctaaccagt ctgcaccggg agtgagttct ggtgcagata ccgacagcct gcacatcaag 20220

ctgattgagt ttgaagaaga ccgcaagtat ttgcgctctt taaatgccat cgctgatcga 20280

attaagcaca agcgtgaagt actggtaccg aagtacaagc cgtatgttca ggcctactta 20340

gaaagtggtg aagcgttcga aaacccaatc tttaccaact tggtggtttg gctattcgac 20400

attaaagagc tggataccgc aatcgaatgg tgcatgaaag ccattgagct cgacttacca 20460

acgccagaga acttccgccg tgactggccg acgttctgtg ctgacgaagt gttggcatgg 20520

gcggagaaag aatcggaacg tggtaattca atcgagcctt atttctctca ggtatttgaa 20580

aaggtcgaga aagactggcg cttgcacgag aaggttcacg caaagtggta caagttcgcg 20640

ggtttatacc tgattcgaaa tgaagaaggc cagccgcaag ctactgccat cggcaacgtg 20700

gaaacgttgg aaaaagcttt ggctctgctt caacacgcgc acgataagaa cagcaaagtg 20760

ggtgtgggta cccaaatcaa aaagattgaa tctcgtattc gcgccattaa cgaaggcaag 20820

aatctgtaaa gactcctacg ccaccgcgcc tcggctggtg aggtaagaga agccaatcgg 20880

ctaactcgat accgtcgacc cagtggctag aggcgcccta atttaaataa gcaaggaact 20940

gttatgagct ttggcggaaa tgttaacagc gcagtcgata tcgccatacc aggtgaagga 21000

tggcccgatt tatctacgga tgaattccgc agtttacgcc gtgttcccca tacgtttgat 21060

aacgactctt tgaactacgc ggtgaccatc gccgcgctaa atattcagga acgactggaa 21120

agcctgattg tagatggcga aaagccagtg ctgagcaatc cgaaaatcat gctctacaaa 21180

cgtgcggttt atggtcgggc tcatgcagaa ctgatgaaag agtttgcgac tcaagaccgc 21240

cgaaaagaag gtgaaagcgt ggcaacggat gaaccggaac aagaggcacg ttttctcgcc 21300

cagagtaaca aagatgtgcg tgcgctcctt gggcgcagtg cgaatgggat tgactcgata 21360

tgagcgaaac cacttacaac aaaaccaagc tggaacacct gacggattac atcgtcagtc 21420

acctgaattc caatgtgctc gataacaaaa tcgatgcctg gcaggaaaat ggctccatcg 21480

tgccaaacgg tgaagaccga gggaacggcg gttatatcgc ttgtcattgg aagtacaacg 21540

cagtgatcag tattgaagag tttccgcacc gacttctgga ccctcgctgt ttacttgctt 21600

tggttgcctg ttggctgagc gattacgaca ccacgcgtaa tgaagatgag ttaggcgacc 21660

cagacctttc ggttgatgtg ataaacagtg aggtagccga cgttgccatt gaattggaaa 21720

tgatggagcc gattgagctg atccctgacc ccgcgggaat gatcacttgg cgaggggaaa 21780

cctaccgagt gcaggccgtt gaaatctaca ccgcagaaga agcggagttg gtgaatgaag 21840

ccgcaaatta aggtcaatga gcgcgatgtg ctcaacatgc aggaaaagct ggccatgctg 21900

gctctgccac ctaaaaagcg agtctggata ctgaaaaccc ttggccgttg ggaaacgacc 21960

aataccagaa agcgcattcg ttcccaaaaa gacgttaacg gacgcgctct gcaaccaaag 22020

aaaggaaaga agaaaggcaa ggttttaaag cggatggcaa agggcttaac gccttatgtg 22080

agaaacgcca accagcttga tttgacttgg agtaacaagt taaccgcaaa aatcgcagca 22140

aggcaccacc ttggtcaaaa gcaaaaaatg accaagcgcc aaatgcaaaa gcgatggggc 22200

aaaccggatt actcagcacc ttgtaccaag gggcaagcga gaaagctaag agaactgggt 22260

tacacggtac cgagaaaaag cggcaaagga cgaaagaaag ccagcttacg tgagttgatg 22320

gcaactatca cccatggaca agcaggacag ctcattcgag aactctctaa tcagccaaat 22380

atcaccagtt gggatattcc attagcagag cgtcagattc tcggtagtaa agaacgtgaa 22440

gtgaaccgcc agctcatcaa gatatttgag caggcaaaac agaggaaata accaatggca 22500

accggaaagg tagaggttaa caatctcaat ttggcacaag gcggtatccc tgagatagaa 22560

cgtcacgtgc ttttcatagg gcgtactgac aaggcagaac tgcaaggcaa agtgacccgc 22620

attaataaca tgaccaacct tgacgaagtt gttgccgatg atgcgcttgg tctgaacgtg 22680

aaagccgccc agcttaacgg caaacaaaac tggacgggtg cgattgttgg cttagatgca 22740

gacgatacct ggcaagacgc tgttgatctg gcgaaccgga ctgactcatt cgaaggcatt 22800

gccatttgtg acccagtcac ggtcaaaact cagtttgatg atatgcagtc gaaagcaacc 22860

gagctgacta gcaaacttgg tcgttgggtg ttcttcctcg ccgcttgtgc gggcattgat 22920

tcaacccctg atacaggcca aacgtgggca gagtacgaaa ccgccatgct tgacctggtg 22980

aaagatgttt ctgcaaacct tgtgacgccc gttcctcagc ttaacggcaa taacgtgggt 23040

gtacttgctg ggcgtctatg tgatcgaaac gtcacggtag cagacagccc gatgcgtgtg 23100

gcaacaggct cagtattgag tttgggagac atgccaacgg acagcgcagg caaagcatta 23160

gagatgagca ccatcgctac gctggcagac gcacgttact cactgccgca atggtatgcc 23220

gatttagaag gggtgtactg gtcggacgca accacattgg aagccaaagg cggcgattat 23280

cagtacctcg aatacgttcg cccggtgcac aagcttaacc gtcgtgtgcg tatcaaagcg 23340

attcgccgta ttgccgaccg cattcttaac tcaacgcctg caagcattga gctgaaccgc 23400

acttacttcc gcaaagatat gcgtgatatg tcgaaaacga cagaagttgg tggcattacc 23460

ttccctggtg aaatcatgcc gccagaagac ggtgacgtca ccattcagtg ggtaaccaaa 23520

accaaagtgt cgatcggttt gatggttcgc cctcataact gcccgaaaca catcgtggtc 23580

aacatcgcgc ttgatcttac taaccctgca gatttggagg cgtaatccat gagaatttct 23640

ggcaagaaca tgcatttctc tttgggtgac tacaagctca aagcacaaaa agtctcgtta 23700

tccattaccg ataattccgc cgtcaataaa acctctggtg tgccagatgg ttacgtcgac 23760

ggagatgttg aggctagtgg tgaaatggag ctgaccacgc agcaatttaa cctgctcagc 23820

aaagccgcta aacaagccgg atcttggcgt ggaatgcctg attttgacgc gctgttctac 23880

ggcaagattg ataaagatga gctcaaaatt gaagccttcg gatgtcgtat caaaatctct 23940

gacctacttg atgccgattc gaatggaggt agtgcattag ttcacaagtt gccgttcgag 24000

gtgacaagcc cagactttgt cagcatcaac ggtgtgccgt acctgcgtcc ggatgaaacc 24060

gaagatttgg ttcaataaca aggaggcatg atggcagatg ttatcgacca tgcctgcggt 24120

cttgaaaccc aattcacaga agtggcgctt gccaaccaac tggcaagggc taagcgaatt 24180

gaagaacggg aaagcgcaca tgaatgcggc gaatgtggcg actcaatccc agaaaaacgc 24240

cgccaaaaag taccaggttg catctactgc acccagtgtc aaagcgaatt ggagcgaatg 24300

acccgatgaa tttagcaaag ctctttgttg agaaaatcat gaagccagtc cttgaccact 24360

tagatatggc atccggtggt aaaggcacaa tgaacactca agcggccatt aatctgatcc 24420

tgatgattat tgctcatgag tctggaaagt ttacttactc aaaacaagtg cgtggtcctg 24480

cattgggctt tacccaaatg gaaccagcca ccttcaactg gcttgtcgag tggttaggta 24540

acagtcgccc tcacttgttg gatgcactgt cgatgttttg ccccgttgag cacttagacc 24600

ctcgttacat ggtggtttca cccgagttcg cagtggcaac cgcacgactc aatttaattc 24660

ggttcccaga agccttgcca gaagccgatg acctagaagg tttggctcgg tacgcgaaga 24720

agtactggaa cacaagcgca ggtaaagcaa cagcagaaga ttacctacag gcatatcaat 24780

ccctaatcgg agaagcagca tgaatttctt aactggaatc gttggcaaga cattgttgga 24840

agtactgaaa ggtctgttct ttcaaatcag ttggtcaatc atccttgaac gtttcgccac 24900

acgctcagtg gtgtggggct tagaaaccct aaaaggctta actagcaatg atgtgatgca 24960

agaaaccgtg gacgacatca tcaacgcatt acaaggcaaa cgcttaaaag aaattccaca 25020

gaaggaatag tgatggattc atcatggata tcggcgattg tggcaacgat agcgctgctt 25080

atcgccatca tcaatgtggt tttcggcaga acggataaag ggcaaagcac cgcccgagac 25140

catgaccgtc gtatccatgc caatgagcta gccactgagc gactgcgcgg tgatgtcgct 25200

gagaagtacg ccaccaagca cgaactacgg gaagccgtag atgatttgaa agattctatt 25260

aacggacgat tcgaccgttt agaaaccaag ttagataaag agagagatgt agcatgagca 25320

aacaaattgc actaacggtt ggtgatgcag atatcacgtt cgtcccaaca gaagcagatt 25380

acaacgacta catgaacgca ttggcacagg gggaaatcgt gaactctgcg cataactttc 25440

tgatgaacac cgttacagaa gaaagcaaag aagtattccg tgaactgacg aacgagaacc 25500

caggtgcagc aattcaggtt gtgggtgaag ttctcaagga atacacgccg aagctgcaaa 25560

tcaaagtaaa aaaatagatg cccttgttcg ggctatggac tccaacgagc tcgaacaaat 25620

gctcacctgg cgccgtaagt ggttgccggg tgagacagac agcgaagaga accttgcaag 25680

ggccatttgg ttagagcgtc agtattggga aaacatgcaa gctgctaccg ctaacggggt 25740

tgccaaggcg tttagctgat aaggaacacg atgttaccag aagcactcag atttcaagtt 25800

ggattgattg accagatttc taaacctctg ggcaatattc aacgcaattt gaatgatgtc 25860

accaatacct accgtcaggg tactcatacc atgatggcag gagcggcagg catggtgggt 25920

gctggttttg cattgcaaca agccttgatg ccagcgattg agatggacag ggcattaggt 25980

gaagtgaaat cacttggtgt tgccgatgac cagctaaaaa cccttaccca aacagcaatg 26040

aagttttcgg ttgaatacgg taagtcggcc actgaatttg tggcggcttc gtatgatatc 26100

aaatctgcta tgggggatat gacaggcgat gaactggcgg gtgtcacaag aagctcggct 26160

attttagccg ctgcgactaa agcagatacg gccaccatta ccaactacat gggtaccatg 26220

tactcggttt ttaaagacca agccgatcgg attggtaaag acaactgggc tgaacaagtt 26280

gcaggtatga ccgcaaaatc tgtcgagagg ttcaaaacca cgggtcaagg aatgtcggat 26340

gcgtttaaag gtgttggcgc attaggtaaa actcacggcg tagccattca ggagcaaatg 26400

gctgtgcttg gcctgctgca aggttctatg actggtagtg aagcgggaac tcgttataaa 26460

gcctttatga gcggcgtggt aaaagcgcaa gacaagctag gcatggcgtt taccgacagc 26520

aacgggaaga tgctgccaat gttcgacatt atgtcgaaac tacgcaacca attcggtgat 26580

ttagattcac tggaaattga tcaaattaag caagctttcg gctcggatga agccgtactg 26640

cttatcaccg acttgatagg taaaacgggt gagctgcaat caagcgtcaa agacctaaat 26700

gatgccagta acttaaatac ggcaattaac atggctggag cgatgaccga ccaatgggaa 26760

cgactcgaac aaggcgtgtt tgcggttcga actgcttttg gtgcggcgtt gttgcctgct 26820

ctcttgccag tgatttcgag cttggccgat ggtgcgatag aaatcatcga atggacagag 26880

atgttcccga acctgacaaa gtacattggt tttgctgcca tggcgatttt aggtgcagca 26940

gcagcaggtg gtgcttttac cttaatgatg ggtgtcggta aacaagccat ggcgacttat 27000

atgctcacca tgaagatgtt tacaggggtg aactttttac taaccaaagg aatggcagcg 27060

cttcgcgttg caatgctagc ggcaaacatc gcaatgatgg cgaaccccat tggattcatt 27120

gttggtgctg tagttgccgc aatcgccgca gttggtgcgt tgattttcta ctgggatgat 27180

ttgaaagcct cgtttggtga cacaacatgg tttcaagtac tagaaggggc aatcacttta 27240

atcacacttc cattcagagc catgtttgag ttcatcaagg caggttggca atgggtcatg 27300

agcggcttta ccgatacgag tggatttgcc tttatcggtg atatggccaa ctcaattagg 27360

gaagtttttt ccggagtgtt caattggatt acccagagcc tagcgggaat ttgggactca 27420

gtaaaaggac tcattgattg gataccaggc ttcggcggtg atgatgaaac tacccaagtg 27480

aaatcgaagt ccgttcaaag cgctacgcct cgtgcacaaa ttcaaccagg tggcgcagct 27540

aagaacattg ccagctacca aaccagctca accaactacg gtggtgtggc gatttatcca 27600

acctacatga acagcccgca agacatggct agtgaactag aaatggcggc aggctaatgg 27660

cggattacaa ataccaagac attttgattg agaacgggga tgtggtgctc gatgcaggcc 27720

gcaacccaat cttgattcaa gaccgagcgg ttatcgccca agacatcaaa cacgccatca 27780

tcgagagcaa cttagcggtg gatttaatcg ctgagcgaag cccgtcaaag aaagccgata 27840

ttcgcaccaa gctggaattg ctagttgaag aagacgttcg attggtacca ggtaccgtgc 27900

gtttggatga accgaccgag ggcacgattt atgtgtttgc cgacaccatg gactttggcg 27960

agttgcaatt agagatagtg aataacggag agcgttaatg actgatattc caaaaccaga 28020

ctattccgaa ctggtgaagc aatccggtat cccaaccgat gaagctgggt ggaagaaagt 28080

actcaaagag gagatggata aagaaggttc catcatctca aacgactcgc cgttttctcc 28140

attttggcga ctcattgaag ccaccgtagt gaaagtcacg atgtggctca tcaatacctt 28200

attggttggt tacgtcttac cgaacatgtt tgtcgcaacg gccgtagacc aatggctcga 28260

cttgttggcg tggcaatgca aactcacccg caaaggggca accaaagcca aaggcatgat 28320

cgcctttcaa cgttccgcgg ctaaaggccc tgcgttggtg atccccaaag atacatgggt 28380

tcaaaccgaa ccgataaacg gcacgattta tcgagtgaag gtacttgccg atacgacgat 28440

gccagaaaac gaaaccatgg tgatggtaga agtcgaggcg gagaacgagg gcgcggctta 28500

caacctaggt gaagggtatt accacatttt gccaacagcc attccgggca ttggtgcagt 28560

gaccaaccct gccgaatggt tgaatgaagc aggggcagac aaagagagta atgacgaact 28620

ccgcctacgt attcgtaacc agtggagcgc agtcgcaaga tggcacattg atgcggctta 28680

ccgttcactg ctcaccagtc gagcaggaat caacgacgac aatgtgtatt ttgagcataa 28740

cgcaccgcgt ggcccaggta ccgcaaacgc gctcatcctt ttagacactg gtgaaccatc 28800

ggcagaaatg ttggcggatt tgaatgagta catccgtgtc gaaggtcagc acggccatgg 28860

tgatgatctg caagtgctcg ccatgccaga aacgacacac gatattgcct gtcgggtttg 28920

gccgctgcgt tcactaacga tgggagaccg agacgcgcta cgcgtaaaag tggagcagtt 28980

cattggtgcg gctttccgtg agaacaccga ctactcgcca acggtcacta atccggttat 29040

tcgattcagt ttctcaaggt tagggcagga gctgcatgcg cagttctctg agattgaatc 29100

cctcgaattt gataacgccg acatcatcaa caatctgacg gtaccgcgaa ttaattctct 29160

gggggtgacg attgaaaatt cctgagatta atctgcgtta ctggatgggc cgaggcgagc 29220

tggctaagtt cgcccgagcc atgcgcaatt actgggaaca tgtaagagcg gcattcgaaa 29280

tgccactgca gcagcatgat ccgttaaccg ccccaatggc attggtgaac attcttgcct 29340

ggcaacgtga aatagaacgc ttaggccaag agccggaagc cctgtttcga attcgggtgg 29400

ctcatgcata cgggtttgcg cgtgacgcag gcacgatagc aggctgggaa gatatgttcg 29460

ccaagttggg ctatccgcac atcgggcaag acgagcggtt agctaatgta gattgggatg 29520

taatcagtct aaaaatccgc gacggcgatt taaccaatgt ccctaagttg ttagataccg 29580

tcattagaca gtacgggcgc acttgccgcc gatatcaata caccagttat gttgaaatgc 29640

cgttggccgc tcgaagcaag aacgtcgaag cgcaatacac aacgtcgcac gttaagacca 29700

ggctcaacgt tggcatgctt ccaagtgtgc tgaacgtcga ctgcgaatat taccaagcca 29760

cagtgaaggg ttaaggaatt actaaacatg gcaaatacca ctgacaagtc aattttaacc 29820

gccgcaggta aagcactgtt ggcacagctc aacgctgaag aaaaaccact tatcatcgac 29880

aagatgatgt ttgcgaatgt cccaaaccgc ccagagtttc cacagccaga tgatgtggta 29940

ccgacggatg acatcgttca tcaagagcag gttgagcaac gcggccgcct ttctgcagac 30000

tcggtgatct acagcaccac attgaccagc gatgttggtc cgtttgaatt taactggaca 30060

ggcgcatact gctctgaata tggcgtatta gtcacgattg accatcacgc gcttacacca 30120

aaaacagctg atgaaccggg tgttgcaggt aataccttgg tgcgttcggt cgtccttgaa 30180

tacaaagata tcgcggaaat caccaacatc acggtggatg catccagctg gcaatacaac 30240

gccaccgacc gcatgaagaa gatggacagc gatgtggcgc agtccatcat cgaccagaat 30300

ggcaaagact ggtttatcga agacggtttc ttggtcacac catcgggcag cgcatacaac 30360

atcaaagcgg gtgccggtta tgtttctggc aaccgtgtga gcatggagtt tgaccgcagc 30420

gttcaggtac ccaacaaacc atcgttcatc tacatcgatg cacaccgcga aggcacacca 30480

acaggtgagc aggtgactct gtttgatttc gttatcacgg ccgaagagaa agatgattac 30540

atcgattcat cgactggtaa agatatctcg catttcgtgt gcaaaattgc caaggtgctg 30600

gctgatggct cggtgagtga tttgaggccg gagagtaatg tagcgacgct tgaatatgta 30660

gagaaagtag cgggagcagc ttggacagaa ggtactttgg tcaccaaccc taaatccatc 30720

tatgtttttg ctaaagactc ggcatttcct cttcaactat atgcgccaaa tgcttcagtt 30780

gaatcgccag ttctcatggg agaaaagcca gactcaaact gggaaactca ctatttccga 30840

tttgctgatc tgggagacat gcgcggtaat gggaacaaac ttggtatttt caatggggca 30900

aaagctgttg tcgaaaatgt gaatttcgtt gtgggggata tcgatgaagg ggaaggtatc 30960

tccatcgata acactccact gtatgccaat tcgataaaag gtggttttgt tcatggtgag 31020

tcttttggtg gagttattgc cagcgaacaa tcaatggtgg aatcaggaaa aatttacctc 31080

aaccttatcg aaggagatga tagataccgt tcatatttgt attttgatgc accagatatg 31140

gcggtctccc ctattgccta ttcggagagt tatggtggtt cggtaaagga tggcattttc 31200

gcagtaaccg accgaaccgg ggaaggggtt cggtctttcc atggttaccg agcaggtcat 31260

aaaagtacca cactcaatgc ctgctttttt ggggctcaag cgggcgaact ggcgaatgac 31320

accttcaccg aacaatggtg gaaaggcggt agtgctggct ttggtcgttt agctcttcat 31380

gattcaacgt cggcgaatac ggcggcaatg gggaatgagg caggcaatca tctaaaggga 31440

gatagagggg cttacttcgg ttcattcagt ggtaattctg cggaagggaa agaaagttgt 31500

ggtgtcggtt ttcaatcatg tgaccacaac ttggggcatt ttgccaacgg tgttggcttt 31560

caatcgctgt ttcagaacca aggtgactac tgtgatgggg taggttatgg cgcgttaggt 31620

aacaatcagg gagatagaaa cggtggtatt ggcgcttatg ttttaaacaa ctcgttagga 31680

gctgataact tcggagtagg gtatcgtaca ttatccgtct gtcacggttc gtttaatgtg 31740

gcaggtggaa accaagccgc ctacaagccg gacggttcac ctttcactgc ttcccatgtt 31800

gcattgtggg gctaccgcgc tggttacaac tccgatggca acgggctttc tggtcttgga 31860

agagaagtct tatctggcgc aacaggtgat ttcttgtcag cacttgggta tttagctggg 31920

gttggagtga cctactcacg ttcaacttgc gttgggcagc aatctacagt gacgggcaat 31980

gaccaaattc agttaggtgc accaggttca tcggtttatg cctatgggcc agttcaagac 32040

cgaagtgatg agcgagataa gacggacttc aatccgatta gtgaggcgtt gaaaagattc 32100

tttcttgacg ttgagtttca aacctaccga ttggactacc gagagtctta ctttgagtat 32160

gagaaagttc aagtgggtat tgatgatgca gctcaacctg tatttgaaac cagaataaag 32220

aaactgctaa aggatgggtc aaaagctgga caacgtcatc acgcaggggc agtagcccaa 32280

caagtcaaag ctgcgatgga caagcacgga gtggactttg gtgggtatca agaccactta 32340

gtgaatggtg gtgaagatgt taaatcatta ggttatcagg agtttatccc tatcataggc 32400

gctttaatcc aagaccatga tcgagagata aatgagttga aagccatcgt aagtaactta 32460

actaaacaag ttgaaacgct tctatgataa ccctcaacgg cacccaaatc tcacttaaga 32520

acctgcgtat tagtgtacgc caacagctcg ccggacaaga tatgtccggc cagtcctcat 32580

cgactgacca agctgaaacg ggcaacaaag gcaaagtgtt gtccgtcagt ggcgtgatac 32640

cgttcaacaa gagtgaaacc ctaagcaacc ttttcacctt ggcaggcggg caagacaaca 32700

gtgcccgtca gatttatcgc atcagcaaca atactgccgc tgcgctaaag attcgccagg 32760

tgaagttcca aggcactatt cgagccgatg aacaagagag caaccgacaa tggaacgtcg 32820

ctttcgagtt ggttgagcac ttgtccgttc ctgagcgtgt tgaacagcga caggaagata 32880

agccagcaac tcaacagcag gtgcaagggg taaccactcc ggttgaaacg ggtcaaagtg 32940

aagacgttcc gccggatacc agtgtcgaac tcactggcat catgaagttt ctcaaatcac 33000

tagacgaaat attgtcatag gggggaggtt atggaaccaa accacaagtt tgtatgccga 33060

gcctatcttg gtagtcaaaa ggttaaagca aagaatcatc gcattctctt tgatgtgaac 33120

acacctggtc gctgctcagt ttccgttgaa ggttctccaa aggtaaatac catcatcgcg 33180

gtggatattg gatggggtga cagcatatcg cgggtattcc ttggctacat cgaacgtgtt 33240

caagcatcag aaaaagggtg gtcagaactg ttttgtcgcg agttggcggc attgttgttt 33300

aagccactcg atatcacgct tcgtcatccg acattgatgc aattacttag cgatgtcacc 33360

aacaaaaccg gattgcagtt cgtagtaccg gaagcggcct acagtaaaac ctccattcct 33420

tgcttttaca gtgatggaaa tggttatcga gtgatggacg agctgtctca agcgtttgct 33480

attgaagacc tgttttggca acagcagggc aacggccaaa tttatgtggg caattggaag 33540

gactcttact gggctgataa gcctgtgact attcccgata gcttaatgac tcaacacact 33600

acggcaaagt cagtgaaaat acccgcgagc ccgaagttaa gaccgggcgc catcgtcaat 33660

ggcttgcgtt tggtgggcgt tgattttcaa ggaacagagg tgaagctgac atggacgtaa 33720

atgcaataaa gcgaatcata tttcgtttgt tcccagaact gaccggacgc tggcatttgc 33780

cacgatgggg tacggtggtt gcgttacctg agttgccgga agagggagat atgtctgatc 33840

gcttttatcc gcattacgca gtggatgttc agctgctcga tgagaagggc gtggagttca 33900

aagataaatc accacttcag gcagtaccac ttccaatacc aggggtgggt gaatacgccg 33960

gcagacttga gccaccggcg atcggaagca ttgtagaaat cggctttatg ttcggccaac 34020

cggataagcc tttcattcgt tgtgtgctgc cgttggggtt caagttacca gcgattaagc 34080

aaggtgaaag ccgttaccaa cagcgccaag gggtttacca gctggtcgac gaagcgggta 34140

actttgaaag caagacagac aaagacaacg tcaccgaatg tttaaaccaa cgcatcaaag 34200

tgttggaaga caaaatagaa gaaatcacaa ataacaaaac cactactgca aagaaaatca 34260

tagaagtcgc tgatctaatc acaatgaatg gcggtaaagg tgttgtgcaa ggtgactgca 34320

tttgtgctta cacaggcaaa cctcactctg atttttcatc gacagttaag gcaggtaaat 34380

aacatggcaa tgaataaaga ctctctcaaa gcaaagttgg aaagtgaact acaagcacaa 34440

gggttcgttc tcactggtga attcgccatg gcagggaaga tggcagaggc gatcgccaat 34500

gctgtatatg atgaaatcac acaaaatgcc gaggttccag ttactggcgg aagctcagct 34560

gggacttatc aaatcactta acaccttgtc tcacagcgtg agacattttt ttggttgcct 34620

tatagataaa tcaacctatc tataggagca aagaaacttg aacatttcac caaacgactt 34680

actgatttac ctaggaataa ttcatacctc tattcttatt attaacgggc tatcaaagca 34740

ttagcttcca acccataata attatcaaag taagcgccta tcttgggcgc ttttctttta 34800

cctcgaacat ctacaactat tactcactct atgcggctaa accataaata ggaccatcca 34860

gtgacggaag gcgacctcta ggtgacggaa tccgcactcc tcctccccac ctgcgacgtt 34920

ttcgatctgg ttttttcgca attcttaaat gtcgaaattc agctgcctac gtaacacggt 34980

agatatcaag cgaagcctta gccatagcgg ggttcgagaa tccccaagac ctctaagaat 35040

cgcttctaga gccattttat aaatttcaaa agttgaaaag aatttcagga aattgcaatt 35100

ttaatgatct ggaaagatca ggtggtgatt ggtaagtgtt tgaaaataat ggagtgaggg 35160

tgtttttcgt cataaatgaa gacgatcaaa agaagaaaga gacgatcagt tgtgaagctt 35220

acgaagcctt gtatagacag gcttgaaggc gaattgccaa gataaataag tttttcaatt 35280

tatgagactg tataattact ctgagatttt gaaatgactt tgaagttaac ggtaagaaat 35340

cgctgttttc gtggggagcg cttgagagga gccaaagcct gcgtttttgc cggacagctc 35400

gttaacttcg aagtctaaac caagcaatta gatttaaatg tgtcgccact tcatcgccac 35460

ttaatgtttt tgtttgattt aaataaatga ttttaaaggt ttaattatta aagtggatat 35520

aaagcttta 35529

Claims (3)

1. BacteriophageVibrio neocaledonicus bacteriophageVneM1, accession number: GDMCC number 61215-B1.

2. In the application ofPhage according to claim 1Vibrio neocaledonicus bacteriophageVneM1 for preparing vibrio lysisV. fortis、V. chagasiiOrV. coralliilyticusThe preparation, the preparation for regulating and controlling the symbiotic microbial flora of the corallinum hance or the application of the preparation for enhancing the high-temperature tolerance of the corallinum hance.

3. Vibrio lysing bacteriaV. fortis、V. chagasiiOrV. coralliilyticusThe agent for controlling symbiotic microflora of coralline plexiform or the agent for enhancing high-temperature resistance of coralline plexiform, which comprises the bacteriophage of claim 1Vibrio neocaledonicus bacteriophageVneM1 as active ingredient.

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