CN114703152A - Burkholderia farci phage vB _ BpP _ HN01 and application - Google Patents

Abstract

Purified bacteriophages having specific bactericidal activity of burkholderia melioidis, such as burkholderia melioidis bacteriophage vB _ BpP _ HN 01; a composition for preventing or treating infectious diseases caused by Burkholderia melioidis comprises bacteriophage as an active ingredient; an antibiotic comprising a bacteriophage as an active ingredient; a feed additive composition comprising a bacteriophage as an active ingredient; a disinfectant or a detergent comprising a bacteriophage as an active ingredient; and methods of treating melioidosis using bacteriophage. The purified bacteriophage of the present application has specific bactericidal activity against burkholderia melioidis. Thus, the bacteriophage provided by the present application can be used for the prevention or treatment of infectious diseases caused by burkholderia pseudomallei, and can also be widely used for the preparation of antibiotics, drugs, antiseptics, disinfectants, and diagnostic preparations for bacterial infection.

Description

Burkholderia farci phage vB _ BpP _ HN01 and application

Technical Field

The application relates to the technical field of novel bacteriophage, in particular to burkholderia pseudomallei bacteriophage vB _ BpP _ HN01 and application thereof.

Background

Burkholderia pseudomallei (hereinafter abbreviated as Bp) is a gram-negative conditional pathogenic bacterium and can be parasitized in human cells. The most significant problem in the current treatment of this disease is the complex inherent resistance of Bp, which earlier clinical data show to be resistant to the action of penicillins, first/second generation cephalosporins, macrolides, polymyxins and aminoglycoside antibiotics. The specific drug resistance mechanism can secrete lipopolysaccharide to form a biofilm to shield the drug, and Bp can also code beta lactamase so as to relieve the effects of beta lactam antibiotics such as cephalosporins, oxacillin and the like, and the interior of the thallus also has the effect of further reducing the antibiotics by an efflux pump system consisting of transport protein, fusion protein and pore protein. Under the long-time action of a single antibiotic, the heterogeneous drug resistance of the strain is difficult to avoid, and the acquired drug resistance can be generated due to the accumulation of point mutation, and researches show that the drug resistance of the strain to ceftazidime can be increased by 85 times when the coding endogenous beta lactamase has single base mutation.

Currently, there are only three phage with potential for clinical applications against Bp: phage ST79 (unmaporn Yordratum, Unchele Tartawasart, Surasaki Wongratanaceae, Rasana W.Sermsan, Novel viral bacteria from soil fat soluble bacterium and plasmid pseudo-psid, FEMS Microbiology Letters, Volume 314, Issue 1, January 2011, Pages 81-88) and E094 (Muangsombut V, Withatanung P, Chantratita N, Charenonsudjai S, Lim J, Galv EE, Ottiwet O, Senye S, Janesomebunon S, Lonnener MJ, Dunnel M, Kosrisate S.Rad. Clinker. K. O, Sengybean S.12. the former was isolated from the laboratory bacterial strain of Lab, Lab.12, Labiatae, Missue, Misso, Missue, Misso, Mitsu, Mitsugaku, Miyashi, Miyae, Miyas, Miyae; phage C34(Guang-Han O, Leang-Chung C, Vellasamy KM, Mariappan V, Li-Yen C, Vadivulu J. Experimental phase Therapy for Burkholderia pseudomoallei infection. PLoS one.2016Jul 7; 11(7): e0158213), isolated from Malaysia, although possessing a narrower lysis profile, animal experiments showed that mice with over 1/4 did not die after Bp infection.

Disclosure of Invention

In order to explore a novel diagnosis and treatment method for gangrene, the inventor of the application finishes the sequencing work (Genbank number CP038805 and CP038806) of a Hainan local reference strain Bp HNBP001 in 2019, separates and obtains a series of phage capable of infecting Bp from 100 environmental samples (including paddy field soil, irrigation water source and sewage) collected from Oriental city in Hainan province in 2021 by taking the phage as a host, screens and obtains the first short-tail phage vB _ BpP _ HN01 with broad-spectrum Bp lysis capability in China according to the form of the phage, whether the lysis state can be stably maintained and the infection condition of clinical strains, researches the morphological characteristics, lysis characteristics and infection characteristics of the phage, simultaneously carries out sequencing and necessary bioinformatics analysis on the whole genome of the phage, and ensures that a cell model confirms the in-vitro Bp resistance capability and the possibility of treating gangrene in the future and antibiotics, has wide application prospect.

In a first aspect, the present examples disclose a purified bacteriophage having a genome of double-stranded DNA comprising a nucleotide sequence of SEQ ID No.1 and having antagonistic activity against Burkholderia pseudomallei.

In a second aspect, the embodiments of the present application disclose a purified bacteriophage (Burkholderia pseudolitea phage) vB _ BpP _ HN01, which is deposited at the China center for type culture Collection at 20/4 in 2022, with the deposition address of Wuhan university, Wuhan, China and the deposition number of CCTCC NO: M2022422; the bacteriophage can be used for antagonizing growth of Bokholderia pseudomallei strains, and can be used for preventing and treating melioidosis diseases, pulmonary infection and/or bacteremia.

In a third aspect, the present application discloses a composition for preventing and/or treating infectious diseases caused by one or more kinds selected from Burkholderia melioidis, the composition having the bacteriophage of claim 1 or 2 as an active ingredient.

In a fourth aspect, the examples herein disclose an antibiotic comprising as an active ingredient a bacteriophage as described in the first or second aspect.

In a fifth aspect, the embodiments of the present application disclose a disinfectant or a detergent containing the phage of the first aspect or the second aspect as an active ingredient.

In a sixth aspect, the present examples disclose the use of a bacteriophage of the first or second aspect in the preparation of a medicament for use in a method of diagnosing a causative agent of a bacterial infection, comprising:

(I) culturing a tissue sample from a patient;

(II) contacting the culture of step (I) with a medicament comprising a bacteriophage of the first or second aspect;

(III) monitoring evidence of growth and/or lysis of the culture;

wherein evidence of lysis of the culture indicates that the culture comprises burkholderia pseudomallei.

In a seventh aspect, the present application discloses the use of a bacteriophage of the first or second aspect in the preparation of a medicament for reducing or inhibiting colonization or growth of burkholderia mallei on a biological surface in contact therewith, said biological surface being mammalian skin, damaged skin or mucosa.

In an eighth aspect, the present examples disclose the use of an effective amount of the pharmaceutical composition of the third aspect in the manufacture of a medicament for treating or reducing the incidence of a bacterial infection by burkholderia pseudomallei in a subject in need thereof by administering the medicament.

In a ninth aspect, the present embodiments disclose a non-therapeutic method for reducing or inhibiting colonization or growth of burkholderia mallei on a solid surface, comprising contacting the surface with the bacteriophage of the first or second aspect.

Drawings

FIG. 1 is a plaque situation in which phage infects Bp HNBP01 provided in the examples of the present application

FIG. 2 shows the adsorption efficiency (A) of the phage to Bp HNBP01 and the final titer of the phage under different multiplicity of infection conditions (B) provided in the examples of the present application.

FIG. 3 shows a one-step growth curve (A) of phage and the lysis of host bacteria by phage under the same conditions (B).

FIG. 4 shows the genomic organization, repeat sequences and tRNA markers of vB _ BpP _ HN01 phage provided in the examples herein.

FIG. 5 is a genome-wide alignment of vB _ BpP _ HN01 with sequenced Bp phage as provided in the examples herein; FIG. 5A shows the results of homology comparison with a short-tail phage, a long-tail phage, and a myotail phage; FIG. 5B is the result of Viptree; FIG. 5C is a homology alignment based on portal proteins; FIG. 5D shows the results of homology based on the amino acid sequences of the terminal enzymes.

FIG. 6 is a graph showing the results of the cytoprotection assay in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

In a first aspect, embodiments of the present application relate to purified bacteriophage having antibacterial activity against one or more strains of the nosocomial pathogen burkholderia mallei. It has a double-stranded DNA comprising the nucleotide sequence of SEQ ID NO. 1.

In a second aspect, the present application provides a bacteriophage having a genome comprising or consisting of a double stranded DNA comprising the nucleic acid sequence of SEQ ID No. 1. A specific example according to this embodiment is an isolated short tail bacteriophage (known under the name Burkholderia pseudolei phase) vB BpP HN01, which targets Burkholderia pseudomallei. FIG. 4 shows a schematic structure of the vB-BpP-HN 01 genome comprising the nucleic acid sequence of SEQ ID No.1, and also the Open Reading Frame (ORF) in the vB-BpP-HN 01 genome and its position within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the designation of putative functional proteins, are shown in FIG. 4.

The phage provided by the application belongs to the morphotype group of the Podoviridae (Podoviridae), the head of the phage is in a symmetrical structure, the diameter of the phage is about 62nm, and the tail of the phage is about 20nm (as shown in figure 1C). As shown in FIG. 1A, after the phage is infected with Bp HNBP001, transparent plaques with the diameter of about 1-2mm can be formed on the double-layer plate, if the culture time is further prolonged, the existence of a capsular hydrolysis ring can be observed at the periphery of the plaques, which indicates that an element with capsular polysaccharide hydrolase exists on the genome (FIG. 1B).

The inventor collects 100 samples of paddy field soil, orchard soil, chicken and duck manure, irrigation water, sewage and the like in a high melioidosis area in east city of Hainan province, separates phage with specific bactericidal activity and the characteristics on 25 melioidosis Burkholderia plantarii, is named Burkholderia plantarii phage (Burkholderia pseudomonad phage) vB _ BpP _ HN01, and reserves the phage in the China center for type culture collection at 15/4 of 2022, the collection address is Wuhan university, and the collection number is CCTCC NO: M2022. Consistent with the information of the deposit certificate.

In another aspect, the present application relates to a composition for preventing or treating infectious diseases caused by one or more kinds selected from Burkholderia pseudomallei, which contains the bacteriophage of the first or second aspect as an active ingredient.

In some embodiments, specific strains of burkholderia melioidis are shown in table 1.

The term-prevention "as used herein means all actions that suppress or delay the progression of the disease by administering the composition. The term "treatment" as used herein means all actions by which the condition of a patient has improved or shifted to a favorable state by administration of the composition.

The composition of the present application comprises not less than 10⁹pfu/mL vB _ BpP _ HN 01.

Examples of preferred infectious diseases to which the composition of the present application can be applied include, but are not limited to, melioidosis, which is a symptom of chilliness, hyperpyrexia, pulmonary infection, bacteremia, sepsis, etc. caused by burkholderia pseudomallei.

The compositions of the present application may additionally include a pharmaceutically acceptable carrier and be formulated with such carrier to provide antibiotic, pharmaceutical, antiseptic and bacterial infection diagnostic formulations. The inventors were asked to confirm that the extension was properly made.

As used herein, the term-pharmaceutically acceptable carrier "refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. For formulation of the composition into a liquid preparation, sterile and biocompatible pharmaceutically acceptable carriers such as saline, sterile water, ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol and ethanol may be used. These may be used alone or in any combination thereof. Other conventional additives such as antioxidants, buffers, bacteriostats, etc. may be added if desired. Further, diluents, dispersants, surfactants, binders and lubricants may be additionally added to the composition to prepare injectable preparations such as aqueous solutions, suspensions and emulsions, or oral preparations such as pills, capsules, granules or tablets.

The prophylactic or therapeutic compositions of the present application may be administered or sprayed onto the affected area (afflicted area), or administered by oral or parenteral routes. Parenteral administration may include intravenous, intraperitoneal, intramuscular, subcutaneous or topical administration.

The dosage suitable for administration, spraying or administration of the compositions of the present application will depend upon a variety of factors including the method of formulation, the mode of administration, the age, body weight, sex, condition and diet of the patient or animal being treated, the time of administration, the route of administration, the rate of excretion and the sensitivity of the response. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.

Examples of oral formulations suitable for the compositions of the present application include tablets, troches, lozenges, aqueous or milky suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. For formulations such as tablets and capsules, useful are binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol waxes. For capsules, a liquid carrier such as a lipid may be further used in addition to the above-mentioned compounds.

For non-oral administration, the compositions of the present application may be formulated as injections, by subcutaneous, intravenous or intramuscular routes, as suppositories, or as sprays inhalable via the respiratory tract, such as aerosols. The injectable formulation can be obtained by: the compositions of the present application are dissolved or suspended in water together with stabilizers or buffers and the solutions or suspensions are packaged in ampoules or vial units. For sprays, such as aerosols, propellants for spraying water-dispersed concentrates or wet powders may be used in combination with the additives.

On the other hand, the examples of the present application disclose antibiotics comprising the above-mentioned phages as active ingredients.

As used herein, the term-antibiotic "means any drug that is administered to an animal to kill pathogens, and is used herein as a general term for antiseptics, bactericides, and antibacterial agents. The animal is a mammal including a human. Unlike conventional antibiotics, the bacteriophage of the present application has high specificity to burkholderia melioidis so as to kill specific pathogens without affecting beneficial bacteria, and does not induce resistance, so that its use cycle (life cycling) is long.

In another aspect, the present application provides a disinfectant or detergent having the bacteriophage of the first or second aspect as an active ingredient. To remove Bp, the disinfectant including bacteriophage as an active ingredient may also be sprayed and applied to any area of livestock activity, slaughter house, livestock death site, cooking space, and cooking facility, but is not limited thereto. In addition, cleansers comprising bacteriophage as an active ingredient may be used on the skin and body areas of live animals that have been or are potentially contaminated with Bp.

Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the present application is not intended to be limited by these examples.

Isolation and purification of phages

A similar gangrene high-incidence area in the province of east city of Hainan province is selected for sample collection, and 100 samples of rice field soil, orchard soil, chicken and duck manure, irrigation water, sewage and the like are obtained. Wherein, about 5g of solid sample is weighed and put into a sterile 50mL centrifuge tube, SM buffer solution with 3 times volume is added, the mixture is fully shaken to prepare suspension, and then the suspension is kept stand overnight at 4 ℃ to ensure the full release of the phage. Then, 3500g was centrifuged for 20min, and the supernatant was collected. For liquid samples, aspirate 20mL to 50mL and add 20. mu.L of 5M CaCl₂After mixing, 3500g was centrifuged for 20min to collect the supernatant. Then using 0.45 μm filter membrane to filter larger particles and part of the particlesThe thalli are cleared, and the filtrate is transferred to a new sterile 15mL centrifuge tube and stored at 4 ℃ for later use.

The recovery of Bp HNBP001 (burkholderia melioidis HNBP001, hainan medical institute P2 laboratory) was performed before isolation of the phage: a single clone was picked and inoculated into 5mL of LB liquid medium, incubated overnight at 37 ℃ and 220 rpm. The next day, shake to log phase at 1% passage. The phage were then isolated according to the double-layer plate method: 10mL of the phage-containing supernatant was aspirated, and 5mL of 3 XLB liquid medium and 200. mu.L of Bp HNBP001 were added thereto, incubated at 37 ℃ and 220rpm, and incubated overnight. Centrifuging the above culture at 4000rpm for 10min, collecting supernatant, sterilizing with 0.22 μm filter membrane, mixing 200 μ L with 400 μ L log-phase Bp HNBP001, standing at room temperature for 10min, adding 6mL semisolid agar medium (55 deg.C, ensuring that the medium is still in a flowing state), pouring onto 2% agar plate, solidifying the upper layer medium, culturing at 37 deg.C by inversion overnight, and observing whether to generate plaque, and the size and transparency of plaque the next day.

Individual plaques were picked and placed into 1.5mL sterile Eppendorf tubes containing 500. mu.L of SM buffer. Soaking overnight at 4 deg.C on a horizontal shaker, sterilizing and filtering with 0.22 μm filter membrane, mixing 100 μ L with 100 μ L log phase Bp HNBP001, standing at room temperature for 10min, adding 6mL semi-solid culture medium of 55 deg.C, mixing, and pouring into 2% agar plate. After coagulation, the cells were cultured in an inverted state at 37 ℃ overnight. Observing the formation of the plaques again, repeating the steps for 5 times, and preserving the phages which can generate uniform size and have the same transparency: 5mL of SM buffer was added to the plaque plate, left to stand overnight at 4 ℃, the soak collected, and the plate was rinsed with 1mL of SM buffer. The two were combined, collected in a 15mL centrifuge tube, and the cells were removed again using a 0.22 μm filter as a phage solution at 4 ℃ for use. For the phage needing long-term preservation, the phage is placed in 30% glycerol and frozen at-80 ℃ in an ultra-low temperature refrigerator.

To obtain enough phages for subsequent experiments, a sufficient number of host bacteria were first prepared: single clones of Bp HNBP001 from fresh plates were picked up at 5mLShaking LB overnight, inoculating to 200mL LB liquid culture medium at 1% ratio the next day, adding 4mL phage solution obtained in the previous step when OD600 of thallus reaches about 0.5, and adding CaCl₂(5mMol/L) and MgCl₂(5 mMol/L). Culturing at 37 ℃ and 220rpm until the liquid is completely clear and bright. For the phage collection, first adding solid NaCl to make its final concentration reach 0.5Mol/L, after complete dissolution, in 4 degrees C refrigerator standing for 2 hours. Then, the mixture was centrifuged at 4500rpm for 20min, the supernatant was collected, PEG 8000 was added to a final concentration of 10% (w/v), and the mixture was mixed well and then left overnight at 4 ℃. The pellet was then centrifuged at 6500rpm for 20min at 4 ℃ and the supernatant carefully discarded, resuspended in the appropriate amount of TM buffer and filtered through a 0.22 μm filter.

To obtain a specific titer, 100. mu.L of the concentrated sample was diluted 1/10 to 10^-9Then, 10. mu.L of the diluted sample was taken out and mixed with 100. mu.L of log-phase HNBP001, and the number of generated plaques was calculated (N) by the double-layer plate method in the same manner, and the titer (PFU/mL) was calculated as NXdilution multiple X100 by the following method.

Determination of the host Spectrum of bacteriophages

Selecting the to-be-detected bacteria on a fresh plate to be subjected to monoclonal culture to 5ml of LB liquid culture medium at 37 ℃ and 220rpm for overnight culture, and carrying out subculture to a logarithmic phase according to a proportion of 1% on the 2 nd day; then 100. mu.L of the test bacteria were added to 6mL of semi-solid agar medium (55 ℃), mixed and poured into 2% of solid medium, and 2. mu.L of 1X 10⁸The titer of phage was dropped on the double-layer plate, dried and inverted at 37 ℃ for overnight culture, and the sensitivity of the selected host bacteria was judged on day 2 according to the plaque production. The results are shown in Table 1. In Table 1, - + "indicates that a certain strain of bacteria was infected with the phage to produce plaques, that is, bacterial cells were lysed; for a certain strain of bacteria which could not produce plaques, the sensitivity was marked as "-".

TABLE 1 host spectra ranges of the phages

a: the 2 Burkholderia cepacia and the clinically common gram positive/negative bacteria standard strains used at this time were given by the clinical laboratory of the second subsidiary hospital of Hainan medical college

As can be seen from Table 1, only one strain (Bpn012) derived from Haikou among the 24 Bp clinical strains screened by the method of lysis in addition to the host Bp HNBP001 could escape the action of the phage. Meanwhile, for Burkholderia cepacia and other clinically common gram positive bacteria/negative bacteria standard strains, the bacteriophage can not complete infection or can be converted into a lysogen state after infection.

Observation of morphological characteristics of phage by electron microscope

Reach a bacteriophage titer of 10¹¹PFU/mL, add appropriate amount of DNase I and RNase A to the concentrated sample, incubate at 37 ℃ for 30 min. Then, residual endotoxin and other non-phage substances were removed using a 100kDa ultrafiltration membrane (Amicon), 10. mu.L of the filtrate was dropped onto a carbon-supported membrane copper mesh (400 mesh), after 10min of adsorption, the remaining sample was removed using filter paper, and negative staining was performed for 5min using 10. mu.L of phosphotungstic acid (2%, w/v). Excess dye was also blotted away using filter paper. The prepared samples were left to dry completely at room temperature for 5 hours and then observed by transmission electron microscopy according to standard procedures.

As shown in FIG. 1, after the phage is infected with Bp HNBP001, a transparent plaque with the diameter of about 1-2mm can be formed on the double-layer plate (FIG. 1A), if the culture time is further prolonged, the existence of a capsular hydrolysis ring can be observed at the periphery of the plaque, which indicates that an element for coding the enzyme exists on the genome of the plaque (FIG. 1B); the microscopic structure was observed by transmission electron microscopy and confirmed to be a typical short-tailed phage with a symmetrical head diameter of about 62nm and a tail length of about 20nm (FIG. 1C). For this reason, the selected and isolated phage was named vB _ BpP _ HN01, which targets burkholderia melioidis.

Phage adsorption assay

Culturing Bp HNBP001 to logarithmic phase, adding phage vB _ BpP _ HN01 with infection complex number of 0.1, centrifuging at 0min, 5min, 10min, 15min, 20min, 30min, and 40min for 2 × 100 μ L respectively, collecting supernatant, and determining titer T by double-layer plate method_AThe titer T was determined for another untreated sample, likewise according to the double-plate method_BThe adsorption rate was calculated according to the following formula, and the average adsorption rate was calculated by sampling three times at each time point, and the whole experiment was repeated three times independently.

As shown in FIG. 2A, the samples were diluted, phage infection experiments were performed at a multiplicity of infection of 0.001 to 10, and comparison of the final titer values revealed that the phage produced the highest titer, about 10, at a multiplicity of infection of 0.1¹²PFU/mL. As shown in FIG. 2B, the adsorption rate was measured under these conditions, and as a result, as shown in FIG. 2B, about 40% of the phages were adsorbed after the co-incubation for 5min, and more than 95% of the phages were adsorbed at 30 min.

One-step growth curve of bacteriophage and determination of infection rate of host bacteria

(1) Culturing the Bp HNBP001 to a logarithmic phase, adding phage vB _ BpP _ HN01 with the multiplicity of infection of 0.1 into 10mL of bacterial liquid, and incubating at 37 ℃ for 10min to ensure the adsorption of the phage to the host. The cells were centrifuged at 12000g for 10min, phage free in the supernatant were discarded, and 10ml of LB was used to resuspend the cells, which were cultured at 37 ℃ and 220 rpm. 100 μ L of the resuspension was taken at 0min, 5-40 min (5 min sampling interval), 50min, 60min, 80min, 100min, 120min, respectively, titer calculation was performed according to the method described in 2.2.2, 3 samples were taken at each time point, the average value was calculated, the whole experiment was repeated three times, and a one-step growth curve was plotted using origin (version 7).

(2) Bp HNBP001 was cultured to OD600 ═ 0.3, and phage with a multiplicity of infection of 0, 0.1 were added and cultured at 37 ℃ at 220 rpm. Changes in absorbance at 600nm were measured every 30min using LB medium as a blank, three samples were taken at each time point, and the OD600 average was calculated, and this experiment was independently repeated three times.

As shown in fig. 3A, vB _ BpP _ HN01 was latent for Bp HNBP001 infection for the first 20min, and then titers increased explosively for up to 40 min. As shown in fig. 3B, the culture broth of Bp HNBP001 strain reached an OD600 of 0.8 at 150min in the absence of phage (MOI ═ 0); when vB _ BpP _ HN01 was inoculated at an MOI of 0.1, the growth curve of Bp HNBP001 cells was significantly reduced at 30min, and more than 95% of the cells were lysed at 150 min.

vB _ BpP _ HN01 phage whole genome sequencing and bioinformatics analysis

(1) Extraction of genomic nucleic acid

Preparation of 10mL of titer 10 by scale-up¹²PFU/mL or more of phage solution, and its genome was extracted according to kit (ZP317, Chuanchui): mu.L of RNase A (20mg/ml) and 6. mu.L of DNase I were added to the solution, and the free nucleic acids were digested (incubation at 37 ℃ C. for 30 min). 5mL of phage precipitation solution (precooled beforehand) was added, mixed well and then placed on ice for 20 min. Then, the mixture was centrifuged at 12000rpm at 4 ℃ for 15min, and the precipitate was collected and dried at room temperature for 2 min. The pellet was gently pipetted using 400. mu.L of phage lysis buffer and resuspended, and 50. mu.L SDS was added to disrupt the phage outer membrane protein structure. Adding 10 μ L proteinase K, treating at 56 deg.C for 60min, 500 μ L buffer solution B, and 500 μ L anhydrous ethanol, mixing well, and separating out phage nucleic acid. Then using silica gel column to adsorb phage nucleic acid by sample-centrifuging method, desalting the genome crude sample with 70% anhydrous ethanol, volatilizing ethanol, and adding ddH₂O the genome on the column was eluted and its DNA concentration was determined and the sample was finally stored at-20 ℃.

(2) Whole genome sequencing

The phage genome nucleic acid was sent to huitong scientific and biological limited (guangdong, china) and the entire genome was sequenced using the illumina platform: a DNA library with an average length of 350p was constructed using the NexteraXT DNA library preparation kit (Illumina, San Diego, Calif.). DNA was sequenced on the Illumina Novaseq 6000 platform to obtain a 150bp paired-end sequence. The original sequence Reads was edited using the NGS QC Tool Kit (Version 2.3.3) with high quality Reads assembled using SPAdes software (Version 3.15.2). Open Reading Frames (ORFs) that may be contained on the genome are predicted using GeneMarks and annotated by Blastx alignment (www.ncbi.nlm.nih.gov) according to gene homology.

The sequencing results of the phage genome were analyzed using PhageTerm software, and phylogenetic trees were constructed using MEGA (Version 7.0) by the Neighbor join method based on the similarity of the terminal enzyme (Terminase) and the portal protein (portal protein) involved in phage replication and structure.

As shown in FIG. 4, bioinformatics gene analysis vB _ BpP _ HN01 genome comprises a double-stranded DNA of nucleotide sequence shown in SEQ ID NO.1, a heavy sequence (Repeat region) with the size of 407 bp is identified from the sequencing raw data as the starting point of the genome, the phage genome structure has 93 ORFs, 22 of which are marked as hypothetical proteins, and the ORFs can be divided into according to the responsible functions: 7 ORFs responsible for the synthesis of the phage outer structure, of which ORF73 encodes the coat protein, homologous to the N4 short-tailed phage. orf70 and orf90 are collectively responsible for the composition of the phage fiber structure and are of a length determined by orf74 and orf80, both of which also share high homology with the gene of a brachyury phage. The surface of the phage was also decorated with the translation product of orf 24; a total of 9 genes are involved in the events associated with the phage nucleic acid, ORF51 is a DNA helicase, ensuring the initiation of genome replication, during which ORF62 (single stranded DNA binding protein) and ORF60 (primer) are involved, the entire synthesis process being responsible for the polymerase ORF53, with the aid of ORF38 and ORF 45. Furthermore, it is speculated that ORF76 is also involved in DNA replication and is homologous to MazG proteins, which regulate the environment within the host bacterium, adapting it to phage proliferation. The whole transcription process is controlled by the phage own transcriptases (ORF23 and ORF 67).

Further whole genome alignment of the phage shows that: compared with the sequenced Bp phage (the related sequence is derived from www.millardlab.org), the homology between vB _ BpP _ HN01 and the Bp phage is not found (FIG. 5A), and secondly, all similar viruses/phages given by Blastn have the whole gene coverage rate of not more than 5% in the alignment process, and are all invalid results. When virome analysis was performed using VipTree, it was found that vB BpP HN01 was recognized as having affinity with phage capable of infecting achromobacter (fig. 5B), and the late analysis was probably due to the similarity of single genes or partial fragments such as RNA polymerase, fiber protein, etc. (fig. 5C and 5D). As judged by homology of the terminal enzyme and the portal protein, it was found that the phage may be somehow related to Erwinia phages or Rhizobium phages.

Cytoprotective experiment of vB _ BpP _ HN01 phage

A549 cells (

CCL-185^TM) Inoculating in RPMI 1640 complete medium containing 10% calf serum, and culturing at 37 deg.C with 5% CO₂When the coverage of the cells exceeds 70%, the cells are passaged according to standard procedures. Then 2 is multiplied by 10⁴A549 cells were seeded in 96-well plates and 2X 10 cells were used⁵It was infected by CFU Bp HNBP 001. After 2 hours, 2X 10 of water was added to the well plate⁴PFU phage, 40. mu.g/mL ceftazidime and mixture of both (three groups in total), 37 ℃, 5% CO₂After 18 hours of incubation, the final absorbance at 450nm was measured using the CCK-8 kit according to the instructions and recorded A_X. The A549 cell group without any treatment was used as a negative control, and the absorbance at 450nm was measured using the CCK-8 kit and was designated as A_B. According to the followingThe cell survival rate of the cells infected by the Bp HNBP001 and treated by the phage/antibiotic is calculated by the formula. Cell survival rate ═ a_B—A_X)/A_B×100％。

As shown in fig. 6, the survival rate of a549 cells was significantly decreased by Bp HNBP001, which was only about 40% of that of uninfected group. The mortality of the cells decreased with the treatment of the v b _ BpP _ HN01 phage with MOI of 0.1, the protective effect was slightly due to the effect of ceftazidime at 40 μ g/mL, and the survival rate of a549 was further improved with v b _ BpP _ HN01 and ceftazidime together after infection.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Sequence listing

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<120> Bokholderia fargei phage vB _ BpP _ HN01 and application

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<400> 1

gggggcgtcc agccccctac acttacacta cccccccggt atgtcttaca cgtacacatg 60

gggggtgccc ctatgttgta gatgctccgc atcttatggc aatcatgcct taactacata 120

ggagtctcat catgtccgct aagctcacta tcggtgctgt cctcggtacg gttgcagatg 180

cagcaggtgc tatcagcacc actctcggta ctgctaccaa ggtagtcaac atcgctgaca 240

actatgtcga agactttgct aacaagcaga agattcgtat tgctgcaagc aatgtcggat 300

acaagaagca gatcatcatg gaaactgcta tgcaagttca gcaacagaag cttgttgttg 360

ataagttcgt tgatgaaaat gactgtgcag atcatttcaa cgatctcgta gcagaactcg 420

aagcagcaat ggcctaaaca tggcctagac ctcaaggaga aatccttggg gtctaaaacc 480

ctttgagtgg atagtccgaa ggacgaactc tcgaagagag ttcagtcaac ttgtagacgg 540

aaagttaaac cagagtctac ttcctgtagg acaaatccta tggaaagtag aatcaaatta 600

gaggaaagac aatgcctttc atcaccaaga tcatcctgat cctgatcgct cttgcgatcc 660

tcatcctgtt cccgattgtg attccgttcg caatctacat gctgctgagc atgggattcg 720

aaggtaagaa cttcggcaag ttcaacatca ctgatgcagc gaggcactga gtatggaata 780

cgattgcaag cactggccta tattctggga agtaggttat gagtttggtt tggatacgga 840

tctatttatg gatccacgag cagaagcaca gtatcaacgt tggctttata cgccgagttt 900

gtcctctcaa gtaaatgcat ttgtacctct cggagactaa ccatgagcat cgaagaacaa 960

ctgaagctgg ctgaagaaga aatcgcagca tacgaagagg atcttcagta tctggatggt 1020

gaggacatca gcgatgaata acttttacga actttactat cagtatgttc ggagccaaga 1080

agagaaagaa ctcttcgcat tctcatttaa cgtgtggcga gactatttcg gccacaactt 1140

cgagtaaatc atgaagaaga taatcgctct cagccttgtc ctgcttggct tagcaggatt 1200

tggcttaatt ggtttgggtg catacttcgt aactactccg cagcaatggg tagtgcccaa 1260

tcaagacatg gtactcatca cactgggtgc atgtgcagtt ggcttggtga tcctcatcgt 1320

gagccaagac aagtaataca gtcagtccaa gtagtccaca tttaatcgtt tctcgggtat 1380

ttagcccatt cattattaat cctttaggag agtcatcatg gcaatcacca acgtttccgc 1440

attcggttcg tcgaagagga ccgcagcagc aaataagcag gaactcaaga agtcccaatt 1500

ctggatcaat atcggctatc aagctgagat ccaaacggaa gaaggaacgg aactccgttt 1560

catcagcctg ccgaatggca tggcactaga cgacatggaa ccggtcaagg tgtccggtaa 1620

caacgaagtc tggctgtatc agcagtccgc acgcaacgat ctgctggccc aactgaaggc 1680

caaggcagag tcgctcaagc cgggtgagga agtcctcatc ggtgaaggcc cgatcgtgat 1740

tcaactccgt cgtgtgagcg acgaagccaa ggagatcaag gcagcagcca acccgttggc 1800

acgcaatctc ggcctgtaag taggcaaggg ggactgacct tcgggtcggt tcccttaatt 1860

ttttagttgg cttgagagtt caactttcta aagactaagt tcaactttta agccaatttt 1920

gactaaagga gcctaagagc tatggaatgt catcaatgtg gtgcttgctg tactgaaatc 1980

agtatttcca cctcgtatgt tggccatccg aacggtaaga aagcaggcac tcgttgcatc 2040

tggctggagg ctgataacaa gtgtttgctg ttcaacaagc cgctgagacc tgccatctgt 2100

agcagctttc aagctgatcc tgaaatctgt ggaaccaatg gggaagaagc taccaagctc 2160

atccgttggt atgaaaagga gactcgtcca tgaagaagat cgccatcttt gccctgttga 2220

tggtcagctt tgcagtgcat gcagagcctc agatggaata ccgcaagggt ggtatcacta 2280

tcgaactgaa caacagcaac tggacgatga cgggtgataa gtcgttcgac aaaggttgct 2340

tcgatcagaa gatgggacgt accaccatcg agcaacgcaa gacgtacatc tgttggtttt 2400

acgacgtgga caagggtgac atcgtcctca agttcccgca cacaaagcat tttaagagga 2460

tctccatcaa cgagttcgat gcagtctgag ttagggaaat cgtatgaaga tgcgtcaggt 2520

agacgacttc ggtaatgagt tctggcccat cttacacaaa tgggcctact acactggtcg 2580

ttggtattac gtgaccgtga agaacaatct aatgtggtaa ctaacatttg agccaaggag 2640

agtgaacatg ctaaaactgt tcgctacgaa caaatatcat cgacgtggtg gggccactta 2700

cgtgaatgtg atgcgtggaa ctccattggg caatccgttc gtcatgaagc atgaaggcat 2760

gcgtgacgaa gtggtggcta agttcagggc gtatttggct gaagccatag ccaacaacga 2820

cgaggccatc tgcgacgaac tgaacagaat cgtagggctg ttgcaacagg ggcacacagt 2880

gcatctggag tgctgctgca agccgaaagc ttgtcacgct gatgtgatcg tagaagttat 2940

caacgacgca gctaagtaat caatcgaacc aaggggcgaa attatgctgg tgcgaatcaa 3000

tgaaggtaac ggcaagttcc gtgtcgtatg gatggcacag aaggatgtgg cacgcattct 3060

ggagaatcag aagtgagcga actcacgctg ttcgaacggg agtatccaat ggtagcaacg 3120

gtagatgatg cattggccgt tgctatcgaa tgggatgagc gagccgcagt atgtaaggag 3180

aaacatctga tggccgctct gggacgtgca gtggcatggc gagaattggc attgagttat 3240

gccagctatc tggagtttaa gcaatgagag cattccttcg tagccaagac aagatgtctg 3300

gccattggcc caatcgcacc tacttcaacg taggactggg tagtccgatc gacgtggagc 3360

gtcatgacta tcctgagtgg cttcaggagc aatgtaggaa cagtgttcct gctgtggtgg 3420

agtatctgga cagcattgca caagcagtgc tggctggtgg tgttatggca gtcgtgcttg 3480

aatacgagac catgcactca cgttccaatg ccatctggct tgtggagttc atcaacgaca 3540

tcatcagtgc agccgaggag caagtatgag ctatcgcctt agagtgaagt tagaggtaat 3600

caacgaggat ggtgatgtgg ttgaccatcg tggactcgac cgattccaca aacgctttca 3660

agcaccgggt atccagaccc aacaggatct gttccgtacg gacgatctgg atctggcacg 3720

tgaacatctc aacaagatgt acgccatcat cggcaacatc aaggagcttc tgaaatgaaa 3780

ggctggaaga acgatggtag ctgtgtgttc gtcttcggat cgaacgaggg tggtaagcat 3840

ggtgcaggtg cagctcgtac tgcatacaac aagtacggtg ctcgctggaa ctttagctat 3900

ggtcacatgg gcaactcgtt tgccattccg accaaggact tcaacatcag caagacgctg 3960

agccttcgtc aaatccataa ctacgtgacg ggtttcttgg catatgccga gggtcatcca 4020

gatctcacgt tttacgtgac gcgtattggc tgtggtctgg ccggtctgga agataaggat 4080

atcgctcaga tgttccactt cgcacccgct aattgcctct tcgatgagga atggattccg 4140

tatctcggaa ccaatggtcg cgaatactgg gggacgttcg aatgaatgta tccattcaag 4200

acatcaaaga agcaatggca tggtggaatg ctctcagccc tgctgagatg ctgaaacacc 4260

gtaagaaaat tgaacgcaac gatgcaatct ccattgctcg ttactggctt aaggaaatca 4320

aatgacagca gtatttgtgg aagaagacca agtggttaat gagatctatg cccttcgtac 4380

gggcatcggt cgcaagatca tggaaggtga cgagcaagct tgccgtaacc gagccaagga 4440

aatccaagcc acggctggca tgaagccgat gctcaagctg gtcaagatcg tgaccatgga 4500

acaagaactg gaaactctct aaggagaatc acatgacgac ccataaagac gcgactgtag 4560

agcagtggga acatcaggaa ctcatcgaga agactttcaa caagaaccag acctactcac 4620

gcattcgtgc tgagttcgag gacgctggtg aagagttgaa gcaacatatg atcgacaacg 4680

gtattgaacc ggatttcggt tacgacctgc tcgttcaaat ggctctccac aagcgtgctg 4740

atgtagctac gctggttggt actctgcgtc atcacttcgc tgaggcacag gatgtgctca 4800

acatggcgat caagtgtgca gaggctgatc tcgtagagtt tgtggtggat ttcaaccagt 4860

tcgtcgtaac actggacatc tctgaagatg tgcaggaaga actggatcgc ttccagtatc 4920

cgctgccttg catcatccag cctcgtaccg tcaattccaa tacggacaat ggctatcact 4980

tcgtccgtaa gggcagtctg atactcaagc agaaccatca caacgatgat atctgtctgg 5040

atcacatcaa ccgtgtcaat gctgttcgat tccgtctgga tcacgacacg gccaagatgg 5100

tgaagaacca atggagcaat ctcgacaagc cgaaagaagg tgaggagaag gaagacttcg 5160

agaaacgtgt acgtgcattc gagaagtatg accgtaccgc aaaggatgtg atggacttca 5220

tccaagagct tggcgatgag ttctatctca cgcacaagta tgacaaacgt ggtcggatct 5280

actgccaagg ctatcacgtg tcctatcaag gggcaccgtg gaacaaggca gtgatcgagt 5340

tcgcagaagg agaaataccg acatgatttc gacccgtgca cagaccctgc ttaataagta 5400

ttcggaggct gcgagcatcg tttacttcct agaggatcaa ggaactaacg atgctgaagt 5460

aaccaaagcg aaaaacgact tgcgtcaagc tgagcaagat ctcaagaaat acattgctat 5520

attggaggcc aaatgagcaa ggcaaagcct tacgatgagt atgcagtcga agggctgtat 5580

accatcaaga gccacataaa aggcaacacg acaaaggagt ggcgtgtgtg ggtcggaaat 5640

atcaagacct acgaacaggc tctggcaacg ctcagaaatg gacgtaaccg agtcttcgaa 5700

tcgagctttc aactccgtat cgtcgggtac atcaaagaga tcatcgaaga tgagtcctga 5760

caccagtggt cagtgggtgc tacaacgtcc tgacaccaag gagtattgga atacgggaca 5820

caagtggtat ctcaaccaag ctgcaatcct cagtgggagt caaatggaag acttgatcgg 5880

ctccttcccc tttctggtaa acgcatacca catcacagga acctgaatca tgcctatctg 5940

gctcctagta gcaatctccg tatttctgag tgcgtggatc gtctgtatgt atttcagcct 6000

caaagatgac acggctcgcg aacgtcgtgc agccatgctt gggcgtaagt acgtaattac 6060

gtattcaccg ctcgggaagt tcttcctcat cggctggctt ccgctcacca tcttctggct 6120

gagctacctg attttcggct acggcaagta aactaaggag caactaatgc ggaaactgtc 6180

cgtagttggc aacgtatacg ggcagttaac cgtagttgct gaagcagagc cacgtatgtc 6240

cggtaaccgg aaaacaccat acgtgctctg tagctgtacg tgtgggacat ccattgaagt 6300

tcttacatgg tcactgacca agggtttgac cgagtcatgt ggatgtctca gacgtgaagt 6360

tacaggagat cgtgcgagaa cacatggtga gtcgcacaca cggctgtacc acatctggaa 6420

gcacatggta cagcgaacca cgaatccgaa caatgagaag tgggaatact acggtgaacg 6480

tgggatatcc atctgtaagg agtggcgaga ctcttatgag gcattcgcca aatggtcacg 6540

cagtaacggc tacgcagatc acctcaccat cgaacgcgat gatgtgaatg gcgattacac 6600

ccccttaaat tgcagttggg ccactcgtaa agagcaggcc aacaacagac gcccaaggag 6660

aaagaaatca tgatgcaatt caccggcacg cagtatctga agatcgacgt agcgaataac 6720

atgggtcacg acaaatggac atggaatgag cgaatcgaat gggtcgatgc acatgaccac 6780

gagttggaac aactgctgga tcaagcagac gtaccggctc tgtactacgc atccgtaaag 6840

gctctgcgtg cagcacaacg tggtgaagta tcgaacttcc ccatctcact ggatgctacg 6900

gcatcgggca tgcagattct gtctgcactc accggtgacc gtaacggtgc agcactctgc 6960

aatgtggtca accgcattgt ggatggtgag gtcaagcgtg ctgacggcta taccatcgtc 7020

ttcgaagaga tgaaggaacg tgtcggttcg tctgccgaga tcaagcgtga catgatcaag 7080

aaggcagtca tgaccgcact gtacggttcg aaggcaatgc cgaagaaggt gttcggtgag 7140

ggccgcctac tggacatctt ctacgagacg atgtccgatc tggcaccggc cgcatgggag 7200

ttgaacgagc acttcttgga gatctggaat ccggagaacc tgaagaactc gtgggttcta 7260

cccgacaact tccatgtgca cgtcaaggtc atgacgaagg tcgaagaaca ggttcatctg 7320

ttcgacgagc cgatcagcac gttccgtcaa gtgaaccaac cgcaggaaga aggtcgctca 7380

ctgggtgcga ataccatcca ctcgatcgat ggcatgatcg ttcgtgagat cactcgcatg 7440

gctgactacg atgtgaacca agtgaagaag ttgtacagtc tcaacactct ggcactcgtg 7500

gatccgatga gtctcaagcc gttaaaggag tcgaaggaga cgaagcttct acgtatcctg 7560

ctgggtcact tccgtgactc cggtttcctg tcggctcgtg tgatcgacat catcaacgaa 7620

gacaacttcg tgttgctgga tgagcacgag cgtgttgccg tcatgaagtt gattgatcaa 7680

ctgccaacga agccgttcaa ggtgatcgct gttcacgact gcttccgctg cctgccgaac 7740

catggcaatg aactccgtcg actgtacaac ctgcaactgc acaagctgta cgcgtcggac 7800

atgctgtcgt tcctgttgag ccagatcatg caaacgaagg tttcgatccc gaaactggac 7860

aatgacatgg ctcaggacat cctcgacgct gagtacgcgt tgtcgtagaa tacggccaac 7920

ttcctgtcaa ggggagttgg ccaacttttt aaatgataag ggatagttat gacgacactt 7980

caagtgtgct acaaaccgga tggtcagaag atcatggtac aagagaatgg cacggcagta 8040

cccggtggtt acaccaagct gggtacgttc gatcacccgg atcctgacga cagtctcagc 8100

cgtaatggtg atagccatgt gtggtatcac cacattcagg agatcatgta ccacaccaag 8160

aactcgggtg ctccgattgc agggttcttc ccggagaaca tcaccaacat gcagatgctg 8220

accatcgaaa cggacatcgt tcgtgcagaa ggtctgacag tggcaccgaa gacagcaaat 8280

gtagctattg ccgccaccca gcaactgacg ccgacgttct tccctgccgg tacgacggat 8340

aagaccgtga cgtacacaac ttccgacgcc acgaaggcaa cggtgaacgc tgctggtctg 8400

gtaacgggtg ttgctgttgg tacggcaacc atcaccgtta cgtccaacga tggtgacttc 8460

aaggacacta ctgtgattac cgttacggca taagtagttc ttctctggtt tttaagtcaa 8520

cgatagcccc tctccggagg ggcttctctt ttataggaga caaagatgac aacccgcgtc 8580

attcatattg ctggaatgga actttcgggg atccactata cagtggcatg cgagttcaat 8640

gcggcagaag ggccaagcat ttcgattcaa gtagacaatc aagaaacaca cggcatgtca 8700

ataactgagg ctaatttact ggctacagcc atcaaaactg ctattagtgt aattaaggaa 8760

gaatgatgaa catgtacgta cacggcgaga acggatccac attcgtaggc ccaatgatct 8820

acagcctctg cgactggcgt gctctggtcc gatcgaaggc aatcgttccc aatgacggca 8880

tgggcttctg gatcatcggt ggcaaggtca gcgagatttc gtgcttcgat cccatgccgg 8940

aaaacgcaac gggtgtcgaa tggcacaaca gatgaaccac aaagggggtg acaaagtgca 9000

caagccatct gtcagcaagg tgcgtatcac actcaccccg tactggcagc agaagttcct 9060

gctcggtgca ctacacgagc ttggtgtgca tgagtcatac agtgtggaca tgatcctgac 9120

catgggtgaa aaggatttcg tccgcttcta tacctctgcc gcaaaagtag ctgatgtcac 9180

accgactcaa ttgaaattcg agtacgtgaa catcaccaat caacccgtac gcattcgcat 9240

acgatctggt ggactcaaca actatgatga agacgcggca tagataaagg agaagcacat 9300

gaaagaactt acattggagc gtctgaaaga actgattaga tacgatccga tcactggtga 9360

gttctttcgt attgcactgt ctccgcgaag taaggccaag ctgggcttag tgaccttgta 9420

tgccaaggac tctggacatc ttcggatgtc tattgatggt ggtgagtatt acgcagccag 9480

agttgcacac tactacatga caggtgattg gggagagatt gtggatcatc gcgacggcga 9540

ccccaacaat aatcgctggt taaacctccg taacacaaca ccactaggca atgtacagaa 9600

tcaagtacgt gcacaccacc ataacaagtc aggcttgctt ggcgcacatt ggcgtgaaga 9660

taaacaaaag tacgaatcgg ctgtttggca caaatccaaa cgttaccgcc tcggttattt 9720

cgataccgca gaagcagcgc atgaggcata caagcaaaag aagcgagagc tccatgttac 9780

ctgcacaatt tgagccaagg aggctgagta atggcgcaag gtgagacttt tctcccgttt 9840

gtgattatgt aatcacgacg caatcaaaca cccatgagcc tgactcattg gctcatctgg 9900

caatgtgctg cacgaacaca tagcgatgca atcgaagcct tcgaagaaga aaatccggac 9960

tgcgagttca ttgaagtagt gcagacggct gacttcaata aagccattca agaagcggag 10020

gctacatacc gtggctaagg aatacgtcaa ggactctgag ggtgaaatga tcgagaagat 10080

caagcctgag accaagaagt acgaagagtc ttaccgcaag ttcatggagt actacgtacc 10140

tgcacacaag gattgcaatc attgtggtgc tgtgcgtcac aaaaactacc ggtgtgtccg 10200

gtgtggatac gaatgatgac cactctccag cttggtcagc gagggatcct cgctaacaag 10260

aatggcacgc tgatgtgtgc caccgttctg tctcttggct cgaactacga tgcatatcgt 10320

atcgacgacc agaagactcc taaacgtgtt ccgaaaggaa ccaagaccta caagttcttc 10380

gttggtgagt ccgcagtggt cgatgcatcg atttggattc acacgagggg catcaaggaa 10440

ctcgatcatg cctgaagcac gaaagacatc tgttgaactc ctgctggaac tcaatcagat 10500

gacacgcaag tacgacgaca tcaccaagac cggccgtatc aagatcggca ctgtggcgat 10560

ggcacaagag atcgtcatca agtcggcaga gttgttggct gctacccgtc acgagtatca 10620

cgaacaaaac ccggtggact gatgagcaag cgaaacagtg gtcaaaccac acgtctggtg 10680

ctgttctacg tacagcagtg tttggaacat ccgagcgatg aaatcgccgt gtccgatcac 10740

cctgccgaga acgcagtcca tgcgaccatc catcagaatc tgatggtggc gaacaaggtc 10800

agtgcaatcc ttaccgcact gaatgtccaa cactacgtga acagcaacaa ggtttgcgtg 10860

ctgccgatca acaaagacca atgagcgaag tcgctgaaag ccccctctcg ctgtaccaga 10920

acggtacggc tgatcaagtc tacaacgacc tgatccgtgt gctgcgtgcc aaccgtgttg 10980

ccatgatcca tggctcaccg ggtattggta agtcagccat tggccataaa gctgctgaag 11040

agtgcaacct gctgatgctt gaccatcgca tgagcacgtc ccgtccggaa gacctgactg 11100

gtctgccgca gttcaaggac ggctatgcac gctttgcacc cttccgcgaa ctgttcccgc 11160

tggaagatgc agaactgccc gagggcaaag atggctggct gctgttcttg gacgagttca 11220

actcggccca caagagcgta caggctgctg catacaagct gatcctcgac cgtatgatcg 11280

gtcagcacaa actgcatccg aacgttcgca tcgtctgtgc tggcaacttg gcaaccgacc 11340

gtgccatcgt gaacgatctg ggtactgcta tgcagtcccg tctggtgcat ctggagatgc 11400

gtgtcgactt cgagtcgtgg ctgaaaaacg tggccattcc gttcaagtac gacagccgca 11460

tcgttgcgta tctgagccgt tacaacgcaa agctgtacga cttccgtccg gatcacaacg 11520

acaagacgtt ctgctgtccg cgtacgtggg aattcgtcaa cgatctcatc aaggacgaag 11580

cagcactgga catggctaag gatgctcgcc tgctggctgg caccattacg tccggtgtgg 11640

ccaccgactt catcgaattc acgaaagtgt tcgagaacct tccttcgctg gaagaggtcg 11700

aggctgatcc gtgcaacgcg aagatggcaa cggatacggg aaccaagtgg gcaatcaccg 11760

catggctctc ggagaaggct gacgaaaaga acttcggtgc aatcacggag tacatggatc 11820

gcatggacac cacgttcaag atcctgttct accgcatgat cgtgctgcgt caaccgggcc 11880

taaagatgca tcccgcattc agcaaggctc tttcgtccat cggcaagtac ctctggggca 11940

acgtctaatg gccaagttcc tgaacgagtg caagcatggt gatcacacgt acatcagcgt 12000

cttcgatgcg gatgctgtga tcatctatgc tcgttcgatg ccgcaagctc accagattgc 12060

catcgagcat ttcaaaccca agaagcgaga caagaagctc atcgagacca agctgcatat 12120

catgagcaag gacttgatga agcacgtgga acgcttcgca aactacgaag gaaacgcgtg 12180

aaaccgaccc cggtagttga ttccgagttc gaccaatacg aactcactcg tgagctggac 12240

aaaacgaaat cggaagtctt catgtcgggg tcttccgcct tctacgggcc aatcatgtgc 12300

tcgctggagt tcatgtggtc acgtgatatc cagacggctg caacggatgg tgtgcacctg 12360

tactggtcgg acaagttttt catgaagacc aagccggtga ctcgcaagtg caccctgcgt 12420

cacgaactgg agcatgtcgg aagactgcac cgtttgcgtg gcatcggtaa agatcccaga 12480

gactggaaca tcgcgtgcga ttacatcatc aacaacgcga tgaagaagga aggttgggag 12540

ttcgatccgg acgacatcaa ggtgtacatc gacgacgact atgcggactt gtccgaggaa 12600

gaagtctacg agttgattca ctccggaaag atcgagccta agcttccgcc gggggccaat 12660

ctcgatgacg acatgctgga ggaagaagaa ccaactccag aagagatagc cgcaaacatc 12720

tcaatcgttg tgcaggccat ccatgctgca aagatgtctg gcaatccggg agcaattccc 12780

gggtgtgtcg agcaagtaat cgacagattc ctgaatcctg tgattccatg ggaaaaggaa 12840

ctcgccaagt tcatgacgga gcttcaggac ttctactaca cgtgggcacg tccggaacgt 12900

cgttacatga gccaagacat ctatcttccg agtatggagg atgacgaagg agcattggag 12960

catctgtgct actaccttga tgtgtctggt tccatcacag acaaggacgt gattcgcttc 13020

aacagcgagg tcaagtacat caaggaacag cttcgaccca agaagttgac actggtgcta 13080

ttcgacacaa tcattcaaca ggaaattgtc ttcaatgagg atgacccctt tgaccgtgtg 13140

gttgtggtgg gccggggtgg tacatgtttg gcacccgtac gcgagcacat cgagaagaac 13200

aaaccaaccg ccgctgtcat tttttccgac cttgaggtca cgcccatggt taaaccttcc 13260

cgcaacatcc ctatactttg ggtgcggatc ggtaacactg ggcacaagcc ttccttcggg 13320

aagatcataa ccatccggag ttaataattg tcggcacaac tggagaaaca gtgccggaag 13380

tgccttcaat catttccggc aacaaccgaa ttcttttaca ggaattccgg tggcaaatac 13440

ggactcacgc caagatgtaa gtggtgtgtg aacatggata acatggaaag ccacgttcgc 13500

cgtttggccg ctgatcctgt aaagatcaag gcgcaagctg ctgcaagagc cgcaaaaagt 13560

taccacaagc acttggagaa gaatcgagcc aagggaaggg aacacgctgc cgctgccaga 13620

gccgatcctg agaaacgtgc aaagatctac gcacgtaagc gtgcaggcgg ggctgggctt 13680

acagtagaag agattgagca gatcaagcag aagcaaggca acatgtgtgc tatctgcaca 13740

agccctaatc ccacagactt ggatcactgc cacaagaccg gtgctgtacg attcctgctc 13800

tgccagcact gcaaccgtgg tttgggggca ttcaaggaca accctactct gctgcgaaaa 13860

gcagcggatc tcatggaaga gattcatgat agtcaatcaa agctcactgt tgaaagcagc 13920

cccgatcaag ggcatggttc aggagaaacg taagcacttg ggcttctcgt ggggtctgac 13980

ggaatgcgga tacgacatcc gtatcaagca atctgtaact ctttttctgg gtcgtcgttt 14040

tcggcttgca tcctctatcg aggaattcga tatgccacat cacttaatgg gccgtgtgct 14100

gaacaaaagc acatgggcac ggaaaggtgt ggatgccagc atgacaacta acgtcgaacc 14160

ggggtggaaa ggatttttga ccatcgaact gcactacaag ggatggaagc cacttaccat 14220

ccctgccggt gtgggtatcg cgcaagtgat cttcgagacc atcgaagttc ctgtcgtcta 14280

cgatggcaag taccagaacc aagaggatcg acccgtagag gctcgtgggt agtagacacg 14340

cagcctttgt ttggggtaca gtaaacacgt accaacaacg gagaccatgt aatgctgcaa 14400

gtgcttctga tcctgaagct cttgttcggc tgtgaagcaa agaccctgag tatccagtct 14460

gtccgctggt ttgcgtcaag agctactcag gataaggagg gcagaagccc tcacgtaacc 14520

gagattaatc aactagaata cggttagtaa cgcttcacat acggacatag agaacatagg 14580

atgatagggt gatgaccaac catcaaacat gtggagtgct cttgcgaaag cgagacccca 14640

cccgagtcca accgcaaatc gggggattcg gaagaccgag agacactttg tcgttgctgt 14700

ccccaacagc cggtctttca aatcccctct acccaacagg cccatgctag tacagatggg 14760

ccttttctat ttctgagcca tgattactca agacgaagtg aaacagatag cggaagccct 14820

gaccgttgct gagggtcagg ttctgctgga ggtacgtgat agccaagggt ggactgtgaa 14880

atcccttggt gagattgcag aagacacctg catgtccatc aataccgttc gacgtacgat 14940

taaatctcta cgtgagaaaa atctcgtcga actcatccgt ctgacgcaag acgaatacga 15000

ccataccttg tgtggctctg ggtatgcacg aacctacgcg ggtgaccgtg ttgctacgga 15060

gatcagccag tcatgatcag caacacttca gggatgttgt tacttcgttg gatgggtaaa 15120

gaaggtggtt atgagcttca tgaccatgcc taccatttca catcagacga cgttgacatg 15180

tactacgacg ccatttatga cgccgtagtg gaacacctca atagcgtaaa gcctgtactg 15240

gccatcgacg atgtgcttcg tatcgtgttc atcggagtgt actcactagt tggagatggt 15300

gaggatgtgg atgagttctt tgatatcgaa gcaattcact acagaaagat ggacatggac 15360

gaactcaatc gcttctacga gcaactggaa atcgacgagg acttacgtga acaagaaaga 15420

gcagaagaag atgcgtcctg actatgtcat cctattcatt gatgacttgg aacctgctga 15480

cgtaaagcaa gcaatcgacg aatgtcagca aggagcactg ctgggtgcat tcgatatcgc 15540

ggaaacacag gctgatgtgc ataacgcaaa tgaacatgca cgtctcactc aacgaaccaa 15600

gaaacctttg ccgtactatc acggcaataa acggagattt tgatggaaca cgaaattttc 15660

accgcagtaa tggttggtat ggctctgtgc gtgtctggat tctgtaagca ctccacacgc 15720

gtgttcgtgg gcttcctgat cattggtgta gcaggtatgg cctacagcac caagagccat 15780

gcagcagact ggggtaatgg tctcaattgc gattcgatcg ctaacctcag ccgtgctgtc 15840

atgtaccaga aggagcaggg caacgaccgg tacacgctga agcagatgac gtactacgca 15900

gcagttggtg caggtgtgac caacctcaag ctgcttaccg atgccatcgt cgactcggca 15960

ttcaagctgg agggcatgac tccgaatctg ttctcggcaa tcatgtacaa cgaatgcgtt 16020

cgtgccaatc aggacgtacg taagcatggg gaaggcatgc tgtaatgcgt aagggaacca 16080

agcgaaagat acgtgttgcc agcaccccga tagtctttcg tgatcgtgct aatgaggcag 16140

aactgatgct tattccgcat ctatgccttg agagcttcac gaagggactc ggcaatgagc 16200

aagcattcga tacgcttaca tatcgaatgc taatggcgac agcactgacc aagcacatcg 16260

aggttgagga agacttcgat tacgcagcag gggtaattgg tgcagcagta caagccatga 16320

agaacatcgg tgaccgatac cttcgcacag gtaaatgggg tatgagtggt ggcgacgtga 16380

gccgtatcaa ggacggttta ggtatcgctg accaacttca ggaactggtg ccgattaaac 16440

agcagtctct cgcttcgcag aaggttggca agcatgtcat tggctcgata cgtgagactg 16500

tccacaacct cgaaagactt ctggagaaga aatgaatcag cacactggcg agcgagcata 16560

cctcgcagca ttggagcaca tcatgatcca tggttcgcat tacatggatc gcaccaaggt 16620

gggccgcaag tccatcaaag ggatgatgat gcgtttcaac atggccgatg gcttcccgat 16680

catgaccacg aaaaaggtga gcttccatgc tatcaagcac gagcttatct ggttcctgaa 16740

tggcgagacc aatatcggtt acctgaagaa gcatggtgtg aagatctggg atgaatgggc 16800

tgatggtcat ggtgcactcg ggccgattta cggtgcacag tggcgtacgt ggccgaagca 16860

agtcgacggg cacacggagt acatcgacca agtcggtgaa ctcatgcgtt cactgcgaca 16920

caatccgttc tcatcccgca acctcgtgtc ggcatggaac gtagctgaac tcgaagacat 16980

gaagcttccg ccgtgtcact tcgcatggca ggtgcatgct cgccaagaag tcgacggtat 17040

ctatctggat ctccagttgt accaacgatc ggctgacatg ttcctcggtg ttccgttcaa 17100

catcgccagc tactcgttgc tgctgcacat gatctgtcat cagctcaact accttccggg 17160

tgagttcatc tggactggtg gtgactgtca catctatgac aaccatcacg aggctgtgga 17220

cgagcaactg caacgtacgc cgggagcatt cccttcgctg ctgatcaacc gtgctgcacc 17280

aaacatcttc agctatgacg ctgacgattt cgaactgctg gggtatctgc cccagcctgc 17340

catcaaggca cctgtagccg tgtaaggcta cgcacccttt cgagggtgct tatacgtgga 17400

gacgggtgcg acccaaagtg gaatccgctc gcaccggagg atatggaggg ccgtctccca 17460

gtataagcat cactcccaac caactgaacc aaggaaaatc acatggaagt caataggccg 17520

gaaacactga tcacccacgc tgttctcggt ggtggtcaat cgcaggaatt tggtatttcg 17580

aactcggcag agttcttcca aatcctttcg tccacgctgt acaccgatca gaaaggtgct 17640

gttgctcgcg aagtgctctg caatgcatgg gatgcacata tcgctgctgg caagactgac 17700

atgccgatcg agcttacgct caatcgcgaa gaattcgtca ttcgtgattt cggctttggc 17760

atcgctgatg ccgatatcgg gccgatctac atggtgtatg gcaactccac caagaagaac 17820

gacgggaagc agaccggtgg tttcggtctc ggctgtaagg ccccgttcgc ctacaccgac 17880

cacttcaagg tgacgagcat caacaacggt atcaagtcca tctacaccat gtcgaagtcg 17940

gctggtacgg tgggcggcaa gccgggttgc actcgaatca tgcaggctcc gactgacgaa 18000

ccgtctggtc tggaagttcg cattcccatc aactcaacgg gtgacgtggc tgagttcaag 18060

cacaacatct tcaagttcgt gcgtaacggt gagatgctgg ctcgtctcaa cggcaatccg 18120

gatacgatgg aaacgctccc gttctcgacg atgaagcatg gcttcctcat cacggatgtg 18180

gacatcggtt tcaatgaccc gatccaagta cgctacggca acgtgatcta tccgattccg 18240

gatcatgaga gctatgcccc tcagtacaag cagattcgtg cactgctgaa gaagttcggg 18300

aagcgtgatt actacaatag cggcatcttc cgaatcatcc tgcaagcacc cccgcactcg 18360

atcgcagtca ccccatctcg ggaagctctg tcgatgcagg agcacaccgt gaactcgctg 18420

aagagtctgc tgaacaactt cgtgtccacc atcaacgtgg atatgaagga gtacgtgtac 18480

aagacgatgc gtgagtcgat cgccagtgcg gtgtccaagg cttcctacaa ggaactgctg 18540

tcggatgacc agaagatccc cggcctgaat gcagacacgc tggcacgcaa caagtatgtg 18600

gtcgatacgg aatcgatctc caagcacgtc gtgtatcaca actacccgga ctatgatggc 18660

ttccgtaagg aagaggtcaa actccgtgtg atctcgatga ttaaggctaa ccaagtccac 18720

cgtggcttgg ctcaaacgtg gctggatgcc tacgatgccg agccggagaa cgtacgttcg 18780

ggtggttggt acaacaactc gcatagggct gatccaaagt cgtggttcca gcgtaatgtc 18840

gttggcctgt tccgtcgcaa gatggtagca gagccggaac tcgacatgca acgacttctg 18900

acgtatggac atgggaacca cgaggctgca cgctatggca atacgcatgg tgtcaccatc 18960

gcccacatgc agtccaagac tccgttggaa agcctgttgc cgtatctacg caacatggta 19020

atcctgtcgt tctcgcgact ggaaattggc cctcgtctca agagctatga attcccggca 19080

gggaaggatt gggagccgtc gtgctgtctt atgtacgtgg ttccgcgtag ctcgcagaag 19140

aagatcgagg ctgctcgcaa cttcttcacc aacatgggct gctacatcgt tgacctgacc 19200

attcgtcatt catgggaaga tcagtcagtg ctggccgtgg agaagaagac tgttgttaag 19260

aagccagtga agaagggtct gccgattctc gcagacggtt acgattcagg tggctatctg 19320

gcaatgcagc ttctgcgtga caaggaagac gctcgtcgta ccgaatcacc ggaatggatc 19380

gtgcgactcc ctaccatgtc cagtagtggc cgtattagca aggggtgcta cgatcaatcg 19440

ctcggcatgt ttgacgaaac ccaatcgaag ctcatcgtga agctgtgggg cgacgttggt 19500

ggtggttgca cttccgagaa gcaaatggaa tcctaccatt ccaaaggggc caaaacagtc 19560

gatgagttcg tgaaggaaaa ggttcttcac tccatgaaga ccaacgttgc aatccggaga 19620

tacatgaagt ttgctctgtg gcgtgtcttt ggttcagagg aatctcgtcg ctatgacaat 19680

aagcaacaag tcatggctga gtttgccaag atcgtctaca acacgcccga actcgccaag 19740

gagtttggca ttgtggacac ccgaaccaag gaagagaagt ggatggtctc gctgtaccgt 19800

cacctcgtct ccaatctgta ttggatcgaa aagaagttcc cggagatcaa gactgtgatg 19860

gatagtatca acaccatctc tcttgatgat ggggcgctaa agctgaagaa aaccctgtcg 19920

gaaggctcca ctctggaatt cctcagtgca gaacagatca gcgaagcaat caagcagaaa 19980

gtagacacgg cacgtcgtga tcaggcaatt gccttcatga aactcgcact caaactttaa 20040

ggagcagtac caacatgact cacaaaatcg tagttgtgtc ggctgttgtc gacacgttcc 20100

ggatgacgct ctacacccag acgggtgcaa cgcatgtcat caaccaaggc gacccgaatc 20160

tggagaagat tctagccatc atcaccccga aactgtcggt taacgaaccg gtggaactcg 20220

acctgacggc tctcaatgcc gagaagccga tcaacgagta ttcggagatg gagaagaaga 20280

cgaatggttt cgtccgattc ttcaaggtgg ccaaggagaa gatcagttcg atctttggct 20340

cggaagagaa gcagaagaca cccgaggtaa agcatgtcgc aagccagact cttggcaagg 20400

ccagcgaaga catcaagccg accattgcgg ctacgccaac ggttgtggat gctaagactg 20460

aggctgtcgc gaagaaccaa gcggcagtcg ctgaagttct gaagaacgca accccagtga 20520

aggacgatcc ggagccggtc aaggcaacgg agacgctggt cgctgtggtc aaggactcga 20580

agggcaatga tcaggtgatc gccggtgtgg aagcactgtc gaaccaactg aagcactcga 20640

tccgtggctc ggagatcggt atccagaact tcctgaagcg tctggccgaa gtcatcagcc 20700

agcgtcgcca ctcggtcgac gacctgatga agttcatgga gcgtggtgat ctgcctgtca 20760

gcgatgctgg tgacatcatc atctacaaga tcctgaacaa atgtaagacg aagctcgaag 20820

gtatcgagtt cgactacgtc gatgtccatt cgtcacgtgt cgtgcagcgt gtcggaacgt 20880

acatccacat ggatcattcg ctggtcgacc acgaccgtcg taacgagtgt tcgaacggcc 20940

tccacgtggc tcgtcgtcag tatctcggtg gctttagtgg caacgtaatc gtgctggctc 21000

gtgttcgccc ggaagatgtc atcgccgtac cacaatacga tgccaacaag atgcgtgtct 21060

gtggttacca catcctgttc gagatgctgg aagaagaccg tcgtgcactg atcagcaaca 21120

agccgctggt caccgaagac ggcaaggcca agctcggtaa ggcactggcc ggtgatcacg 21180

tgggcattct cgaacacgtg aagatcggtg gagccaaggg caccaatctc accgtcacca 21240

agaaggtgaa gggtgagcgt gtggccgacg aactccagtc gaacgtggtg caggcaggtg 21300

atcgtgtgga atcggctatg caagtcgatg acgacgacaa gctccaagct cctgctgtgg 21360

atccgaagac catcgcacag gtggcagacg cagccaaggt agccgtggca gcagaagccc 21420

cgctgtcgcg tgcagaacag gctcagaagc tttggaacac ggtcaacagc actgccacca 21480

tgggcgttcg tgcaacggct ctggagacgc ttatcgagct gaaaaagaag gcgaaggtgt 21540

cgtggaactc gctgggtctg aaagaccacg agatcaacga gcagtcgagc aagctcctga 21600

agcaaaacaa agtggccgaa gcaaaggtcg ctcccgcaac cgcagttaaa gaagcgaagg 21660

aaggaaaagt ggcatctacg caagcatcgg gtactccccg tgaacgtatc gcagcactgc 21720

tggccatgcc gggtgcgatc gatcgtctcc gtgccaagca gatctatggc atcaagcaag 21780

acgccaagaa gggctggtcg gccctcggtg tgacggatag cagcatcatc aagaccatcg 21840

agaagaaggc gaaggactga tccatggggg caaagaagaa catgcccccg gacgatcatc 21900

gcctcacatt cggcaagtac agggggcaaa ccatcggata cgttcgatcg gttaaccccc 21960

tgtaccttga gtgggcgagc gagaacatcg ccgggttcaa agacggtcag catggcacca 22020

gaccatcgga gcttgctcac cgtcgcatca ttgccaaggt gatcacacac tctggcaagg 22080

ttgagtggta cacggagtga taatatagag tcttctttaa ggaggctcaa atgtctactg 22140

tgtatcgccc taatcgaaag gcgacagacc atgacattat tcgcatgaat agtgtcggtc 22200

tgtcccttca aaccatcgcc cgtatcatcg ggtgtcaccc caccaccgtg accctgcgtc 22260

tcaagagtct ccagattcca cctgcagaca cacgacgcac gttcatggag gacattttca 22320

agtcactcac ccctgatcat caggagtggc tggccgatcg gcttggcccg actgaatcga 22380

tcaaatggta cgtgcgtgac ttgctcgtag aggaatacaa gcagcatggt cacaatcaaa 22440

caagtaatcg actggttcgc caaggcacgt cccaaccgaa ccaagcagca gttcctgtcg 22500

cagacgggag tgcacttcga ggagatctcg gagatgctgg agcagatggc tcctgaagat 22560

agccatgcag cacggctgat tcatgacgcc tacgtggcga acaaggcact cggccaagcc 22620

ctcaaggcag gtgagtgcag tttccgagtc aagaacgatg tcctgttctt ggacgccctg 22680

attgaccaga tcgttacggc gactggcacc ggctacggca agggatacaa catggatggt 22740

gcactcgaag aagtgaacgc atccaactgg tcgaagttcg acggggagac cccgatcttc 22800

caagacaacg acccggacaa gaagatcctg aaaggcccga actacttcaa ggccgatctc 22860

agcagtttca tctaagcccc gaaaggggct tttcttattg gagccaagga aatatgacga 22920

ccctgaacgc agggcaacaa gccgcagcag atgggatctt cgatttcctg ctcggtaatg 22980

agaaggaact cgtgatcagt ggccccgggg gtacgggtaa gacccacctc atgggccaca 23040

tcatcgacga agtcctgccg atgtaccaca agacgtgttc gatgatgaac atgcccccgt 23100

tgtaccgtga tgtgtacatg acagccacga cgaacaaggc agcagacgtt ctcggcagca 23160

gcactggtcg acccacgtcg actatctaca gcctgatgaa cctgaaggtc gaacagaacc 23220

tcaacaatgg ttcgcagcgt gtcaccaaga cgaagtcttg gatggtgcac aggaacaaaa 23280

tcattttcgt cgacgaggct tcgtttgagg attcggtgct acgcaactac atacttgaag 23340

gtacgaacaa ctgcaagatt atccatgttg gagaccattg ccagttggca ccgatcatgg 23400

agcagaagcc ccctgtattc tcagcaggac tccccttcta cgaactcacc gagcaaatgc 23460

gaaccagcga tgccaatctg caaggattga atcagcagct tcgccgtacc gttgaaacgg 23520

gtgagtttct ccccatccaa cacgtgccgg gagtcatcga ctgggtgacg gacgaggatg 23580

agatgcaggc tctggtggag gaacactttg tggatagtca ccacaactcc cgactgttgg 23640

catacacgaa cgatcgagtg aacctgtaca acgggttcat ccgtgacttc cgtcagttgc 23700

ccacatgtcc ggtggagggt gacatcctcg tgtgcaactc ggcaatccag ttgaataccg 23760

tgcagaagtc gatccccacg gaaacgcagt tggagatcga gaagatccat tcgcagtaca 23820

aggtgacgat cagtccggat gtggagttgg atgtgttgct ggtgaacgct cgttcgcagt 23880

atgcactcta cgagaatctg gaaatcccag tcgaccgtga gcacttcaac gccctcacca 23940

agtggtatgc aggtgagaag aactgggaac gctacttcta tctgaagaac acgttccctg 24000

atctccgtcc ggccgatgcg tgcacaacgc acaaggctca gggcagtacg ttcgataccg 24060

tgttcataga catgggcgac atgagcaact gtcgcaatcc ggacacggca gcacgcttgt 24120

tgtacgtggc cgcatctcgt gcacgcaaac gcgtgatcat gtttgggcat ctggctcaga 24180

aattcggagg catcatttaa gaggcagcta tggcagagac ctattcattc gggaaggcag 24240

caggagctat tcgtgatgcc ctcatggaca agctcagaga gccagaggaa agacgctggc 24300

agctccagat agacaagctg gttgatgcca actttgcttg tgatccaagt aacagcatga 24360

aggggttcat gtactccggg caacgctata ttcatagcca agctcgaacc aagtacaaga 24420

gctacccgat gctgtggcac acgctctggg gtgagatgga tgcgatactg ttggatcaat 24480

ccatgcatga cctgaaattt tctcaggttg gtcagatgat ctatcgactg ctttacgagt 24540

gcaacgatcg aatcgaagct cgtaataacc ttccggagtg tctagttcag ttgctgaacg 24600

aagatctgag aaacacgcct agaaagtttg tggaggggta catactccgt gaaggctgtg 24660

attggcgtgc acagaaacag tttgaagagt tgcttcccac catggaggca atggttgtcg 24720

ctcgcatgat catgtgagcc aaggaggata aatgcgtcac ctcgtgttcg aacagaacga 24780

tcagtacccg gtgtgcctct tggttcaaca gatcaatgag gacgccatcc gaaaagagta 24840

cttggatccg catccggaca tcagccctgc tgatgttctc gtactcgatc tgcacactta 24900

tccggggaag aagaaagtac ccaaggctga gatcgtgcag tacatcacgg aaatgctcgt 24960

gcctgtgtgg caagagcaga agtgccagta catcatgtgt tccgatccgg agtacttcaa 25020

ggcactaacg ggggaggcaa aagcggatgc caacctcggg tacgctctcc cgtgcgtata 25080

catggaagat gtaaaagtgg tctacatccc gaactacaag gcagtcttct acgatcctga 25140

aaaggttcgc agcaagatca ccattgcagt cgagacgttg atcgcatcag caagggggca 25200

gtatcaagcc cccggtgtcg ggattatcca gcaggcccac tacccgaaga ctctgggaga 25260

gattcaggag tggctccgga aactgttgga catgaatgtt ccgcttaccg tagacattga 25320

ggcatttagc cttaaacact acgatgcggg catcggcacg atcaccttct gttggagcaa 25380

gtctgaaggc atcgccttcc ccgtggatta tgtacctatc cacggtgctg cttcagcacc 25440

ctatggcaga caagtgcgca acgaagagat tcgctcgctt ctgcgttggt tcttcgaaga 25500

gtgcagccaa cgcatgatct accataacat cgcgtacgac gtgtatgccc tcatcttcca 25560

gctttacatg gacgatatca ctgacaacgt gggactgctt cgcgggttgt ccgtcatgtt 25620

gaagaactgg gattgcacga agttgatcac ctatctggcg accaacagtt gcagtggaaa 25680

taagctcggg ttgaaagacc agagccaaga gtacacgggc aactatgcgc aagaggacat 25740

acacgacatc acgctgatcc ctctggatca gcttctccag tacaacctca ttgatggact 25800

ggcaacatgg tttgtctacg agaaacactg gggtacgctc gtcgctgacg atcaggagga 25860

tgtatatctg aatgtattta agcccgcgac caccgacatc atccaaatgc agttgaccgg 25920

catgccgatc aatatgccac gggtgcttga ggtcgaagtg gctttgacgg cagtgatgaa 25980

tgatgcactc gctcgtatga accaaaccga ggcaatgcag aagtttgcat accggttgaa 26040

ggaaaagtac atcgagaaga agcacaagga gtggaaggtg aagcggatca ctctggcaga 26100

ggtaccggac gatgtggtct tcaatcctcg ttccccgctc caagtccaag acttgcttta 26160

cggcatgtta ggactgcccg taatctcgtt cacggatagt aagcaaccgt ccgtagacgc 26220

cgacacaatc gggaagctca agaaccatac gaaagacgct gacacgttag cgttgttgga 26280

tgcactcttg gacttcgctg ctgtggacaa gattgtcggg agcttcatcc cagcgatgaa 26340

gaatgcccag cagggggcag atggatggtg gtacttgttt ggtaacttca accttggcgg 26400

cactctgtcc ggccggttgt cgagtagtaa tccgaaccta caaaaccttc catcgaatgt 26460

tgccatggca atcagtgatg cattgctggc catgtttgac atcctgaagc agtacacgaa 26520

gaagggcaag ctgagtctag gcaagctcat caagtattgc ttccaaccac cacccggttg 26580

gctgtttgtc gggttggact tcgcgtcact ggaggatcgc ataagtgcgt tgacaaccaa 26640

ggatccgatg aagctgaagg tgtatatcga cggcttcgac ggccattgtc ttcgtgctta 26700

tgcgtacttc ggggatcaga tgccagacat cgatccgaat agcgtggaga gcatcaacag 26760

tatcgcagag aagtacaagg tgctgagaca agacagtaag gcaccaacgt tcgcactgac 26820

ctatcaaggt acgtggagca ccctagtcaa gaactgtggc ttctccccgg aagtcgctaa 26880

agccattgag gctcgttacc acgcactgta cgaagtatcg gatcagtggg tgcagagcaa 26940

gctggatgtg gcagcgaaag tcgggtacgt cacagcagcc tttggacttc gtgttcgtac 27000

gccactgcta aagcaagtca ttcgtggaac cagtaagact ccacatgagg cagaagcaga 27060

aggtagatca gcaggcaatg cgttgggcca gagctggtgt ctattgaact cccgtgctgg 27120

ctcggagttc atggggaagg tacgtgagtc cgaattcagg aatgcaatcc gaccatgtgc 27180

acagatccat gatgctcagt atttcatcat caaggacgac attgctccgg tgtcgttcac 27240

gaacaagcat ctcgttgatg cagtgttctg gcaggagcat ccggatatcc aacacgacac 27300

cgtaaaactc ggtggcgagg tatccatttt ccaccctgat tggagtgaag agatcgtgat 27360

cccgaactac cttcagggcc aagagatttt cgatttcatt gacaaggaag cagcatgagc 27420

aacaaggcaa agttctacca cctgatcgca acgaaggttc tgttcgtgat cgagggccaa 27480

gaagctgcac agtcggccga cctgaacgcc atcctgtaca cggaacagaa cttcgtgaat 27540

gcgaagatgc tggcacgtgc tcagaagaac gtccagatcc aactgcacca agtgatcggt 27600

gaggacgtga agtacaacgt cgcgaacgtg atcatcaccg gcgtgtcgag cctcggctac 27660

atgacgccgg aagagttcgc aggtgtggac gaagaaggtg atggtgctgt ggccaaggtg 27720

ttcgcatgag taccgggccg aaagaaactc actcagtagt cagccctacg acgtacaaag 27780

acatcgctac tggtgagtgg ttcatggaca atcaagacga ggtctacgtg aagttggaca 27840

atggcactgc ccgatggatc ggacagtatg tcgaaggcat caagccatca tctttccacg 27900

acgacaaggt agtccgtcgt gtagtaggtg tccactacac ggtgaaagca tgagcaagga 27960

acgtaccggt ggcaaggtga actactacct tgcccaagtc acgaaccccc agcgtgaaga 28020

acaagcaccg tatcaggctg agtgtgagga catcatccaa gcactcagca tgaccttcga 28080

cgaagggtgc gagttcaagg ccatctggcg tactgctgcg gcccgtatgg gtctggagaa 28140

ggacaccgga aaaggtttgg tcgagaacgc cttgtacgac gcacagaagc gcgtgcatta 28200

cgctcaacgt agcgtgaaag agtacaaggc taaactggat gccaaggcaa ctcaaccgac 28260

aggttggatc acttgggaaa gcaaggataa taagtgtccg gtgtttccga gtacaaaggt 28320

ggaagtccga ttccgatcgg gtacttcgat cacttcaatc gcacctgaac agatcaactg 28380

gagtcatcta aacggctccg gtgatgtggt tgcctatcgg gtaatcgact gaaccaaggg 28440

caggaactct gagtgttttc ttggctctta gcctgccctt cattcaatcg attaaggaga 28500

attaagtgat cacactagat gatctcaagg agcgtttgca ctacgatcca gagactggtg 28560

tgttctcgtt tcgtaagaca ggtaaggtag caggtcacct aaacaaaaag agtggatacg 28620

taacgatccg cttggatggg attagctact acgcacaccg tcttgcttgg ttctacatga 28680

agggcgagtg gcccgaacgt gcagaccatg agaacaacaa ccgtagtgac aacaagtgga 28740

caaacattcg tgaagctact caagcccaga acaatcgtaa tgggcgaaag cgtgaagcaa 28800

acaagagtgg ctttaaaggt gtgtcatgga attcccaaag ttccaagtgg aaagcaagca 28860

tcacattcaa tcgcaagcaa gaaacgattg gacagtctga ttgcccaatc gaagcggcac 28920

gtatgtatga cgaacgtgcg atagaactac atggtgagtt tgcaaaaacc aatgtagcac 28980

ttggcttact aaaggagttg ccatgagtat cacaaacgta aacgatgtac cccttgccct 29040

tgcagtttgg gcaatgtttg acgaatacga ttacgtatcc ggtgtcgaga attacatctc 29100

agtgacttcg ttgatgaagc cccttcgaca gatcgtcatt ccgccacgca ttccccaaga 29160

gaagcagggg cgtgctgacg tgatggactt catcagcagt gcactgggta aatccctgca 29220

cgactcggtg gaaaaggcgt ggacgaaagg ctatgcagac aatctgcgag ccttgggtta 29280

cccagattct gtcatcgaga agatcgtagt gaacccggca cacacgccga gtgacgatca 29340

gatccccatc tatctggagc aacgtgttct gaagaagttc atgggcttca ctatcggtgg 29400

caagtacgac ttgatcgctg acggtctgtt gcaagacctg aagtcgacta cggcctacac 29460

gtggttgtat ggtggcaagg acgacgatta ccaactgcaa ggcagtctgt accgctggtt 29520

gaaccaagac aaggtgacag aggacttcat ccgcatcaac ttcatcttca ccgactggca 29580

gaagatgagt gcaaagcaga atcccaagta cccgcagaag cgtgtggagt acaaagacat 29640

tccgctgatg acggtgcagg aaaccgagaa ctgggttcga gccaagctca accagatcgt 29700

catgcacaag aacacgcctg agcctcagtt gcctcgttgt accgatgagg aactgtggcg 29760

ttcggctccg cagtacaagt attacagcaa ggtcgagact gctcaggcag gtggccgagc 29820

aacgaagaac ttcgacacct tggtggaagc caagaccttc tggatggttg agaagggtgg 29880

tgtcggaacc gtcaaaaccg taccgggtac tgtaaaaaga tgtgggtact gccccgcatt 29940

tgaagggtgc actcaacgac tggagtatga gttggaatga ctgtgacact agaggtattg 30000

aaagagtacc ttcactacga cagtgcctct ggtatcttca catgggtcaa agctgcatcc 30060

agacgtgtga aggtggggga tgtagcaggt tctgcgcata gtgaaggata cgtctgtatt 30120

gggctaaaag gcgaatcaca ctatgcccat attctggcat ggtggatgta cacaggagtt 30180

attccaagtg ggcttattga ccacaggaat ggcattaaac atgacaatcg gataggtaat 30240

cttagagaag cgtcttacac cagtaacgcc caaaactgtg gtaagcactc agacaatgag 30300

tgtgggtaca aaggtgttgc taaaaaccgt aaaaagtttt ctgcaaaaat ccatgtaaat 30360

ggcaagaaaa tacaccttgg aagttttgca accccagaag aagctgcaag agcttatgat 30420

gcaagagcat tacaagaatt cggggaattt gcaaaaacca acgcgaatct taaattactc 30480

aggagttagt atgattgatt tgactggtgt gcaacaccat gcagccatcg aagagatggt 30540

caacgttttg tgcagtaaaa ctcagaacac agatcgtggt ttcttccgtg tggaagctgc 30600

ttactttctg ggtaagctgg ctgcgaatat gcgtgcagtg atcgtcacca aggatcgtgg 30660

cgaagtacct gtgaacatct atgccttggc tctggcaact tcaggctttg gcaaaggcca 30720

ttcggtgaac atcgtggaat cggacttcat gtccggcttc aagaagcgat tcatggagga 30780

tagtttcggt gtcatcgctg aaaagcattt gtgggaaatg gctcgtgctc gggctgctcg 30840

caatgtctcc gacgagcaag aagagtacga cacggttgaa tccgagtatc gtcgtgcagg 30900

ggcgtttgcc tacacgttcg acagtggtac tcccccggct gtaaaacagc ttcgtcacaa 30960

gcttcagctt gcgaactgtg gtgcagtaaa cttgcagatc gatgagatcg gctcgaacct 31020

gtttaacagt caggagttgc tgaccctgtt cttggagttg tatgaccaag gtctggtgaa 31080

gcagaagctg acgaagaaca cggccgagaa cacccgaggc gaagagttgg atggcaagac 31140

gccgaccaac ctgctgctgt tcgggactcc gtcgaaactg ctggacggtg gccccaatga 31200

agacatgttc tacagcatgt tggaaaccgg gtatgcacgt cgctgtctgt ttggctgggg 31260

ccaagtggac aagaaggctg gcaacgtcat gtcggcagca gaggtctatg ccaagctgat 31320

cgacaagagc aacgtgtcat cgctccagaa gtggaatgcc aagttccaca agcttgccga 31380

tccggcagta ttcggctgga agatggaagt cgaagacgac gtagccatca agctgatcga 31440

gtacaagatc gcatgcgaac aagcaagtga caagctggca gagcacgaag agatcaagaa 31500

ggcagagatg aaccatcgct acttcaaggc tctcaagctc gctggtgcgt atgcgttcgt 31560

cgacgagagc aacaaggtcg agatggatca ccttctgtca gcaatcctgc tggtggagga 31620

atctggtgtg gcattccaga cgatcctcaa tcgtgagaag acctacgtca agctggcgaa 31680

gtacatcgca gcagtgaaca ccgatgtgac tcacgcagac ctgcacgaag cactgccctt 31740

ctacaagacg ggtaacgcag ctcgaaacga gatgatgcag ctcgcaactg catggggcta 31800

taagaagcac atcatcatca agaagttctt catcgatggg atcgagttct tccgtggaga 31860

gtctctcaaa gagactaacc tcagcgagat gagtctgtcc tatggcgatc actgggcgta 31920

taactatacg cacgaactgg tgccgttcga tcagcttcag acgctctgtc aagcgagcca 31980

agaggacgga tcagcgatgc actgggccaa tcacggtttc aagaacggtc accgtgccga 32040

agagaacgtg gtggccgggt tcaacctgat cgtcatcgat gtggatggtg gggtgtctct 32100

ggacacggtg cacgacttga tgaaggacta caagtttgca acgtacacca ccaaacgtca 32160

cggaattccg gatgatcacg gcattaccca tgatcgtttc cgtctcatca tcccgaccaa 32220

ctacgtgttg aatctggatc atgacgacta caaggagttc atgaacaacg tgatggagtg 32280

gctcccgttc gcctcggacg agagtgccaa tcaacgagcc aagaagtggg aaacatatgc 32340

aggtggtacg ttcttccaga acctcgaagg cgagttgctg gatgtgctgc ccttcattcc 32400

gaagaccagc aagaacgaac agcatcgcga gaagatgtcc gagatccagt cgatggacaa 32460

cctcgaacgt tggttcgctc aacgtattgc gtcgggtaac cgaaacaaca acatgctcaa 32520

gttcgcaatg gccctcgtgg attccggtct cgatctgctg accgtccaac gacaggtgct 32580

ggccttcaac ggtaagctca acaatcctct cacggaacaa gagttgaaca gcacggtgct 32640

cgtctccgtg gccaagaagt accaccagta atcaggtggc tgggagcctt tcttggttct 32700

cagcccatca aggagaaaca catgagtgac acgcaagacg atgcaatcga tccggatttg 32760

atcaatgacc agttggtctt gatcgcaggt ttttcgggcg agggcaagtc ggcttcgttg 32820

cgaaacattc gcgaccagaa agactggttg tatctgaact gtgaggcagg caagcgtttg 32880

ccgttcgcca acaagttcca gagctaccgt atcaccgatc cgtatcaagt ccatgacgca 32940

ttcgatcatg cgacgaacga tgatacgtcg atcaagggca tcatcatcga ttcatccacg 33000

ttcttgatgg acatgatgga atcgatgtac gtgctcggct cggccaacac ccaagcagca 33060

tggggcagct atgcccaatt cttcaaggtg ctcatgcagc agaaggttac gttgttcggc 33120

aagcccgtca tcgtgatcgc tcacttgctg gacatctacg atgaagcgag ccagacgatg 33180

aagaccggtg tgccggtcaa gggttcgttg aagaacaacg gcatcgaagc gtatttctcg 33240

acagtggttc atgccaagaa ggtcacgttg aaggaactcg acaagaccgc aaacggcatg 33300

ctggaaatca cggaagacga gcaggaactc ggctacaagc acgtgttcca gacccgcaac 33360

accaagggaa cggtcggctc ccgtattcgc agtccgatgg gcatgttctc caaggcggag 33420

acctacgtcg acaacgacgc ccaaaagctg ctcgaccacc tcaccgcgta ctacgcgtaa 33480

ctttcaccac gcagtaaaaa ggaattcaca acatgttcgg taatctcaat tcgcaaggca 33540

cggaagcagt caaggactcc ctcggtggtg gtggtttcgt catcgacacg gatgcgtacc 33600

cgctcacgat caaggcactc tacggtggca agtcgaagca cggttcgctg ttcgtccaac 33660

tgatcgcgac gaaggaagac ggcaaggagt acaaggaaac gatctacgtc acgaaccaga 33720

agggcgagaa cttctacacg aaggacaaca agaagtatct gctgccgggc tacattctcg 33780

tcaacgatct gtgcaaggtc gtggcagaca aggaactcca cgagatggat caggaagaca 33840

aggtcttcaa ggtctacgac gctgacgcca agggcgaagt gccgaagaac gttccgatgc 33900

tcgtcgaagc agtcggtgcc aagttcggtg cgctgatcca gcgttacaag gagttcaagc 33960

aggtcaagaa cgaattgacc ggtgcatacg aagacacggc agacacccgt gagggcaaca 34020

acatcgaaaa ggtgttcgac ctcgattcga agtacacggt tgtcgaagca acgaccggtg 34080

ctgagtcggc cacgttcgtc gatgcatggc tcgaagccaa gaagggcaag gtctacgaca 34140

agaccgcagg caagacgccg aaggcaggtg caggcaacaa cggtgccccg ccgaaggcag 34200

gacaagcagc aggtggcaat gcacgtccgt cgctgttcaa gaagtcgtaa tgcctgaacg 34260

ctggcaaccc atagccgggt tcgctggcta tgaggtatcc aacttgggga acgtgagaac 34320

gttccgtccc aagaacggtc gtggaccttt gaaagaagag tcacgacctg tccgacaaga 34380

taaagcaaaa ggtaaggaat acttccgagt atctcttagc gatggaaatg gacacagcat 34440

gcacaggcca gttcatcaac tggttctgga agcgttcagt gggccaagac catcacctga 34500

gcacgatgct tgtcatcgag acggaataca tacgaacaac gtcagcacga atctgtattg 34560

gggaaccaag caggagaatg ctgacgatca aatccgtcat ggcacccaag tgaaaggcga 34620

acaagtaggc ttggccgttc ttacagacga tcaggtgcga gagattaagg aagcattgcc 34680

cacgtggaaa cgtggtatgg gtcgttactt cgcagaaaaa ttcggcgtgg gtgacacagc 34740

aatatctgaa atcaaacgcg gcaatacgtg gagacaccta tgatcgtatg tggcctcgat 34800

ccaagtttac gaaattgggg gatagcctat ggttggctag acctcaatac cggcgagtta 34860

acagaccttc acttggatgt gattcaggtg aaggacgaca cttccaagca ggttcggaag 34920

aactcgaagg atctccatat cgcccaatct ctgtttgagg gcgtacttcc ttatgccaag 34980

gaagcgaagt tcatcttcgt ggaagttccg attggctccc agtcagcgag agcgatggca 35040

tcgtatggca tttgtgttgg tgtccttggt tccttgcgtg caagcggcta cacgctcgtt 35100

gaggtaactc ccagcgaggt gaagatggct ttcaccggtg ataaggaagc atcgaagcac 35160

aagatgatta cggcagctat gtcgtactac ccggacacaa actggcccat gcaattgcag 35220

aagggtgtga atcgggtcat cgagtccaag gcagagcaca tggctgatgc catcgcctcc 35280

atccatgcag gggtgcaaac acctctgttc acccagacct tgcaggtctt gaaaggaatg 35340

tgatccatgc agatcatcta caacgcagca gaactcgcag caatcgtgga gcgtaacctt 35400

cgcatgaagt tcggtgtccc ggacggtcaa ggcgaaggcg atatccatgt ccaattcgcg 35460

atcgtcgatg gcgaaacgaa agcattcgtc ggtgtcggtg agttcccgac cgtgccgcag 35520

aacacgctgg caccgtggga agatcgtacc catgtgtcga tcggtgtcga tcacggcaag 35580

tcgatgtcgg agacggtggc cagcctcagc acgtcgctga gtacgactga accgcaacgt 35640

cgtacccgtc gaaccaaggc cgagatggaa gctttccgcc agcaacaaca ggcggaagct 35700

gaagcgaagc aggccgaaac gggaaacgaa tccgaggttt cgaagagtgg cgacgacgcc 35760

tcgaagcaat cccaagccac cgatgcgaac cagcagcagg aagctggatc cgaacaggct 35820

ggatccgatg gtgagcaggt gaagggtgac gcacaggagc agccaacgta taccccggcc 35880

gctgaagtgc cgatcgctga tccgaacgac acgcctccgg cacaaccgga tgaaggttcg 35940

acgggaaacg cccccgtgga atcgacttcg cagcaagaag gcacgcaagt accgaccgct 36000

ccgcgagtct cgctgttcgc caagaacaag cccgacgcgt cgacgactgg tacggctggt 36060

gcccaagttt cgggtgacgc accgcctgca tcgccccgtc caagcctgtt cgcaggtctt 36120

aacaagaacg ctggctaaat ggtaagagcc ctactttgtg gggctttgct gttggtcgtt 36180

gtaagcgtga ttgcgacggc catcaccatc atagccccgt acgtagcgat cgttgccgta 36240

ctggctgtcc tgatttggtg gctaggtagc acacctgaga agtaacgaag ccccatccgt 36300

aattggatgg ggctttttaa atgttagtta atgacgcttg cccacggatt cagcatcggt 36360

gcatggaaca actggccggg gccagctgcg taaccaagag tgccattggc catcttggag 36420

aagaagttgt caccagtcgg cagaccaaca ccaccgaaca catggggcac cggaaccaag 36480

gaagccagaa gcgtatgcac tggattgtta cggatcatgc tgactgcttg cttcgtcgag 36540

cggagcttga agttcatgaa ccagagcagg cccatttgct ccaagtaccc acggttacgt 36600

cccggcaggc gatcgtagtt aacgaactct tccgagattt ggcccagagc tttctctttc 36660

gaccatccct tcttctgcgt caggtgatcg aacaagatgg ctttaccgag gaaatcgccg 36720

tactcaacca tcttctggag gcccgtgtac agggcagtat ccttggagac gattgcgtac 36780

ttaccgacag tcttagcacc ttccggaagg cgatcaacca gaccttccac atacgaacgc 36840

catttacccg atgccagctt tacatcatcc ggcatcatat ccgtagcatg cgagataccc 36900

gagaactcac cagcttcgat caggggagcg atcgacatac gcttgaagct gtcgttgatc 36960

gattggatct cagcttgcag cttggtgcgt tggttacggt cagtagcagc aaccagttca 37020

gcctctgcct caacgatacg cgtacgaccc ttcacgtatt cgttgatctc attcgtctta 37080

gccgggatag caccgatggc agtcttgaac ggaacaccac ggccaaccag ttgaagaatg 37140

ttcgacatag cgttggcgac cggtacgatg acagacttga caacgatcgt cgacttagct 37200

tcaccgacca cgttcttcac aaacttctcg gcattggtga acaccttgta cgcatcaaga 37260

ccaatcgcac cgatgagagc acgcttgaca gtatcttgcg tctcttggct ccaacgcgag 37320

ttgccagtcc atgcgtcacc aaccgacgct tggcgatagc caagtgcatc gttcagcatg 37380

tcacggcgaa ccttgaagcc gtccgggaag cgatcagcga tcagggtacg catctcaccc 37440

gagatcagct ttgcagcatc acggagaacc ggatccttgg acgagaacac atcttcgtac 37500

tggttctcgt tgcgtgagtc tgccttgatg tcagcttcgt acatgtcagc cagcttgccg 37560

atcagagctt cgttatatac cttggctcgg acttcttcaa cttgacgacc agcccactgg 37620

cccatgctct tggcaaggtt acgttccggg ttcagcttgc tcatcatttc cggatcgatg 37680

gaacgctcgt atgcaaccac cgtgccgttt tccgagtaga ccgggagcag aggctctttg 37740

gtttgacctt cgttggcgta accacgggtg atcgcagcaa cctcgttgcg atccgtgata 37800

cggccagcca gcagttgagt cgagtaaccg gtgacagcat caacacccga tgccgtgcta 37860

cggacgttct ggaacagacc ttggtcgaat gctgcacgac cctgcaccgg tgcgaagtag 37920

tagcccttct tctgcaccac gtcagccgac gaacccttgt acgaaccaat acgcttatgg 37980

ctcatctcag accacttcgt atgttcgctg tccggtgcaa cttgcagcga cagtccttgt 38040

tgaccattct ccggcgacca acccttgtag tggttgatca gagcagccga cgtttctgcg 38100

acacggatgc tctcgtcttt acgcagaccc ttcaggtagt tcagggagaa gtccatacca 38160

tccgactccg aaccaaccag cgaagcgaga tcagtcttag ccgatgccga ttgctgatca 38220

agtgcgtaga gcgtaaccag atggtcgatc tcacgttgag ccgcacgatc cgaaaccttc 38280

aggttcttcg tcttctcacc gaacatcttc gagatggcgt aggagttacg aagcagattg 38340

tgaccagctt cacccgtcac catgaactta gcaagctgtt gcatcttcgc ttgcttagca 38400

ccccagttag ccgggtcatg tgcttggatc gatgcctcaa ggttgttgat gcgtgcatcg 38460

atctcagctt gggagccaag aagcttacgc acgtcttcaa ccgagttgcc gtcgagcaac 38520

gaagcgatgt ccgtcttggc catgccttca tgcagcgtct tccattcacc ttccgtcaac 38580

tcacgggtga acttcgaagc gatgatgttt ggcacttgct cacggtagat ctggcgagtc 38640

gcttggatgt tcgacttaac cagcttgatc agatcgatca cgctggagtt ggactccgta 38700

cgaccaacca agtcattcat gatggagcgc agaccgtcat gaagcttcgt cgtgttcagg 38760

tgtgcattca gtccttcagc aaggatcttg ccacgttgtt ccgtagcaag gccgagcgtc 38820

agacgaccaa ccttgtagac cgtcttggca aggttgctgt tcgactgcga ttccagttga 38880

ccgagcttgt cgatacccgc ttccgagtat tccgagatca tctcaaccag tcgatcgttc 38940

gcgttgtcga ggaacgaatg tgccttgtcc gacgagttct ggatgaacga gttttcgtcc 39000

tgcgtgatgt ccatcaggcg atcatgcagt gcatccatag cagccgggac agtacgagcg 39060

tccttggttc ccgtcaggcg acgcgacagt gcatccatca caccgtaacc gatgttctcg 39120

atcaggcgat ccgggctacg ggtagcatcc gacttcttgg aacccggcat accgatctta 39180

gccagagctt tacgagcatc cgggttcgtc agagccaagc ccataaacac tgagaggttc 39240

gtgctgcgac ccgatgcgtc cgtctgacca accgtacggc caaggagtgc attgaacttg 39300

gcattggcag gcgtgaacag gttcgtatgc tgtgagtctt ccgggttgtc taggaagtgc 39360

tcaaccgtca ggttcttcgc cacgtgagcg aaatactctt gagccttggc catcacgctc 39420

ggatcgaagt tggcttcagt agccatagca gcaacgatgc gacggaacgt gccagcttct 39480

tgcatagtca tcgggaacac tgcttccact tcgttcgaga ctgccacagc cttcgccact 39540

gcctcacgaa cgttgccctt tgcaaccact tgcaggggag cgttcttcac gtagtccgtg 39600

atcttgcggg tgaacgtgtt acgtacttcc gtgagacggt catccgtacc gaacacacgt 39660

gactggaaca gcgtcgactg agcagcttga gagttcagag tcggctccga gttcatcagg 39720

atggccgagt tgaacagcag gtgcgagaac atgtccgtac ccggcttcgg ttccggcaga 39780

gccttccgac cccacaccag ttgtttgatc gcagcgatag cagccttcgc cacagcgacc 39840

agacccttgg cctcggtctt acccgtcaca gcagccagtt ccttgttcga cagaccccat 39900

gccatgaact cgttcagacc agcagcctta cgggtgttca cgtcatatgc cgggttgttc 39960

aatgcagctt cgatcgatgc ttgagcatta cggaacgact catacgtctt ggaatccgtc 40020

gagaacgact cttccatccc acggaattga tccatgagag tttcgagatt gccgatggct 40080

tggctcacta ccggcgacga ctgaccgttg tagtgcgatt gcaccttatc gaacgtggca 40140

gcatgcacca tttcgtgcag taggttctct gccgtacctt ccgtcgagac gatcaccttg 40200

ttggccgtat ccgtaaagcc tgccgtgttc tggtcgagct ttgcagccag ttccggcgag 40260

taggcacgaa tctgttccgg cgtaccgacc agcaccttgt aacccttggc agcttcactg 40320

cgttggattt cacggagcag gttcgtttgg atcgtgtcga ggttgttcga cttggcgagt 40380

tgacccagtg ccgtgtacga aagcacacga acacccgact tgtcttcacg accgaggttc 40440

gagagatcca tcttctcaac cggagcagca gcttctttct tgctgtcgag cttcgcacgt 40500

tcttcagcgt agatctcgtt caggcgggca agcttctgtt ctgccgtacc aaccagcgat 40560

tccttggacg tgacacggta cggagcacca gcaccagcca tctgatccat ggagatattc 40620

agacgatcga tagtcttgtg acgtgcttca gcatccttcc agcccaagtc caattcgttc 40680

tgaagcagag tcagttgagc atgcagcatc tcaggcgacg gcgtttcctt cgtaccgagg 40740

ccattaaggg cttggttcag ttggttgtac atgtccgaac cttccacgat catgtccggc 40800

gtcatgttgt ccatgaactt gctgaaggag tcatacacag ccttcagcgg gttgccttgc 40860

cacgaatcgt agacagcctt gttcgccact tccgaaccct gtcggatttg atccaagggg 40920

aagtgcacac cgtcgaagat tgggagcgta cccttcggca tcttcggatc gttgtacatg 40980

ttctggatca tcttgccatc acccataccg atgttcagga acgggatacc agccacacca 41040

ctgtcagcag ggccatagac gaaacccgga gtacggaaac gatcatcgag acccttaccg 41100

agaaccgacg actgcacttc cgagttctga gaaccggaga tgaagaagcg ttggccgttc 41160

gagtcgatca tcggagcgta cttgtccagc gacttgagca cttcagcttg ttcgttctgg 41220

ctcaggaatt ccgtcttctt acggccagca gcaaccactt catccagctt ggtgttgatg 41280

gcttgcttga agagttcacc gaggacgatc gactgagctt gcgtagcctt gcgaaccatc 41340

tctgccgagc ccatgaccga gtcacccacc gtcgtgttga tggcagcacg aagcggatcg 41400

acgaacagct tacgcatgtt cttcgtcatg ttggctttct ggttcttgcc gaacgtaaag 41460

gtttcaaagt tggtgatcgg attgtgctcc gatgcttcct tcttaccgta ccagcccttc 41520

ttgccctgct ggagcgtgtt ctcgctcaga tagttcactg catcgttgaa tgcagcccac 41580

ttggcttcac caccttcacc gaacatggct tcagccatcg acagcttgga gtcgttcgct 41640

tgggcttgca gggcttccga catggcttcg tagatgttcg tgtggagagc ttccaccatc 41700

ttcgcagcaa taccttctgc acccgaacca tagacggtaa tcgtcagcgg gttcttggcg 41760

ataccacggg acagttccag cttgccatcc ttgatatcgt agtcggacat gaacatgtcc 41820

atcagcgtcg agaagtggct attgaatacc gagccttcgc cttgcagcgt ttgagccaag 41880

tcagccagat tgcccttcag cacgtcagtc gttgcttggt acagatcctt gctatccacc 41940

gtcgagcgat agacttgcga attctggtct tccgtaccac caaagaagaa gccacccttg 42000

gccaagttgt cgatctgacc ttgcgtgaag gagccacggg acagcaggcc catagcgttg 42060

atcgggccat tggtcatacc gtcagcttcc atgtatttcg tcgtcacgaa tgcttgcgga 42120

tcgttcgttt ccagcgagcg tgcgtgttcg agcagtgcat ggagatacac cggagcttga 42180

cccttaacag cgtcgatcag gacatcagcc gagaagtctt ggttgttgag gaacttacgc 42240

agttcttcga tagccggagc attcttcgcc agttcagcag cgagcttgtt ctccattgct 42300

tcacgttgca tgttgtggat cttcacaccc agagcttgag ccaagccaag atcgaaggca 42360

tgacgatctg catcatcagt caggtcgaga gtcgcacggt tcggaagcag gacttcacga 42420

gcagtcttgg ctgcttgcgg gccatacttg ccaagttgtt gcagacgacc aacacgcgac 42480

atgttgaagg cgtagttcac cggaacctga tcttcaggga taccgttctc ttcagcatag 42540

cgacggactt cagccatcgt ttgatccagt tgaaccagag cacccatagt ggtgatgttc 42600

tggccatcga gggtcgcacg atgattgacg ttcaggggcg tattacgaat gtccccagca 42660

gcgaacattt ccatcagacg atcacccagc gactcgtaca gacgatagac attcgagtct 42720

gcgtagtacg gggtctcttg gttcgctttg atggctttca tatcctgtgc cgtattctgc 42780

acagcttggt tacgcatctg cgtcttggca accggcactt ccgtgttgat gtacaccgga 42840

cgttccgtct tcacaccaac cgagtcagcg atagcagacg gcatctggtt gatcgcattt 42900

tccttgtcga tcgagttcac cgtaatacgg acgaagtcct tcgggccacc atcaaccgac 42960

aggttgatcg tgttcacttc gatgtgaccg gtcgcttcca tagccgacag gagttcagca 43020

gccatcgaca agggaatacc cttagtgtag ccatccgggg cagcattgtt cgtttgcaga 43080

ccccagaagg tcggcagcag ttgagccaag gactgagtag cttcgagagc cgacatacca 43140

gcagacatct ggttgaacag ctcatccgac acatcggctt ccttcatgcc gagcatctga 43200

gcaacatcgc tacggtcaac gatctgaccg tacaggttcg agttggcgaa ccagtgcata 43260

gcagccagcg ccgagttctg gatgatctcc gggttgtacg tcagcttgcc accgttctct 43320

tcaacgaggt tcagaagctt gccatctgcc cacgtatgca cgtcttcgcc ttgcataaag 43380

cgtgcaccaa ccttgttctt cttcaggaag ttggcgagtt gggtgttcag ttggtcaaca 43440

accagaccag cgtcatccag cagacgaccg aattccttag ccacttcctt cggaagctta 43500

gccttcagat cagcacgtac cgtatcgatc ggagcttccg taccgatggt cgtgctcttg 43560

gcttccttcg gcagacggaa tgcttcaacg aacttgttgc caccttgggg aactacgaga 43620

cccggataga cgtgctggat cgaatccttc ggagcagccg gatcagttgc cttgggatcg 43680

gacgtaactt cagaagcttg ttcaacaacc ttcggagcag tttccgaggt tgtagcaggc 43740

gtaacatctt gttgcgaatc agatgcttcc gacttacgtg cgacagtagt cttggtatcc 43800

ttcggggaaa tcgtttcttc cttaaccggt gcggcttctt gctgcacacc agacttgaac 43860

gactccacga cttgcttggc attgccagca gtcagacgag cgtccaaggg agcctgagcc 43920

atatgtttgc caccaagttg cgggaaagca tccaccagcg agttgtgaat atccgtaagc 43980

gtctgagcct ctacgccgac tcgctgagcc agagcaaccg aacgctcgtt gttcgtattc 44040

acaaacatgc cattacggga cttcacccaa ccacggtcgg cactgctgga caaagcctcg 44100

tagtgcaccg tcgagttcac acccggaacg gccgtgtcga gatgctcatt gatcgccccg 44160

actttgttct gcatgtgttg gacgaacttc gaaaagtccg caagctgggc cgaggcaccc 44220

tcgatatcac ctgcacgtac agcatcgcga atgatacgag catgttgcaa ggctgagtac 44280

tgacccggcc cacggccgtc atcagtcttg atttgcttgc ccactacgtc ttccggcttc 44340

aggccgagac gagcagtttc ctcatcctgt accttggctc cattcagcag agcagcagcc 44400

gtacggagag cattgatctg ggcatccgac aggttcagct taccgtcctt gttctgcttc 44460

aggatcgtag ccacatcatc cgggcttgcc ttcaggggag cgttctcagc cacagcaatg 44520

tggttgttga tgtttgcatc acccagagtc gtaccggcct gaccatccgt aaccggcttc 44580

aggttatcag ccaccttctc cgtgagcgtc tcagcagttc gttgagcacg ttggatagcc 44640

ggggattcgt tcagggatga gataacatca gccacaccgt gtgccatctg cacagctggg 44700

tgatctgccg gaagaccttc cagagccttg ggattcgtct gaacgaattc gctgataccg 44760

ttcagcgtct ggttcaggat tgcacccaca tgggcacgtt cctcttccga gttcttcgtg 44820

tcgttgacaa actggctcat cttctggaga gcgtcgactt ggttcgatgc accatccgta 44880

gcacgagaca gaacatccga accagccaca gaagcggggt cgaattggaa tgcaccagcc 44940

agagcatcca cgtgctcttg tgcagcttgc ttctgttccg ggctgagttc cggtgcagct 45000

tggatatcag ccttggccag atcagcttga accggcactt cttgtgcaac ggtagcagca 45060

gcttgcgaga cgttcgtatc cgagacaggc gactgttctt tagcagcagc aatcttcttg 45120

tcgccacgag cagccagcca gttggtcaga gcagacgtgc cagcaacgag aggcttggta 45180

gctgcaatgg ttgcagcctt gcccaccgta gcagccttgc cagccagttg tgcaccaaca 45240

cccggagctt gggtgagacc agcagaacca agaccaccga cgagaccctg aacagcctga 45300

tcaccaacac cttctgcgag gttctggttc acgttcgaag tgagctgctt agccttgttc 45360

tgggacagtt gaccagtgat cgactgaagc gtttcttctg caccttcctt caggacgtta 45420

cgacctgctt cacgcagcga cggaaccgag aacggatggg cttcgaactt cgacgcgatg 45480

acacccgtag cagcagcgag aggggtctga atagcagcag ccagaagacc agcacggtta 45540

gcgacaaccg agcgtgcttc ttcttgcgtt gcaccaccag ccacgagttc gttgaacatc 45600

gggctgttct tcttgagatc agcaaaggaa gcaccttgga tgtcgccagc attcgactgg 45660

tacgcaccac caccttcttg cagaccgata gcagcaggca tcgtccactt cgtaccgatg 45720

aagttggcga cacgtgcagc cgaccacgta cccatggatg cgtcgatagc cgctgctgct 45780

tggataccgg cttgcgttgc cttcggcact acagccgatc cagcagaagc caaaccacga 45840

gaaagagggc cagcagtaag gatagaacca acaccttgag cagcaatatc tccgacaagc 45900

tcagggctat cggccaagcg accggcagaa tcaagagcat cacgaccaac acgcttgaga 45960

ccagccatga agccagaatc accagcagct ttatcagcct gttgttgggc ttctcgtaca 46020

ggggtttgga gtgcactgag gttctcgctg atgttgcgag cagccttcat ctggggagac 46080

tgggtggact gcccgaattc attcaggtct tgcaggctct ttgccatagc agggccagca 46140

cccgtcatcg gaagagtcaa gccagcaccg agtgcggcaa gtccaccgac agagttcgca 46200

agaccgaggc caatacccgt aaccgtatca gcagccactt ggctacccga acgggggggt 46260

tgggattggt cttgacggag tgcacgtgca ccggcagcac ggtcagccat catctgggtg 46320

gcttgattac cgtacttggc atagagggcg agaggatcca tggtggcagc atcacgctga 46380

cgttcatcca tggcagaaag accaacacca gtctgaccag caagagccgt ctgatagttc 46440

tggggagtgt tttggccttg gttcatcacg gccattttct gatcagcagc attcgcaaca 46500

tctaccttct tttgctctgc cgtcagtgcg acgttagcgg tagaagggag gtagctgctg 46560

gcgcttgctg gttgcggtgc cgcacctgcg tactgggcca acaggtcgag tgcattcgac 46620

attgctgttt ccttattaga agggagtttg aggaaggact atagctgaag tcctagaaag 46680

aagaaagccc ctcactagga ggggcttcga ttttacttct tattaggctg gaagttcatc 46740

ggcaacatcg ccggattctg ctgagtctga gccagtgaca gagcaagtgc ctgctgtgct 46800

gcttgcaggt ttgcctgata cgtaggcagt cgatcacgca gggacgggtc gaactgagca 46860

cgacgagtag cttctgcaag ctgagcagac gcttggttga aggcatctcg tgcaccctgt 46920

acagcagctt gctgatcctt gataccttgg ttgtccaagg tctgaacgat cgtatcaccg 46980

gacttaaggc cagcgatatc ggccttgatg ccttccatgt tgaaacgctt gccaccggca 47040

aggttcgacg tgtgttcttg ggaagtccag tcacccgaga agaactgacc gaggtttcga 47100

gcagcacgac ggaagccaat gccattcgga cgccacgacg agttgttgta gctgtcgtca 47160

gcttgatcga tgttacgagc caagacagca gcagccgtag cagcgttgac gttaccatca 47220

cgcatgatcc gattgagctg accgacaatc acaccttccg gcacattctg gagcacagtc 47280

ttacgaagct cctgtgcagc ttccacattc gaggcagtcg agggcagagt ggtggtgaga 47340

tcaggagaca caccttgggc ctgattctgc gacagacggg actgcatgag agcaccggca 47400

gcgttggcag cagtacccaa gtcacgttgg ttggcacgaa tctccgggag actctgacca 47460

acttcaccac cagcgatgac gttacgcatc tgttcccacg gaacatcctt gtatgcaccc 47520

ggagtcgcat tcggaagaga tgcccacgtc gacttgaggt tgccaccctt gttgtcgtcg 47580

aagatagcct tggccagctt gtcttggttc tccggggtca tctgggtgtt cttccagttc 47640

gcaccaaaga ccttcgggcc atagttctcc atcgtaccgg ccgtgatctg gaaagcaccc 47700

atggccgagc tacccttgtc cgtacctgcc aagccgaggc ttgcattgtt acgggtattc 47760

gggatgaggg tgttcttccc gaaattgatg gcatcaccca tgctcatcga agtcaggggc 47820

tgctgaggcg tgccgaatgc cccattgccg atcatggtgt cgaaagcatt gcctcgtgca 47880

ccacctgcac ctgcaccacc cggagcaacg ccggacgaac tcggtgcacc ggcaaacaga 47940

gcacccccag cttgggccgt accaatcggg ccaatcgtac cgggaaacat ctggttggca 48000

cgaccgagca aagcttgcaa agcacccgga gacaggttgg gagccaagcg agcaatacca 48060

gcacgcatgt catcggccgt aagcgaagta cgggacagtt cgttgaaagc agcagcagca 48120

gcttggttgt cgttgtcctg acggtaagcc actgcattgt cgaagccggt acgttggttg 48180

ccaaggtcac cacgaaccag accctgattg ttcgatgcaa tctgagcctg ttgatccgga 48240

gtcaaccccc cgagaacgcc ttggttctgc gcatatgcgc tctgcaagcc aacacgatcg 48300

cctcgttggg cagcaccaag cagttctgca actgccgggc ctgctgcatc cgtggcagcg 48360

ttcagcttct gctgacggcc aaagttgtac gcacccttgt ccagttcctg ttggttcgtt 48420

gcttggttga tcagttgacc aacacgggaa ccaagagcag cagcaccatc agccgagata 48480

cgcgaggtgt tgatgccatt gaacaacgaa ccgtccacca gagcattccg gtagtcgttc 48540

ggattctgga aacggagagc agccaactgt gcagcagcgt cagcctgtcg agtttgctca 48600

cggccaaagg caccgattgc atccgacaga ccactagctg cacctgcaaa ctggttggca 48660

gctacacggg aagcgtcaac ggccgtaccg agattcggtg cgtctacgtt acgccaagtc 48720

aaagccatag cactttctcc ttatcgcgag atacggttac gatcgacata cgaggcgatc 48780

tggtcagcag tctggccttc cgctgcacca cgtgcacgtg ctcgatcttc aagagccgta 48840

ttgtacgact tgatcgagtt gttcaggttc gtattggtga tgcccttcgt gaaatcgaac 48900

tgatcacgag ccagcttctg agcttggaaa ccaccccaga gattagccag agagccaatg 48960

gcacccagac caagctgaag ggtaggcatg ttcaggccga ggccaccaaa gctaccggca 49020

ccattaccga gagcaccagt ggccccaccc agagggccac cacccagagt cggactggcc 49080

atgccatagg tattaagcga accgtacggt actgcaccaa cgggggcacc acccccaaaa 49140

ttgaaggaat tggcacccaa cccccactgg ttgttttgac cgaagtcgta cataaaatct 49200

ccgtttaagt aatcccttgg ctcggatcca aggaggttga agcaaaatcg ctgatcatcg 49260

catgggagag ttctgcgata tcagatccgg tgagaagcgt acgggacaag aaagtctccg 49320

gcttctcagg gtacatctgg tcatgcgtag gatcgacaag ctgaagcggg ttgatgttgg 49380

cattggcata accaatattc tgtccccaca aacgctgaat ctcttccatc tgcgtattgt 49440

acgactccat gagtcgagca gtgttattta ccatctccat ggcactgatc tgctgatact 49500

ctccgatgcc cttggcagta gcactggtaa gggaaagaag actcatggga ctggtgagtg 49560

cagccatacc accagccaga gaaccagtgg tcatgtactg ctggcccatg acgacagcga 49620

taacgccagc gatagcaccg atcactgcac caagcttgtc accgaacacc atggtggaaa 49680

cttgcccgat gatcttcatg atcaacatcg cagcgatggc attaacagca gcaccaacca 49740

caagagcagc cacaccggta agaccaagag cagcccctgt agccatggca gcagtggcga 49800

aaggcgtacc agtaccccac gtgaagtacg tgataacgac cacgatcacg accatgatga 49860

tctggaagaa tccggactgc caccatttct tcttcttctc aacgtagcag ttgaagacca 49920

agaaacaaga ggcagtgctc atctgggttg catccttcag agtcatttgg cgatacattg 49980

cctcgtgcaa tgggatgatg aaacccgatt cgtcactgtc tgctagggca tccttagcgg 50040

aagtgttgac cgaatgcccc ttgtaaatca tgttctggtg ccatgcaccc cagatatcga 50100

gacgctcata ctgagtgtcg ctgatctggc gactgagtcg aatatggctg atggactgtg 50160

taccaacacc gatccatggg ccaagagcac caccaacagg aacaagctca tcctgtggac 50220

tgatctcgta catggtgaac gcgtcatctg ggagtacatc ccatttgtac tcacccttac 50280

gcccgataga gccattgatc gtggtttccg agatgcccac ccagaagatg gtcatgtcgt 50340

agttcagtac acggttatta tccgaggata ctcgaatgga cgatccggga acattgggat 50400

atggcttgat ctcaggacgt ggtttgatag gctgacccca gccaacaaac ccaccagtct 50460

tcttccactc caagtagtct ttcagcgatt gtgtggctgc ttcccagtct tgcttgaact 50520

gttcgtacaa gccaaggttg gctggactct gatccagaag aaccttgaag aagttataaa 50580

tgtactgtcg ggatgcattc tccttaacgt tcagtgctac gccataaaca gcataggcgt 50640

agtcgatatc cccaacttgg ggattcttct tgatctcgtc catcacgtct tggaacttgc 50700

caccagatcc cttcttgaat gccttcttgg cccaagggta gagactggcg tactgaggtg 50760

agtcgacgtt gatgttgtcg atacggaaag gtacgaacgg ataaaactta cccatgttcg 50820

tgggacgagc gaacatcgag tccaatactg cattgccctg cccttgaccg tagatgaaca 50880

cgttctgagg gccatacgta ttgaccaccg tagtctgctt gtccactcga taggatcgag 50940

tgacctgcaa ggtctgggtg gttgtcgtag tggtagtggt tttcttggtt ccatccggaa 51000

gagtttcttc cttcgtctca accacagggg aaccaatgac cacattaccc acctgatcct 51060

gatacatggt ctcaaccagc atcgacgtat agttgggatc gatctgggtg ttggtctgac 51120

gaccgtacac accatgcgtt tccgaatacg aatacggagt ctgagtagtg tccgtagtgt 51180

gctcatccgg tcgaccatcg ctatacgtga tcttggtatc caccaccttg tccagattgc 51240

cagacttgga gatattcgaa gtagacacga cattccatcc cgagatagga gggaacggat 51300

cacctgaacc aaggatgaca gtattgcccg gaatcagtgg gccaacctga tcagggccag 51360

tctccatgta cgtgacatag aggtatcgga tcgactggtc gtaaccaaca ggtgtgaacg 51420

tgtccttggt cttgtccgag tacgtaatgg tgacaatgcc agtagcatcg tcgtactcag 51480

cagaccatgt atcgctcacc ttattgggtc gattctcacg aagccacttg tccacccaga 51540

tgttgaggtt agccaaccca atcgtagctg actggatagt tgccttgtta ccgggcgaga 51600

caggaagttg ggttgcgata acagtctggt tgagcgtatc ccctgacagc acagtgccag 51660

taaccaaacc gatgatgttg ttgtatccgg agtagtcagc ccatcgtgca aatgaccgga 51720

acttgatacc ggggccattc agatatgagc caacgatcga atcacccatg gaagaggatt 51780

gaccgagaac atccccaact accgtctgac gaaggtagtt ggggcgtttg ttctcatctc 51840

cggcgaggtt gtataccaca ctcgaaacaa cgatgacgga accaccaccg aagagaccca 51900

tggttagacg aacccgttat tgaccttgat ggtctggaac accttgtcca ccgaggcatt 51960

ctcaaatgcc gggggaggca caagaccctc gtcgaccgtc ttctgcgtga tccaagcatc 52020

cgagaagatc ttggcagctt tgacttccga gtcacgcttg tagctggtga tctgctgatc 52080

gtacagatcc ttctgcttac cgagtacacc acgaactgcc tgaccatccg tacggacatc 52140

gagggtctgt gcacgttgag cttccgtttg ctcttgggtc atgaccagtt gcttatccag 52200

cagttggcta tttttcggaa ggatgttatc cacctgaaat ttaccagcac cgaagttggt 52260

cgattccgta gccagcttgg ccttggtgag agcgtagttg gccttcgaag tcagggcatc 52320

gaactgcttg ccgatgagtt cgaacaacgc agtcttcagg ttcacttgtg cagtgacagc 52380

ctgaatctga gccagttggc cttgccagaa tgcaccatca cgttgggtga ggtactggac 52440

ggcattctgc atacacgcat ccgtgagtgc agtgtatgcc ttcgtgtact cggcaccggt 52500

gatacgcccc ttggaaaact cttccttcag gtgacaggac accgactcca tgagtgcatc 52560

gaacgtacca tcacccttgg gatgacgttg cgtgagcgac tcattggtga gaggtttcag 52620

gcccgagtac atgggcgagt tgggatcgta cttgatctgg aatgccgggt cgttgatatc 52680

gacggtgggg aatgagacat cctcacctgc cagcagagcg tcatacatct gcttggcttc 52740

aagatctgca ccgtacatcg tgctcatata tttctccttg gttcagaatg gattaagccc 52800

gtggagtcta cactctcacg ggctttttag atacttccta gtggaagctt agtccaccga 52860

tccggcagcg agttgggccg tagccagttg cttgatctct tccggagtca gcggatcgag 52920

cacttcgagg ctgtattccg aaacccagcg ttgttcggtg tgggtcgtac cggtacggcg 52980

atctttacgg agcttgatgt cgaggaactt gcgatccttc atcatctggt acagacagta 53040

cgggacatgc caaccttcgt ccgtcacttc accgtagggg atgtacttgc ggatcgtgcc 53100

gatgtattcg ttcgccaccg tgaggatctc acccgggaga tccttcttct tggggtcgag 53160

gttcgtgata cgcagacgga tgagcttcgt agcttcatcg atcagttgct ggcggaatga 53220

cttctgacca gcaggcttgg cacctgcaac atcaccagcc agtgggttca gttcagcagg 53280

ttccttgtct tctgctttcg gggcttgtgc ttcagcaatg cgagccttca gagcttcgac 53340

accgatgttg ttgctgaact tgacgccgag cagcgtggca cgttgcttca gcagatcgag 53400

ttccgagaat tcttggactt gttccagcga ttggccgtct tggccattgt tgagatcttc 53460

gctcatttct gtaccttgtt gggtgagggt gaaccaaggg gaagattatc cccttggatc 53520

cttagtcact aagtccgatt agatcggggc gacggtcttg accagagcga tacgctccgg 53580

acgcttgatc agaataccgt agtaccactt gatcgagctg aacccggttt caccgtacgg 53640

gtcgttacgg tcagccgtag ctgcacccgg catcttcgtc gtcaccgaga acttcaccga 53700

cttgccgtcc gtctggaagc cgatcgtggt gaacgagtca tcaccaatac acagcatcgg 53760

gaacacgtcg tagtgttctt cgccaccctt cgtcgaggta cggtagcccg gattagctgc 53820

cgtagctact gcaccgacac ccgcccagtg cagcatttcc ggcacttgga tgatgtggaa 53880

cttgtcgatc gagccgatct caccgttcag caccgtacca gcagccgagt agtgttggat 53940

ctcgatgaac gccttgttgc cgaacagatc cttcatttcc ttcagggccg ggacgagttc 54000

cgagccgaca tacaggacac gcgttgcacc cagaaccttc gtgtcgatca gacggctacc 54060

cgtgatgatc gtcgtctgcg tcggagtacg gttgtccgtc aggatctgat ccagacgcat 54120

caggttcttg tacgagacca tcgagacggc agtaccttca cccgtgatcg tggcatccga 54180

cgtagcagca cctgcgtaca ggatcacgcc agcagcagcc agtaggtcac gttgcagaac 54240

agcttcggtc agttggactg caccggtcat cagttcacgc gacaggtgat ccatcaggcc 54300

atcgtccgaa tcgaagtcca gagcttcttg agtgaattcg gtgaagaaac cgaacttgtg 54360

gatcgaaccc tcacgttgca gacgggtgaa cccaactcgg ttcactcggc caccgttttc 54420

cgtcagcgtc ggcagcttcg acgtgatcgt acccacgtcc ttgctagaac catacaggtt 54480

gccgttcgcg atcttcacgc cgttggcatc gataccttgg tcgttgatgt tgcgatcgtc 54540

cagcagcggc acgtattcgt acacgcggat cgacttgccg tagtgcttcg gcatgttcgt 54600

ggtcgacgcg aggggcatga aatactgctc cttacgtgcc gtgatgatcg ccttcttcag 54660

ccagaagaag gtgagcatct gatccgagcc attgccgtca atgctcgatt tttggccctc 54720

ggccggtgcg ttgtagttca acatttggtc tgtttccttt tagacgcggt tgttcatttg 54780

cttcaagaat gcatcgtcat ccatatcgag atagttgacg atgggtttcg ccttaacagc 54840

aacggcacga ttgcccgatg ctgcatttgc cgtgtcactg ttcgctacct tcgtaaccgg 54900

cttcggtgca gctacagcga tgggttgctt ggtggtcgtt tgagccacgg agggcttaac 54960

gatatcagca aacttccctt cactctgcat tgcgtcacca actgccttat acgcttgcag 55020

gaacggagtt ccggcaggaa tttgcccaag caccgtgagt cgagtgatct cagtcgtaat 55080

gcgatcgtag atcccattac tacgctgttc atcaaacact cgcagaagcg aggggtcttt 55140

ccagaggaca tccttgctgg cttgatccca cgtcgtgttg atcgtctgta ccgtttctac 55200

gccggactgg gtggatacga gatcgtccag tgccgattga aacgctacgt gctcatcact 55260

gacggcatga ttgcccggca ggtaagtcga gtctttctct acatccagcg tcatcgggtc 55320

gatgttgctg tccttgagca gcttctggat cgcctcggga ttcttcttat cgatatcgat 55380

cagataagaa agtttgcctt cgtcgagcag accgttctgt tccagcatca tcagcacctt 55440

acgatggggc tgaatgtctt gcatcttgcg agtgtagttg gcacccatct gcatcaactg 55500

gattgcctcg tctgcgttac gcagttcgat ggtcttcccg tttgccttga agggagcaag 55560

caacttcttg tactcggctt cgtagtcgac ggtcgcttca gtcgtcgtag cagccgcctt 55620

actggcagct ttctgctcgg aattcccagc gttcgaatcg aggggcttcc cgttggcatc 55680

tactttcgat tctgcgttat cgtcagcatt tgccgcaacc ttaccaccat tcggatcatt 55740

tttggaagct tcttctgctt cctgagccag acgggcagct ttttcctctt ccgtctcttc 55800

cttgttggca gcttgctcag cagccagtcg atcggcttct tcctgatcca gacgtgcttg 55860

ttctgccgca gcagcttcag cagcttcacg tgcagcagcc tcttcggcag cgatctcttc 55920

aggggtcttg gtggggactt cgaacacggg agccgtgctc atgttttcga tctgctcgtc 55980

cgacaagtca aagatgtcgg acggctgatc cagtttaagg tcagccgtcg tcatcgttta 56040

ctccgcttcg tcagtttcgg tgttggcttc tgcgagtcgg gcctcttcca gaccctcacg 56100

cagctcttcg agttgtgcac cacaggtttc tgcttgcatc agttggatca cgatccaacg 56160

cttcagatga cccggtgctt ggctcagagc cagtgcatca gcacgttcac gatcggtcag 56220

ggccgggtta cctgcttcgg cagcgtatcg tgcagcttcc ttggtcgaga actcgtcgat 56280

gatgaccttc ttgaattcct tgttttccaa gagtttcgaa agggctgcac cgagggccac 56340

ttgtgctttg gcaccggcca actcgttctc gaacttctgc accagactgg tattgatcat 56400

ttttgcaatt ccttaactga ggttgattgc tgggttaaga gcagggtcaa gactcgggtt 56460

aaattcctga gaaccaaggt tcaaaggatt cgtccccgct agtccccctg ctgcatcacg 56520

cgagatagta gacgaaggtg cagaagaatt caagttaccg ctaattttat taaaacccac 56580

ggctgcttga atatcaggag ccttctcacc ttccttggtc ggggatgtaa gagccttcgt 56640

aaccgcaagg ttctggttac cttccgactg tgccttctga agctccatct cacgagcatg 56700

cttcgtaccg gtttcggttt ccacgtactc cagattcttg agatcgccag atgcaccttg 56760

catcttagct tgagccaaga agagttcagc ctgtgcctga ttcttgccga tatccgattg 56820

aagcttctcg atctccagtt gcttcttctg ctgttcaaga ggatccggct ggggttggta 56880

agtcttgagg gcatgcgcca gttcaggcat acgcttcagc ttagcgatct caacgaggat 56940

gatctgaacc agtccccaat caccattcgg ccccatggtc tgaagcatga atgccagatc 57000

ttgcgacttc tggttatcca cctcagcagt ggagatatca gcgatcactt cgaagttgcc 57060

tttcagatcc tcacgagaga cttctacgaa ctgttcgttg gtcaccttga ccgtttcttt 57120

ctcggacagc cagacagcat tcatgctgat cagcttcatc gcgatctcag ccatcccctt 57180

agccatgcga cgaaggatgg ccatttctcg cttagaagat gcatcgagca ccccacgaat 57240

accagcagca acatcaccat acgcatctcc ggaaacacca ccagcaaagg atttaacacc 57300

ggtaagtgct tcagcctctt ggttctgaag gttgagcatg agcatcacgg attgtggcag 57360

ttccgggtac ttgtgctcca tcaaaccacc ggcagcagga ttctggttgg ggttgaactc 57420

atagtcctgc ccgttatcga agcgacgctt gttcacagca tccagcatgc ccttggcaat 57480

accagtctga gcattggccg acttgcccat caggtcgata gcacctcggg taactgcacc 57540

caagatcgcc tgattgtctt ccagcagttc cgcatccggt tcaccgtaaa gctcacgctt 57600

gaccggcaga tacgggatca acacgaacgg aagcttctca tccgggaacg ggttcttctc 57660

cagtcggatc atcacatcac cgatccaagt ggcgacgatg ggggtcagtg caccattccc 57720

ctcgatatcc cagaagcccc agtactcata agccacgacc ttcttacgtg ccttgtcttt 57780

gaactggaac gagtcaggag tagtggtttc atgatccggt tgagccaagg gggtattacc 57840

ttcccagttc acacggtcga gattcttgta ccgcttcttg tccttcttca gttcagacca 57900

gcttgtttcg aaggagatca ccacgaactg tgccttctcg aaatccccgt tacatgaggg 57960

gtcgatgaag acgttgtggg gattcatgat ctccagagac ggctgattga agagaatctt 58020

ctcttcctga atggtcgtgg tctcaccagt agcttgagcg taaacaggga tctggttttc 58080

ttccgtgtat tccactgcca tccgaacttc ttccggcagt tgatcgtatc cacggggatt 58140

tgagttcttc aactccacgg cctgttggag ttgctgaagc tgatcttcag tctcgatctg 58200

gtagtacgcg tacagaggca cttccttggg aagcatgatc gtctgacgat tccaaccaag 58260

cttgatgatg cacgtaccat catccacagt ggaacgcacc atgtcgtcga tgaacttgac 58320

cttgttcagc ttggttcgga attgccagtt cagtacaagc tcgttctgct gggcagcgtc 58380

tacatctgcc cacgtagccg gactcacctt gaacagctta tccgagccaa ggaaaggctc 58440

agtcagggca gagtaccgcc actcagcctg acgacgaatc agcttcggct gtacttgcga 58500

tcggcccttg actgctttcg gtttggcagc tccccgtaca ttccggagat cgttccatcg 58560

ttgaatgtgc cccgtctgta cttgttgaga aggctgtgcc tgctccaagt cgtgcttcag 58620

gtcttgaatg ctcggttcct tcccccagtc cgtcaacttc tgagggagca aagtgctcgg 58680

agcgagttgc agcttttcgt ccattttggt catccgtaaa aaggtttcgg tcgttgatca 58740

gttgtttgcc gactgcttgc aactgagagt cacgcagtct aagagtttcc ccgagttctc 58800

gtaccagact tctgccttct tcaagagatc ggtcgagttc ggctgtatgg cttgcaagat 58860

ctcggcagga atcggatccg ccttgggctt gacgttgata taccgaggct ctggcttcag 58920

actgctgcac cctgagattg tactcactac gagcagcagc aaggtcaact gcatgggact 58980

cacgagcttt ccggagtgca ctctcgatct gagttgtttc agcacgatgg atttcctctt 59040

tcacaacaag ttgttcttgc aggtcttgga tcttggcatg ggtctcaatc ttctcttggt 59100

tccatgcagc ttggacgcta gtcttaccag catccctgcc aaagtagtaa ccaagagcga 59160

agaccgtaag ggcaatgccc acgtagctga acaccttcag tacggtctct ttcatcatcg 59220

atccccttca caagccttgt cgatgttgga gtcgattcgg ttatcgatcc agccgaccgt 59280

gaagtccgac atcttcagac catcgtagtg accggtctgc ttggcattca ggagcttcag 59340

ggtaagggta catgccttga acttccctcg ctttttctga agggcttggt atgcacctac 59400

cgttccgggg ccgactttac catcgacctg aatttgagga aagtccttac caccacggga 59460

cagggagtta agactctctt ggaaccaacg ggaaggacga gcagggccga catttacacc 59520

ggagtcgacc agcttgtgca tgatcggaac cgagacagtc tcgaaacgga tgaaacccgg 59580

atcgacgatg tacttctgga catagatgtt ggaagcatcc tccttggtca gatccttcat 59640

ggagccgata tagccagccg aacgtgcgac agacaccgtc accccatgat tggtctcacc 59700

acccggatcc ttcgggttgt tcacgtatcc accttccact gcgaagatcg atgccaccat 59760

ggcagcaacg acagcagaga tggatgcctt ctgtttaaga ttgattgcca ttggaacctc 59820

cgaacttgat gatccctcgg atgatggcag caccaaacag cacaatcgag attgacttgg 59880

tgtgttggaa atccgctttc agttccggtg gaattgcctg ccacgcttgg gtcaggattt 59940

cagggagagc cagaacagct acccctgcgt aagaaagcca agtagcaggt tccttgtgaa 60000

cctgacgaac cttggacttc ttctttaccg gtttgacaac cattggatga tctcccctcg 60060

aatagtgaaa aggaacgaga tgatcccccc aaggatcgcc cacgtccact taccagccag 60120

tcctgcacca actaccttat gcttgatggt gatgaattct tcgatggtag gggcttgttg 60180

cttcaagcta ccctccacaa cctgaactcg gttatccatc tgagttaccg tctggctcat 60240

ctcttcaagc ttctcgtact gaatcttacg cgaggctttc gcctcgctca gttcagtagc 60300

gatgaagttg aagcgttctt caagacgtgc aagacgcact ttttcgtcct cgttcatagc 60360

cccgaccatt ggttaaagta aagaaaacgg gagcctaagc tcccgtgcga atagtacctc 60420

aagttacgtt ttgctacaga tcaaatggtg gacgggtctt cggcttctcg taatccggaa 60480

ccacagtgat gatgtcctca cgatctgtct caagggccac tcggctggag tggggcagga 60540

agaagttctt gggttcttgt ttccagtcag cgaatggaac aaaaccccag aacgagcgaa 60600

ctggggtttt catggttaca ccgccgggtc gatcggatcc ggatcatcga ccgcatcttt 60660

gtattccgga agggtacgca gatagtcgta cgcttgacgg aaggggttgg ccccctcgat 60720

gtcgtacttg atcgagtaca tctccgaggc gaaagcgttc ggcgcaccag cacgaacata 60780

cttgcggatc gagaacgtgt tgatcgaacc agccagacga acttcctcga ctcgaacgta 60840

tgcaccgttt accgggatat cgcccggtgc cgtcgtattt acgataagac ccattttggt 60900

cacctcttag aaccattgca cattgaatcg tgcggtgtta gtgaaactca agggcactcc 60960

cgagagttgg ttaatgcgta ctgagagagt ttgtcccggg gttactggga tgactccagt 61020

gacaagggag caattactag caccactgga cattttctct tcttcggcta caggcccacc 61080

atcaaggtag atacctacgt atcgttgccc agtgccattg gattgccacg atccgttgag 61140

ggtagcagat gcccacttaa cattgggcgg tacaacaatg agcatcttac cagcctcatc 61200

aaatgcttca agacggtcgt actgtgaacc cgaccagact acagtttgcg gtacgccaga 61260

ctgaactgtg atgcctgacg agaggtaaca gttcgtatgc gcaggttgtg tcccccacgt 61320

catacaccca tctgcattgg cgaaatccaa cgtatcccca tagatagaga tgtctgcacc 61380

agcgccaaca actacttgtt gccgaacgct gaaccccggc agattacctg aacgacacca 61440

cgcaccgtgt ccaaacgatg ggtggtttgt gagactcttt ggctgaatcg tagtcaaaac 61500

ttccatgcgg ccaacgcaag catagaagaa atggttctgc gccagtgaac ccgggtaatc 61560

tgctgacacc ggattcgtac cagtatccga tgcatgcaag gcgatagtac cgttccaacg 61620

acagttaaag aacgtattat cgtccgagtt ctcgatgtca acgtgtcgac tttgatagtc 61680

tgagttgaac cattgacagt tgtagaagat gcactggttc acgccaccct tcaacccatt 61740

accgaacatc aacacagact tagctgtatt gcccggaagc atagcagtgg acacacagaa 61800

gttgtcaaac tcacacagtg agatagatgt gttagttccc gatccaccgt catacaggtc 61860

gtgggtagta acacccaata ccagccccag ctgggtgtgt cgaaccacag agctgtttac 61920

gtacttagct ccacgtgtgg ccagaatctt cacaccgtgg gttgctcgat tattgcagtc 61980

aaacattacc ccttcaatac ccccaccctg aagtggtgca gctttaagac ccggttcatc 62040

tttaggggaa attaccagca tgaacgaggg agtagttgcg ccaacccact ggagagtcga 62100

acctgcgtta gctacgatgt tgtcccaagt gtcttggaac ggctggttac catcgattgc 62160

cccaccgtat ccacggataa gaatatacgg ggaatcgaca ataatggtct gggaaatctt 62220

gataacgcct ttaggcaagt taatagtggc acccttaaga cgtttagcct cagccactgc 62280

acgaataact gcacctgacc aatctgcttc accagcgatg aagaatgaat ccacataaag 62340

ctggttatcc agaattgcag caacagtctg gacttgtcca ttaggacgac gataaccaat 62400

aagaccagca cctgctgaag aatgcaatgc atcaattgcc tgcttgttct gactgatctg 62460

atccagatac ccagacagat cgaagatgtt acgccaagca gaatcaggct cgttcgtgta 62520

cttccactgg atatgtgcat cagcagacac tcgaatctcc ggggactgcc cagtgatgtc 62580

gatgagcgga agcaagacct gccacgggcc tttcgtacca tccttctcga tgtatcgcca 62640

atccagcgtc ttgttggcga cgttctcctg aagctcaatg tccttgggac gaagctcgtt 62700

caggttctcg gccagatact tgatgtgttg aagctgacgg taaacagctt ccacaatcgg 62760

gaagctgttg cccagtcgct tatcaacaga catcgaagca ccaccgaagg ggttgtgtgc 62820

tccacgttga ttcatgtgtt cctcacagtt agacggatac ggcaagtttg cctgtaccag 62880

tgccagcact tgcaaacttg atctgtgccg tgatggtagt ggagttcgtg ccaatcacag 62940

ccatccaagc aataggcgga atgctaccag attcatatga gaaggtaaga gatacatcct 63000

ctcggttggg ggttcgtaca agcttatgag agagggttac cgtctgtaca gcagtagagg 63060

acaagtcaat agccccatac ccaacaaact tgttggagaa gttgtccgtg ccatcgtaat 63120

tgctgatatc agcaaccttg tacatacgag ggcctaagcg ccccaagtta ccgaaagcag 63180

tggtaaccgg tcgatccgaa cgcagacgat agatggttcc gatgtcggca ttcacaccac 63240

ccggaaggtt gatgttcgta tcacagaaac gagccgacac atcaaggtcg ttacgtacgg 63300

tagtcgaagc ttcttcgtac ccaacttgca tggtgttggc atccctcttg gatacaaccg 63360

tagccttgat cttgctggac gatgcttcgt tcaggaagcc cttgccacca ccagtgaatg 63420

cgttcgaata cccgtacacc tgtgcagcga tgtcatggtc ggaaccaacg agatggatac 63480

cgagactacc gttctcagga gcatcacgga acacgtcgat cttggcctgc ccaccgagtg 63540

tagaaatgat cttcacttgg ggattggctc cagcgagagc atcattcggg ccacccttta 63600

cagggccacg tcgattcgag tgagtctcca gaaggttgat gttgtatcgg acagcatcac 63660

cgatgaagac gttgccgtat gagttctcac ccatcaggta gttggcccgg aagcgaatcg 63720

acgggtttcc tgccttacgt tcgatgtgca ccgcataccc atactcattg tcgtacgagt 63780

ggataaagcc cacctcgcac gaacgaagga ggcggatacc gtggatcgga tcaccgggga 63840

acagcttaga ctgtttcggg ccggtggcat caaacgtatc aaaacgggaa ccacccggcc 63900

aaccaacgaa gatgttgtcc agatattgat cagttgggcc gttgaagatg aacccttcca 63960

tgcccgtttc cagcacttcg atatcccgga tctcggaaaa cttcgagtcc gtgatcgagt 64020

cgaaattggt gtaagaaggg atagcaggga actcggtgta cagaccaata cctgctgtct 64080

tgccgatgac cagactcttc agtcgaagca tacggccata gatgcgaaca ccgtaaccgg 64140

tcttatcggt aggttgacct gagaacacgg caccattcac ggtgcagttc tcaacaccat 64200

acgagatggg gcaaccatca gcagtacctg ccggttgggt tgcttgaatg gcatcgaagt 64260

tttccgtctc cacaacgttg cctgcaaagc ctgcttgagt gacgatctgg gaagatgcag 64320

ccccagagcc aaccaaggta acggtagata ctagtttgat accagtgtat accttgagga 64380

ttccgttgaa atgaacttcg ccaatgccaa gagacttagc agttttcatt gctatattgg 64440

cagcaaagtc agaatccccg taccactgaa gttggatgac cccactgaac tgacggcgga 64500

aggtctttcc ggaattcgta gtcagaaaca cgccaccatc atctgtcccg gtagctcgaa 64560

cgaagagacc accagtaagc acgtcagtaa cgatgagagc atctgcagta ccagtgtact 64620

gacgaagtgc cgtgtaatta gccacacgag gtgtggaggc agcaattgct gatgtagcag 64680

cattctcggc aagggtagct gaagcttgtg ctgccgctgc cgcatcaagc gcctcttgtg 64740

ttgcagatcc agtatcacct ttcggcccct tgaagaatgg gtacaactgg atcacattat 64800

catcacacga cataaatacc tcagttggtt acgtgatcaa ctacgtcaat gaaggaggta 64860

gtcgtatgta caacttcccc attcgagtcg accaccttga aatcgatgta gtgtacttgc 64920

ggacgccact tctccgtatc tttaggaggt gcgtacacac gtacggtatt ggtctgtgca 64980

tccagcactt cgacctgaag atcttgtacg aactcaaacc gtaccttacg acggagtgct 65040

gccgttacca cacaatcctc aagagtgaat ggggcacact cacccggatc ccaaaggaag 65100

ccgccatagc tgaaggagcc acctcgttta aatttgttca tgtgtatccg gggacagagt 65160

ccccgtcccg ttttaggtga gggtcacaac tttaacagca ccggtgttgt ccacaaagct 65220

cagggcattg gcattggcag agtcaacgaa gaaggcgttc ttcttgccac ccaagtctgc 65280

aagcacagcc ggagccaact ccatgaactg cttcacggaa gcaaccgaag tgctaagcga 65340

actgacacca ctgttcgtcg aggaaagccc cgtggacaaa gaggcgacag aactggttgc 65400

cgtactgagt ccagtcgaag cagacgtgga cagagacgta gtggtgctca gtgctgtgga 65460

cagggcagtg gatagcgaac caatcccact ggatactgta gtggacaggg atgccaccga 65520

actgacggca gacgacagac cagtctgcgt cgaggtggac aacgattcaa ccgaactgtt 65580

ggtcgaggac aaaccagtcg aggtagctgt ggaaagcgac gagatacccg tagctgtgct 65640

ggtagacagg ctaacgatgt cttgggttgc cttatcggca attacacgag tctcttgaag 65700

aagagccacg taaccagaaa ggtcgaagat attgcgccaa gcactggcag gctcatccgt 65760

gtatttccac tgaagcatgt acccggcact gattcgcatc tcgggactct gccccgtgat 65820

atccaccaga ggaaccagca cctgccaatt acccttggtt ccatcttggt tcaggtatcg 65880

ccaatcgagg gtacggttga ccgtattctc gatcagctcg atatctttcg gacgcaactc 65940

acccagatgc tcggacacgt aggtgagctt ctgaagattc ttgtaaacct cacggacgat 66000

tgggaatgca ttgcccagac caagatctac catggccgac ttgttctcgt cgtcttgaaa 66060

tgcattacgc atcagaccca cccccctttg ttgaaacgga tgttcgtagt cgacgaggaa 66120

gtggacacca gatccttgtc gatcacgtct tggcagatag cctcgtacat cgcaatgtga 66180

ctctgtgcga tgccttgggc ttcctgcgta ttcttgttac tgtacagctt gtgggctacg 66240

taggccaaca gtgcttcctc aagcacgtcg gggatctcga tctcttgttc ggtctcgtcc 66300

cgatcccagt agaggatagg gtgcttcgcc tgatacacga ggctcagtgc ctgaccggat 66360

accggacggg gaacctgaag cagtgcacct tgcggggaga ataccgattc tggttgagtg 66420

tcatcattca acgggatctg atcaccgaag ttgttgtaca ccgacaggat tcggatgatg 66480

tcatccgtga acggttcacg ggtcaagtcc ttgatgtaca ggaagtcctg accactcgtc 66540

tcccactggc tctcggcaaa cttcgacttg agatggtagt tcgtgatgtg gtcgtggagt 66600

tcgatcacca agttgctttc cttcagtgcg aaccgggagt acagacgaag cagtgcatcg 66660

ttcaagtgac gaacgacttt cggcttgaag tcatcccgga gtaccccaga accttcatca 66720

gcttcataaa gattctggag ttcaccgaag gagagctttt cgtaaagctc ggcaagggtc 66780

attggttcct cacacaatat aggaagaaag tcctgtagct tcctcgggtt ctgcatcgtc 66840

gtcgtacacg tccggtgcac tgtgtgcacc taccggtgcc gactccgatg gcttccacgg 66900

tttgaagtaa ccaagcatgg atatggtgtc gagtgcatca tccttaccct tcaggccaga 66960

ctgcgtggcc aatcggatct cggtcatgaa ctcggccatg atcttgctgg tcttcatctc 67020

ttcaggccag tagaacttgc ctgccttgaa ccaaggaacc acgaggttga atcgggacag 67080

cttgtcgacg acaggtcgga tacccggagc accggacttc tcggaggatg caaagttgaa 67140

ccagatattc cggttcatca tctcagactg gagccaaggg ataaatccac cctgctgacc 67200

tgttacttca atcccaacca tctgaggttt gtagatagag accaatcgga agagatcatc 67260

cacgttcttg tccatggtct gacgttcgca taccccatct acccagaacc aatcgccgtt 67320

cgcattatag gcccagaccg agatgacgga gaagtcggca gtctgcttgg ccgacgtggc 67380

gaagtcagtc gtgatgtaga agttgaagtt gctacggtac ttgagcaagt cagcacgttt 67440

gtaccacttg atctctgcat cctgtacaag acgttcttct tccgacgtaa tccggagcat 67500

aagctcctgc atgaatgcag caaccttacc ggtcttcacc gatcgttcgt atgcgtcgag 67560

aacgtagtcg tagctgaacc gatcttccca agctcccacg aactcttcgc gggtacaggg 67620

gaaacgttcg cataccggcc agacgttgac atcccaagca ccagattcaa cggcttcaat 67680

gaggatgtcg tctttgttaa agggcgtccc attgaacacg actttccgtc gagtcggatc 67740

gagtgcatag tcaatgcctt tgtaaacagt gtccttgata gcggccatgc tggtcttgga 67800

cttggcatca tcgtcgctga ccaagtcatc gagaatacag aagacaggtc gcttaccgaa 67860

gatcttggaa ccacggatac cggtcttggc accgaacatc ttgaccccaa gcttcaggcc 67920

atcgacgttg gtgaactcca agtagttgtc cgtgaacttg gcatcgggaa tccacttctg 67980

aaggaattct gagttggcgt atcgatactc gatgttctta cgggcagact tcaccccgtt 68040

gtccatggag tccgagacgt agatcatggc gtcgatacgg ccgaagttcg gcaggtagcc 68100

gaagactgcg atgtacagag tcatatactc catgaacaga gtcgtcttag cagcacctcg 68160

gaaacagagg ttagccagat acgcactgtc tccggagccg atcttgtcca gcatcttcag 68220

gtggacaggg ggagtcttgt tcgactcacc ttccttacca ttgaccagct tgatgaagtt 68280

catgaacgac aaagcgaact cggacggcat gtacgtatcc gagttcagct ctgcgtagtt 68340

cacctgatcc agccactcat cgagggactg ggcgatcaat gttcgctttg ttttcggggg 68400

ttggctcata cgtcttcagc atccacgatc ttctgagcag ccagactctt cgctgtgacc 68460

ccagcagcaa tgaggtcacg ttgctgttga gccaagctac caagagttgc cttcaggtct 68520

gcgagaccgg acgactctcg catgtcgatg ttcaccagcg ggcctgcttc cttgggcttg 68580

gccaagtggg acagcagact gttggcagct tccatgcgta ccttctccga ctgtgccgat 68640

cgcatcagtt gggcttggac gttgatggct tcctgatgca gatgctgatt cagtacccac 68700

gaaggaacca gactcttctc gatgatcgag ttgaccagct tacccttggc atatgcagac 68760

acataagcac tgatgtcctt ggacttagca ccacgtgcca gcagtgcagc atatcgctga 68820

ggaaaggtct tgaagtaggc atcttggttc gtgtcaccca tcagcttgaa gctgacgtac 68880

ttcactgccg agacgtagtc ttccgtcttg tacttcccct cagccatcac actggagtag 68940

ctgacgaagt tatcagcaat ctgttgagcc agcaccggat cactgacagc attattgatg 69000

tcgtcggcaa tcgcttgcgt atccacgtta cgcagtccgg tcgggagtgc cttggctacg 69060

agttctttgg tcagcatagt tgttctctct tacaggttca tctcagtaac acgcaggttc 69120

agccaatacg cataatcctg cattgcttca cgttgtttca ccatgtgacg gtgttttctg 69180

ggttcgagcg tatccacgaa cgtcttgttg gccagagctt tattcagacc ggtaagtcgt 69240

tcctcaacct gccgtagttc gacgaccatg cgtgccttgt agtctttcgg ctcgttgaaa 69300

ctttggtagg ccagaaggaa cccaaagaac ttccagcact ggctgaatgc gtcttcatag 69360

gcgtaacgca tgcccagttg agcattgaag ttctcgacga atacacacga ggactctcct 69420

cggatagtga agcgatcgaa gaacttgatc gaacagatgg tcgtcttacc atcgtgaagc 69480

acatcgaagc tcacgtcttg catggcttcg agcatcatca gttcagttac tttcggtgca 69540

gtcatggtct tctccttgga aaatgggata tactttaggc tgtgtttagt cgtcagtcaa 69600

actctatctg taatgggatg ggaggcagag aactatggtg ttccacacgt ccccctacaa 69660

atatagtttg caaattcacg agagcacgct tacactgacc ctgttgttgc ggttggtgag 69720

aagcgtgagc ttgccatgag accccagcta gtggtgggga taataactac tagcagtcag 69780

agccaagacc aagaagtaac tcctacttgt gtgatctgac cagtcccctt gctgcctcaa 69840

ggtgtattcc ctgatagctc agtaggtaga gcacttgact gttaatcaag gtgtcgcagg 69900

ttcgagccct gctcggggag ccaaagaaag aaggattgga tatctagtct ttccctcttt 69960

ggtaggtata gatcaagaag agatcaccgg cctatagccg gagaccgttg gtgagatccc 70020

aacacctctg ccaaacagaa atgggaagac gttccctaaa gtcctcggac acaatagccg 70080

tagtacacga gagtgtggcg aaaagacggg gcaactattc aacagaccac cttagggtgg 70140

tcttttcatt tcaggagcca agatgctaga tctcacagcc ccatggtaca aggatgacag 70200

cagcaactca ggcatctggg ttcctgaatc cttgggccat ctgcttgatg taacgaacac 70260

cctatatacc tcgtaccact tctactgtac tgaccgtgac acgtatgaag ttctcaaagc 70320

cctccttctc gtacaaggct tggctcataa ggttgagtac ggtaagccag acctgatccc 70380

acgtaaatgt gtagatacgt atggcatctt gcacatggcc gcatgccagt ggcaaccgga 70440

agatggatcc cttcacaaca acccacgctt cgacgtgaaa ttcgcaaagg ttagccatga 70500

ctacatggac atctgatcga gtccgcagtg caggaggatg aatgagtatc catgaccagt 70560

ggtacatctc tggtggggac atccaaccga tcagtatcca agaaggactg gagaacgaac 70620

acaccttcat cgtcagtaac aaggacgaag aggctgaact ccgtgcctat gcaatgttgc 70680

tcggtcacac tgctctcgtc aaagttatgc tgggagcaag acagtacacc cctgacttga 70740

agcataagga tggtgtgttc accttctact taacccagta cagaggaaga gatgacagat 70800

tcaacttcat ccccctataa gttcgaccga tgggtagggt actgggcagg cgaggtacgt 70860

aacaataacg acttcgcctt atcagccctg actacatcca accctgacgg tgacgtattc 70920

gtagtacgga accaagaaga caaggaagag tgcctagcct tcttcgccct gatgggcttt 70980

aaccgtaggg tgatcctcta cttagaagac ttcccccaag gtactgcacg agcattcata 71040

ccttgtgcag atatgccaac ccataaacca agaccgtacc atccatgacc ccaatcctcg 71100

aatgtaagtg gaaggtagcg tctggtggca ctacgctatt taaactatgt gaccttatgc 71160

gagttaagga catagtcttt gtcgtactgc acgaaggtga ccgcaacacg cttaaagcgt 71220

atctcttagt tgagggatgt acgaacccag tcgtggatag ctacgacctc gtacctatga 71280

ataagatgcc catggctccc atgcactagg gtattagaac tatctttgta taggatgcga 71340

ttctggctaa aattttgcaa gggcagaatt ttgatagcta tgttccacag tagggtat 71398

Claims (9)

1. A purified bacteriophage having a genome of double-stranded DNA comprising a nucleotide sequence of SEQ ID NO.1 and having an antagonistic activity against Burkholderia melioidis.

2. A purified bacteriophage (Burkholderia pseudolei phase) vB _ BpP _ HN01, which is preserved in China center for type culture Collection at 4 months and 15 days 2022 at the preservation address of Wuhan university in Wuhan, China with the preservation number of CCTCC NO: M2022422; the bacteriophage can be used for antagonizing the growth of Burkholderia melissii strains, including Burkholderia melissii of different hosts, and can be used for preventing and treating melioidosis diseases, lung infection and/or bacteremia.

3. A composition for preventing and/or treating infectious diseases caused by one or more kinds selected from Burkholderia melioidea, which comprises the bacteriophage of claim 1 or 2 as an active ingredient.

4. An antibiotic comprising the bacteriophage of claim 1 or 2 as an active ingredient.

5. A disinfectant or a detergent comprising the bacteriophage according to claim 1 or 2 as an active ingredient.

6. Use of a bacteriophage of claim 1 or 2 in the preparation of a medicament for use in a method of diagnosing a causative agent of a bacterial infection, comprising:

(I) culturing a tissue sample from a patient;

(II) contacting the culture of step (I) with a medicament comprising a bacteriophage of any one of claims 1 to 3;

(III) monitoring evidence of growth and/or lysis of the culture;

wherein evidence of lysis of the culture indicates that the culture comprises burkholderia pseudomallei.

7. Use of a bacteriophage according to claim 1 or 2 in the manufacture of a medicament for reducing or inhibiting colonization or growth of burkholderia melioides on a biological surface in contact therewith, said biological surface being mammalian skin, damaged skin or mucosa.

8. Use of an effective amount of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating or reducing the incidence of a bacterial infection by burkholderia pseudomallei in a subject in need thereof by administering the medicament.

9. A non-therapeutic method for reducing or inhibiting colonization or growth of burkholderia melioidis on a solid surface comprising contacting the surface with the bacteriophage of claim 1 or 2.

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