CN112342200A - Pantoea agglomerans bacteriophage and medical application thereof - Google Patents

Abstract

The invention discloses a Pantoea agglomerans phage and medical application thereof, which is named as a Cophage vB _ PagP _ QF 1; has been preserved in Chinese dictionary in 2020 and 11/2Type culture Collection under the name ofPantoea agglomerans phage vB _ PagP _ QF1, accession number: CCTCC NO: m2020676. The bacteriophage has high lytic activity and stability. The invention also provides a separation and purification method of the phage and general biological characteristics thereof, and provides a potential alternative therapeutic drug for treating infection caused by multiple drug-resistant pantoea agglomerans; can be used independently or in combination with other substances, and provides a safe and nontoxic bacteriophage sterilizing product for sterilizing and purifying the environment. Has higher lytic activity to the human-derived pantoea agglomerans, does not find virulence genes related to lysogeny, resistance and drug resistance in the whole genome, and has safety for drug treatment research.

Description

Pantoea agglomerans bacteriophage and medical application thereof

Technical Field

The invention discloses a Pantoea agglomerans bacteriophage, and also provides a separation and purification method and general biological characteristics thereof, belonging to the field of biological engineering.

Background

Pantoea agglomerans (Pantoea agglomerans) are members of the Enterobacteriaceae family, and are gram-negative aerobic bacilli. Its "clumping" results from the unique property of the organism to form unique elongated aggregates surrounded by a translucent polysaccharide sheath that plays an important role in the colonization of the host. Pantoea agglomerans (Pantoea agglomerans) are widely present in airborne dust, plant pollen, vegetables, soil, foodstuffs and feeds. Pantoea agglomerans is not an obligate infectious pathogen of humans, but is a opportunistic pathogen of humans. Generally, infection with pantoea agglomerans mainly includes two cases: (1) wound infection by plant material (2) acquired infection in hospitals. Pantoea agglomerans infects when host immune function is low and causes a series of serious diseases, which can cause arthritis, synovitis, endophthalmitis, periostitis and systemic bacteremia in human. Endocarditis and osteomyelitis have become common cases in adults and children. At present, effective drugs aiming at the Pantoea agglomerans are mainly antibiotics, but due to the generation of drug-resistant genes, novel drugs need to be developed to control infection caused by drug-resistant Pantoea agglomerans.

A bacteriophage is a virus that specifically kills bacteria. In recent years, antibiotic therapy has been carried out with the continuous emergence of drug-resistant strainsThe effect is limited, so the phage therapy is paid attention again by extensive researchers. Phage are reported to be about 10 on earth³¹Large number of about 10 kinds⁶The phage is a very abundant gene library, so that the phage is a good genetic research object. Phage therapy is an important alternative method for overcoming the occurrence of bacterial drug resistance, and more researches show that phage has good treatment effect on animal disease models caused by multiple drug-resistant bacteria, and obvious adverse reactions on organisms are not found. And studies have reported cases of successful phage use in human therapy. In addition, a new direction has been extended in the study of phage therapy, namely the study of phage lytic enzymes as therapeutic agents. Lytic enzymes, encoded by the phage genome, are expressed late in the phage infection of a bacterium and can hydrolyze the cell wall of the bacterium, thereby causing the death of the bacterium. It has been shown that lytic enzymes from bacteriophages can kill bacteria efficiently and exhibit a more rapid lytic activity when externally acted upon by the bacteria. In recent years, bacteriophages and lytic enzymes have been increasingly studied as therapeutic agents. Compared with antibiotics, the two are characterized in that: specificity, does not affect normal flora; different from the action mechanism of antibiotics, the antibiotic is also effective on drug-resistant strains; high efficiency and can quickly kill specific pathogenic bacteria. At present, the research on Pantoea agglomerans phage is very little in China. Therefore, the feasibility research on the Pantoea agglomerans phage is beneficial to providing potential alternative drugs for the treatment of drug-resistant Pantoea agglomerans.

Disclosure of Invention

The invention discloses a Pantoea agglomerans phage, namely a brachypanosomatidae phage which is separated from the nature and can specifically kill human Pantoea agglomerans, and is named as vB _ PagP _ QF1, and the phage has higher cracking activity and stability.

The invention also provides a separation and purification method of the phage and general biological characteristics thereof, and provides a potential alternative therapeutic drug for treating infection caused by multiple drug-resistant pantoea agglomerans. Can be used independently or in combination with other substances, and provides a safe and nontoxic bacteriophage sterilizing product for sterilizing and purifying the environment.

The brachyury bacteriophage of the Pantoea agglomerans is named as a kosher vB _ PagP _ QF 1; the strain is preserved in China Center for Type Culture Collection (CCTCC) at 11/2/2020, and the address is as follows: wuhan university, Wuhan, China, with a preservation namePantoea agglomerans phage vB _ PagP _ QF1, accession number: CCTCC NO: m2020676.

The invention also provides application of the Pantoea agglomerans phage in preparation of medicines for preventing, diagnosing or treating infectious diseases caused by Pantoea agglomerans.

The invention relates to the use of the active ingredient of the Pantoea agglomerans bacteriophage in preparing medicament additive, spray additive, disinfectant or cleaning agent for killing Pantoea agglomerans in human body, body surface, ward, medical apparatus or medical space environment.

The Pantoea agglomerans bacteriophage is applied to killing Pantoea agglomerans in space environment.

The Pantoea agglomerans bacteriophage is applied to killing Pantoea agglomerans on the body surfaces and in bodies of livestock.

The Pantoea agglomerans bacteriophage is applied to killing Pantoea agglomerans on the surface of a plant.

The present invention provides a composition for preventing or treating infectious diseases caused by Pantoea agglomerans, which comprises the bacteriophage according to claim 1 as an active ingredient.

The bacteriophage and the composition thereof are used for preparing feed additives, feeds, drinking water additives, drinking water, disinfectants or cleaning agents for killing Pantoea agglomerans in bodies of livestock and poultry, body surfaces of livestock and poultry, livestock and poultry feeds, culture devices or culture space environments.

The invention has the positive effects that:

provides a Pantoea agglomerans bacteriophage: vB _ PagP _ QF1, and the phage has high lytic activity and stability. The invention also provides a separation and purification method of the phage and general biological characteristics thereof, and provides a potential alternative therapeutic drug for treating infection caused by multiple drug-resistant pantoea agglomerans; can be used independently or in combination with other substances, and provides a safe and nontoxic bacteriophage sterilizing product for sterilizing and purifying the environment. Has higher lytic activity to the human-derived pantoea agglomerans, does not find virulence genes related to lysogeny, resistance and drug resistance in the whole genome, and has safety for drug treatment research.

Drawings

FIG. 1 photograph of phage vB _ PagP _ QF1 plaques;

FIG. 2 Transmission Electron micrograph of bacteriophage vB _ PagP _ QF 1;

FIG. 3 optimal MOI plot of bacteriophage vB _ PagP _ QF 1;

FIG. 4 is a graph of one step growth of bacteriophage vB _ PagP _ QF 1.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless otherwise indicated

It is noted that all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In an embodiment of the present invention, a novel bacteriophage having a killing activity against pantoea agglomerans is provided.

Bacteriophages are bacteria-specific viruses that are capable of infecting specific bacteria and inhibiting the growth of bacteria, and are viruses that contain single-or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as genetic material.

Specifically, the bacteriophage of the present invention is a novel bacteriophage isolated from sewage by the inventors, having a head in the shape of a regular icosahedron and a very short tail; the phage can form transparent plaques on a BHI agar culture medium, the periphery of the plaques has no halo, the edges of the plaques are clear and regular, and the diameter of the plaques is about 0.5-2.2 mm. Has been preserved in China Center for Type Culture Collection (CCTCC) in 2020 at 11/2, with the preservation number of CCTCC NO: m2020676, address: wuhan, Wuhan university, China.

Example 1

Phage isolation and preparation

The host bacteria of the liquid manure sewage sample are Pantoea agglomerans pag; collecting sewage, filtering with gauze, centrifuging at 6000r/min for 10min, taking supernatant, and preparing BHI culture medium (100 mL) by using the treated sewage instead of ddH 2O; adding 1mL of overnight cultured host bacteria pag into the culture medium, and culturing at 37 ℃ for 10-12 h; 1mL of the culture was centrifuged at 12000r/min for 5min, the supernatant was filtered through a 0.22 μm filter to form a phage stock solution and stored, and the resulting filtrate was used in a plaque test to examine whether or not phages capable of lysing into Pantoea agglomerans pag were included.

And (3) plaque testing: pantoea agglomerans pag was inoculated in 5ml of BHI broth at a ratio of 2%, and cultured overnight with shaking at 37 ℃. Dropping 0.1ml of the prepared bacterial culture liquid in the middle of the flat plate, and uniformly spreading the bacterial liquid by using a spreading rod; after the phage is dried, 20ul of the phage stock solution is dripped into one area; after naturally drying, placing the mixture in an incubator at 37 ℃ for culturing for 10h, and observing whether plaque is formed in a bacteriophage dripping area.

If plaque formation occurs, the presence of phage is confirmed. A series of 10-fold dilutions was made from 100mL phage stock. Get 10^-2、10^-4And 10^-60.1mL of each diluent is uniformly mixed with 0.1mL of the host bacterium culture, after the mixture acts for 15min at room temperature, the mixture is added into about 7mL of BHI semisolid culture medium at 45 ℃, the mixture is quickly poured into the upper layer of a 1.5% BHI agar medium flat plate, the mixture is uniformly shaken and horizontally placed for 10min, after the mixture is solidified, the mixture is placed in an incubator at 37 ℃ for 8h and then observed, and a double-layer flat plate forming a single plaque is obtained.

Example 2

Phage amplification and purification

On the double-layer flat plate with the formed plaques, picking single plaques which are relatively round and bright and have larger diameters by using a tip of a sterile pipette, inoculating the single plaques into 5ml of BHI liquid culture medium, adding 200 mu L of phage host bacterial liquid, uniformly mixing, acting for 15min at room temperature, culturing for 10-14 h at 37 ℃, centrifuging for 10min at 12000rpm and 4 ℃, and taking supernatant; the double-layer plate experiment is repeated, so that 4-5 times of single plaques are picked repeatedly, and the phage is purified into plaques with the same size.

1mL of freshly cultured host bacteria was taken and 300. mu.L of phage lysate (as single extract)The ratio of individual phage cultures to host bacteria was 1:1, 1:10 and 1:100, respectively). Incubating at 37 deg.C for 20min to make phage particles adsorbed to host bacteria; adding 800mL of BHI liquid culture medium, and adding CaCl₂And (3) shaking and culturing the mother liquor to a final concentration of 1.25mM at 37 ℃ for 6-8 h, centrifuging at 12000rpm at 4 ℃ for 10min, and taking the supernatant, namely the phage lysate.

PEG purification: adding RNase A and DNase I into the phage lysate until the final concentration is 1 mug/mL, and standing at room temperature for 30 min; adding NaCl to the final concentration of 1mol/L, uniformly mixing, and carrying out ice bath for 1-2 h; centrifuging at 4 deg.C for 15-20min at 8000r/min, and collecting supernatant; adding 10g PEG-8000 per 100ml, stirring gently to dissolve, ice-cooling for more than 2 hr (preferably overnight) to allow bacteriophage to form precipitate under the action of PEG-8000; centrifuging at 12000r/min for 10-20min at 4 deg.C, recovering precipitated phage particles, adding 2mL SM solution, washing precipitate thoroughly, and acting at room temperature for 1 h; adding chloroform with the same volume for extraction, and carrying out mild oscillation for 30 s; the organic phase and the hydrophilic phase were separated by centrifugation at 5000rpm for 10min at 4 ℃ and the hydrophilic phase containing the phage particles was recovered to obtain purified phage.

The phage titer is detected by adopting a double-layer plate method: diluting the purified phage solution by 10 times gradient, and collecting

0.1mL of each of the phage dilutions and 0.1mL of the host bacterial liquid are mixed well, spread on double-layer agar plates, cultured at a constant temperature of 37 ℃ for about 10h, and each agar plate is subjected to plaque counting. Selecting a plate with the plaque of about 100-200, and calculating the initial concentration of the phage according to the dilution multiple to obtain the titer of the phage. The purified phage can form clear plaques with no halo-rings around, clear and regular edges, and a diameter of 0.5-2mm in 1.5% BHI agar medium, as shown in FIG. 1.

The purified phage was self-named vB _ PagP _ QF1, deposited in the chinese type culture collection, deposit unit address: china, Wuhan university, zip code 430072, deposit number: CCTCC NO: m2020676, deposited under the accession numberPantoea agglomerans phage vB _ PagP _ QF1, with a collection date of 11/2/2020.

Example 3

Transmission electron microscope observation of phage vB _ PagP _ QF1

The phage purified by PEG in the embodiment 2 is taken for electron microscope observation, and the specific operation steps are as follows: dropping 10 μ L sample on copper mesh, precipitating for 15min, removing excessive liquid with filter paper, staining with 2% phosphotungstic acid (PTA) for 1-2min, drying, and observing with transmission electron microscope (Hitachi H-7650); as shown in FIG. 2, the head portion was a regular icosahedron, the diameter of the head portion was about 64nm, and the length of the tail portion was about 20 nm. The vB _ PagP _ QF1 belongs to the family Bravirus (Podoviridae) according to the eighth report of the Committee for Classification of viruses, International Committee for Classification of viruses (ICTV), 2005.

Example 4

Optimal MOI assay for phage vB _ PagP _ QF1

Optimal MOI determination: adjusting the concentration of the bacterial liquid cultured to logarithmic phase to 10⁷cfu/mL, then mixing the phage and bacteria according to the ratio of phage/bacteria of 0.00001, 0.0001, 0.001, 0.01, 0.1 and 1, transferring into BHI liquid medium, and culturing at 37 deg.C for 8h with shaking. Centrifuging the culture solution at 4 deg.C for 15min at 10000g, filtering the supernatant with disposable filter with pore diameter of 0.22 μm to obtain bacteriophage increment solution, and measuring titer of the increment solution by double-layer plate method to obtain optimal MOI (phage to bacteria ratio) with highest degree, with the result shown in FIG. 3.

Example 5

One-step growth Curve assay for bacteriophage vB _ PagP _ QF1

One-step growth curve determination: mixing the host bacteria cultured to the logarithmic phase with the phage according to the optimal MOI proportion, standing at 4 ℃ for 15min, suspending the precipitate with a fresh BHI liquid culture medium, carrying out shake culture on the suspension at 37 ℃, and taking a sample every 10min to determine the titer of the phage, thereby drawing a one-step growth curve of phage-infected bacteria, wherein the result is shown in FIG. 4.

Sequence listing

<110> Jilin university

<120> one strain of Pantoea agglomerans bacteriophage and medical application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 44318

<212> DNA

<213> Enterobacter agglomerans (Enterobacter agglomerans)

<400> 1

vbaggtgggc tatctgggta tgtcacggcg ctatccgtgg gctaacgctg ggttgtgctg 60

gctatcctgt gggcgtgcgg tcgcgtgcgc ttgccttatt gccttttgtt gccgtgggcc 120

ttgccgggct atcgtgggct attgccgggc tatgctctgg ctatctcagg gctatctatc 180

aatccatcat ggttagactc tggttagctc tgggttgtgt cgtgggacat agccgggtta 240

gtgctgggtt gtgctgggac tctgtgagtc gtgctaacca tccttaaagg ttagttgtta 300

catcgtgcgt cctgtcgtcc ttactctata acgtcaatca taattatctt tgaacgtaat 360

cggattattg ataggtaaaa ccttgtttac ggattgcgtt tcatcaggta tcatcgtgcg 420

gtcgccgcag gcccacaacc actatccaac ggttaaccgg gactatgctg tataggctgt 480

aatgccttct tgagcgtagc gaagcattac cagctatagc gatgggtaag cggtggcgta 540

acatggctat acagtgagaa tctgccttgc ttaactattt tcaacaaagt gcttgcaatc 600

tctaaatcat tcatctagta ttcaatccat cgaacggcga cacgggaaac cactagccga 660

acgatgcgga gctaaaccgc taagtagaca acctgctaag tcatacgaaa aacaggtatt 720

agcaaataag tttcacaaag tgtttgacaa gtgaatccag acggattaaa ttgattacca 780

agttaccgca ctgataagta aacaggttca gcgggaccag ttgagaatcc gggtttgata 840

cctgcttaag tgcaggcggt tgtaagagca acgtcaataa gcctcttaca gtcgatactt 900

cgaagattcg cagcgaaacg aaataaacgc ttgacaagtt gaacgataac gattaactta 960

gatagcaagc aacaacaagc agccttgcgg cgcggtctga gtcctaacgg atgttgacga 1020

ccaagcctaa acaggatgca ggcgaaagcc agtgttcccg acacgttaat cacggggccg 1080

aactgaggca agtgcatata agccgaggcg tgagcagcac gctactctgt atcgagtgaa 1140

caaggcgcgg agtgtatacc gcaagcgact ttaacaaagc gtcagtgatt atgctggcgc 1200

ttgattaacg tcactaagga ggcattatgc gtaaacgtaa acgccagatg ttagcaggca 1260

tcaatgatgc gctgaaacat ggcaagctat cagaagtgca gggcgctatg gcgactcctg 1320

agcctttaaa gaaactgtct cacaagtcac aaactgacaa ggtgagaggc aagcaaaagt 1380

gtaagggcag cagcaaggct ccggcaggct ggtattcacg ggggatgttc gcatgagatt 1440

agcaattatt ggcagcgcgt ggttctgggt aggcattggc ctgaccgtgt acggctttgt 1500

gcagtatctg tagtgttatg acctgctact tgcgagtggc aggcaataac tttaccgata 1560

acctgatagg tgattattat gacattcaaa cttgtagcag taggcagcat caaagcaaat 1620

ctggcgaaag cggttgagct gaaacgcaat accacaatca gcgctctgtt tcatggcatg 1680

gttgcaaaca acgtatcgtt tgtaactgac atgcgtaaag aagacatggc agactttgac 1740

actgtgttgc gccagctgtt gccagtggca tgggacaagg ctaacgaccg ttacgcctat 1800

tctgacaaga aggcaatcag cagcgccgac aaaatgggtc tggacctgtt cggcctgcgt 1860

cagcagttta agcacggtga caaggcaagt cgtgaagaaa tcgtgcaagt attctttggc 1920

gtggtgatgg attactacaa cgccaacgaa gcggagaaga agggcaacga cctgacagcc 1980

gaggacaagg ctaagcaggc tctgagcaaa atcaagtccg gcgtgaaggc agcgaaagag 2040

caaggcgcaa ctgacgctga aattctggat atgctggctg ggatgggcgt gaacgtgact 2100

ggctatgtgc cagtgctgga gcgcaaatga tagtaatggc aatggcagcg ccagtcctag 2160

cgactggtgt actaacaatc tggttaatca aatggggtag tcaatggcac ttcacgtaac 2220

taagcaagta gcagcacaca cggcacacaa gatgcacttc ctgtgtcatg gtttctacct 2280

gacagccgct gtgctggaag ggcatcttct gtactcaacg gcagcggcaa tcctactggt 2340

tgtaacggta gcaggcgcgg cattaggtag ccccgaatga agataactcg gagtcacatt 2400

gagacggtga tactcgtcaa ggtggcactg agcattacat tcgccgccgt atactttctc 2460

ccggcaccga tggcaggcgc tgtaggagtg gcaagcaatc tcgtctggtt gtggaaagta 2520

tagactgagt gactaactaa tgctgctgca agtaccgggc ggcattgtga tattcattca 2580

acgaggagca acaacatggt acaggcacca ttcacagtat tcgtatccgg ctggcgcaac 2640

gatagcccac tgtttgacaa cctgaaacgg caggcactgc tgggcgacgt gctgcgcaac 2700

catgcctata acccagaggt tgtgctgggc aagtatgggc aagagctgga gcaatcctat 2760

gctgttgagg tttacagcga ggatgcagcg gagcgcatcg ctaagcacgc cggggcaatc 2820

tttcagcaag cgtgtgtcgg gctgctggat aatcagggcc gtgagttccg gcttgtgtat 2880

ccgaacggca agcatgaatt gctcggcaag ttcgaggaga ttacagagca gcaggcaacg 2940

agtgagccag cctgttcatt ctggcacggc atgtggtggc gtgcagcgtg attaacttaa 3000

tgcaggtcac ggcctgcatt gcggcaatca tgccataaca actgaggatt acaccatgac 3060

taaagcaact atcgcaatcg tatctatcaa caacctgcca gcttcacgtt tcgctgacgg 3120

tgagccagtg gaaatgctgg gtctggcata caagaaagac ggtgagctgt tcgccgctgt 3180

gccaatgctg gtccgtgagg atgaagaagg caacgtccgc actgatgaag aaatggtggc 3240

agcggtcagc aagcctttcc caactgcaca ggatgtgccg cctccgatgt tcatgcagct 3300

gatgactcca ttcagtgtca tcccggcaga cgtgctcgac gaagaaggcg tcacggtgcg 3360

tgagctgggc gagctgggta gcttcgacct cgacgaagac gaggacgaca gcgaggcaag 3420

cccggaatat gtaggcgagt aacctgactg tcagcgcatc ctgagtggtg cgctgtaggg 3480

catgttgcca accaactaaa cgaggattta ccatgaccac taccattcgc ttcttcttca 3540

acgctgcaaa caaagacgtt atcgctacct tcaacggcgg cgaaggcatg accgtgggtg 3600

atactgcact gtgctttgaa ggcgcacccg gcatcggtct gttcggtgct gacattgaga 3660

aggcaaagca atcctgccag tctgttgttg acgacttcaa agagtcaggc gcattgctga 3720

tactcatgaa cggcggtgag cttaacgcgg tgccagaggg ttacgagttg attaacacta 3780

tcgaagcgtg atttctgtct gcatccgggc aagccgggtg cagtagggca atcatgccaa 3840

ccaaccttta aggaatatgc tatgactaag ttaatgacca tcgttgatgc gccagccacc 3900

tctgctaccg ttgtccgtgt aacctcggca gacggtgcaa agaccgtgct caccctgctg 3960

ctgcgtgctg atgacgctaa gttcgagcca atggcagcgg gtgtaagcct gcgtgacgac 4020

aaggtggagg aaatgactaa ccgcttccgc accagtccta tgctggcaac catctttgtt 4080

cagggtgttg tgcaggagcg cggcgctggt atgctggctg atgacgtgaa gattgaggag 4140

ctgtccgaag ccttcccaat catgccgatt caggcagacg gctacgagaa gcgcgacaac 4200

cgcactggcg aagtgaccac tgtcccaacg tccggtagcc gcaccctgca ctaagcgtgg 4260

cctgctgcac cagcacgtca aatctgcgtt aaccacaagc gtgcgtcaat ggtgatgcac 4320

gttatagacc aaccaaagag agcataatat gaccgcattt aaaatcgttg aagcattggc 4380

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ggttgacgca gagcgtgagc atctgcaaga ggaaattgac cagctttcgc agcagctgca 4500

cgttgagctg gcgaaggtca aggcgaagta tcagggcaag caagctgagc tggcgcaagt 4560

gctggagcag catgacgcag cagtagacgg tcacgtagca gcggcaaccg cagcagcgac 4620

caaagcacaa caggttaaag aggtattgcg ctaatgtttg aagtcaatca gaaggtagca 4680

atcaagctga aatccgacgc cactggcgac atgcgggaca tgaccggggt tgtgactggc 4740

gtattcccag aacgtcctta cccggtgctg gtgctggcag acggcatcga ccgtgaagac 4800

gagcctgctt atcaggattc tgaatccggc atcgagtttg atttcagctt cacagctgat 4860

gggcagctgg tggttgattc tgacgtgggt agcatcacgg cagcagagtg attcggtgat 4920

gcgtcctgcg gggcgcatcc acgttaccac ttgaaagagg caagcatgaa aactatccaa 4980

cgtgttatcc tgcgctataa gagcgcaact ggtgaggagc tgtctgtgca gcgcaccgtg 5040

ggctacccgg ctgagttcgg ccttccaaaa gtacagagcc tgcgtattcg ctgggctggt 5100

ggtgcaggtg gcacgttccg tctgtaccag atgcgtgagg atggtatcct gagcacgctg 5160

catatcccgc aggctaacgt agtctctgtt gaaacccaat atcagaagga tgcgaagtaa 5220

tgtggatttc cgctaaagat ttacagtcca tgatggatga cgcgaagcac gctggcaagc 5280

tggaaggtga gattgccgga cgccgcatcg gacaggctga gggtgaggag cgggccaacc 5340

gcgccatgcg taatgccgac cgcaatgaca agctgaacga tgtttaccgt tcgctgggta 5400

tgcgcactta tcgtggtgtt gagtgtgacc cgcgtcgtct ggtgtccgtc tcgtttgagt 5460

acaagggcaa ccgcaccacg ttcggtgtca acgacaaggt aggcaagttg acctgcgcaa 5520

ttcgtgagaa tcgcttagtc gttacagtct tcaagtatga ccagtcgtac gtcacctatg 5580

actatgagct gtcacgtatc aattctgaaa tccgtaagga gtacaaatga cacacggtgt 5640

agcagcaacc aaaggccacg tagcgggcaa cggtggactt cgcgggcata gctgcggcga 5700

agtattccca ttcgtaatct acgcacaggg tacgtttgac gacctgtggt attgggtatt 5760

gcagcctaac ggcctgcgca ttggcccgtt caaagactgg accaaagccg agggcttggc 5820

aaagcggttg aagatgttcg gccagctgga cccggatgca ccagagacac aggctatcat 5880

ccagcaggaa accacgcagc agtacacgta ttaccgggac cgcaacgctt cgctgcatga 5940

cagctggcgc tggcgcgtcg cagtgggttc aggtccacaa gtgcgggccg agtacacctg 6000

caacaaaggt gggctgtggg aaagcagcgc atacactgcc agtgaagtac ggcaggatat 6060

tgatagccag ttcgtagcaa tcgggagcga cccgctatgg caagagcgtt aacagacttc 6120

tggcgaatct atcagtttcg ccttcacttt gaccagcaag gtcatatcaa cgccatcaag 6180

cacgcttggc gtgcaacggt ggctcgtcac tggagaataa agtaatgggt aatcctgagc 6240

tggtgctgat gcgcagccgg gccgctgtag agtgtaatgc tgatgcagtg caggcagagt 6300

tgaaagcgat gtattgccgt aaggcatggc gcgtgggtat gtcactggat ggttactgcc 6360

aacgctttgg cattgagaaa acgtgggagc gtgaataata tgggctggcg tctgagcaga 6420

gttgagaatc aacaggcgtc agccgaggtg ctgtgggatt gtctggaggc gcagcgcgac 6480

cccggattga atgcgccacg gctgatggac atggacgacg cgctgcataa gctgctgccg 6540

tttcgtcgta gcacagtgaa gcgccggacc cgcgagggtc tggagctggt ggaagtagtg 6600

cgccactttg attcggccaa agctattgaa atgctggaag ccatagggta tcccgattgt 6660

attgcagacc tgcggactat cgggtatgaa gcaactaagg cacggttcat caggcgggcc 6720

gtgcgctatg cacaagcaga acaaatcaac gcttggtatg cgaggtagca aatgagtttc 6780

aaacggttca gtaaggtgtg cgactgcggt acaggctacc gcagcgagtg ggacttgaag 6840

tgtggacact gccggggcaa gacaggcgag cagcgtctga gcatgttcca gcgtgagctg 6900

atgaagcagg agcgtgcatt gcagatgcag ttctttggat tctatacgcc ggacgcacag 6960

cccggcgacg gcacaatcag agcggagttc ccggaatgag cgacttaaag gtaatgaagg 7020

gcagcgcata cagcgagaag cgtctgcgca aatggttgaa ggaagacggc gcggtcgtta 7080

tccagactaa gcgtgacgag ttccgctgca tggtgaagat tgactggagc gataaggcta 7140

agccagtcgt cacctatacc agtgcgcagg gcaagccact ctacaacctg agtcagttcg 7200

atagcctctg ggccgctgcc agccacttct acggcacgaa cgtattcgac acgggtgttt 7260

gtgtcaatga cagcttcgaa ctcaccagac gcacgctgag agcgtctaag aaggtgtatg 7320

acctgcgcgg tggcacgaag catcacatcc tcgacaagaa gaaaggcaag gtgctgttcg 7380

aatactacgg cgaattgcag gcgcacttct ggctgtatga cctgccactg attgagctgg 7440

actacacctc gcgccggatg gatatggcta agctggcgct gaacttcccg gagttcctgc 7500

gtatccctga atcagagctg gttatcgttg atgacttagc gacgccagag cacggcatcg 7560

acatggctgt ggttgatgtt gagcgggcat tcgagcacgc tctactggca agccacgaag 7620

gtctgatggt taagcgtgcc gaccacaagt gggagccgcg ccgctgcaat gacagctgga 7680

tgaagatgaa accgagcgag gaaatcgacg gtgtaatcgt gggctggaac ccggcgaaag 7740

agggcaagtt cttcggcctg attggttcgc tccaatgcaa ggctgaggac ggcagtgagt 7800

tcaacgtgtc cggcatgact gatgcagaac gtgccgactt cactgagaac tttgagcagt 7860

accgtggtca gtggctgacc gcaacttaca tgaaccgcga cgcacagggc ggctaccgtc 7920

acgtccagtt caaccgtatg cacgacgaga agaacctgta ggagatagga tggacaactt 7980

aaataaccgt cgctttaacg agcgtaatgc acctgtgtgg gctaacttcg tagctcgcca 8040

caatgggaat tggtattggg aggagtcttt gcgtcaggag aatggcaagc gcttccagaa 8100

catactgagt gaaggcggcg cgattccacg ttacagcaac agcacttcga aaggctggga 8160

caaagcgtac cgtcttccag ctgtacccgc tgtggcacct gagccgtaca tcgtggacga 8220

gctgccgcca gtaccaacca atgagctgta cctgtccaac acagggcggc gctgggtaga 8280

cctcaagctg gagcagacct gcatcagcat cgggcatgac tcaaccgggc ggcgcgtccg 8340

tgtctcagtg gccacggccc gtgacatggc gcatgacctg attcgtatgg cagcagaact 8400

tgaacgccgg gaggcagaat gattttagtg aacgatgcac agattactgg tacactggtt 8460

gttcgtaacg ggacgttcgt taatcccggc gacacgctgg aaggtcatct gtatggtgac 8520

agcgccacgc acggcaacca tcccttcccg gatggcaccc gcgtgaatac cagccgtgtt 8580

gagtctgtga ttgtccggga tagccgggtg ttcgtgaaga ctcgcaacag catctaccgt 8640

atcaagactg tgggagagat tacaaaatga tggacgcgcc gtggcttagg gcagcaaagc 8700

gattacctgt tggtggcgtt gagcgattcc gttgctgcgg ctcgacaaag gcagcggtga 8760

tattcaacaa gcctgacgct tgggaaatgt attgtcaccg ctgcaagtcg tcaccgaaag 8820

agcgcaagca gtttgtgcaa atgcagcggg tgcagcagga ggcccgcgtg cagcctgcac 8880

cagcagacgt aatcaacatt gccgacgcga gacagtcggt gcaacagaac gtgtactcgt 8940

tcttggctac caaaggcatc atgcctgata tggtggagga tttacagtgg tccgatacag 9000

cgcaacgtct ggtgtttccg gtatcatctg gactatccct tgggcgtgca acgacgacat 9060

ggcagaatcc aaagtggatt cagtattcag gaaagagcag ctttgtcgcg tgctcaccga 9120

gccagaccgt tacgggaatt atattgaccg aagacttatt atccgctaag aaagttcagt 9180

acgcggtgaa ccgcttcggc acaggtgata tggcggcggt ggccttactc ggcacccgtc 9240

tggataaacg actcaagctg tggattgcgc agaacaactt ccctactctg atgatgcttg 9300

atggtgacga agccggacac gcaggggtgg ctcgtatcaa acgagagttg cgcccatacg 9360

tgaaaactgg tatattctac gttgacggga aagacccgaa agacctaacc attaaggaga 9420

tattagatgg aatcgaactg catcgaccac ggctttaaag gtgacgcgca gggctactgc 9480

cgcaccacca cgaagaccca ccgcaacacc cgcaagcatc gcgtggtgtt tgcggaggcc 9540

aacggagtaa cgctggccag tctggatggc aaggtcatcc ggcacaagtg cgacaacccg 9600

cgctgcatta atcctgacca tttggaaatc ggaacgcagt tacagaacgt ggctgaccgg 9660

gtagcccgga accgctgtgc gaagaagctg acgcaggagc aagtggactt catcaggatg 9720

aattacaagt tccgagacaa agagtacggt caggtagcac tggcccgtcg ctttggagta 9780

acgaaagaag ccgtgtacag cgttgtccac ggtcgcaact ggaggtaatt tggacgcttt 9840

aattatccgt gcaatgtgta atcgcaaggt gttcgaccgc ctgcgcaaga gtattccggg 9900

cagcatgatg gcaccggata ccaactcgat gctcggctgg gtagctctgt actggagcac 9960

gtacccggac gatgcagagg tgtcatggag cagtttcggc acgatgctgt ccctccgtgg 10020

aactcacctg tcgcccgaag aaatggcaac aatgcgattg ctgattcagc aggttcagaa 10080

ggtatccgat gccgatgtag cgggaaccgt gcgtatgctg gcagagctgg cgtactccgg 10140

cgaggtggcg gctgtgaacc agcgctatca ggacggcgat gatattgaca ttatggccga 10200

actcaagaat ctccagcgca agtacagcga gactgtgaag acgcaggact cgctgctgca 10260

atgggagaat cgcaacgtcg atgatattct ggcggcgaac gatgaacaag gtggtttgaa 10320

gttccgcttc ttgcctgtgt tgtctgaacg tatacgaggt gcaagaggtg gagactgtat 10380

cgctgtggct gctcctgtgg atgctggtaa aacttctctg cttagctgca ttgtcgtgga 10440

ttgggtggag cagatggtgc agcagccaga tgtgtatggc gaccgtccga tattgtggct 10500

cgtcaacgag tctctggctt cgcgtactgt tccacgtatc tatcaggcgg cgacgcactg 10560

gacgttacag cagattcgtg aggctcacca aaagggtgag ttcgcgccgg agtatttgaa 10620

gaaggtcggg acgtgggacc gcatccgtgt gaaggatgcg cacagcctga cgatggcgca 10680

gattgccacc ttgatggaag aaatgaaacc tgcggcggtg gtcatcgaca tggtggcaaa 10740

cgtcaaaggt gggacgatgg agactgagca ccagaacctt gaggcgaagt ggcaggagct 10800

gcgtgtgctg gggtgtgagc atgactgcat tatgctcggc accatgcaaa tgtcagctga 10860

gggttacgac ctgctgtacc cgccgctcac tgccttgaag cagtcgaaga ttggcgtaca 10920

gggtgcgctg gacctcgcgc tgtttatggg acgtctggac ccgcagagca agccagagta 10980

tatcgacatt cgcggcatca gtacgccgaa gaacaagatg cctctgtctg gccaacaggc 11040

ttcatcgcaa ttcgaagtcg tgtttcacgg cggtacatgc aagtttgaat aaggagtacc 11100

catgtcctgc ataatgcaca ttgacttgga gacagagaac tatgaatggt atggctcaaa 11160

ggcgtcgccg tattgcccgc agaactatgt tgtcgagtcc gcatggcgtt atgaccgtgc 11220

cttatcagac ggtagcacag acgtcggacc cgtacagtat gttcgataca gcagccggga 11280

ggagtttatt gcagcggggt cagagtggtg tccaatccct gatgactgct ggcttatcgt 11340

cgcacacaac gcggcgtatg agataagctg gttccttgag cacgaccgag cgaacttcga 11400

agcgttcctg aaacgtggtg gccgggtgtg gtgtacgatg cacggcgagt atctggtgac 11460

ggaccagcag agccaatacg catcactgga cgagacggca ccgaagtacg gtggtactca 11520

caaggtggac ggcgtgaagg tactgtggga gcagggcgtc cgcaccagcg agattgaccc 11580

gatgctgctg catgactatc tggtcaacgg cgacattccc aacacggcgc tggtgttcta 11640

cggccagtat cagaagttca ctgagcgcgg ccagttcccg ttagtgtggg aacgtatgga 11700

tgcgctgctg gcgtttgcat actgcgagtg gttcggtctg tgggtggata tgcctacggc 11760

gaaagcgaat caggttgagc aagaggcacg aatcgcagag ctgaaaacag agctggaaca 11820

gctgctgcca gagctgccgg aaaccttcga gttcaattgg ggttccaact tcatgatgtc 11880

agccctgatt tacggcgggc caatcaagta caagaagcgc gtcagctatg acccaatcaa 11940

gtacgagaag gaggactact actacttcgg cacccgccgc gtgcctgttg cacaagcaca 12000

tgaaatcggc atcgaagtgc tggaggagcg attcgacagc aacatcgcca cgtacaaatc 12060

tgggaagaac aagggtcagc cgaaggtgtt ccgcgaggac actgacgtcg agaagctgaa 12120

atgggaagat gacctgttct acttccccgg ccacatcgac attgaccgac tgcccggcca 12180

cgtagcggag aagtacggcg accgtggtga gttccgtgga gcacagacgc tgccagatgg 12240

tagcccggtg ttcagcacca gcggcgatgc aatgaagggt ctggcggcgc agggctttac 12300

cgctgcgaag ctggtgaacg agctggcgtc tttggagaaa gacacaggca cctattacct 12360

gcgaactgag tacaacaaag atggaagtgt taagaaactg tctggtatgc tccaatacgt 12420

tatccctatg gacgatgacg gttcgggcat tattcatcac cgccttaata catgcgctac 12480

catcactacc cgcctcagct ctgggaaccc taacctgcaa aacctcccgc gtgacggaac 12540

ctcgaaggtt aagcaaatgt tcacctcccg atttggaccg caaggccgca taatcgaggt 12600

tgactactcc gcgctggaag tggttatggg ttgtgttcat actggcgact tgaagctgct 12660

ggggctgcta cagtccggca ccgacatgca ctgctaccgt ctggcgttcc gtcacggtat 12720

gccgtatgag gaagtgctca agctggctgt cgaagacgag gacggcccgg actacaagat 12780

gtggaagcag cgccgtaccg acattaagcc accgtcattc gccgcgcagt atggtgcaac 12840

agcgaagggc atcgcgtttg cgactggtct gtcagttgag ttctgtcagg agttccttga 12900

caacgaagcg aagctgttcc cggactcaat caagttccgt gacgtgattc gcaaagaggt 12960

cgagcgaacc gggaacctgc cgggccagct caagcgtgag cagaacgacg atggttcatt 13020

cagcgtgtac aaggtgggct actggacttc accatctggc cagcgttaca gcttccgtca 13080

gaagaagcag tggcgtgaag gtcgtcaggt catggactac aaggccactg aaatggcgaa 13140

ctactggtgt cagggtgagg cgttcttcat gatggccgta gcgttcggta tgctgattcg 13200

tcacctcatt gccaacgact tctacggcgg caaggttgca ctgatgacca acgtacacga 13260

tgcggcgtat ctggatgcag cagacgaagt ggtcggtcgt caggcggcgc tggacactaa 13320

gcacatcatg gagacggcgc gggaacgtat cctgtcattc tggccggact acggtatcct 13380

gaaagacgta ccattcccag cagcagctga aatgggtccg agcatgtaca gcaaaacaca 13440

cgttaagtga ggcatcatgg aatacacaaa gcaagagtac aaccgattca cccgactggt 13500

ccgcatgtcg ggcagtcctg ttcagatgga ccgcatcacc gcccgtctcg aaatgcctaa 13560

gctcgtcaag gagttaggca acgagaagtg tcagaagatg tgggatgaca tggaagcgaa 13620

cggggatggc gacccggatt accagtagga cgtgggcctg cggcgacggc gcggagcata 13680

ccatgccgtc gccaactcgt caacaccctg catgaaattt aaattaagtg ttgacagtta 13740

gacgaatctg tgagtttgca gatattaaag agtaacgtta tagagagaca acaactggag 13800

aattaccttt gagaatccgt gaactattta ccaacaccga tattcgtaac gctatgcagc 13860

gtgcagagaa cgtaatcagc aaggcagcga aagacctgtc ccagctgaaa gacgtgtcag 13920

tgtcgccgca gctactgcgt tattgggtat cctgtatgga cgatagcgag gtcgaaggca 13980

gcattacgca agaggttgag cggacccgtg agctggcacg tagccggaac atgagcacca 14040

gcaacaacac tctgcgccgg gacatgcgtg ctattctgga cgagaatcag gtactgcgcg 14100

attaccgcac gctcctgaac gacctgagca ttaacccact gcctatcgta ccgcgccgct 14160

ataacccggc tggtaagaag atgacagtcg aggcgctgtt cagcgacctg caaatcggta 14220

agctgtctgg caactacaac agcgctgtgg cacgacaacg tgtgtccgag tacggcgatg 14280

cgctggtgca gaagattcag cagcacgtcc atgcaggcta cgacgtcgag ctgattaagc 14340

tggtcgttct gggcgacgtg attgaatctg acaagaagca catcaactct gcccgtggct 14400

gtgacatggg tacggcagag cagatgaagg aagcgactga gctactggtc gagttgattg 14460

gtaagctggc catgactggt atccgtgttc actgcgtaat ggtgacaggc aaccacgatt 14520

gggacggtca cggtatgaat agctacatgg cgggtcagga gcatctgagc tggccgatgt 14580

acaacgcagt gaagatgata agcgaggcag tgtacggtga gcaggtcatg ttcagtatcc 14640

cacgcggggc gtttcacctt gacaatattt acggcacgaa tgttctgtac gagcatggtg 14700

tcggggtggc gtcgagccaa gcaggtctgg agaagcgccg ggaccagcgg gcgcagcagc 14760

tgggcgttca tgttggccta ttccgtatgg gtgataagca cagcatttgc cgctttaaca 14820

atgaccgcct tatcgttaac ggtgccttct ttggtacgga cacggttggt ggcgagtaca 14880

gcagcttcct cggataccag aacgaaccat cccagatagc tctgttccat gtgcagcgtc 14940

gtgagggcga cccgcgcagt accatctttg ataccctcgt aatccaactg gcccacattc 15000

gctaaggagc gaccatgaaa tttctgactg cacttgcaat tctgttcatt gccctgaaac 15060

tgactgacgt tatcgcgtgg tcatggtggc tggtcctgct cccgctgtac ggcggcgcag 15120

cgattgtgct gggtctgatt gtactggctg tactcggtgg ggcaacgcac ggcatcgcca 15180

agcgtctgct gagaaagaaa tagttgacag ctggctgagt ctgtgattca gccgactttg 15240

aagatatacg ttatagagag cacggagctg gtgccgccta caccagcaca acaaaacttc 15300

attgaccaaa agagagcaca aatatatgac tactgaaaac accgcgttta acccactggc 15360

tcaactgaat gcactggtag cacaggctgt cgagacgcaa gacgttgaca tgaccgaaac 15420

gggtactggt gggtttgaga aaatcattct cccaaagggt gtttactacg gacacttcgc 15480

ggaatacatc gagctgggca aacgtacccc tatgtttaag ggtaagccta ctggtcgtcc 15540

acctgtcctg aatgtcaaag ttggcttcct gatttacggt ccgaacggcg aggttgtccg 15600

caaccgcagt ctgggtctgg ctgtgggtaa cagcgagaag tctaagttca agcatctgtt 15660

cgaccgtctg aacagcaaag gtgacattaa gcacgctgca cagcgtctgg gtcaggcatt 15720

cagctttgag gtgactgtcg agaagaccag cgcaggcaaa gagtacaacg ccatcaacta 15780

tgcgtcagta tctgacctgc cgaagttcga cccgaacact ggtgagaagg ttgccattcc 15840

tgagctgtct ccgactgacg tgaagctgtt cctgtggaac gcgccgacca aagagacttg 15900

ggatagcctg catatcgaag gcaccaacga caaaggcgag agcaagaact tcattcagga 15960

agacattctg aaagctgtcg attatgtcgg ttcacctctg caagctctgc tggaaggcgg 16020

tatcccgatg ccgcaggata tggccccggc caccccagct gcgccagcgg ctcctgcgac 16080

tccagcggca ccagctgaac caacaccgtc tgctccggca gcaccagcgg ccccggctgc 16140

acctgccgct ccagcagcgc ctgctgcacc agctgcaccg actgcctgaa agagcgatgt 16200

gattaaccag cgggattggg acgagccagt cccgctttaa taaggagcac gaatgcacca 16260

gacactattc caactgctgt acgcagaggc cacggccatc ccttatgacc gcacccgcat 16320

tcagggcgca cgctctgcac tgtaccgact ggctaacctg tacggcattg accctcagcc 16380

gctcactgag cgcatcaagg gcaacaaggc tgcactacgt gacgcctcga aagaactcgc 16440

agcacagggc gatttaggcg cttctgtagc gactatggga accatcgcca ctcagctggg 16500

ttggattgcc ggacacgttc atgcaccact ggagcagacc aatctgccgc agctggccac 16560

ggacattttc ggattcctga atactttggc tgtactggac aagctgccaa cggctaaggt 16620

ggcgtaatgt ttgaagaact gctggctaac ctgccgcctc agtttgaggt ggaggagcag 16680

aagaacggtg acaaggttct tctactggat gctgacttcc tgatttatca ggcagcagcc 16740

acggtgaaga cgttaccgac tgcaatccgg cgcttctaca aactggtact ggagttcatg 16800

ttcctcactg gcaccaaaga gtgccgcatc tttatcactc ccacgaactg tgcgaagtgc 16860

aaccgatact tctatcctac cgtcaaggtg tatcaggacc agcgtaaaaa gcgtcaagag 16920

ttaccgctga aaggcccgct caagggccac ctgctgaaca accctgacga gtacgctcag 16980

catggtatcc gggttgccgc aagcgattgg ttcgaagcag atgacctgat gattatccat 17040

gctcatgagc tgggtgatga cgggattgtc tgtagcggtg acaaagacct gcggctgact 17100

cccggcccgt ggtgggataa cgagataggc cgggtcaaca caatcccgaa ccgtttcggc 17160

tacctgtatt gggatgaaga cgcaccgagc aacgcacgct gcaaagggcg tgggacgaag 17220

ttcttctggg cgcagatgct catgggtgac agcgccgata acgtgaaggg tatccaaacc 17280

tacaacgggc gtctctgtgg tcctgccgct gcgttcgaaa tactgcaaga aatagaaagc 17340

gagtccgaag ctgccaatct ggtatgccgg gcttacgccg cgattggcca gaacgtattg 17400

gccgaagcgg aaatggtgtg gctgcgccgc tccgaagatg attctgctta cgcctacttg 17460

atggaggtcg tgactgacga agccatcttg gagtggctga tgcagttgca tgaatatcat 17520

caagctcaca ttcagtgggt catggagcaa gcagacaatg gcgaaaatta aagacttagc 17580

agcgaaggcc cgtgaatact tctacgcaga taacttcctc gaccttgagg acgttatgct 17640

gctgctggcc gagcgctcac cagcgacgca ccagcggctt acgcaggagt tcgctgacat 17700

tatcatggag caggatttga tggacgacat tatgagtatt cagccatgat gcgcaagatt 17760

acccgtggtc agttacgggc agtcggaatg caactggcaa aggcgcaggg tggggtatgc 17820

ccgctctgcg gtgaacctct ggactttacg attaagggga ataaaggtga cagcgttgtt 17880

gttgaccatt gccatattac tggaaggatt cggggtgctc tgcacaggtc atgcaatggc 17940

ggtgagggca aagttgcatc agccgcaggc cgctggattg ttggttccat gcaggactcc 18000

cgtagaattg cagccgcgct ccaccgagta gcgaactatc tggagcagga gcctaccgac 18060

atgatttacc acagtcataa gtcggaagac gacaagcgcg agattcgtgc agcccgtgaa 18120

cgtaaacgcc gcgccgagat taaagctcgt cgtgaagcag cgaggattct gaaatgaaga 18180

acgtgattat ctgggacttg gacggtacgc tgtctgacgg cacgcaccgc atccacctgt 18240

tgccgacgaa ggattacggc aacacaaaga gctgggaggc attcaacctt gccagcggtg 18300

cggacacgcc gtttcaggac aacatcgaac tgatgaacag cctgcgtgct atcggctacg 18360

aaatcgtaat cctgaccggg cgtagcgaca ttgcccgcga gattacagag aagtggctgg 18420

cagaccacgg ctgtcattat gacgcactga ttatgcgtcc gttcgacgac caccgccgcg 18480

atattgactt caagcgcgag gagctggagg ccattgggat tgacaatatc ctgtgcgctt 18540

tcgacgatgc cgagcacgtt gtgaagttca tgcgcagtct gggtatcacc tgccatcagg 18600

tgacgcatta cgaaatcaaa cacgctcatg tcaagtcaca tgaggagaac gagggcgaag 18660

catgacaggc catatctgtg acaatggttg tcagtacgcc aaagatgttg gtatggaaaa 18720

tcatagctgc gagggtaagt gcatgtacga tagtaaacca gctgatgcaa ttggtctggg 18780

tatgaagtat gacggcgaca agccacgtat ggacctgttg gtcgatggtt gccctaacgc 18840

tctggaggcc gtagctggcg tcctgacgtt tggtgcgaag aagtacgcag ctcatagctg 18900

gcacacagtg ccggaaggtg agacgcgcta caaagccgcc ctgctgcgac acctgaccgc 18960

ccgtggcaaa ggtgagctgc acgatgacga gtccgggctt ctgcacttag cccacgccgc 19020

ctgtgacgct ctgtttatcc ttgagttgga gatgcgtcgc cttgctaagt ccgagtgaat 19080

ggtgctataa tcagtacgat aaagcggtcg aagaaggccg cacggaggac gctacggcgt 19140

acctccagtt gtttgagctt tggaaatcac gtaaccagta ggagatacga tgcagactgc 19200

cgagaatagc atgaacatca aagaagcaaa agttgaacgt caattcgaaa ttgagaataa 19260

ggcgcgtacc cgtgccatcg accgcagccg caaagctgtg aaagatgcgc tcgacagcgg 19320

tcgtgcatcg gaactccttc cggtatcgcg gttaatcagc gcagcattta gctctgtatc 19380

cgacgagatt gaaaagatta aagctgagaa agctccgggc gttggtggaa agtatcgtaa 19440

gttcctcaag ctggtcagca ctgatgtact ggcaacaggc gctctggtct atgtactgga 19500

cagcctctgc cacgaatcac aggcgaagtc atcagctcag gctctcatgg ccgggctggg 19560

ccgcttcgtt caggctgagg tgctgaaccg caaccttgag attgcagcgc cagcgtacat 19620

caaccgtgtt cacgaataca tgaaagagaa gcacactcgc agccagtcac acatcatgcg 19680

aacgcttcgg gcctctgctg atgcagtgaa gctggctcat gacccgtgga gtaacacgga 19740

gtgtatcgcg gttggtcgtc tgctgatgca ggcagtctgg gaaactggtc tgttcaaatg 19800

gcacagccac agcaatcaga tgaactatct ggtgccgggt gagcagctgg agaaggtgct 19860

gactgatgtg gtgacacaca gcagcatgat gatgattgcc ccgccgatga ttgtgccacc 19920

gcaggaccac accacgattc acgatggtgg atacctgact gacattgacc gtcgcgggac 19980

ttacaagaac cgccacatca cgaacaagca gcgccgcgaa gtggctgcgg cctttgccag 20040

tgatgatgca gctgacctgc gtgctgcgat gaacaaggca cagaacgtac cgtaccgggt 20100

gaacaagacc atcctgtcat gggtgcagca ggcacgggca gcgggtatcg gtattggtat 20160

gcctagcacc aaaggccgtc cgaaacctga gtggcgtctg gatggtatcc cgaaagagca 20220

gtatgacgcg cgtgagctgg aagactttga ggagtggaaa gctgttactc gtcaatggta 20280

tacagaggaa cgcaagcgcg tctcacagct tcgcgggatg gccatgacca ttgatatgtg 20340

cgaggaattt aaagatgaag acgtgctgta cttcccgacc tgtgtggatt ggcgataccg 20400

cctgtacttc aagtcagcgc tcaaccctca aggttctgac ttgcagaagg ctctgctcga 20460

atttggcgtg ggcaaaccac ttggagaaag aggtctgcat tggctcaagg tcaacgtcgc 20520

cactacgttt ggttatgaca agcccttatt cgaggagcgt gcagcttggg ttgatttgca 20580

ctatgccgag attgagcggg ttgcagatgc tccgtttgag acggaatctt ttgcggacgc 20640

cgattccccg tggtgcttcc tcgctgcctg tatcgagctg gttaatgctg tacggtctgg 20700

ttgcccggca gagtatgtta gccacctgcc aatcgctatg gacgctacga actctggtgg 20760

acagcacttc tcagcaatgc tgcgagacga gattggtgga cgcctaacca acctgttctg 20820

gaacggcaac acagagaaag cggaccttta catgaacgtg aaggagcgca cagatagcaa 20880

ggtcatcgtg gctcagcgca atgctgacac ggtggtgcag gcgacctact ggcgtgagaa 20940

cgagattacc cgctccatga ccaagcgccc ggccatgacc ttcttctact ctgcgactgt 21000

ccgcagctgc tccgactaca tcatgctcgg tgcgctggac gagggttacg agccgctgga 21060

agatttcagc atgatgaagt tgtctgggtt ccttgctccg ctgatgcgtg agtcgattga 21120

ggaagccatg ccagccgcag cgaaagcgat gaagtatgca cagcaggtgt gccggacgat 21180

tccactggag aaccacttgc agtggaagac gttgctgggc gggttaatca tcaaccgcta 21240

cacgacgacc gaggagaagc gagtcaacat ccgttcaatg ggtctgactc aggtggtggc 21300

gtataaccgc aactatgacc tgaacaaccg ccgtaaggca gcgtcaggga ttgcaccgaa 21360

cttcgtacac ggtcaggaca gtacgcacct gatgatggtc atcctgcgtc acagtggcta 21420

catcgtgccg attcacgact cagtggctac tcatgcctgc gacgttgacg aaatgcacaa 21480

gcatctgcgt gaggcgttct gtgacctcta caccagcaca gaccctctgg aaaccttacg 21540

cgaggcggca gaagcagctg gcggtgactg ctccgaaatc gacatgccag agcacggcac 21600

attgaatctg gaactggtca aagactcacc attcttcttc tgctaatcaa atgatgatgt 21660

acgcttaggg ggttaaggtt ttatcctccc tcctaagcac tacatcttgt gcatgattta 21720

accaatacat ctaaatgatg atgtacgtta tagagagata accatgaaac attcctcagt 21780

ccttctggct tcgtcagaga ttgaacccta tgttcgacag ttccacatct ttggtcacgg 21840

tatcgccaat ggcgttcccg aagacgtcca cttgcagcgt atgctgaaag agacagaagg 21900

ccagatggga ttgttaatct ctgaaaattc tgaacctgtc gcactggcga ctctggtgcc 21960

cggtatcgtg gaagactcac acttccccgg tgccgggctt atctgcgttt atctggctgg 22020

tcgtctcgga gtctccgggg tgcgctacct ccatcgctgt ctgcgccaac tggccgctga 22080

taatggcggg agttggtact gcatcagcag ccgggtgtcc acttacgaat accgcaatcg 22140

gtattacatg ataggagagc atccatgagc ggtggtgtat tcaaatccat tctcaaaccc 22200

gtaaacaaaa tcctgcaccc gttgaagaag ctgggcggtg acattatggg tacacgcgct 22260

gcacgcgaac aggctaaggc tgtgcaacag cagaccgaag cgcagcagca gcagtatcaa 22320

cagtctttgc agcaggagca gcagcagaac cagctggact cgcagggtga cttgagtaac 22380

gtcgctactg taaccgctgg cggtacggct tccgcagccg atactgagct gagtgacgcc 22440

acacgtaaga accgcaagca gctgatttcc agcacgctcg gcttataagg aggtgtcaat 22500

gtccgctcca atgacttgcg ctggactgtt tgaaaagttc aaggatacgg cggtattgac 22560

ttcttctgag cagtttgctc gttggaccaa cgccggggta ttcccgaaca ccagcgcaca 22620

agacacaggt aatcgacagg ctttacagcg tgattatcag tccaaaggcg ctatgctggt 22680

gaacaacctc gcatcgaagg tgacacaggc tttattcccg cagaactccg cgttcttcga 22740

gattagccag actcctgaaa tgttgcagat tgcacagcag atgggtgccg atgcgaaagc 22800

agcagcctca ctgtttgctg gtctggaggc ccgtgcttcg gcgcgagtgt tcctgaacgc 22860

aggctactct gctctgagcc atgcagctaa actgttaatt atcacgggta atgcactggt 22920

ctatcgtgac cctgtatccc accaatttca tacctactct gtacgcgatt acgttgtgaa 22980

acgtgatggc agtggtaagg tgctctgcac tatcctgaaa gagcgtatcg ccctgatgga 23040

tttgccagag gacttccgat taagtcgctt gccgcaccgc acagaaatgt acgaggatgt 23100

taccctgtac accaaagtag aacgcaagac cctcaatggt cgagtcatct acgaggtgtc 23160

gcaggaagtc gaggagcacg ccatcggcac tccttcgatt tatcccgaat atctctgtcc 23220

gtacattccg ctcacatgga acctcgtcac tggtgagaac tatggccgtg gtcacgttga 23280

agacttcgct ggcgacttcg ctcgtctgtc tgaactctcc gaatcgtctc tgctttacga 23340

agtggaaatg atgcggttaa tcaacattat tgaccccggc gctggtatcg accttgatga 23400

tttcatggat gccgactgcg ggaaagctgt atcaggcaaa tcaaacgctg ctggtaatgg 23460

tgtcgtggct cacgaaggtg gtaacgcgca gaaactggct gctgtccaga atgatattgc 23520

cgcactgatg caacagcttt ccattgcgtt catgtatacc gggaacaccc gtgatgctga 23580

gcgtgttact gccgaagaaa tccgcgctaa tatctccgaa gcaaaccaga cactcggtgg 23640

cgtatatgcc aacctgtcag aagtgcttca cctccacctc gcccacatcc tcgttgttga 23700

ggaagaacca cagatgctac agcttctgat ggtgcagggt gttaagctgg acgtatcagt 23760

gggcctcgcc tctctgaacc gtcaggccaa tgtggagaag ctgcaatacc tcgccaacgc 23820

cattcagctc gttatcccag cacttactca gtcttcacct cgcttcaacc cagacctcgt 23880

tatcgacgct ctgtgtcaag gctatggtat cgaccgtgag gcgctgtcgt atacggatga 23940

acagctcaag cagttgcagc agcagcagga tgcctcagca cagcagtcaa tgctgaatgc 24000

acaaggtgca gcgaatggcc aaggggctga ccctcagctt gccgcactga actcgcaaca 24060

gttaggatta acacaataag aggattttat ggcagacgaa caagtattag accaaggcgc 24120

agcagcaggc cagccgggag cagtggagcc tcaacaggca gaagcgaagc aaccaacaat 24180

ggccgagcag ctggccgcgt ctcagcaggc ccagaaagag caagagggtc aggtcgaggc 24240

tggtaaaccg cagggcgata ttccctcaga cggtaagcca gagggttcag aggataaaga 24300

atccgaaaag cccaagctcg actctgacaa tctggcacag actggtgatg ctgtactgga 24360

tgccagcatt gaaatgatgc agcgcaaaac cgggttgaac tccgaagatg ccgagaagat 24420

tctgtctcgc gcctttgagg agggtgatgc gtctcttatt gatgttgcgt acatcaaaga 24480

gcgattcggt gaagacgctg gctacattga gcagttggcg aaggcgtata ttcagcacac 24540

tcagacccaa gtcaatcagg tggtcaaaga ggtgtatgag gtcgccgggg gcgaagaaaa 24600

ctggaacctg tataatcaga ccttccagca gcacgccgac gaagacctga aagataccgt 24660

ggctactctg tctgatgcag ggaagttcgc aaaagccgca cgaatgattg tgaagttcgc 24720

acaggaatca ggaatggtcc cggtgcatgg acagcacatt cagggtggtg gtgcagtagc 24780

taatggcgct ctatcggcca aaggcttctc agaagaactg gcgaaactgc gtcagacgta 24840

caggggtcgt tcccttgagt ctggtccagc ggctgctcaa tatcaaacct tgctgaatcg 24900

ccgcaacgcg ggccgtatgc agggaattta atctcaatta agaaggaagt aacaaatggc 24960

taatacccct tacgctggtg ctctgactcg tcctcattgg ggcggtgcca attctgacgt 25020

agacattcac ctcgaagtct accagaatga agtcgatacc gcattccagt acagcgcaat 25080

ctttactggt atgtctgcgc agcgttctac cgctgagcgt tccaacacct accgtattga 25140

ccgtctgaac aactctgttg tgaagggccg taagtccggt gaagcgctgg aatctcagag 25200

cgtgaagaac gacaagctgc tgattgttgt ggataccact ctgtatatcc gtaacccaat 25260

cgactatcag gatgactgga ccgctccaga tttcctgacc gaaatgggcc agaacaacgg 25320

caccagcttc gcggaaatgt tcgaccaagc gcacgcaatc cagctggtga aagcccgtgg 25380

ctgggaagcg cctgaccacc tgaaaccagc attcggtgac ggtcaagaga ttgacgtgac 25440

cctgaaagct gacgctcaga ctcaggacga tttggaggct aacgccatcc ttatcaacaa 25500

ggcgcaccgc gacgctatcg accatctggt caagcgcaaa gttccactga acgacatggt 25560

aacgctgatt ggcaccgatg aatacagcac cctgctggag catccaaagc tgttcaacaa 25620

cctgtatggc cagaccaacg atgacggtta caaaggtcgt cgtgtcgtga ccatgaacgg 25680

tatccctgtc gtggagttcc ttgagttccc tgacggtgcc atcactgacc acccgctcgg 25740

tagcgcgttc aacgtgactg ctgacgatgc gaagtgcaag atggtcgtgt tcagccgctc 25800

taagactctg gtgactgtcg aggcgaaatc cttcacctct aagttctggg acgacaacgg 25860

taacttctct aacgtgctgg actgctacgc gatgtacacc gttggtgtgc gccgcccaga 25920

taccgccgta gttgtgaagt tcacctacgc ctaatccaca gggagccttc gggttccctt 25980

atttaccaag gagctataat ggcgctgttg aaacgctaca ccaaagagca tcatgacagt 26040

atcgcggctt ctcgtcgcaa tctgcgtgag gccacgaatc gcatcagctc agagacgctg 26100

gttgcacaaa agccaaaggc gaaagccaag gcaaaaccca aagctgacga agccgagggc 26160

gagtaatatt ttcagtgctt ctgcggaggc acttataaat gttattcggt ggtgtcgcat 26220

agcggcaatt gcaggagact gtaaatctcc cggccttcgg gtctacgctg gttcgagtcc 26280

agccaccacc accaattttg gggaattagc aagttggaca tgcgccggac tgttaatccg 26340

cgaataccgg gttcgattcc cggattcccc gccaattcaa tcaggagaca ccaatgcaat 26400

tacttgaagc aatcaatatc tcgcttcggg caattggtga atcggaggtc gtctccgaaa 26460

gcaccagtaa cccgacagca ggcatcatca aatctgctat cactcagcac cgtcgctctc 26520

tgctggcgac aggctggtgg ttcaatacgg ttgagcagac cattactccg accgctgata 26580

accgcatcac gccgccaagc gctgcgctgg ctatctacgg ccagaccggg aataagttcg 26640

ctgtccggga cgggtggctg tatgacctga tgaaccagag taaccgcttc actgaaccga 26700

cctacatcaa ggcgtacatc gactatgact tcgaagacct gccagaatac gcggcgcagt 26760

acatcgccaa ccgtgtagcg gctgagactt accgaaatga cattggtgtc gattccaact 26820

ttcaatcgct gacttctgac tccgaggagt gttacagcct tctgttccgt gagcatacgc 26880

gcaaccaagc gcggagcacc gtgcagtcgc gccgcttccg caaagtcaac tcagctcgtt 26940

tctgctaagg agtacacatg gcttccagct tagaaggtgc atacaaaagc ctgctgcaag 27000

gcgtatcaca gcaggtgcct cggcttcgtc tggatggtca ggtgagtacg caacagaaca 27060

tgcttgctga cccggtgacg tccctgcgtc gtcgtccggg cgctccgctg actgcgacct 27120

acaacttcgg tggtatcaac accacgaacc tgtacactca gtacgttgag cgtggttccg 27180

atggccgtac actggtcatc aacaccagct caggcgcgtg gtgggttttg aacaaagacg 27240

ccactgctgt attgaagtcc ggtcaggaca actactttat cgcctctggc ggtgccagct 27300

ccctgcaatc cacctctgtc ggtggtgaga cgttcatcct gaacatccag caggctccgg 27360

tatccaacac aggtggaacc agcaagaaag acccgaacac taccgggtgg tacttcacca 27420

aagtgggtgc attcagcaaa gactacacgc tgtctgtgtc acacggtggc gtaaccacga 27480

ctgtcaccta caccacacca agtggctcag ccacgggtga tgcggagaag tccaccccga 27540

cttatatcac aacgcagctg gccaatctga tgcgtaacgc tgtgaacagt ctgaacgtgc 27600

agcagcagga catgtacctg tatatctcag ggaccgctcc gattattgtc agctctacct 27660

ctggtgtcag ctatgtcggg tacagtggtg cgcacaacgt ggcgcttatc actgacctgc 27720

ccgctgtcgt cccgaatggt ggtgacggta tgatgacctc agtaggccgc gacggaaact 27780

ctctgacgtg gtatcgctgg gaccaaggct ctcaatcgtg ggaggaagac agctcctatg 27840

gctctccgtc ctcaatcacg aacatgcccc gcgtactggc cgcagatgac acaatcactg 27900

caccgccgtt tgagggacgt ctcgctggtg atgacattac gaatgagctg ccaacgtttc 27960

tggaccaagg tgtgattacc gggatgacca cgtatcaggg ccgcttagtg ctcctgtctg 28020

gtgcctttat cactatgtcc aagtccggga acccgtaccg attcttccgt tcgaccgtga 28080

ctgagttgca gaactctgac cgtatcgaca ttggtatcgg ctccagccag aactccattc 28140

tgcgccgggg tatccagttc aaccgcgacc tcgtactgtt tggcgatgct attcaggcgg 28200

tggtgtccgg tggcgggaac atcctgacgc cgaccacggc tgcaatctct ctgaccagtg 28260

aggaatcctg cgtctccaag attgcaccaa tgcaggcggg ccagacggtc ctgtatccgt 28320

tcaaacggag ttcaggctac tctggtatgc ttgagctgat tccatcgcag tacacgtcct 28380

ctcagtacgt ctcacaggat gccacaggtc acgtaccgga atactttgct ggtgacgttc 28440

gcgttacagc agcttccaac gtggtcaata tgtgtgtgtt caccgggacg ggtgatacga 28500

gcgctgtgta tgtgcatgag tatcagtggt cgagtgatgg taaagttcag gctgcgtggc 28560

atcgctggtc tatgccgcag acagtcgtct ctctgcactt cgcccgtgag aagctggtaa 28620

tcttcaccac tgacggtacg agcgttatgt tgaccgtgat tgatgcgcgt gagggctacg 28680

acaaccagac tacatccgag ctgccttatc tggacctgta ctcacaagtg caagtctctg 28740

gtggtcagtt cactgtccct gcacagctgc gtgcgccatt caactcaggt aagaagctgg 28800

gtctggcgtt cgctactggt acagctgcaa cagaggaggc tggtatcgcc agcatcaaca 28860

actcgtcatg ggttggtctg gtggagctgg gtgtgccgga cgggacgtac tggattggcg 28920

ttaagttcat gtcacagttt acaccgaccc cgcctatctt gaaagacgag aatgggaagg 28980

agattggtgc aggccacgtc cgtctggtcc gcatggaggc cgctgtccgc aacactggta 29040

tgttcaatgc ccgtgtcaag gatgtgcgta ctgacgtaga ccacacaggc gattacacag 29100

gcttgttcct gaacagccca gagctggtgc cggattatcc gctcattatc tctcaggcta 29160

acgtgattgt gccttgccgt acactggccg acaccactga ggtgacgttc agcacggacg 29220

gtactcacga catgaacctg ctcgatatta gctatctctt acgctacaac caacgacgcc 29280

ggaggattta acatgggttg gtatgttgtt gcggcgaaag tagcgatgca gcttatcaat 29340

ggggcgtcgg ctgcaaagtc ggcgtctcag cagaacatcg ctcagaacaa gatggttcag 29400

acgtacaaca gcaaggtgat gtatgagaca ctgcaacagg ttacgcaaat taaccttgag 29460

cgtgctcagc agcgccaaca gacaacagcc gccctgttca atgccgggct gcaaggccag 29520

tctgcccgtg accagattac gacgcaggcg gcggctaccg acacaatcgg tgccagtgtc 29580

cgggatgcta ttagcacggt gaaccagaag cagagccaag ccgctggctc tattgatgac 29640

cagtacatgc gggcaatcga ccaaagcaac ctgatgttgc agaagacgat tgaccaaggc 29700

agcaactcgt tgaaagactc agtagctgac aactcaacag cgattatgaa ccaagcagga 29760

tggggcgcta ttggctctat cattggacag gccgcaggtg actatgccag taaatctggt 29820

ggtggtgacg ctaagcccgc tccaatctct caggcacagg gtacgcctac aggctcctat 29880

gactatcagg gattgaacac gtccaacagt ttggccagtc agaatccatt tggactcagc 29940

ttcggcttcc agtccagtaa tctgttaagc aaataaggag ggcagcgaat gcctgtatta 30000

cgccaaggtg tagacaccgg gagcgcacag ctccagattg gtggcactaa ctatatggtt 30060

ggcaccgctg cccctcaagt caatgaagta cagccgaagg tagactcgcg tggcgctgcc 30120

atccagagct tcctcggtaa cttcctcgac acgtttactc ctgcgattca gcagggccaa 30180

cagcgtgccg cagcgcaggg tcagattgat gccagcgcgg acccgaatgc actcaagaac 30240

tctgacagtg cagcggacaa acagaacatc ttcatgcggg atgcgtatca gcgtggctac 30300

ctcggtgccg ctattcagca atccgtgacc gatttccaag ccgggattaa cacccgtgct 30360

caacaggctg gtttgaatgg aatctccgac gaggagttct tgcagaccga acgccagcag 30420

aacgcacagc tgatgcagag ccttgccggg tatctaccgc acgctgcacc agagacagtt 30480

gccgccgttg ccagctcact ggacaacacg cgcacctctg cgcttaacct gctgcgtaag 30540

acccgtctgg gtcaggccaa ggtaaacaac aaccgtacag tcgaacaggg tggcttccaa 30600

gcgattcagt cgttcaatga ctcaatggcc gcaggtaaca acttcgacca gtcgtggcac 30660

tatctggaag accaagccaa catgattggc agcaacgcca tcatggacga gaaggagaag 30720

aagcagcagc tgcataacct gttcctgacc gtagcgcagg gtgtgaatga ccctgacgtg 30780

attaaccagt tagctggtaa agcagcaggc atcatgggcg ttactgaccc gtccctgacc 30840

agcgcacttc actctgagtg gaaccgcgcc gggacacagc aggcgggtgc taccctgatg 30900

gacttgcaga accgctatga caatatcggg acactgccag cgtatcagca gcaggaggcc 30960

aaaggtaact ttgagcacgc gctgattgat gcacagacct ctggccgtat cagcactgga 31020

cagatgatgg agttctataa caagctgcac aaacagcaga ctcctaagct ccagatgaca 31080

ggtatggtat ctgccgtcgc atcacagggt ggtggcctgt cagtcgaagc actgcacgct 31140

gccgcgccgg gcgtaacccg tgagcagatt cactctgcta tcacagagtc tttcccagac 31200

accattgagg gtaacaccaa gatgctggct gcgggtgctc agggcaatga cccgtgggta 31260

atcaagactg cgctgggccg tgtcggtaca cagatgactg accagctgga aacgctcagt 31320

accgtgatga agcctgtcac tgacgagaac ggtaacacgt cctatcagat tccacaggcg 31380

gtgcagcaga acgtggtggg cttcatgggg atgtatcagg cttctgatga cattaccaag 31440

cagaccctga tgaacactct gccgcaggat tggcaaggcg ttattaagtc ggctatcgag 31500

caagacccta cgaacgtcaa caacaacgtg ctggacacta tcaagcgcgt ggcgtccgag 31560

aaagcgtctg gtatgtacaa agacgtgtct gctacaccga cagataaaat gctgaacact 31620

gatgccgccc tgcgctggta tcagcgtatc aatccaatca ccacggattc tgaggagtct 31680

cagcgtgccg cgatgaacca gcagttacag gccgagtaca accgcatcta caacaccgac 31740

aagggattgc tgaccgggaa gtctgccgac accatcaaca agatgctggt cggtaacatt 31800

caggcccgta ctgttccggt gtcgattggt cggttcgctg ccaacgtcac attgcccgca 31860

ggaacgtctc tggacagtta tgcgagtgca gctggtgtag acgccaacac gtactcaaaa 31920

gccttccaga atgtcgctga cagcgtattt aaggcacagg gtatcaacgt cgataacatg 31980

gattcagtcc gtattgaacc gaacacaggt ggtgctcagt cgaaagactt caccctgtca 32040

gtggttacga agggacaaaa tggcctgtac acgacgaccc gtatcgccat gcctgccagc 32100

agcatcaacc gccgcgcaca gtctgactat gcagcgatgg tggaccagca gcgtgcagac 32160

ggctctcaga aagcgggtac tcagattgcc acgttcatgg accacacccg tggtggttat 32220

cagacgatgg aagtctcagg caccaacaac tctggattgt ctccgacagt cttcaatagc 32280

atcctgtcta acaccatgcg atatgagggc ttcaaggcca ccaagtcaaa tggctctgta 32340

gggtacggct ggcacgaagc ctctggcgac agtgtgccgg acagcctgac cccgcaacag 32400

gcacagatgc acctgaaaga cctgtacgag aaacgctaca tcccgatgac taagggctac 32460

atgaaggatg caggaatcag tggcgacact gcgacctcac ttctggctga ccttgcgtac 32520

cagcgtcctg ctgatgccaa ggctctggct accgcgatgg gccagttcaa gcgtggcgaa 32580

atctcaccat acgagctgtc gaaagtgtta ccgtccctgc cgtcctacaa agatgctggc 32640

ggtacagaga agactgtccg taatatggac cgagctaacg cactgcttcg ctggtcaaac 32700

ttcgagggta atccccgcgt caacaactgg taacaatgag ggcggttcgc cgccccttta 32760

tcattaattt tggagattta acatggtaga ctttacccgc gcaccgggca acccggctcc 32820

ggccaaggct aatgataaag ctggggctgc acaagacagt accccgatga tggattacaa 32880

caacgcgact gacctcggta ttcatgccgt agaccgccgc gctgtcatgg ctgcatccag 32940

tcaagcaaac gtctctatgg gccagtcgta tatcgctggt gcaaagacct ctgtgctggg 33000

tggtttaatc accaagctgg aagcaccaga ttttagcaaa gacccaagct ggaacaacca 33060

agctgcactg gatgctgacg agcgtgtcaa agatatgcgc ccggatgcag aggagctgga 33120

ttggttgaaa ggtgccacgt cttatgacga gtaccagtac cgctgggcgc agattccaaa 33180

agtgcgtact cagatgcaac aaatggccgc taacccgatt tccgggattg ctggtgccgt 33240

tgtagctgat gccccgtcaa tgctcgtacc gttcgtacag gaaggctcta tcggtcgtac 33300

cgctgccacc gctctgcgtg tagccgctga cacctacgat attgccagct ccgtctacac 33360

agctgaccag ctgggacaga gtggctttgc gaacgctatg gttgctggga ctggcgtact 33420

ggacatggct catttagtgt cgcgtggatt tagccgcgta gcactccgag ctgacgaatc 33480

aatcgaccgc accgcagaac gcgcagagcg tactgctgca caggatggaa accgtggcgc 33540

aactaacagt gaccttaacg ctgatgaagc tgctcatgcc tctgatacag gcgtacaccc 33600

acctgttgag cctgacgttg cagcgacagc cgaaggtaaa gcagatgacc ttgttgtgga 33660

cccggataac ccgacagtcc caaccagcac ccgtgacatt gaagacgacc tgactactca 33720

cgactcaatc ccagaccgcg atgtacgcga taccggattc agtgaccttg agcagcagag 33780

cattaacact aaccacgcct tagcaggcac caagcccaac aaccgacaga agggtaaagt 33840

agtcggtggc gacctgaaca tccagagcac tatggcgcgt gcctctgagc gcattgacga 33900

gctggcgcag accgggcgct tacccgctga cttcccgccg cagtaccgca cactgatgaa 33960

tgcgattgca gataacgctg ctgatgttcc tgtacgtctg gaccggaaga ccacattccg 34020

tgctcacttc cagccgggct acggtagcgt cgatacgcac atccgtctgg ccttcccaaa 34080

ggctgctgct ggtaaaggta agatgacagc tgctgacgta atcggtagca tggatgccgg 34140

agaactccgt accttcctgc atgagtctat gcacgcggcc acaagccgtc agattctcaa 34200

agctgagcgt gatgccagtt ctgtgcatcc gtcagtgctg gacgccatca aagacatgga 34260

caccctgcgt gctcacctga aagaccgtct tgctgacgaa gtgaaagcag gcacagtaga 34320

caaagaaggt gcgcactaca tcggttacta cctcggaaac aatcatgagc tggtagctgg 34380

tctgggcgat tcccgtggag gctacaccga gttcctgcaa cgccatacct ctgtgactgg 34440

taaagaatct gcgctgcgta agctgggtaa atccatcctg cgtgtgctgg gcgtcaagac 34500

tgccgagcgt actgcgctgg ttgaccttgc tgatgccatt gaggacttac tggccgctgg 34560

tgccgagggt aacggtaagc tgggtaaaca ggcgctctcc cgcgctgctc cagagtttac 34620

ttccgacacc atgaagggta tgacagaagc tggccgtaaa gctgctatga atgctccagc 34680

tggccgtgcc gagtctcgcg ctgctgatgc catgtatgga cagttccgtc agaagatgaa 34740

ccgtttcttc tctctgcgtg atgacattgc ccgtggtggt gccgacactg ccgagttcgc 34800

tgattatctg gttgccgatg gcacccgtat tggtgagcgt caggaatcca tcgctgacct 34860

gaaacgcctg tacaaatccg aaatggatgc agtgactgct caggttgaga aggctatcac 34920

cgacaagctg accgagaatg gcgtagggcg tctgcaacag ttcttctacc gtcgcaactt 34980

tgttgaggcg cgtcgtgagc tggaacagaa gctggcgcac tatctggatt atgctcatgg 35040

cgaataccgt cagggccgtc cggtagagcc gccgtctgcc gatattaaat cggtagtcga 35100

tgcctacacc caatcaggct ggggtgagcg ttggttcgac cacatgcaga atgcaggtat 35160

caacgtggaa ggtcggttcg agaaatcacc gtactacctg ccgcgtcggt acagctctga 35220

taagctgcgc cgcctgtcgc gtgagaaggg cttcgaacag aaagactttg tggatgtgtt 35280

ctctgctgct atgcgtgatg cgtacccggc tatggaccgt gacctgtctc gtcaggttgc 35340

acgtacatgg tatcagggtc tgacctcagc acagcctaag cagggcgcta tgtggcgtcg 35400

cgctatcaac ggtatgagca atgacgagtt cgttgacatg ctggttgaga acggtgttga 35460

tgtagctgat gctcaggcga ttctggagaa atctgtgttc gcttctgagc gtactggctc 35520

caacccacaa cgtaacctgc gtaagcgtaa tgaactggat atgcagaagg tgtacacctc 35580

acagtctggc cgcacactgc gtctggctga tgtaatggac actgacgtat ccaagctcat 35640

gaaccagtac aacaaccgta tgtcgggccg tacagcgttt gctgctaagg gtcaagccga 35700

catgaaggat gtggccaacc gtatcgacga gctgcgtaac acgctgccag aagatgctga 35760

tggctggacc tccaaggttg atgacacgat tgatgccctg atgggttatc ctgtctctgg 35820

tgacactcca gattggcttc tggcctcggg ctacctatcc aacgcaatga tgctcaagaa 35880

ctcaggcttg taccagctga ctgaccttgc gctgtctgcg aaggagttcg gtatggcccg 35940

cgtcgtccgt tccatgttcc aatcaggtct gttccgtcat gcccgcgttg agctgggtaa 36000

agaccagaac ctgcaccagc gtctgcacaa catcctctct ggggcaactc agaacgatat 36060

gcgcttccgc tggctgcata ctctggccga cgataacact gacctgacac gctctgcata 36120

catgaccaac ctgactcgca acttgtctca agcggcgtac tcagctaacg gtatgcgtgc 36180

catccaccgt gctatggtga atatgaatgc tggtatgatt caggattcca tcatggcggc 36240

gctgggcggc tctgagaagg atgctaagct gttgcagcgc ttcggcctgc gtagcgatgc 36300

tctggaccag atgcgtgctg cttaccgcga gaatcctaat gctgcgttca accctcagct 36360

ccagacacat ctggaagccg tgggccagcg cttcatggac tacgtggtgc agcagaaccg 36420

gacaggtgaa acctcacact tcgcggaaat gaacccggtg ggccgcgtgc ttatcggcta 36480

ccagtcattc gctatggctg ggaccaacaa gattctgcgt cgtcacatcg acaacggaga 36540

gtatctgggt ctggctatgc tgatgatgta tcagttcccg atgatggctc tggccactta 36600

cgcccggtat ggcgctgacg gtaaatctga ccagaagaca accaaagacc tcattatgga 36660

ttccgtatcc ggtatgagtg caatcggcgg tgcctctctg gtgatggacc tgttcagcga 36720

taaccagcgc ggcggtggag tatcaatgct gggtggtgca aactctgtca ttagcacgct 36780

ccagagcgtt gcgaagaaag gcgagttgtc tgcgcaggat gcaagtaaat tggtgccgct 36840

ggcgcaggag ttcatcccgc tgcgaatcct cattaacaac acgggcgatt aagttcgccc 36900

ttcaccaagg aaacagaatg gctaattcat ggcagaccga aacaggtaca gccgggcaaa 36960

tcactattaa tgtgaatatc ccgtacctct cccgcagtga catttacgtg tatatcgctg 37020

gtactcagaa atcctttacg tgggatagcg ataccgtggt tcgtctacag accgccctga 37080

aaggtggtga agaagtgctg gtcatgcgcc gcactgcacg cgagaagctg cgtatcctgt 37140

tctcagaggg tgctgctttc agtcgtgata atctggacga acagaacatg cagttcctgt 37200

atctgtctca ggagctggta gaaggccgca gtatcgaagg cttctacggt gacatttcaa 37260

tgaacacttt ccgcatcact catttgggta cacccatcaa tgacactgat gctgcgaaca 37320

aactctacgt tgacgaggaa gtcaacactg tgaaccagcg tgcgctgcgt gtgccggagt 37380

ccagcgttgc tgtgacgcct accgtggccg gacgcaagaa taagctgctg ggctttgact 37440

cagtgggtaa tccggtatcc gctacgccga ttgaaggctc agccacggag ctggagctgg 37500

acctacagga tattgacggt atgagcattc tgggccgcgt ggatagcatc gctaccctgc 37560

gtaccatcgc tccgcgtcag gataagcgtt tcgtgtcagt acagagctac aacgctggct 37620

gggccgcgac gtccaaagct cctatgggtg gtggcttctt cgagtatctg gcgaatgaca 37680

caacctctgc cgatgatggt ggtatcgtca ttgtgggcct gaacggtgct cgttggcgtc 37740

gtatgatttc caccaatgat gtgactgttg agcacttcgg tgcaatggtc gatggtgtaa 37800

ccgatgatgc cgctgcatgg ctgcgtatgc acaactggtc ccgcaccgtt gatgctacgt 37860

tcggtccggg tattgtactg ccaccgggta agagcgcaat ctctcacatc gacactggca 37920

caaccgaaca gcctgcattc aaggtgcgtg gtccagaagc agcgtatggc cgtatcccac 37980

gtgcttacgt gaagctgctg gctactgact ctgactatgc catcaatgtg aaagcgcgtc 38040

gtatggaagt ctcctgtctc tacgtggatg gtgcatccag tacgaagggc ttcttccaga 38100

acaccgtgac ccgtggtgac tactgccgta tcgactgcgt tcaggcgcgt aacatgaccg 38160

gacgtgtgtt ccacgtttat gacaccatcg acaccgctgt aacgcagtgc tactcgactg 38220

gtggtaaagc gtcgttcttc cgcactgact ggtcgaacga gaaccccggc gcatgggacc 38280

acccaaccgc aattctgatt tctgactgta acttcgaagc gcacactggt gagttcgccg 38340

tgtcgtgtat ccgtgcaggt cagtctgtga tgcgtaactg ctggttcgac cgtaacgagc 38400

agggcttcga catttcacag ggcggctgga ttctggagaa cgtaacgcag gagaacagtg 38460

ctaaaccgtc tggttctcag tacgccaaaa tcgtgcagat tcgtaaccga ttcgcacagg 38520

gtaaagggat ggatgatacc gtgtccggtt atgacccatc gcaagacccg tccggtaaac 38580

tgccaagctg ggttaactct gggtatgaga acggtacagc tgagctggct gtgttgggct 38640

tcaagaacac tggctccatg tcttatggct acgagtcgtc tcagttcaag tgttcaaaca 38700

tgacagccaa tcaggtctgg attgacgtag gctcattctc tcttgcgggt gcagtcggtg 38760

agacgctggt gctggagttt gtgggtactg gtcactttga cgcagcaact gcgaacccac 38820

gtccgagcgg caccaactac ggtgcaggta agacaaccat cctgatgcag cagaaggact 38880

ccgatgcgaa atcccgcgca agctggttct ctgtcggtgc taccccgatt gttgcagtgc 38940

gtgttgccat gaaccgcaac cgtccgcacg tctacatcca gctggcgcag tataccgggc 39000

ctgttgcgat gactgtgcat accagctcga agtccaactt tgagggtggt cagcactatt 39060

accacaacat cgttatgacc gaagtcgatg cctctgttgc gacagctttc ccagctgcac 39120

ctgcaacgtg gaacgtcagc aacggtaact acggtttggg tatggacatg gacaccgggc 39180

gtatgtctct ggatggtccg ggtctggtca gcagcaacgc cagctcccat ctgccaatcc 39240

agtacaacgg caccgacatt ctactgcctg tgcagccaag cccactgagc acccgcgtac 39300

agcgtgtgac caaggcacag ctgccgaagg caacagagaa cccgtatggt gtcgtgctgg 39360

tcacggatgc tgtcacaggt aacggtgtca cccaatggcg tctggccttc tgcgatggtt 39420

ccttctggta caccgcagac ggtagcgtca acttaggttc aggctctgtc acataaggag 39480

gcaatatgag caagccgaag gccgggagca aatcccggct ttctgcactg cacgcaatgt 39540

ttactgaggc actgattagc gagttgcagg aagcgagtga ggagaaaatc ccactgcccg 39600

ctgccgacaa gtcagtcatt gccaagttcc tcaaggacaa cgacattacc gcagatgcag 39660

atgatgaatc aatgaaacgc ctcaacgacg agtttgagga tgaactcgaa gaacgccgga 39720

aggcccgcgc cgctgcaatc atgcagaagg tgggaggcga aggcgacgac ttagcaggaa 39780

tcatttaatg aaactcaatc cgtttgttct cagccgtttg aagctcattg ccgagcagac 39840

gaacgagctg aactttaacc cgcgcactat cccacgcgat aagcgcacag agctggctat 39900

gatgctggcc attacgttca aagagttccg ggacttcgcc tacgttggta tgcgcttcct 39960

cggctttgag ctgacggata tgcaggccga cattgcagat tatatgcagt acgggccgcg 40020

taagaagatg gtggccgcgc agcgtggtga agcaaagtct acgctggctg ctctgtactc 40080

cgtgtggcgt cttatccaag accagcgtac ccgtgtcctg attctgtccg gtggtgagca 40140

acaggcgtcc gaagtagcga cgctggttat ccgtctgatt gaaacatggc cattgctgtg 40200

ctggctcaag gctgacagca cccgtggcga ccgtacctca tacaccgcgt atgatgtgca 40260

ttgcgacctt aaaccactgg acaagtcacc gagtgtggcc tgtatcggtg tgaccgcatc 40320

cttgcagggt aagcgtgctg atttacttat cccggatgat attgagacaa ccaagaacgg 40380

tatgacccag accgagcgtg agaagctgct gacagtctcc aaagacttcg ctgcaatctg 40440

tacgcacggt gacaccctgt acctcggtac gccgcagacc aaagacagta tctacaagac 40500

gctgcctgcc cgtggctttg aggtgcgtgt atggccgggt cgtatccctt cacttgaaat 40560

ggaagaacgc tatggcgaaa cacttgcgcc ttatattcgg gagcttattg cttctggcta 40620

cgctcggagt ggtttcggag tcgatgggac tctgggccag tcaaccgata ctggacgcta 40680

ttctgaggac gacctgatag agaaagagct ggacttcggg ccagagggct tccagttgca 40740

gtacatgctg gacacgtctc tgcttgatgc catgcgcacc aaaatcaaac tgtccgacct 40800

gatgattcac ggtggtgata cagacaccgc gccggaccgc ttcatgtacg ccgctgaccg 40860

ccgtaatctg gttgaggagt acgaacctat ccggggcgag aagctgtact acccagctgg 40920

cgtcggctct gaaatgctcc agtacaagca caagctgatg gtgattgacc cggctggttg 40980

tggtggggat gaaatctcct acgctgctgg cggtgccgta tcgtcctaca ttcacctgtt 41040

cagtgttggt ggcttccaag gtggtgtatc caccgagaac atcgacaagg tgattgacct 41100

cgctaccgaa atggatatta gcaatatcct cgtcgagagc aacatgggtc acggtactgt 41160

ggaaatgctg attatgaacc gccttgccga gcgcaagatt gcgggtatcg gtgtaaccgg 41220

gatgtataac accactcaga aagagcgccg tatcatcgac actatctccc caatcacgcg 41280

ccgtcaccgc cttgtggtgc atgagcgtgc attgcaggac gataccgcgt cgtgcatggc 41340

gtacagccgt gagaagcgct ggctctactc cgcgttccag cagatgcaga acatcacata 41400

cgaccgaggc tgtctggcca aggatgaccg cattgatgcc atcgctatgc tgttgcagga 41460

gctgaactcg caccttgtcg aagacgagag cgtagcagcc gagaaagcgc gtgaggagga 41520

cgctaagcag ttcatgcgtg accctatggg ttacttcgat aataaaccaa aacctcgtca 41580

gggtgctcgt cgtgagcgat tcggacgtgt ttcaaataga aggagtcgtt aatgaccttt 41640

aagcaaacct ctgtcgataa tcgtgtcgct atccgtgaca gctctgtgaa gctgggtaat 41700

ctcgcgcagc gtctgactat tggctctggc ccggttgatt ccgggctggc taccgagatt 41760

gatgcgctgg cgcagtctgt ccagctggcg ctgagtaccg ctggttatcc tgtgtctctg 41820

ccgtccggca ccgcagccgt caagaactct gatgcagtgt cggtcagctc tggcggtaag 41880

tcggtcccgg ctactgccgc tgtctctggc aataccctgt ccggtgtcaa cctgaccacg 41940

gctaccgata ctgtcgcatc tgatggtgac gctgtatcca ttcaggaccg caataacgtc 42000

accctgctgg gttccagcgt tgcggcccgc gtctctggtg gtaagctggt atcagcctac 42060

ttcacctcac tgacgaacgg cctgaactct gatggcaacc cactggacgt tactactgcc 42120

cgtggcggtt caaccgtctc tggccgggtg cgtgtctacg ttacgaacaa cgtggtgcgg 42180

aacgtgtacc ttgatggtgc cgctgctctg gtaacgggca acacagtgcc tgtgcagaac 42240

tctgcgggcc tgaacatcca accgggtact gtcacagtca caaacaacct cgtcgcctct 42300

gtgaagctcc cagcgaacta cacagccttt ggtgacggta ttgcacataa ggtatcagac 42360

atggctggta agaatgctca ggctaccgct aaggtatcca acggctcttt gaccgaccaa 42420

gtgctacctg caacaaatgc tatcgtagca aatggacaga ccatcgctgt aacaggcggt 42480

actgtaacac tggcagtcgc caacggcgta ctgaccgcaa cctacactgc ctcgtaagga 42540

gaatcctcat ggcatattca gcagcaacta aggcgcagaa acaagctctg cgtgacgcaa 42600

tcatcaatgc caccaaagtg gtagagcagg cgcaggagtt cggtcagccg ggcgctgcat 42660

cgcaggtgga cgctaaactg tctaccctca agactgcact ggacgctgta gtgtccgctt 42720

cgagcgggag ctaatcatga ttagagcact ggtgttatcg ctggtgctct gtctcagtgg 42780

ttgctccgct acctcggcac ttaccgccgc caccgggctg attggttcta aaccggacct 42840

ctcagctcag gttggagcag agaacacgaa acagggcatt ggcctatccg gtaagtcaga 42900

cagtagcagt gagacgagcg tcaaagacag tacagtcgga accgtcgata gcagccgctc 42960

aaaggcagct aaggcacaga acattagcac aggaagcatc actgcggacc ggattgaggt 43020

acgcaacagc gatagcacag caatgatatt cgcgtttctc gccggacttc tcccgctact 43080

cagtgtggaa gtgtggctaa gaatccgcaa gaaacaggag gcggcgaaag ccgacaacca 43140

aagatgattc acaagaacct tgaaaccgca acctatggga ccagcacagg tttgacaatt 43200

ggaggttgga ttgtgcagca cctaccatca ccggacgtaa ccgcccagat tgcaagttgg 43260

gtcggtatcc tgacgggtgt cggtatgttc atctgcacca tctactacaa gcgtcgtaac 43320

agtaagctgt acgcccaagc cttgaagaag gggtatatga atgagccgtc cagtgaggaa 43380

taagattata gcgcgtgtca tggggtcact gttcgcagtg gccgcaggca ccgtagtgta 43440

tcatgaaggg accgggatta gcaaggatgg tcttgcgcat ccctaccttg acagtgcaag 43500

cgtctggact atctgctcag gcgacaccag cggcgttgtg ccgggtcgtg cagagacacc 43560

tgaccaatgt aaggtgcgac tgcgtaaatc cattgaggag cacgcacagg cgctctccgg 43620

gctaccagag cgtacacctg actatgctgt gttagcagcg gtggactttg cctaccatgt 43680

cggcgtgtac ggtgcgaaga actcaaccac gttcaagctg ctggaagctg gcgaccctgc 43740

tggggcagct gcggctatcg gctcatggaa gtacatcacg gatgactcga aaaggggtaa 43800

gccgggctgg agcttcatca atggccatta ccgctatgac tgctcgactc cgggcaacaa 43860

agtgtgcgct ggtatctgga agcgcaggat gtggcagatg gatacgatgg ctggtaagct 43920

cggctctgca caggatgctg aggctgcact agctcggtac gaccgtgcgt acgcagagaa 43980

aggccattgg gtcatcccgg ataagtgatg gagtcatcct ccgtcatccc ggttagaacc 44040

cggttagaac ccggcaggca cggagaggcc aaggatggca agtacggaga ggctggtgta 44100

atccagcacc tgaaatttat tatactcatg agaggccacg cccctgactc acaacgcgct 44160

tttcccccat cggccctccg ctggccctgc tgagcgaaag agttgacagg gtgggccttg 44220

ctgtgctacg tgtgcgcgtg cgccctgtgt tgtctcgcgc tgcgcgtggg tcattatggg 44280

gtcatcgctg ggtcatccgt gggttgtgct gggtagcc 44318

Claims (8)

1. A brachyury bacteriophage of Pantoea agglomerans, which is named as a Cophage vB _ PagP _ QF 1; is preserved in China center for type culture Collection (CGMCC) at 11/2/2020, with the preservation name ofPantoea agglomerans phage vB _ PagP _ QF1, accession number: CCTCC NO: m2020676.

2. Use of the Enterobacter agglomerans phage of claim 1 for the preparation of a medicament for the prevention, diagnosis or treatment of infectious diseases caused by Enterobacter agglomerans.

3. Use of the active ingredient of pantoea agglomerans phage according to claim 1 for the preparation of a pharmaceutical additive, spray additive, disinfectant or detergent for killing pantoea agglomerans in the human body, on the body surface, in a hospital room, in a medical instrument or in a medical space environment.

4. Use of the Enterobacter agglomerans phage of claim 1 for killing Enterobacter agglomerans in a space environment.

5. Use of a panto-phage according to claim 1 for killing pantoea agglomerans on the surface and in vivo of livestock.

6. Use of the Enterobacter agglomerans phage of claim 1 for killing Enterobacter agglomerans on a plant surface.

7. A composition for preventing or treating infectious diseases caused by Pantoea agglomerans, comprising the bacteriophage of claim 1 as an effective ingredient.

8. Use of the bacteriophage of claim 1 or the composition of claim 7 for the preparation of a feed additive, feed, drinking water additive, drinking water, disinfectant or detergent for killing pantoea agglomerans in vivo of livestock and poultry, on body surface of livestock and poultry, in livestock and poultry feed, in farming implements or in farming space environments.

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