CN112760296A - Vibrio parahaemolyticus bacteriophage RDP-VP-19010 and application thereof - Google Patents
Vibrio parahaemolyticus bacteriophage RDP-VP-19010 and application thereof Download PDFInfo
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Abstract
The invention discloses a vibrio parahaemolyticus phage RDP-VP-19010 and application thereof in aquaculture. The pathogenic vibrio parahaemolyticus ZHBV-18001 is obtained by screening pathogenic oyster viscera from a Qingdao, Jimo and aquatic farm by using a double-layer plate method, and then the vibrio parahaemolyticus ZHBV-18001 is taken as a host, and the vibrio parahaemolyticus phage RDP-VP-19010 is separated from the sewage of the farm, wherein the preservation number is CGMCC No. 19384. The vibrio parahaemolyticus phage RDP-VP-19010 provided by the invention has strong lysis effect on pathogenic vibrio parahaemolyticus ZHBV-18001 in aquaculture environment, and provides a phage source for industrial production of phage for prevention and treatment of pathogenic vibrio parahaemolyticus in aquaculture environment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a vibrio parahaemolyticus phage RDP-VP-19010 and application thereof.
Background
In recent years, the aquaculture industry in China is rapidly developed. With the increasing expansion of the mariculture scale and the continuous popularization of intensive culture modes, the frequent occurrence of fish and shrimp diseases poses serious threats to the healthy development of the large-scale culture of aquatic products. The pathogenic bacteria of aquatic animals mainly comprise bacteria, viruses, parasites and the like, wherein the bacterial diseases have serious harm degree to the aquatic animals.
Vibrio parahaemolyticus is an important zoonosis, not only causes serious vibriosis of marine animals, but also causes infection and even death of human beings. The summer and autumn of each year is the outbreak period of diseases, and fishes, shrimps, shellfishes, crabs and the like are easy to infect. At present, the main method for preventing and controlling vibrio parahemolyticus is to use antibiotics, although the antibiotics have the advantages of convenient use, broad-spectrum bacteriostasis, quick response and the like, the abuse of the antibiotics also causes serious bacterial drug resistance, the effect is worse and worse when preventing and treating diseases caused by the vibrio parahemolyticus, and the development of novel vibrio parahemolyticus resistant drugs is urgently needed.
The bacteriophage is a bacterial virus, has higher abundance in nature, strong host specificity and can not destroy the micro-ecological environment. After the phage infects host bacteria, new phage can be released during cracking, and a small amount of phage can play a role in preventing and treating. The action target of the vibrio parahaemolyticus phage is on the prokaryotic microorganism cell wall, and no corresponding action site exists on animal and plant cells, so that the use safety of the phage is improved. The bacteriophage and the pathogenic bacteria can evolve together to prevent the occurrence of bacterial drug resistance. Therefore, the natural enemy phage of the bacteria is utilized to solve the biological control approach with wide prospect for the bacteria.
The invention provides a safe and reliable lytic proteus mirabilis bacteriophage for treating the proteus mirabilis disease of poultry, aiming at the order of comprehensive banning of resistance of corresponding countries, solving the problems of bacterial drug resistance and antibiotic abuse.
Disclosure of Invention
The invention aims to provide a vibrio parahaemolyticus phage RDP-VP-19010, and aims to solve the problems that aquaculture animals are not treated timely due to acute onset of infection with vibrio parahaemolyticus, and drug taking is ineffective due to drug resistance of pathogenic bacteria and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a vibrio parahaemolyticus phage RDP-VP-19010 is characterized in that, the phage plaque has the diameter of 4mm-5mm, is round and transparent and has no halo observed through an electron microscope, the head of the phage plaque is icosahedron, the length of the phage is about 61nm, the transverse diameter of the phage is about 67nm, the tail length of the phage is about 112nm, the diameter of the phage is about 10nm, the phage belongs to the family of metanaviridae with uroviridae, the phage is preserved in China general microbiological culture collection center in 03, 24 and 2020, the preservation address is as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 19384.
The bacteriophage is a virulent bacteriophage, has strong lytic property to vibrio parahaemolyticus, has a lysis rate of 99 percent, and provides a bacteriophage source for industrialized, large-scale and programmed production of the bacteriophage and treatment of aquaculture animal bacterial diseases caused by vibrio parahaemolyticus.
Further, the bacteriophage RDP-VP-19010 is cultured at 37 deg.C for 1 hr in the range of 5.0-9.0, and the titer is still maintained at 109pfu/mL or more, and the titer reaches 10 at pH 7.010pfu/mL or more, and is resistant to weak acid and weak base conditions.
The invention also provides application of the vibrio parahaemolyticus phage RDP-VP-19010 in preparation of a vibrio parahaemolyticus resistant preparation.
The invention also provides application of the vibrio parahaemolyticus phage RDP-VP-19010 in preparation of a medicine for resisting vibrio parahaemolyticus ZHBV-18001.
Another objective of the invention is to provide a bactericidal composition for preventing and treating Vibrio parahaemolyticus infection, which comprises an effective amount of Vibrio parahaemolyticus bacteriophage RDP-VP-19010.
The invention also provides a bactericidal composition for preventing and treating vibrio parahaemolyticus infection, wherein the content of vibrio parahaemolyticus phage RDP-VP-19010 in the bactericidal composition is more than or equal to 109PFU/mL。
The invention also provides a bactericidal composition for preventing and treating vibrio parahaemolyticus infection, and also comprises an antibiotic for resisting vibrio parahaemolyticus.
The invention also provides an aquatic feed additive, the effective component of which comprises the vibrio parahaemolyticus phage RDP-VP-19010.
The invention also provides a cleaning agent or a disinfectant, the effective component of which comprises the vibrio parahaemolyticus phage RDP-VP-19010.
The invention has the beneficial effects that:
(1) the pathogenic vibrio parahaemolyticus ZHBV-18001 is obtained by screening pathogenic oyster visceral tissues from a Qingdao, ink and aquaculture farm, and the vibrio parahaemolyticus ZHBV-18001 is taken as a host, and the vibrio parahaemolyticus phage RDP-VP-19010 is obtained by separating from the sewage of the aquaculture farm, and the phage RDP-VP-19010 has strong cracking effect on the pathogenic vibrio parahaemolyticus ZHBV-18001 in the aquaculture environment, so that a phage source is provided for the industrial production of phage for the prevention and treatment of the pathogenic vibrio parahaemolyticus ZHBV-18001 in the aquaculture environment;
(2) the vibrio parahaemolyticus phage RDP-VP-19010 provided by the invention can maintain better activity in the range of pH5.0-9.0, the phage titer is not obviously changed, and the phage titer is highest when the pH is 7.0; with the increase or decrease of the pH value, the titer of the phage is obviously reduced, and the phage can tolerate weak acid and weak base conditions;
(3) the vibrio parahaemolyticus phage RDP-VP-19010 can be used for industrial production, and can be specifically amplified by host bacterium vibrio parahaemolyticus ZHBV-18001; the bacteriophage can also be used as a safe biological disinfectant for environmental disinfection and animal body surface disinfection, thereby treating aquaculture farm pollution; the bacteriophage can also be used as a medicine for preventing and treating diseases caused by Vibrio parahaemolyticus.
Drawings
The above and other features, properties and advantages of the present invention will become more apparent from the following description of the embodiments with reference to the accompanying drawings:
FIG. 1 is an electron micrograph of the Vibrio parahaemolyticus bacteriophage RDP-VP-19010;
FIG. 2 is a graph showing the one-step growth of the Vibrio parahaemolyticus phage RDP-VP-19010 of the present invention;
FIG. 3 is a schematic diagram of the acid-base tolerance test of the Vibrio parahaemolyticus phage RDP-VP-19010 of the present invention;
FIG. 4 is a graph showing the lysis profile of the Vibrio parahaemolyticus phage RDP-VP-19010 of the present invention against the host bacterium ZHBV-18001;
FIG. 5 is a schematic diagram of the heat stability experiment of the Vibrio parahaemolyticus phage RDP-VP-19010 of the present invention;
FIG. 6 is a schematic diagram showing the genetic stability curve of the Vibrio parahaemolyticus phage RDP-VP-19010 of the present invention.
FIG. 7 is a schematic diagram of RTD experiment of bacteriophage RDP-VP-19010 of Vibrio parahaemolyticus of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention screens and obtains vibrio parahaemolyticus ZHBV-18001 from pathogenic oyster viscera of a Qingdao, ink and aquatic farm, and determines pathogenicity and drug resistance of the ZHBV-18001 through experiments, and separates and obtains vibrio parahaemolyticus phage RDP-VP-19010 from the sewage of the farm by taking the ZHBV-18001 as a host; and the genotype of RDP-VP-19010 is determined by the whole genome sequence.
Example 1 isolation and identification of pathogenic Vibrio parahaemolyticus ZHBV-18001
(1) Sample treatment: collecting diseased Concha Ostreae, washing with clear water, taking out viscera, and grinding.
(2) Vibrio appreciation: 0.5g of the sample was incubated at 37 ℃ and 180rpm in 5mL of a growth medium for 18 hours.
(3) The bacterial suspension after the overnight culture is streaked on a TCBS culture medium and is placed under the condition of 37 ℃ for 12h-18 h.
(4) And (3) carrying out secondary plate streaking on the bacterial colony with the vibrio parahaemolyticus bacterial colony characteristics, purifying for 3 times, and then preparing bacterial suspension for storage to prepare for subsequent identification.
(5) And (3) identification: the prepared bacterial suspension is sent to Huada gene for identification, is identified as vibrio parahaemolyticus through molecules, one strain is named ZHBV-18001, and is stored in a refrigerator at the temperature of 80 ℃ below zero.
Example 2 isolation and characterization of the Vibrio parahaemolyticus phage RDP-VP-19010
Taking sewage samples of different places of a diseased farm, centrifuging for 10min at 5000r/min, and taking supernate to pass through a 0.22-micron microporous filter membrane; the filtrate, 3 XTSB and mixed strain were incubated at 37 ℃ at 100r/min overnight. Taking supernatant, carrying out phage separation by adopting a double-layer plate method, matting and purifying bright plaques in SM buffer solution by three-time plate-laying to obtain a phage, and naming the phage as RDP-VP-19010; phage multiplication fluid was mixed with 60% glycerol according to 1:1, and storing in liquid nitrogen.
EXAMPLE 3 Electron microscopy of phage RDP-VP-19010
Dropping 20 μ L of liquid containing crude phage particles on a copper net, naturally precipitating for 15min, sucking off excessive liquid from the side by using filter paper, adding a drop of 2% phosphotungstic acid (PTA) on the copper net to dye the phage for 10min, sucking off the staining solution from the side by using filter paper, and observing the form of the phage by using an electron microscope after a sample is dried.
As can be seen from FIG. 1, the phage plaques have a diameter of 4mm to 5mm, are round, transparent and have no halo, the heads of the phage plaques are icosahedron, the long diameter is about 61nm, the transverse diameter is about 67nm, the tail length is about 112nm, and the diameter is about 10 nm; according to the ninth report of the organization of viral taxonomy of International virology, the phage can be classified into the family Myoviridae of the order Narivulales.
Example 4 phage genome sequencing
After enrichment culture of a single phage, centrifuging for 15 minutes at 8000r/min at 4 ℃, adding 10% PEG8000 and 0.5M NaCl, standing overnight, adding equal amount of chloroform, mixing uniformly, standing for layering, centrifuging for 10 minutes at 5000r/min, removing a chloroform layer and a PEG layer, adding restriction endonuclease for digestion treatment, suspending the phage under the condition of gradient density cesium chloride, dialyzing for 3 times at the later stage by TM buffer solution, and performing whole genome sequencing by Chihuada gene biosequencing company for 30 minutes each time. Sequencing results show that the phage has no virulence genes and lysogenic genes.
Example 5 mapping of phage growth curves
Taking the Vibrio parahaemolyticus suspension in the logarithmic growth phase, inoculating bacteriophage RDP-VP-19010 according to the optimal complex number of infection, carrying out water bath for 10min at 37 ℃, then centrifuging for 5min at 12000r/min at normal temperature, removing supernatant, removing free bacteriophage which is not adsorbed on a host, and washing twice by using 2216E culture medium. The pellet was then resuspended in 2216E broth at 37 deg.C and quickly placed on a shaker at 37 deg.C and 200r/min for shaking and timing. And (3) drawing a growth curve by taking the time as an abscissa and the titer logarithm value of the phage as an ordinate, obtaining the incubation period and the lysis period of the phage, and calculating the average lysis amount. Mean lysis is end phage titer/initial host bacteria concentration.
As can be seen from FIG. 2, the titer did not change significantly within 30min after the phage infected the host bacteria, indicating that the incubation period was about 30min, and the titer of the phage increased significantly within 30-80min after infection, then became stable, indicating that the phage lysis period was about 50 min. By calculation, the phage lysis amount is about 163 PFU/infected cell, which shows that the phage has extremely strong lysis and replication capacity.
Example 6 determination of phage pH stability
Adjusting pH of normal saline with dilute hydrochloric acid and dilute NaOH solution to obtain buffer solution with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and diluting phage with prepared buffer solution to about 1 x 1010pfu/mL, the diluted solution was incubated at 37 ℃ for 1h in a water bath, diluted 10-fold with physiological saline and plated on double plates. Counting after inverted culture at 37 ℃ for 4-6 h.
As can be seen from FIG. 3, after 1h of incubation under different pH conditions, the phage RDP-VP-19010 can maintain better activity at pH5.0-9.0, the titer of phage is not significantly changed, and the titer is still maintained at 109pfu/mL or more, and when the pH is 7.0, the titer of the phage is the highest, and the titer reaches 1010pfu/mL or more. The titer of the phage decreased significantly with increasing or decreasing pH. The phage RDP-VP-19010 can tolerate weak acid and weak base conditions and has good acid-base tolerance. The optimal growth pH of the bacteriophage RDP-VP-19010 is neutral environment.
Example 7 determination of optimal multiplicity of infection
The phage multiplication solution and the host are added into 2216E culture medium according to the proportion of the multiplicity of infection of 10:1, 1:10, 1:100, 1:1000 and 1:10000, and the total volume of the culture system is ensured to be the same. After shaking culture at 37 ℃ and 200rpm for 8h, centrifuging at 12000r/min at normal temperature for 5min, and spreading the supernatant on a double-plate to determine the titer. The results are shown in Table 1.
TABLE 1 phage RDP-VP-19010 optimal multiplicity of infection results
| Serial number | Multiplicity of infection | 8h phage titer (pfu/mL) |
| 1 | 10:1 | 6.13×109 |
| 2 | 1:1 | 9.62×109 |
| 3 | 1:10 | 2.69×1010 |
| 4 | 1:100 | 7.96×1010 |
| 5 | 1:1000 | 3.21×1010 |
| 6 | 1:10000 | 6.61×109 |
As is clear from the data in Table 1, when the number of infection was 1:100, the growth medium was relatively clear and the titer was the highest after 8 hours of culture, indicating that the optimum number of infection of RDP-VP-19010 was 1: 100.
Example 8 phage vs host lysis curves
The lysis curve of the phage on the host is obtained by adding the phage to the bacterial suspension, and the OD600 value is changed due to the lysis effect of the phage on the host. The host bacterial suspension cultured for 6h is inoculated into 100mL2216E medium at a ratio of 1:100, and cultured at 37 ℃ for 2h (OD about 1.5, about 6.0X 10) with shaking at 200rpm8cf/mL) were divided into 4 sterilized Erlenmeyer flasks, and phage multiplication solution (filtered through a 0.22 μm membrane) was added to 3 of them at the optimum multiplicity of infection, while taking the average value with no phage added as a control.
As can be seen from FIG. 4, the Vibrio parahaemolyticus phage RDP-VP-19010 is added into the suspension of the host bacterium ZHBV-18001, after 30 minutes of action, a certain lysis effect appears, after about 40 minutes, the OD600 value begins to decrease, the action lasts until 150 minutes, after 180 minutes, the mixed bacterium liquid begins to become turbid, and the OD value gradually increases. As the phage is in the adsorption and infection stage of the host bacteria in the first 40 minutes, only a small part of the phage starts to crack the host, and therefore, the amount of the cracked host is far smaller than the increase amount of the host, the OD600 still keeps a certain rising trend in the first 40 minutes, the number of the cracked host is larger than the proliferation speed of the host along with the exponential increase of the number of the phage, the OD600 value starts to be gradually reduced, and the mixed bacteria liquid is clearest. After the continuous action for 2.5 hours, the mixed bacteria liquid turns back turbid, the original sensitive bacteria are cracked by the bacteriophage, and a flora replaced by the mutant tolerant bacteria is alternated.
Example 9 phage thermostability assay
The phage stock solution is subpackaged into EP tubes, incubated at 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C and 70 deg.C for 30min and 60min, diluted with 10-fold physiological saline, and spread on double plates to determine its potency.
As can be seen from FIG. 5, the activity of the phage was kept stable at 30 ℃ to 45 ℃ and the titer was kept at 1010pfu/mL or more, and is substantially inactivated at a high temperature of 80 ℃. From this, it was found that the phage RDP-VP-19010 is suitable for survival at 45 ℃ or below.
Example 10 genetic test for stability
RDP-VP-19010 was passaged 29 times continuously, and the same amounts of RDP-VP-19010 and ZHBV-18001 were added to each medium generation for 6 hours, followed by plating on double plates to determine the titer.
As shown in FIG. 6, the titer of RDP-VP-19010 was measured after 29 consecutive generations, and the titer stabilized at 1010And pfu/mL or more indicates that the phage has better genetic stability and is suitable for industrial production.
Example 11 cracking Rate experiments
Under aseptic conditions, 1mL of sample and 1mL of host bacterium solution (1X 10)6CFU/mL), incubating for 15 minutes at 37 ℃, mixing uniformly, and diluting to 10 with physiological saline-1-10-3Each gradient was plated at 100. mu.L onto TCBS agar plates and incubated at 37 ℃ for 24 hours, with each gradient repeated twice. Simultaneously taking 1mL of physiological saline and 1mL of host bacterium liquid (1X 10)6CFU/mL) as a blank, repeat the above steps. Plates with 30-300 colonies were picked for enumeration. The test was repeated 3 times and the average was taken. The phage lysis rate was (1-number of treated colonies/number of control colonies) × 100%.
The cracking rate of RDP-VP-19010 reaches 99%, has good cracking effect on host, and is suitable for use in aquaculture process.
The cleavage rates of RDP-VP-19010 and the existing Vibrio parahaemolyticus bacteriophages RDP-VP-19003, VP-HYP2, SX-2, SX-3 and VpJYP2 were compared, and the cleavage rates were determined by the above-mentioned cleavage rate experimental methods. The results are shown in Table 2.
TABLE 2 comparative experiment of cracking rate
| Name of bacteriophage | Cracking Rate (%) |
| RDP-VP-19010 | 99 |
| RDP-VP-19003 | 96 |
| VP-HYP2 | 63 |
| SX-2 | 58 |
| SX-3 | 80 |
| VpJYP2 | 74 |
As can be seen from Table 2, the cracking rate of RDP-VP-19010 is significantly higher than that of other 5 strains of bacteriophage, the highest cracking rate of the RDP-VP-19010 to host bacteria is 99%, and the RDP-VP-19010 can effectively inhibit the growth of Vibrio parahaemolyticus, and shows that the bacteriophage has the potential of replacing antibiotics to treat diseases caused by Vibrio parahaemolyticus, and has wide application prospects.
Example 12 RTD experiment
Dividing the region on the plate, adding a drop of host bacteria (about 100 μ L) to the center of the region, air drying, adding a drop of phage with different dilutions to the spot of host bacteria, air drying, and culturing at 37 deg.C16-24 h. According to the growth condition of the host bacteria, the CL concentration is called as the CL concentration (the CL concentration is the concentration of the host bacteria in the environment which can be completely lysed, and can be used for guiding the dilution scheme of the production practice). The results are shown in FIG. 7, where RDP-VP-19010 is diluted 107After doubling, the bacillus subtilis still has better lysis effect on host bacteria.
It should be noted that the specific embodiments are merely representative examples of the present invention, and it is obvious that the technical solution of the present invention is not limited to the above-mentioned examples, and many variations are possible. Those skilled in the art, having the benefit of this disclosure and the benefit of this written description, will appreciate that other embodiments can be devised which do not depart from the specific details disclosed herein.
Sequence listing
<110> Qingdao Runda Biotech Co., Ltd
<120> Vibrio parahaemolyticus phage RDP-VP-19010 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50642
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
aaagataaca aagaaatcca gaaaaaccgt caaaagcgca aggatttaaa gacagctatg 60
cgtaaactgg atgaagctaa caagcaggtt tacaaagacc gtcaacttgc tgagcgcatc 120
aaagctaacg tagctaaaat cactgagtaa tccatcaact agccacagga caataaaatg 180
aagattgaag atattgtaat tggtcaagag taccgtatcg ttcgaaaagg tttggtaaac 240
acaggtcgta ttggtcgtgc tgtaggcaag aatggcttta aagtcaaact actaatgcac 300
cacaacaacg ctgagcttac actgaatgcc atgtctgttg aaccagttga aaaaggcgta 360
aaagctaaga agcaagttga cgttaacgaa aaccgtaagc ctgttgacaa cgagcttgtt 420
aaacctaaaa agaaaccagc taagaagttc gtagaacgtg acgtatttat cgagccagct 480
ccagatgcta tcgttcttat tgaacaaaat ggctctgttg tgtcggtatc aagaagaaac 540
ttagagttct ttgatgaccg tctgctgtca cacggtgtag actatacaaa ctcttacctc 600
ggattcttgc ctcgtggcgc taagttcaaa gttaacaagc ctgacaatat catggagtat 660
cccttttgga ataccgatga aatgcaagat ggcatggaac tcacatttac taattggcgc 720
aatgacttcg gtgctacaat aatcaaaacg gaagaaaact ttgaaattct ggctaaatgg 780
gttgatgtag gtagtctgca caaaatgggc aaggctaagg ttgaagttga tgacgatggt 840
aatgaagtag ttccaccatt ggaagaacaa gctcttaaac gcatggaaaa agatgctaaa 900
gaagctagtg gtcttgcatt cgctctttac tgggattatc gtggtaagct tgagtctggt 960
actcgtaacc atgcagcatg tcactaccac atgcgttgtt tcgttgatgg tgataagcgt 1020
aaagatgcta aggcattgtt tgcagctcag tttattactg accaccacta cggcaacctc 1080
aagaaagacc ttgttgaccg ttacctaact tgggtaatga acgactctcc ttggtctgac 1140
tacatcgtca ctaaagattt ggctgagggt aaagagaagg gtatacgtgt tcgcactgat 1200
attccagcta acgcaatgat gtgtacattc tacgctctgc gttaccctac tgaataccgt 1260
gacgtaatca ctctgtgggg acgttacacg gacgagggct tagacccgac cattgccttt 1320
tacaaagctg aaactcgcgg ctatgaccgt gacacggctt ctcaccacgg cttgttggat 1380
acattctcaa tgacattgca agatgtgatt gattttgcca ataagaaacg tggtaagatt 1440
gtggatgaaa tctatggtgt taaatacttc aactacaaga acgtatctgt acaccacggc 1500
tcatctgtac gccgacttga aaagactgct ctaactgagc ttaaagataa gtttaagcct 1560
aagaaagtca aagaaaaaca ccctgttctg ggcgaaatcg agaaagaaat cgctgcaaca 1620
gagcaaatcg accaattcct aactagcaaa ctggagaaat aagatggaca tggataaact 1680
accgcaaatt atgaaagtta tggtcgtagg ttctggtatg aacctcgact accatcgtat 1740
gctcaacgac atgggtcaca caacagtggc ttgtcgttat gttgagcaaa ttcgtccaat 1800
cttgtacaaa ggtgatattg atgttgtatt atttgtgggt ggggctgacg ttaatcctga 1860
gctttatggt catacgttac atcccacgtc cggtatttct aaagccgaag atgagttgta 1920
ccaacaagct ttctgtcaag ctcaagaatt tgatgaaatc aagtttgtgg gtatttgccg 1980
tggtagtcag ttcttgtgtg ctgctagcgg tggggttctt gttcaacatt gtgataatca 2040
cgctgtgttt aatgggcata gtgttgaaac taagtggggt cacgaattta atgtcacatc 2100
gacgcaccac caaatgcaag acccacggga agtggaacac gaactaatcg cttggactaa 2160
gcgtactacg ttcaaagatt accacattga tggtgagaca gttaccattg aaactcaggc 2220
agaccctgat gagctggata ttgaagttgt ttggtatcca catactaagt cgctagcttt 2280
ccagcctcac cctgaatttg caggagctgg tgagacgtta gcattgttta aacgttgttg 2340
gaatgagtac atgtaatgca gaatgtaaga attgaattgt tgcaaggtcg tcgttttgag 2400
gatgtgccta taggtgatgt gtttctatat aatagctgcc cttacatcaa attgaacgat 2460
gacttaaact ttaactgtaa gaaattcttc tctaaaggta ctggcagtat ttctcgtcag 2520
acttgtgtta tagttcctga agaaattatc attaaataag gcaggtagtt atgaagctgg 2580
atacaagtta gaaatgtaaa tgattactgg ggtggattcc atgtttattt gaagatattt 2640
aattaattac agttttgtgt gtgatttgat gaattctcct taccacccca gtggttatcc 2700
ttattagtat taagtagtaa taataagaat aacttagtta ttagcgatag caaggctatc 2760
aatctgtaaa accattctct tgtatgtaca tactaacaca aaaaagtgtc actgtcaaga 2820
ggtaaatcaa tcaaagttaa gaaaaggtat taaattatgt gtggaattgt aggcgttgta 2880
ggtcttatta cggcaaacga agaaaaagta ttcaagcagt tacttgaagt tgactctcta 2940
cgtggtcgtc attcgactgg cgttattaag ctttcttctg gaggtgtagt agagacacga 3000
aagaaagcga ttgatggtat ggactttgtt aagcttgagg gcaactggat ttcatctggt 3060
gttaaccgta tgcttattgg tcataaccgt tatgcaactc gtggtggtat taacgatgct 3120
aatgctcacc cgttctcaca tggacacatt cacggtgttc ataatggtac actaacaact 3180
caacttggtt tgaaagacca acataaattc cctgttgatt ctgataacct tttctacgat 3240
atggcacatg gtagcctacg tgaaacagct aagaaaatct caggtgcttg gactgttgcg 3300
atgtatgatg aacaagagaa aacgttcaac atgttccgta acactgagcg tccaatgagt 3360
gttgctctct cagaagatgg tacagtaatg tacttcgcta gtgaagctac tatgcttgag 3420
tgggttcttg agcgtaatgg tgttaagtac aaagacattc ttaatctaac tccagccaag 3480
cttatcacaa tcgaaccgac tgccgtagca aacacagacg gtaaacgtat ttctgatttg 3540
atgtctatca ccgacattga gtacaagaaa gttcctgctg ctccggctcg tggagcaaaa 3600
aagttccttt ccgacttcgg gttaaacatt caaaaaaagt atccgttgtc gttaattgga 3660
cgaaaaaaga caaatgcagg gtatgagtat gaagttgaac ttatggttcc tccatacaac 3720
tcatggactg tgtatgctta ccaacctaag actgcggaag ttcttggtaa gttgtacgac 3780
aacgacgaaa ttgcatgttc tgtattaaca attggttaca acagcaatat gccacattgt 3840
tggggtatgg acatcgagcc agctaccgaa gaagctctta aaatccttgc tgatgaagaa 3900
gcggcagcgg cagcggctgt agttgaccaa aacgacgttg atgatttgac agaagatgag 3960
cttagtcaaa tttttgatgc gtatggcatc acccaaatca aaaaggaatg gtatgaccac 4020
aacggtttag ctatgacaga agctgagttt gaaaagactc aagcaggtca gcatggttgt 4080
tgtatgtgtg gcgaccactt ccaagaacca gaagatgtag tgtggaacga tgatggcgac 4140
actgcatttt gtacagattg tgtagaagca tacaacgcta tctacggaac tcaagcacaa 4200
gctaagtaat tcactaacag agagagattt aattatgtca aattcagtat tcggtaaagt 4260
aacactaggc gcagacccag aaatcttcgt agtaaaacct gatggtactc cttggggtgc 4320
attaactact ggctgtaaag gtactaaaga agtgcctgag cctacagagt acggtgcatt 4380
acaagtagat ggtctagccc ttgaatacaa cattcatcca gttgatacct taaatgattg 4440
gatggatttg cacacagcag ctttgacaca cattgctgac acagcagata aagctggtct 4500
tgaaattgac tcaagctctc gtttggactt tacctcatat attgatggta atggtgccac 4560
tgaagatgag ttactatttg gttgtgaccc tgactttaac gcagacacaa tggaagaaaa 4620
caccatgcca gataacgacg gctctatcaa gttccgtact actggtggtc atgttcacat 4680
tggtttagca aactggggtg aactacatgg cggtgatgat aagattgcac acatgatggc 4740
atctaaaatc atctttgtct gtgacgcaat ccttggtcta cgctctgtcc taacagatga 4800
cggcgtagct cgtaaagaat tgtatggtaa agctggtgct taccgtgtta aaccttacgg 4860
tgttgaatac cgtacactgt caaacttctg gctgtttgat gaagaagaaa tgaaattcat 4920
ctttgatgca ctaaacaata tctttgcatc cgatgctgag tttgaacgtg tgtcagagtt 4980
tgcaacatta aatcgttctg atattgttaa agcaatcaac acgaacgata aagagcttgc 5040
tatcaagctg ctagctggtg acgccccatt aactgataaa taaaggagta ccaaatgaag 5100
aacttccatg ataatgcaca cgctaataac cgactatccc gttgctttgt gtgtattaag 5160
attaatggca agtgggtaac taaacgaatc tcagaagcca ccaaacagag tattgtttgt 5220
cgtgatggtg agatttacga cctacatagt gatgatgtta agattacgtt tgataactta 5280
ggttatgtta acactcagca tcaagctatc ttcgttagtc gtactcctaa gcgtatgtgg 5340
aaagctggtc tagtacctga gaatattcag gcagagcaca ttcatccaca gcatctacgc 5400
cacggggata tgattgagtt tgacaagtgt ctagataagc tgatgaataa tgtgtaccca 5460
tcttaccgca tggcttacct gcgagttaag aacggcaagt gtgcagcatc agctttccat 5520
cgtaacttcg ctgtatcatt gagcgacttt acagaacatt tggttcttta ctacaagaac 5580
tacgaaatcg gattcattga taatgattct gaacaaattg tggtgggtga ctcttacacc 5640
catctacgtg aacaaatctt tgaagcaaca ggtatggagt gtatctcatg aagaaaatcg 5700
cagacatctt taaactaaaa cgtcctcgca atgatgactc ttttggtgta gagctggaac 5760
ttgagtataa caaccgtcct gctgcctcag atgaatggaa aatggagcat gatggctctc 5820
tgcgtgtcgc tggtatcgaa tacgtaatga aagagcctat gagcctagct gacagtaaag 5880
tagctttaca gaacctctca gataagttca aagctgttaa gaatcaccct aaagctacta 5940
acttggcatc atcccacatc cacatcaacg tctctcacat gacgttacaa gagtttatcc 6000
agttcgttag tcttgtacta ctattcgaac cacatatcgg taagcatggt ggtcaagacc 6060
gttggatgaa ctacttcgca gtgagttctc aagagagtgg tatcctaatt cgtgagttgt 6120
taaaagtgcg taccgaagaa cagttcgcta aattcatctt acgtgtaaat cagcgtgatt 6180
ggcgctacaa cgggattaac tttgcatctg tggcaaagta tggctcactt gaaattcgct 6240
acttaggggc tatgccagag cctctagatg ttattccttg gcttgagttc tacgatacac 6300
ttcgtgaagc agcaatgaac gacatccaat ggggtatggt gtttgaaaca gtatcggcag 6360
gtgagacatt cttacttgag ggtacattca aaactccatt ccctctaaat caagatgacc 6420
tattacttgg tgctcgtatg gcacaagatt tggtctaccc agtataccat gatgcaaaag 6480
gcgctccttt cgcagaaaca tggaaactta ctaactacta cgcaaacgta taaaggaaat 6540
ttaaacatgg ctatcccaat aaaaggctat gaggggctgt ataccataga caagcatgga 6600
aacatttgga gttacaatgc ttcaagaggt ttacgtaagt ccccaagaca ggttaaaaca 6660
acaaacacaa acggtgcagg ctactgtata gttagcttgt acaaagacgg gaagaagact 6720
tcacactctg tacatagatt agtggcagag cactttatcc ctaaccccaa caactacact 6780
gatgtaaatc atattgatgg ggataagaca aataatgatg tgtcaaacct agagtgggta 6840
acacgtcaag agaatagaat ccactacatt aaatttttaa aggtataata ttatggcgat 6900
taaattacat agctacaaaa acggttctga atctgctaaa ctacttcgtg atgcacttgg 6960
tatcaagatg ttattacctg ccggtaaagg caaatggaaa ggtaaagctg gtgataaact 7020
aatcaactgg ggttgttcta caccacatcc aaacaaaggt ctagctacct acgttaacac 7080
gccagaagca gttcgaagtg cagctaacaa gcttattaca cagcaacgct ttgatgcaac 7140
tcctgcaatg gctaaccata cattgccttg gacaacagaa cgtgttacgg cacatcagtg 7200
gattgagaac ggcgatacag tagtatgtcg tcagaaatta actggtaact ctggcgaggg 7260
tattgtaatc gctgagaaag tggaagacct tgttgatgct cctctataca ctttgtacaa 7320
gaagaagaaa gatgagttcc gtgttcacgt ctttgatggt gaaatcattg atgttcagcg 7380
taaggctcgt aaacacgaag tagaagatga caaagttaac tggaaagtgc gtaacttgga 7440
cggcggtttc atcttcgctc gcaacaacgt tatcgctcct atgccagtgc gcaaagcagc 7500
tattgaagct gttaaagctt tgggtctggt tcatggcgct gttgacattg gtcacggtcg 7560
tcatgggact tacgtttacg aagttaatac cgcttgcggt ctcgctgggt caaccttggt 7620
tacttatgct aaagcatttg ctaagtacct aggcatggac gaagctaatc taaaactaac 7680
taaggaacag ttagacaatt acgttccaat gaacgaagaa aacaaacaac ttgagcaacc 7740
tgagcttgag ccacttccac aagaagtaca ggttggcttg gctcaaggtg aagaaatcgt 7800
agctgttggt gctggcatta atgttggtcg tccaatggct cagatggaag aagaagtagc 7860
ggcatttatt gcagcacaag ccgaagaagc ggaagaagaa aaacctttcc ctgagcaaga 7920
gcttggcaaa aacaagatgc gaatcacagc agacttgaaa gtgaaagatg ttcgtatccg 7980
tttcggcaac attgttgtag atgttaaccg caataacttc gcaaaagttg tacaggcact 8040
gaaataatac ttgacagtga cgcaaataag gtggtagact tgttgaacga aaaagaaaaa 8100
gttctgattc ctaatgagct tcttccacct aagtatcaaa ataaaggcta tcggatgagt 8160
cctgacggtt ctgtatacgg ggtgtatggt aagaagctac gccctaatga ttactcacct 8220
aatggtttta atcatccgat gctttttaat cgcaatttat ggtacattcg agcagccatg 8280
ttcgtcccta accctaataa ttacacaagg gttgtcgaga ttgacaacga agttcactgg 8340
ctgagtgaaa agaaataccg tgaaatggtt gctaaatact taggtcgtga agtagtggag 8400
cgaaagccct tgaatgattg gggtggggta ttgatagaaa tgcttgaaaa gggaacacgc 8460
atagctgatg tagctaaagc tcttggctat tcccgtgcag caatttacaa atacctagag 8520
aagaataact atgaatacaa aaaccgtaga cttaaaaaac gaaagtgaat acgttccatc 8580
agtacgtggt aatcctgctg acgcattgaa tacatacatc gtcacaattt atgataccaa 8640
aactggtaaa tatcacgaag ggctacgaac tcatcattac agtggtgaag cctgtgcttt 8700
ctgggtaatg caaaaccttt cagacggata cgaatttgta tctgcttaca acccagacaa 8760
taatgtaaca atcactaaaa aagacatcct aaaacaagaa gaggtaaaat aattatgaaa 8820
aaagtaatca tgtacgcatc aaacttctca cgctacggta aacttcaagc agttaaatct 8880
actctagctc acctatctcg tgaagcaggc ttcatcttcg ctttcgttaa cgacccagag 8940
cgtggcaaca cagctaagat taagtttggc aacaaacaag tctttgtacg agacttcaat 9000
cctgctcaac taaacaaaca cctagaagaa atgcaagaca tccacgctgt ataaggagat 9060
ttaaatgtca ttaaaccaaa tgttcgatga gttcaactcg ctataccgtg tgaaaggcac 9120
agacatcaac aacgaatcag ccaagggttt tgttaccctt gtgtgtgatg aagtagtaga 9180
gttcgctgag gagttcgaca aagaaactct tgataaagca gcggctgcta aagagggtct 9240
tgatgccatc tatgcaggta tgcaacaact acgaactatg ggtattgact tggaagcagg 9300
gcttaaggct ctgcattcct ctaacatgtc aaaaactgtt aaacgagcag atgctgagaa 9360
agaacttgaa atcgctcgtg aacgttaccc acaagcctac attgatgagg ctcaagccta 9420
ctgtgttatt cgtgatgcag ctacaggcaa agtaattaaa ccaacaactt acactccagc 9480
aactataaac gaggacatct acaatgcgta aacttacaaa agctcacttt aactacctag 9540
cacgtcatat caacctacgt ttccttaact acgagaaaaa gattctcgaa gatattgata 9600
ataaattcaa gggtgttcct aacttctctt ggattatgaa agttcaaggt acatttaatc 9660
cacatcagac tccagaagtg gataaaacaa tccgtgctat tcttgataaa ctgtacgaag 9720
aagttaagta caagaagtat gaacacttca accattcatt caactgcttt atgaagcgtc 9780
tgtgtgtaga acttaagaaa caaaatccta agttcgttga gtctaaattc cgtgaagcat 9840
gtacaaaagg gagcattcag taatatgaca gcattgaaag aacaagtggg aggcgaccac 9900
tacaaacgcc tacaatacca acctcacaca tttgcaatgg acttgtatgc atctggtggt 9960
gtgatgaaag ttgctaagta tcttagccgt gacaaactag accgcacaga ggacttgatg 10020
aaagctcgtc atgtaatgtg cctagatgaa gattgggtta agaacgaatg tgagaataac 10080
gtattcacat tattcgggga aacaggattt aacgctctgt gggaagattg tgttgtgttc 10140
tatgacccag acgaggacga tgagctggta gaacgattct tcgaacagtt cgctggtact 10200
ggtaacgact tcattgtttg gaaactaacc aagcaagcat tctaccaata cctaaaaggt 10260
aatctacctg cggctaaagg tcttatgact aaagcaatca acttagaaaa atcatacaag 10320
gcagtggcac taaagtcact gcgtagttca gaggaagttt aatgacaatt gaggtactag 10380
attttcatgg aagcgataac gccattgcta atgctgctcg tgttagcttt gctagccacg 10440
gcgattggaa ttcaattcca gaagcctaca gtgaagaaaa acgagataag cttattttgt 10500
atcttgctaa gcatcgccat cttactcctt ttcgtcatac ttcagttact atccgatgca 10560
aggagcctat cttccttgcc cgtcagctag gtaaacatca agctggctta agctggaatg 10620
aagtatcaag acgttacgta aaaggtgatg ttgaatttca catcccatca gcatggcgac 10680
tagcccctga tggtagtatt aaacaaggta gtgctggagt tcatcctagt tctggtcaat 10740
tctctgctgc ttatagtgat ttcattaata aagcaaaaga tttctacgat agcatgattg 10800
aaaatggcgt tgcccctgag caagcccgta tggttttacc acagtcaatg ctaactgagt 10860
gggtttggac tggtaatctg ctagctatgg ctcacatcta ccgtgaacga attgcatcag 10920
gagcacaact tgaagcacaa tatttcgcaa gaaaactgga cgacgcctta cagccgcttt 10980
tccctgtggc ttgggcagcg cttacaaaac ctaacctact caaataagga aacaagtatg 11040
gcaacccttg acatacgtaa tcaagatgct caaataaatg atatagtatt tgcaaacaaa 11100
gagggcggcg agggtcgcct ctataagctg gagcgttttg tacctgtaca atttcacctc 11160
gtcacatcca atggctttaa agcccatgtg aaaatggagg atgttgacaa ccttattgaa 11220
gccctaaaga cagcgaaaaa gctatgggca taacaaatta aacgagagaa aaatcatgga 11280
aaagagaaag cacttcttcg gtaactttaa aaagattatc gacagcttgg taatagacac 11340
tcacccacac agtgataatg gcggtgttgt agtagttgcg gggggtttcc tccgtgactt 11400
acatctaaga cgacaaccta aagatattga catcttcttt agtggaccac agtcgtcaaa 11460
aaccatcttt aagagtctca aggagaagaa ccctgaattg tttaaagact gcaagcataa 11520
gttctactgt gatgaaaact atggtatgga agaagaagaa gttaaaggta actttgctgt 11580
ctacgaccta gttaaagttg aacttacaaa tggtttaaac cttgacatca ttgcaacaca 11640
acgtgtgtgg gaacaacctt gggagttgtt aggtaatcat tttgactgta acctaaacgc 11700
cctagcatat gacggtaaga ctttgtttgc acctaatggt atgcctaaca aggatggtat 11760
cttcttccct cttggtggtg catacattga caaccaagtt actaaagagc gtatgattcg 11820
cttagtacag aaagttcctt tcggtaattt ccaagccttt agtcagttct tccctaacga 11880
ttgggatgag attctagaag cacgtaagaa agcacaagaa gttgaacgta aagaactggc 11940
aaaagcagcg caagctgaag ccgcagcaat ggcagcggta gtgccaggta ttattcaaga 12000
tgaagcagca caactggagt tttaataatg gataagcagt ataacaaatc tcgttacgaa 12060
cgtaagcgca aatacaaaaa caaacaacaa caacgtcgtc gccgttatgg tgatgactat 12120
gacaaaggct ttggaggtaa ctgtggagaa taatttcact gttcacacta aacgtacact 12180
agaagcgctc ccattggtgg agcgtttgta catccaagag aaacacaaaa ttcattttaa 12240
gtataagcgt ggtgatattg tttaccattg tttcatgttt taaaggagaa tagatggcaa 12300
aaattattgg cgatacggct tgtcctaagt gtagagaaaa tggtagagac aaaactggca 12360
accacctaat gattttcgat aatggaaacg gctactgcaa tcgctgtggt tttgtccatc 12420
gaaaagataa gatgggtgag gttaagccag ttcctcgacc tattaaatct cctgaagaaa 12480
tccaagctga gttggctgag gttaagacct acccaattca agctcttggt agtcggaaga 12540
ttagtaaaga gactgctgac aagttaggta tccgagtagc gttgagcgag gaagatggta 12600
aaacccagac tcaacattac tatccgtata ctcgtgagag taaactaagt ggctataacg 12660
ttcgtaacct aggtatgaaa tcattctacg gtgttggtga acgtaagggt gttgacccat 12720
tcaactggtt taatgctaga cgtgtagcca cacgtaaaaa gattattatc tgtgaggatg 12780
agctatctac agcatctgtt gttgaggtgt ttgatagatt caccaaggat aagcaatggc 12840
ttcctgctgt tatcggcttg gcaacatccc cacaaggtat tctgcaagac ttgggtcgta 12900
cctatgttaa agatttcatc atgtcatttg atgagattgt ctttgcattg gataatgatg 12960
aggcaggtga ggagggtctt aacttagcac ttcaactatt ccctgatgct aagtttgtag 13020
atttacctct taaagaccct aacgacatga agatgtctga tatggatgta gagttataca 13080
accttatcaa ctataatgca cgtcgtaaac aaccatctgg ttctttgact ttacacgacc 13140
tcaaagagcg tatgcttgct cctgttgaga aaggttactc attaccttgg gagaagctaa 13200
ataaacacac taacgggctt ctgatgggtc agatttggac tatcggttct ggtgtaggta 13260
ttggtaaaac attcttggct aactctatta cagcttggtt atggaaagaa caccgtatca 13320
agtctggtca gattatgttg gagcaacctg ctgaggaatc tggtcgtgct attgtaggtg 13380
cgatgacagg tcagcatttc aatgacccta agattccaat gaacagagag agattggctg 13440
atgctatcga cgacgttggt aacaatattt atttctatga tgctgtgcgt aatgctgatt 13500
gggacgaaat caaaccggtc attagacact gggtggtggt ggaaggcgtc aaagttatac 13560
agcttgacaa tcttactaca ctcactgctc atcttgatag ttctactgca aacaccgaag 13620
taaatagaat catggctgag ctggctatga tgtgtaagaa atatgaattt tctgcattcg 13680
tatacagcca cttgaacccg cctaagactg gcgatagcca tgaagatggg ggtgaggtta 13740
aggaagtaca attaacaggc tctcgtggcg ctatgcgtta ctctagctat gtgattggct 13800
tcgaacgtaa ccgtaaccca gctcttccac aagaggagca aaacctaagc cgtattcgac 13860
taatgaagtg tagacctatt ggttctactc caaccattgt cggtacatat tataatcctg 13920
caacaggatt gattgaacaa cgagaagaaa tgcacctacc taaaacacct aagcttgtgg 13980
gtgggggcga ttaaacgagg aaacatacac gatggctatt tataaaattc aacgagagaa 14040
aattggtaat atcacacacc tagtacgtgt tcaggttctg ccttttgatg tatactgcaa 14100
agtatgtgag gagtatcatg acggtgttat gggaagtgag gatgatttta ctatccaaac 14160
cgaatgtaat tgtgatattg atgacttctt cacagatgaa gaatacgatg atgtcgtaga 14220
agaacgagac cgttgggaaa tcaaagctga agagcttgaa gaagagcggg acgaagctgt 14280
tgcagacaaa gataagcttg aacaggaata cattcaagag attgaaacac ttcgtgctaa 14340
caacgagcta actctaaaca ctctcgatat tgagcgtgaa gaacaccaag aagccatcga 14400
gttacgtgat agtattattg atgaactgga agctaagctt cgtgaattgg agggttaatg 14460
gaatcatttg tttctggact aggtgaggtc tggtgtactg acatcgaatc aacaggtctc 14520
atcgaggaca tgaagaagca atcaaatcca atgcttcact gttttggagc gattaaccca 14580
gagacgggtg agagacgttt gttctctcgc tctaatggta aactaggctc tgaactacag 14640
gcatttctag atagcggtgt cactctagtt atgcacaacg gaatttgttt cgatggtgaa 14700
gcactcaaat atttgggtta cagtgtggac aagtgccgta ttcttgatac tcttgccctt 14760
agttggtacc ttaatcctag gttaacaagg catggtcttg aatactgggg cgaggtattt 14820
ggagtaccta agccacctgt atatgactgg gttaacgaag acccatctgt atacgaccac 14880
cgagtcatgg aagactgtga gattcagtta agactttggc gtatgcagca acaacaactc 14940
aatgagattt atgatacagc taagagcaaa ggtaagctta ttgattacct catgtttaag 15000
atggaacttc aacgtatcca agctcttaac aaatggaaac tggatattga tggctgtcat 15060
aaacttattg cagaacttga ggcacgtaag gatgaacgat tcgatgcact tcaatgctca 15120
atgccaaaag tacctgttaa aagtaagaaa acaagaccag ctaaaccttt caaaaagaat 15180
ggggatttat cggcgacagg tcttaaatgg aaaactctta ccgaggaaca tgggctacca 15240
ttcgaatatg acggcgttat tgaagtcatt acaggatacg tcgaacccaa tgctggctca 15300
catgtacaaa tcaaagattg gttattctcc ctaggatggg aaccacaaac attcgacttt 15360
aaacgtaata aggaaacagg ggaaacacgg aagattcctc agattagtga tggcaatggc 15420
ggtatctgta agtctgtaca gaagctgatt gagaagtatc ctgacaaggg tatcgacaac 15480
ttagcaggtc taggtattat cagccaccgc ttgagtatcg ttaaaggttt cttaagagag 15540
gtagacgatg acggatacat cgcagcaagg tgtaacgggt tcactaacac gttgcgtctc 15600
aaacatgctg gactggttaa tttaccatcc actcgtgttc catacggtaa ggaaattcgg 15660
ggactactcg gctgtccaga cggataccag aacattggct ctgacttatc gtccttggaa 15720
gaccgttgca aacaccactt ccaatggaaa tacgaccctg agtatgttaa actccagatg 15780
caagaggact atgaccccca cctacaagta tgcttactcg gtgggttgct cacggagaaa 15840
cagattgcag cgcacaaagc aaagcaagaa gaccactcag acacaaggca catcggcaag 15900
ggcggtaact acgcttgtca gtacggtgca ggcatcgaaa cgctgatgcg tcagctaggc 15960
attcctctag aggttgctaa agctgtacac tcaggttatt ggaaactaaa ctggtcaatc 16020
aaaaagattg ctcaatcaac aacggtgaaa catgcaaatg gagaaatgtg gcagtataac 16080
ccagtatcag gtctatggta ttggcttaaa gcagacaagg acagattctc aactctttgt 16140
cagggacttg gtgcatacat ctttgacaag tggtgcgaag ctactggtaa tatcgttaga 16200
gagcgatacg gagcaactat gccgttcgtt gcgcaattcc atgacgaact aatccttatg 16260
gtaaaagagg gtacacttgg tgtatggcat gacattctta aagaagccat caaacgtgta 16320
tcagattcac ttaaactaaa ccgagaacta gactgtgatg ttcaaatcgg cgataactat 16380
tcggaaatcc actaatgtac acagaacctt taaattgtac cgtagttaaa gactattcgg 16440
tagaagactt taaacaaata aaaagtattg aagaatttat tttcagtttt gatggtgtag 16500
agtactacac taaccaagat attgatagtg taggtacact aaaatcttat atagaaaact 16560
atgacttaat acttgtacca tctgggagta ttaacacagc aatttttgta gattatatac 16620
aataactact tgacagtgac gctattttct gttataatag ttgtcctttc acgagaatat 16680
ttaaattaaa gattaagtta tacggagaaa ataacacata tgaatatcgg cattccacaa 16740
atcaacaaac caaacaaagc atttaagaaa atcgtagagc aagctgagcc tgtagaagct 16800
ggcatgtaca aagctgtgtt caaccgattc gtaactctag gtttccaaga gagcgacttt 16860
gaagagggta agattcaatt cattactgtc ttcggctttg agcttgttga agatatgatg 16920
ggtaacaaag tacctaatca ttggaaagtg tttgatgacg gttcacgaga agagggtgct 16980
aaaactctta cacgagaata tccattaaac gaccaactac atcacaagtc taacggcttt 17040
gctattgcta aagctctaga cccacaagtt cctactatcc gtaagggtaa ggaaaacgaa 17100
cacgaataca tcgttgactt caactgggac gctcagttag gtaagacggt gttactacaa 17160
gtcaataaga agaaagctaa ggctggtcac ttctacaacc agattcagtc tgtaatgcca 17220
ctaggtgctc cgcttgatgc tcagctacct acagtattct tcaacctata caaccctgac 17280
atgcaagaag tttgggagaa agaagtgtac ggttgggaga agaaaaagat tacagaacgt 17340
cgtgtaatcc caggtgagcc accagttgta tatggtgatg cccctgcaca agaagaacaa 17400
ccacaagagc agcctaaaga agaagcacct caagctatcg tagatgagga agctgatgcg 17460
gatgacgacc gcccattcta atcctaagac ccaccccttg cgggtgggat tttttgaccc 17520
tgcaaaatga ataaaccaca tacactatta cttgactgtg acatcctacg ttatgagttg 17580
ggtagtatct tatcaaaagg agatacaatc accataggag gtactaaaat tgatgtacca 17640
caatcaagag ccaaagttga ggagcttgtt aaagagaaga ttgattatat tattgagaga 17700
actggatgta cttacgttcg aggctacctc tcacaaggaa agaattttcg atttaaacga 17760
gccactatcc aaccttacaa gggaaatcga aaagactttg ttaaaccata tcactggaaa 17820
acggttggag aggttatccg agattacttg gagaacacaa ttgactgtga gcttttcgaa 17880
gcagatgacc atatggcaaa agaccaagat gtggatgaag ggacaacctg cattggctct 17940
agagacaagg acttacgagg tcgtcatgga tggcactact cgtgggcggt gggtgaaagg 18000
agtcctgaga aaccgcttca atggattgat aagcttgaag ctgatagatg gttctataca 18060
caatgtctta caggagattc caccgacaat attcttggat gtgccgttaa gctacctgac 18120
aagaaaggaa atccaagacg caagggcgtt ggtcctaaag cagcagaaca actattagct 18180
gaagcctcct cagagcaaga gatgtttaac atcgttgctg cacaatacca actacattgt 18240
ggcaacttgt ggaaagaaca cctgcttgag aatggcactt tactatttat gtcctacgaa 18300
cttattccgt gggaagaact agattcaaca aaaaggttat tagaaaacta cgaaagagaa 18360
ttatatgcta gataaatatt tccaagaaga aacacaacgt acagttatta agcgcaatgg 18420
tcgtaaagag cctttctctg aggagaagat taaacgatgg tcaaagtatg cagctaagca 18480
tgacgttaat tggaaagagt tatacggtaa aaccttagat agactgccgc ttgaagttac 18540
tacagaagaa attcatagta caatgattaa ggtctgtcta gacaaagaag aacttaaatg 18600
gtctcgtgta gctgctcgtc ttgagtatgc aacactacgt aagaatatgg cacaggttgg 18660
tatctcagat aaggatagct tcctaactat ctaccacaat atgtgtgaac taggtctatg 18720
ggatgctaag tctttccctg aaccagaccc aatctgggat gaatggtaca aggatttgta 18780
tcctaactac ttcgagttct ggcaaatcaa acaatggaat gacaagtatg cagttaaaat 18840
tgatgataaa gcagttgaaa caccacacat gggagctttg gctatggctt tggctatcca 18900
cggtactact caagaagcgt atgacacagc taaagcggtg gtgtacggtc aactcaatct 18960
cccgactcca gttcttaatg ggtgtcgtaa cggcgacttc gattctatta gctgttgcgt 19020
cattactggc ggcgatactg ttgattctat cggtgttgca gagcatatag cttacaagat 19080
gacggctaag aaagctggta tcggtatcga aatggctaca cggtcattgg gtgaaccagt 19140
taaaggcggt cgtatcaaac acttgggcaa ggttccaatc ttcgcaacgg ttgataaagc 19200
tgttaagatg tttacccaag tatctcgtgg tggctctgct acagtggcta ttgacgtata 19260
tgaccttgag gttgtggatt tactactact taaatctcaa cgtacccctg agaatagacg 19320
actcgacaag ttggactatt ctcttgttta tgatgatgag ttcttagatg cagttatcaa 19380
taacaaagac attactctac gtggtaaggt gtctaatatg actaagaaca tcaatgctcg 19440
taaactattg gctacgttct taactgtacg tcaggaaaca ggtcgtgtat actgcttcaa 19500
taaatcagaa gctaaccgtc acacaccatt cattgacgag attcgattaa gtaacctatg 19560
tcaagagatt tgtctaccaa ctaagccata tgaaaatatg tctgacttgt atggtaatgg 19620
catgtctaaa ggtgagacag cattctgtag cctatcagct attaacgtat ctcgtgttca 19680
tacacatgaa gaatatgaac gcctagctta cttggcagtt aagacagttg ataacctaat 19740
tgaacgtgcc ccaatgatga caaactctat gaagatgtct atcactaaac gtcgttctat 19800
tggtatcggt attactggtc tagctggttg gttacatcgt gagggtcgta cttactctga 19860
tgttaatgca attgaacgcc tagctgagat gcactactac tacctgctta aagcttcaca 19920
aaagcttgta gaggagggtc gtacaccttg tgagggtatc aatgaagatt ggctacctat 19980
tgacaccaag cgcactaaga ctgaaccatt catgaattgg gaagcattgc gaggtaaacc 20040
acgtcgtaac tctgtacttg tagctcatat gcctacagag tctagtgctg tgttctctga 20100
tgcaactaac ggtctatacc cagttcgtac tcgtgttatc aacaagcaat cacgctacgg 20160
taacattcaa tacattgcac caaatgtgat tgagacagct tgggatattg ataaccttac 20220
actagcgaga gcttatgcag cagtacaggc ttacactgac caagctattt ctgctgacta 20280
ttacattaca ccgtcacgat ttgagggcgg caaagtacca atgtctaaac taattaaaga 20340
ctgggttgca caagctcgtc taggtaacaa gacaatgtac tactctaaca ctaacgatta 20400
taatggaggc acattctcat cttcagaaga agagggttgt gaagggggtt gtaaactgta 20460
atgactgtat ttaatgcaaa taacaaaggg tactctgagg ggtacccact tttcctaggc 20520
gaagaactag gtctattcga cacaattaac cgtacatatc ctgagctgga agagctatac 20580
caacagcaaa tggctcaaat ctggaatgag tttgaggtag acttgactca agataagatg 20640
gatatgcaga aagttgaccc taaagttaca gagcttatgg ttaaaactat tagttggcaa 20700
catctagctg actctgtagc ttctaaatct atctctggtt tgttaatgaa atatgttaca 20760
aactctgagt tggagggttt gattaactgt tggagcttgt tcgagactat ccatgctcgt 20820
acatatagcc atatcgtcaa gcagacgttc cagaatcctg ttgaaatgct taaggaaacc 20880
tatgacaacc ttaaagtatt agaccgctct caagcgattg tagaggcctt tgatagacta 20940
gaagcactgc ctaaagattc aacccgtaaa gaggtcatgc aagctatttt aggtgcaata 21000
tttgcactgt ttgcacttga ggctattgca tttatggcaa gcttctcagt tacatttggc 21060
attgcagaga ctggtgtatt ccaaggtatt gcacaagatg ttaagcttat ctgtcgtgat 21120
gaggtaatgc atacacgatt tgattatgct atcttatcta ttctacttaa agaccctgag 21180
tggagtcatg tctacaacag caatattggt gagtacaaac gtatcctaga ctctgttgtt 21240
gagggtgaat tagtatggac agattatcta ttcagtgagg gtcgtcagtg tattggtctt 21300
aatgctaagt tggttaaaca gtacgtattg tatatggcac agcctgtgta tgaagcagca 21360
ggtattacct ttggtttcga agcacctaaa gagaacccac taccatacat ggaaaattac 21420
attgatggtt cacgagttca agtagctcca caagaactac aagttaccag ttacaacatc 21480
ggtgctatct cagatgacac cagtgattta gatgatttag attttgagga tttataatgt 21540
ttttaattgc cgaagaaaag ttttacattg aatttaaaga tggttcaagc atgacaggct 21600
ctagtgctga tggtcacaaa ttaactgagc tggaggatag attaattgtt gaggctggct 21660
ctaagcgtag agtgttcttc aatgatacta tcagctatta tgaaattgaa aagacagacc 21720
tagataagac aggtatggac ttagagattg gtattaaata atgtttgata ttgaagatgg 21780
tgtttgggct aaagtacaag cacattggaa caaatacaag aatgatttgt atgtcagaac 21840
agaactactt aaaaaggtta ttgaattgga tattagggtc aaagacttag aaaagaaatt 21900
aaatgaaacc aaatagacga tggatgcagg gtcagcgaaa ggctgactct aaatttgaac 21960
aggagttaaa agatggggtt cttaaagact ttgatttcca gcacggtaag attccttatg 22020
ctattcccca tacttatcgt cctgattttg ttaaagataa tcttatcatt gaagcaaagg 22080
gaagatttag ggatagcgcc gaagcgagaa aatacttatt tattcgagac gccttggagg 22140
aaaaccaaga actcgtattc cttttctata acccacaaac cccgatgccg tttgccaaga 22200
agcgtaagga cggtactaag cagacacatg ctgagtggag tgaaagacac ggatttagat 22260
ggtttacaaa ggagactgta ggtgaactta ttaaccaagc aaataatggc aaggcagaga 22320
agtaatgcca aagccagagg catagagtgg aaacttactt ttgatgagtg gtgttctctt 22380
tgggaaggta aagctcattt aagaggtagg ggttctgatg atttatgtat gtgtaggtat 22440
ggagatactg gtgcatatga aataggcaat gtctttatag gtactggtct tgacaataga 22500
agagaacagc aaaagcgtag atataaagag tttagagata aacataatat tgaacagttt 22560
attaaagacc gtttaagtgg tcttacttac aaacaattaa gagataagta cggcatttct 22620
gaaggaactg tcggtaattt tataaaggag tttattaatg aatccaacac taacacaacc 22680
actagaccta tcaatcaaag cttataagat ggctcaaatt gaagctcaac gtaatggcac 22740
agactgcggt acatatcctc aatggcgtgc tatgatgatg aagaaagata agtttcgtca 22800
ggaactggta gaggctaata agcctaaaga agaagtgaag cctaagcgta agccacgagc 22860
taagaaagaa aaatctgaat aataaaaagc ccccactcta gttaattcta ggtgggggtt 22920
attttttgtt tattgtttag cctcaagacg actaacacgt tgtaatgtat caatcgtacg 22980
gtcgtctatc ttgtctacct ttttctcgat acgtgttagg atttcactaa atccacccca 23040
ctgtgaagct acggtaaggg ttggaatagt gaaagccgca actaacacta ccgcatattc 23100
cacccatctt gccataaaac acctcctata atgctccctt aaatgccgac atcacaggca 23160
agtcctctaa ttccgcttca ccactaacag ccttattaag caagtttgct tgctggtcgt 23220
agatactaaa cgaaacagga accacaactc gtgaaagctg tcctgtagcc gcatcctgta 23280
gataacccat atggattggc atgagagtat tacggtaagc cttcatgaag atttcctcat 23340
cacttaggtt attacctcga acttcgttct ttaaactctc ctgtaagaag ccaacaaaca 23400
tgatggctcc tgtagtagca ccaaatcgtg caaggtctac agccaattct gcaccactct 23460
tacctcgtgg gtcgattaag tcgtacagct ttggtaggat agtgttagca aacatggttg 23520
gataaccacg aagctgggtg aacatagcac cccatcctgt agaaaaccaa actggtttat 23580
ctagagcctc tgggtttagt gctacgtctc tgttgaaccg tgctaccata gatggtttat 23640
actcatcaat gatattcggg ttcttataca tcgctttagc aatgctctca ctgataccta 23700
aatcatttaa tcgttccaca gcactcatgt aagctcgtga gtttttagga cgactattga 23760
ttgtagttag gtcagtttta agttgtgcat ccattgcatg tacagccata ctgtttacca 23820
agtagttgat agcagtctga cctgtagcat ggaagaatgc attgttaaag ttttgcattg 23880
cagggcttaa gaactcaccc tgaatacggt tgttaagtgc agtagttgca ttcataaaac 23940
tacgaccagt cattaatagc tgacggtttg tttcagattt atctacaata ccaccagtaa 24000
atgtactgat taagtctcgt gatacagaac ctaatgcacc tataagggct ttagtacctg 24060
ccacattgcc tactttaata aacaggttaa aaggttctgt caatgatgac agtgcagtaa 24120
gtggtagagc tactacgtta gagatagaac gagcaacaga agctagtttc ttagctgact 24180
cactctggaa ttgaccatat gtaccttggt aagcatccat agcattgtag aatgtatcta 24240
cttctgctgt agtcatagcc tgagcattac cacgctgaga gtttgcttga gcaataagac 24300
catttaagat ttcattgttc ttgcctaact gctctgcata tgtaatacga tgggctgctg 24360
attgtgcata gtttaaaagc tgctctttaa agtttctaga tacagagtaa tcatttaaaa 24420
tatgctgagg aatatgagcc agttgtcgtg agttttctag ttgaccgtac ttaagaggag 24480
aggaagtctc tactctattt ggagctgtaa tattaccatt ctcatcaata cttacacgag 24540
gcatcttagc agcttgtgta tcctgcttct ctaaagctac tttaaatgac tttagttttt 24600
gaaggttttc ctctggagtt actaaaggtt ttacattacc ctcattatct acagttgttt 24660
tatctacctg ttcattatac caacgagtat attgattttg caacccatca atatcaaatg 24720
tttcagggtc cataatagta ggcaggtagt tgttaatata acctgtatct aagttggcac 24780
cttgcttact attagcacgg gcatgaatgt cacccaatac agaatctaac tctctaagtt 24840
cagccgtatc tcgacgtccc tctgcgtaat ccttacgtag tttgtcagca tctttaactg 24900
aagcaaacaa ggtattaata cggttggtta ggtctgctgc cataacctca cgttcaacac 24960
gcatagtctc aatacgttta ttaagttctt gacctgcctc tgttccctct aagaaacgtc 25020
gtacaggacg taatgcagta cccatagtga agttaagggc tgattgcatg aagccatctg 25080
cacctgcaat atcacctgtc tttactgcat ggtcactagc catctctgct agctcagctt 25140
gtttagcttt atctacagtt gcctgagcca cacgtaaagg agctgataca agcataccac 25200
ctacgatagc ctcatcaata tcctctgtgg aaatctcacc agttacaccg tagttacgta 25260
atacttgttg ggcaccatta gtaccaccat caacagcgaa gttcttagct aagtctgtaa 25320
gtacattctt cttagcttct tctgtcaggt tagttttatc tagtttagcc aacataccag 25380
tcatgccttt agctagtgga atatttaata gtgattcagc accagccgta accattgcac 25440
gacctacatc aatttcctct cctgcttcta cttgttggtt aactgtatca ccaaagtatg 25500
aagctgcatt agcgacaaga cctagcttac caaagaacat tgctgccact tctggagcac 25560
ctgtagctag accctcacca atataatcca tcaattgacc aaggctttta atatctttgt 25620
atgaagctac tttagggtta aacttacgcc actctgcata cttctcttct gagtattgtg 25680
caaggtcact agctgtttca tctagaccaa gcttacctgc tagtacagaa gcagcacgag 25740
attgagaagc cagtgcctta tcgacaccag cctcaaaagc cccaacacgt tctactcttg 25800
gctcaaagcc taaatcaact tcattagtag tagcaccaaa atttacgtta gtatctagtg 25860
ggctgccctc accaaacaat tgtggcgtat tgccaagatt atcactcatt aaaactcctt 25920
aatcaactaa tgtggttttg ccaaagaaca taggttcatc atatgctcct gaagcaacac 25980
cttgtcgaat cgcattctta atcgtagtcg gtgtaacagg ttgtttgatt gacttcaaac 26040
ggataatagc atcttctact acagactgtg caaccttgtt atctgctgca tatgacttag 26100
caatagagcg gtcactggtt gatagttcca atggtttatc ctgactgtct agcaatgaag 26160
cttgctgacg ttgacgatta gttaatgcgt tatactctct cacattttgt tctgctgccc 26220
acttagcatc ttcacgagct tgcttagcgg ctgctgcttt ttgttgttgt cgtgcttgta 26280
caccacctgc tagaccttta agtacagcca cacctacatt gttagtttgt gtagcagctt 26340
gagcacctac ggctagcatg gtaatgaagt ctaccttgtc atttagaaaa gaggcacctt 26400
ccgcaacagc atcgccagca ctaccaagta caccagtaac acctttggca tagccacctg 26460
ttgcttcgtc gaatgcatcc caagaattac gtagatactc cagaggtaca ccaagcaagt 26520
cagccgtacg ttgccacgca ttcttacctt cctccgtatt tgggtcagac ttttcaagaa 26580
tcatagctgt cttctgagca tctgtatatt cttcttgtgc ctgtttaaca gcttgctgtt 26640
gttcttcctt agtcaactta ggagttggtg cagctaatac agtacctaca ccctcagtgg 26700
atgtatcagg agctgtatca ccgaagaaac ctttagcaaa cttaatgcca tctttaattg 26760
cagggacgat agcttcccca ccctctttaa ctaatttctt agttgtctcc aaactaaagt 26820
cctcaatacc tgctgcatta agtgcttcct tagtttgttc tttagtttct gtaataaggg 26880
tagaagcttt actagcagcc tcagttactg gctgactacg cttcaaatac tctttctctt 26940
gctctgcggc tcgtgcagca gcagcatcgt aagtactacg aataccctca tctgcttgtt 27000
gtaggagacc agtaatggct ttatcagcag cttcattagt ctttacaggc ttacgttctg 27060
ctgtctgagc tagtaatgcc caagggttag acttgcttgc ctcctgtggg atgaattgtg 27120
ccattaaatt aaacctccat atgttttctc tggtgtaggt gcttcattag tttgatatga 27180
acgggcacct ctcaatggac tctgtaattg catttgtggt ggttgacctt gtaggttctg 27240
tagtaagttc ataccttggt taatgttatt aaggtaatca ccaccaccac ttagaaagcc 27300
tgtagaagct cctgagctgg ctgctggagt taatgaagta gtgctagcag cacctgctcc 27360
aagacctcct gaagttgctg tagcgcctcc tgcgggtgca gacatagaac ccatagaagc 27420
aaatgcccct gagcctagac cacctgaacc aacagcagca ccggaaccaa atccaccacc 27480
tgctgcggca ccaccaccgg caccacctcc agctccagcg gctagggcac cgccacccaa 27540
aaccgccccg aagatactgc ccacaatggc atcagagttc tttacaattt tattgtcttt 27600
atttaaacct aaagagcctt gcatctcagt cattgcatct tggtggttac caaatgcttc 27660
atcagcatca ccactaaata catccttgac aaagttgaaa tcagcttcca tctgggtgat 27720
gggcatcatt gcaatctctc ctaggtcgaa attatcgaaa tcgaagatgt tactgaagaa 27780
gctcattaca atagacctcc actacctgct gctgtacctg tggttgtagc tgtttctgta 27840
ccacccatgt tgcctgaaat aacatcttta taagcaagca aggattgcat gtagctgtta 27900
gctcggctag cttgatattc agacagacct tgatagccac tcattaagtt acctagacct 27960
tgagcacctg ccatacgacg ctgaatgtta ttctgttcag cctcataagc gagctgtgcg 28020
ttcgtctgag cgatttgagc atttaggtca cccattgcta agccttctgc aattgcactg 28080
cgtgagcctg cccctgcgcc tgcctgatta gatgtttgct gaatacccgg aagtacatta 28140
cgttggaagt tagtagtagc tgcctgagca gaagcatcca tagcattgtt gagagcatta 28200
cggtcaacat tactattcat catttccatg tacatgtttt ggctagcacc taggatgtct 28260
tccatctgtc cggtataatc tgaacctaac tgaaagccac cttggttaag ccatgcatca 28320
gcacgattaa gtagttcctc gtagttacca gattggacgg gcttccatac tgaggtacta 28380
tcttgggttt gggattgttt ttggtcaccg cccattataa ctccttataa atagttgtgc 28440
agtaatggtc gtagtcttta tgaattttac gccatgcatc tctgccttta ataatgataa 28500
tatcaatgcc agataatttg gcttcatatt caatctggtt tagtgcttct ttgaagtcac 28560
catcaaattc tcctgctaat gtagctgtat ccagttctaa gattaactgt cgttcatagt 28620
tacgtcttgt tttacctaca attagagcgt tattataacc ccataaaatg taagtaccat 28680
tttgaagtaa ctggacatat tcatctttat ctgtttctag agctggagaa ttatcatata 28740
atctgtctac taactggtaa gctagttcat ctgtaataag ttctttagga atgtatgtaa 28800
gcataagtaa taattattaa taaggacgct taaggaaggt aagtaataag gaaaggaaat 28860
aaataactaa taattaatta tcagtcctta tataaggata ccatcaaaaa gcgtcattgt 28920
caagtattaa atcaaataaa tttgaaatta gttgataata ctgatacctg agttgacttt 28980
actaacctct acgttcactg tactgttagg aaccttgaat gttacaaagt tagatacagg 29040
gaatgactga ccagttgcat gtaggaactg tgctttaagg tcaattgtta agttacctgt 29100
tgcagttgta gcaggaatga atacttccca agctagtgtg ttagttagtt taaaccatgc 29160
ttggttagcg ttctgtgcta caaactcaaa tgcagatgca gcattacctg tagttgaata 29220
ccctgcccca gattcagtaa cagatgttac acggtagtgg taattaaggg tagagcctgt 29280
actgttagca gcagtccata caataccaaa cctatcaacg ttagtacgtg atgtagcaat 29340
caacagcgat gggatacatg taacaacagc cacacggtca tagttctgac gtggaatagt 29400
tacacgagcc ataatagccg taggagtagc tgcaccatta tcaacaaacc attcagacca 29460
atcaggtgta gagctagtaa gtgaactatc tacagcacca ccaacagcac cagtaatgcc 29520
ttggtaagta acctcaccac caaagtaacc attgttagca tataagtcac cagtaggtgt 29580
tactcggaag ttagccgagg ttgatgttgt attacccacc caaatgcggc tgttagagtc 29640
gctagcgtct agaacaacta catcttgacc tgatgtacca ccaatagtga ttcgtgtccc 29700
tgagttaatc tcagtgcctg taatcgtacc tgctgcaatc tggttagctg taatagtatt 29760
agatgcaatc ttactagcag ttactgcacc tgcttgaatc ttattagcag tgatagcatt 29820
ggcagcaatc tgagcagcag tgatagtgtt agctaccaat ttattagctg cgattgtacc 29880
gttgatgatt gcatcaccat ctacaaacag ttctacaggt tgccatgtac ttgatgaacc 29940
agtaccacct gtatagtagc gttgttctga gaactggttt acagagttaa ataatgtaac 30000
agtatcccaa taacgaacta caccatcaga gttagggtca ttgacaatag cattatatgc 30060
ctctaaattg ctccatgagc ttcctgagat agccttagat acattaaggg taccacgggt 30120
accatctgct ccatcctgac catcttgacc cggtggacct tctggaccga taggaccaac 30180
ttcaccttgt tcaccatcct taccagtaac tttgataggg aatgaataag ggtactgagg 30240
caagttacca tcctcttgta ccagaatcca cgtcatccaa actggtagtg ttgggttgtt 30300
aggtgttgtt tctgaccaac ctgttggtgg gtaagtatta cccggaactt gtggttgtgt 30360
agcagcctct ttaaaaaact gcttagtaat cttaccatca atgccatctt gtccatcttc 30420
acctgaaatc ttaacaggga aagaccaagg atatacaggg tcagtgccat cctcacgaac 30480
cttgataaaa gtcatccata caggtagagt aggattctct ggtggagtct tagaccaagg 30540
agatggggct ggataaacat tactaggtac tgatggagtt acagaagctt ctctaaacca 30600
ctgctcaaag atacgaccat ctaaacccgg agcaccatcc tgtccgtctg caccatcttg 30660
tccatcctca acacctgcac taaatggagt tggttgagac catgtatgag tgtctgtatt 30720
ctcatcgtaa gctgctgaca tagtaaccca aatccattca gacttgtcat tcttagcagg 30780
agccacttca gaccaaccgc ttggtggagc agatgatggt gatggagttg atggaggtgt 30840
accatcagca gtaagacggt aagcaaatac tgcaccagta atacttgatg ggtcatcaat 30900
gattttccac catctctcgc gtctgtaaag ccacacaccg gctgaagtga taatcttctg 30960
acctttgtat tgacctacat tataaacgtc gtctagaggc tctgtgaagt acatcatcat 31020
accctcacgc caacgtactg gcatcttacc ttgaggtggg aatgccccac cctccaagat 31080
accattaaca ataccaccta acgccaagag ttcatcattg atatatgttg gcattgactt 31140
ctcttctgta ggtgttggac cagcttggta gtgacctgag cttggacctg aaccaatcat 31200
atctgctcta cttgacatta tctagctcct gttgctttat attctaacca atagccacgc 31260
aattctgcac gactcttttc tgggatttcg aacttaacat gaatgaactt acctgttgct 31320
gtacaaccta ggttagctcg tttgttaggg tcaaacctac caacatatct ccaagctgga 31380
gactgtgtag ttgtctcact accagctacc cacacatcaa cttcaccacc ccgaacgaga 31440
ggccaaatct tattgagtgt tttcgtagtt gtatagtctt ccatgtcaat ccatttcttc 31500
tctacactag ctttaaagtt agcaccatcc caagtaggct catcaatagt tgtatataca 31560
gcacccttac catctgaggt aacaccagcg aacgctaagc cccactgtga gctgttatac 31620
ttaaagtatg aactaatcca tgtatcagtg gcttcttccc atgaagcgct ttctgatggc 31680
gctgtccatg taatatcacc ctctgtaata gataggatag tataagtaga agtcaatgaa 31740
tctggcatat ctcgtttaga ccagtgacgt tcttcccagt tatatacgta tgctacgtta 31800
caacgaccat taacatttgt tgatgcttct ggaatacata cccatgcctc tttctcttga 31860
taacgtacag ctacgaatac cttgtcgata tggtctgggt caatagagtc aaagattctg 31920
ttacgtacaa taccgtctgc tacagattct ttatgtacac cattatgcat atagatgtct 31980
gattgaccaa ccacaaaatg gtagccatca atctcagcca cacaatctgg accaagtaca 32040
cctacgtcgt taaacaattc cttaaaggcg aatactaaga taccgccaat gtatgacatc 32100
tgccatactg ctcgttcacg gtaaatgatg aacgactctc gaagagattt acctccaaga 32160
atcggtccac cattaccgcc tagaaagttc caaccggact ctgtagttgg gtctgcatct 32220
gcccaagaaa cacttaagtc ttttgtgttg atagcactag accaccatac agcagagcgg 32280
tgacgtccct cgttccaagg actgaactta gcttggtatt cttcttctgg cgtttgtggg 32340
tcagccggag gtgtgaagtt atctaccttg aagtccattg ccaccatgtg attgccgtag 32400
gttcgcataa cctgagcctt aacatgtccg gggaatgttt ccagttcttc aaatttactt 32460
actccatccc acacgtaagg cttacctttc atgaagtatg gaacaccatt gattgtatca 32520
ccaaaccaac ggaactcagg ttcatctggg atacctgctg ttggtgtaac atcttggtgt 32580
aagtctttag tgacgatacg gtagatttca ccagtaccat cttgtttggt tgaggcataa 32640
gcccaccagt agttctcatc agtatcatca cgcaatggga ggataatttc aggatatacc 32700
gaaccatcat taagaacacc agtctgacct aagccttgtt cataaccaga taccttctta 32760
gtcttaccat gcttaaacga gacttcatta gcctcgtccc acatagtaga atctaattgt 32820
gatggctcta gtgaaaagtt gacacctttt ggtttctcaa ctttataaac ttggtatgcc 32880
atttatcctc cttatgctgc ttgaccaacg tatacaatga atgctagtac aacgtactct 32940
ggtcggttat caaatgttgg tacgccttcg ttatcaactt ctgctgtcag tccgtgactg 33000
tgtggttgac caccaccaac ttcttcagtc tcagcaccag aaccaccttt aacatctgga 33060
ccagtaccac caccaccaga acctgttgca ttataatcta gtgggtgttt gtgagctgga 33120
atctctgcta gaacaagagt atgttcttct gaaaccaagt gtgttgatag gtcagggttg 33180
ttctcaccac cagttgaacg gttaggttga gctgtatctt taaggtcgtt ataacccatg 33240
ataaacttat cacgtaagtc tacagtctgg aaaccattct gaatagtacc atcagatagc 33300
acccaaccaa ctggaatgtc ctcaataaga ccttggaaca tagtagtctc accaatgaaa 33360
cgaccttgtg taccatcagt cattttgttt agttcagcta ctgtagcagt aacctcacca 33420
tcaatatttg gaaatgattc ctgcaccatt cgctttacag ctttaatctg gtcagcaccc 33480
tcatagcgag ggtctgtagg tgcagggatg ttaatgttca ttccagcaat gtagttacca 33540
ttatcaattg ccattattta cctccttgtg gaatcatacg acctagagcc gtaaagaaac 33600
cagcagcagc catgataaga ccaatctgcc attgctgtag gtatgcctca aagtaaggca 33660
ttacactgat tacaacgccc gctagggacg tcgcaatact cagttgaatg gaatatttct 33720
ttagccaatt catgttattc tcctaaacct aggtgtgctt caatagctgc taggcggtct 33780
ttaagttgtt tgttctcttc agatagttcc tgaatagcct taaatgcaat agcatggaag 33840
tcattaccct tgcgagagat gttcttcata ccgtcctcac cctctatgat aaggtgtggt 33900
agcactgtct ctacttcttg tgcaatgaaa ccatattcaa actgcttaac tacttcatct 33960
tcttcagttt cgtcattacg ctccagtaca tctttttggt atgtatatac aggcaagctg 34020
ttaacaacac tcaatgcatc ttccaccgga ttaacttcag acttgaggcg ttcatcagaa 34080
gcaacactat ctacgtatgc tttacgagca gccatgttag aggcagtagg gttagtaccc 34140
ggtaagtata ccggaactct agcgataacg tttgtggagt taaacagaat ccactggttt 34200
cctgtatcag ataagttctg agaagcaccc atagttagtg cagttccagt tctaccgaag 34260
tataaagggc tacctgtatt agagttaaaa cggatgttta cgttagcatc accgtctgaa 34320
cgatacaggt ctagtttagt agcacctgta gattctactc tcaattcttc tgtattgaag 34380
atagctttgg tacggtcacg gtaaatactt aatgcacgtc ttactacgtt attgtcacga 34440
atacttacgt aatactcgtt gtttactgaa tccgcagcat aacccatctc cgcaccataa 34500
gagccattag actcaaaaca acggaaagct gtgtttacgt ttggctggtc ggcatacaaa 34560
tccatgtaca tagggcttgt accgtagtaa gccatacgca tttggttagg ttgcaagtaa 34620
gtaaagttta catttccgcc ttcagggtag aaacgcatgg tattaccacc ctcaccaaga 34680
gcctgaatct tagcaccatc atcaccaatc tggaaagcat tatatgcaaa attagagttc 34740
gtacctgtaa gtttaagagc tggactaatt gaatgacccc aagagccacc tgctgcaccc 34800
gatgcttggt atttgtcaga aatacttgta ccacttaaga tgattgtatc accagtacct 34860
gcatttagct gtacgttacc atcagaacgt agtacaatat taccattacc attgtcgtta 34920
aaccaagtca tgttctcatt agcagaagaa ccaaaaccaa taaaaccacg acgtgtggcg 34980
tcatcatgag tccattggaa atatgaactt gaaccggtac ctgatgatac accatcttgg 35040
aatgtatatg cagcaccacc tgagtgttca tgtataatta ggccattttc ttgcactacc 35100
aaacggcgag acgggtcaga taaacctcta ggtcgaatct ctacattgtc accattacca 35160
gacaggaatg tgcgaccatc tgcataagcg aacacaccat gttgtgcgcc tgcacgggat 35220
agaccaaaat tattagtacc ctcatactct actagacctc ctggttggaa gctaaagtag 35280
tctgtatcag aagctacatc cataccattt ggacgtactt gaacacctgc cacagagcca 35340
gagccaccaa tacgagtaga acctccatta aacattacaa aaccttgttc tccaccaaca 35400
gtctctcgga ttgtggcagt gatgatatta gttgtaaatg tcttagcacc actaatggtt 35460
tggtctccag tagtcttaac tacttcacca tctaatgtat caatttggtt ttgaagtcca 35520
gacacgagag tatcggtttg tgtagtagta tatgcaccaa cgtcttgagc agtaggttta 35580
tttgctgtgg tgtaaaaata aacccaatca ttaaacacgc cactataacg agtacgatat 35640
gcaacattac cattattacc tgacacacca taaggccaag caacttgggt aatattgcct 35700
gtagagccgt atgtatgtac ttgcgggtaa tggtaccctg agccaccagt tggtttgtta 35760
gcagcagtag taccagataa aacattataa aaacctgcat cctcaagggt atcccaatct 35820
acacctgtac tacctccagt aactttagct gctgccttaa aggtattacc catagggata 35880
ccaacattag caaaagtagg tcggttatca atctgagatt gtaatgatgg tacaatagct 35940
gctgcatcat cagcactttg ttgtgcatct gtagcactct gtgcagcgtt agcttctgaa 36000
gtagctgcat tgctagcact tgttgctgca ttagactcgc tagtggctgc attacttgct 36060
gaagtagctg ctgcattttc acttgccaat gcattagcct cacttgtagc agcagcagat 36120
tcactgttag ctgcatttgt agcagatact tgagcactat cgcttgcagc tacagcagtg 36180
ttagccgctt gtgttgctgt ctgttcaggc acatcccatg tagcaccatt ccagaaacgt 36240
aggtcatgtg ttgtagagtt ccagtatgta gcacctactt gtagtgggtt gccatcatta 36300
tcagttgtag ggtcaactgg cttagcacca aggtattggt cgttaaagtt atcaaaagca 36360
gcttcagcat cagcagcaaa accagcagcg ataacagcag agttagctgc gctagtctca 36420
gagctagctg cattagattc tgattgagca gccgcagcag cgctgttagc agcattggta 36480
gcttgtacta ctgtatcatt gtaaacttgt acagtatcgt tatagacttg gtctgtttgg 36540
tcattgataa cctgagtctg gtcacgaata acttcagcct catctcgata ctgcttagaa 36600
tcttggctat attcgtaaga cttggctgcg gcttcttctg ccttagcaac caaatcctca 36660
atgtaagctg gacgaacagc aacgccatag aagcccttta ggttatctcg ttgagatacg 36720
ttagggtcat tctgaaattg tgtttggttt gtaaagaaac cgttttctgt agccatttat 36780
ttaactccct agtggaactt cccacaaacc accttggtca tcagctcgaa tactccatgc 36840
ttgaatctca gcaatagatg tttccatacg tgagttccag aaagctgctg tctcataatc 36900
acgtacaaac tcataagcat gagataaagc accatacaag taaacttcag gagctacctg 36960
taatagccag tttgtgtcgt catcaaattg tagtgtagtt ggtagagcaa agtagtgtac 37020
ttcaacttta ccgtacttat caaatgtacc agtaaagcca cctacaatat cttcactata 37080
agctttgaga gggaacactt tgtaaagaga accttcacga gcaaacttct gtggaacacc 37140
cccaatgcca ctggtaaagt atttatctac cgcatcttta aattcgtata ctgaaccctt 37200
cttcaaagag tgtctacgta ggactacttc ttcgtcagga ttactttctt taggttctgt 37260
caccagatag tctatagaga taagctctac taaatctgtt ggaatctgta cagaaccatc 37320
tgcatataag tcaacaatct gggttcgttc catcgtagga actcgtagct gtcgagcagc 37380
tttcaatgta actaatcgga taaagtcttt agcacgagga cgaatatcct cacggtcaat 37440
ccaatcaatt acagcatctt ctagttcttt aaaagtgtca tacatgtttt acaccatttt 37500
gcctttccaa acacgcaggt tagcccactc aggactctgt agtttcttcc agaacttagc 37560
tgtcatatca gggtcaacac ccacaagagt catatcaata ccttcttgtt gccactggaa 37620
gataacattc ataggaatac gtgcagctag tttcatatcc ttaccgcctt gactctcttt 37680
ctgaagttcc ttgttagtgt cgataatgtg gtcaactttc tgtacgtgtt taagttctaa 37740
tgaaccatct gcttgttctt tatattctgt acggtaatct tttttcattg tcatttctat 37800
actctccaaa taaaaatagc ccctcccata aagagagagg cttcatcgtt agttattaag 37860
ctactgggac agaagcgaat gggttacctt tcgcaaagtc gataatcata ccagagctta 37920
gttcgttgtt acaacgtaga ccccactcaa cgttaagtgc cttacgcatg tggtcaccag 37980
tcttaggcaa gtcgtaggtg ttgaagctac gtaggaagtc gatagaccac tgtgaagtgt 38040
ctagcgctag tgccatacca ccttgtgtca agtcaccgcc gatgtaacga gaaggcatga 38100
tacgtagagt accaaagtcc gagatgtaca catcaacaac tgactgaaca gtcttgtcct 38160
tagctgattg ctgtagctga ccagtcgctc gacctacgaa agtattcgag attttggtct 38220
tagttttagg gtcaacaatc agtgtgtttg gtgaacgacc tgactccata tagatgccag 38280
tcaataggtc ttgtaggtga gtctcagtaa gctctgctgt agaagcagat gctggctctt 38340
gccatacacc acgaccatta gcaacacctg tcactggagc tggagtatta ccagtcagtg 38400
tagaagtgtt ggtgtcatct acttggataa cgttcttaga aatccagcta gatacagatg 38460
gagtcttacg agctactgca acgttaccac cgttatcagt gtactggtgg tgaccacctg 38520
caagctgcat ctccatatcc atcttcagtt tcttagaaac cttagccatt tggtaagcaa 38580
gctctgagcc acgaccagct ttcttgattg cttcatcagt accagtgata ccgaaaccac 38640
gctcgttaat ctgcatgatt gagcttagtc ggtcagtaga ctgttgctct tgttgagtaa 38700
agtcatgacc ttctggtacg atggtatctg tcgcatgacc tttaccaatt tcatctacct 38760
gccaatccca tacgatgtta tgtgctgaac cacgaccaat cgagttcatt actggggttt 38820
caaacgggtc aacgttgtag attacgtccg ctaggtcttc acgtttaccg actgatactg 38880
tactgttaaa atctgccata attaattatt cctcttatag aaacgctgca aaagcgtcaa 38940
ctgaatcttt gatattacct gttttacgaa gacgtgccat tgactcctga tatttcttct 39000
gagatttatc ttgagtctta cctgtagcac tctttcctga tttgactact ttaggaactt 39060
tacgcttcaa tttcttctct actactttag tctgagaagc tttgtagtcg tcatattcct 39120
tagccttttg gagtagcttg ataacaatag catgtttaag aaggtcttta tcagaatctt 39180
caaaacctag cttttgtgtt tgttcttgta gtccctgcac caacttgtcg tgctcaggtc 39240
cacgccaccc ctcaacaatc tctgctgctc gtttttcctg agctgctaga tagtctgcct 39300
ctttcaagcc tttctctgag gttgattgct caatagcttg tttacgggct tccatcttgg 39360
attgtaacgc tgtacgtgcc tcattcaatt cgtactgaag aattaggaat tgatttgggt 39420
cttcctgttt aagtttattc cagtcaacct cattatactt ctctagttct ttagattctt 39480
cttgaatgag ttggtcaaga gcggttacat atttagtacg ttcagcttct agctcagcat 39540
aatgctcttg tagacgctta cgttcttctg caacttcttg agttttacgt gtatagtctt 39600
gctgacgttg ataaccttga accagttcat caccagttac ttcgatttcc tctccatcaa 39660
ctttgaccaa gaagacttcg ctatcggtag tttcatcttc agattcgtcg gcttcctctt 39720
catcggattc ttcttcggat tcttcatcct catcttcctc ctcagagacc tcttcctcct 39780
ctgattcgtc ctcttctagc tcctcttcaa tttcagtttc atcctcttcg gattccactt 39840
cttgttcgac ttcttcctct tcaatgccta gcatttcgtt agccatcgac tggaatacgt 39900
cgtgctgaga tgccgctgac aaatcgagtc cttcatcaag gttgttcggt ccgtgcccaa 39960
taacaggcgt agcgtttgta ctcataaatc ttactctcct ttatattcgc taagtttgat 40020
ttccaattgg gtgatgatag catcaaccgc cttagcttct cgtttgtatt gaagggagac 40080
ttcatcagcc tccgcaacca ttgagcggat taatgcatcc gcccttaaat catccattgc 40140
ttcttgaaag accttactgt ccagtattgc ctgaatctgt ttggcctttg tttcgtcctt 40200
tgtagagctt tccatctccg attgagacag ctttctctgt ttccttagta aggacgactt 40260
cagcgatgtt aacttgttta gcccactcaa acttttctcg ctccatgtct tgaatgtctt 40320
cttgaagtgc ttgctcacgt tcttgcaacg caatctcacg ttctcgaagt tcaagttctt 40380
taagcttaat tcgactttcc acatcattct tcttcgcttc ctgttgtacc ttaagctggt 40440
caacttgcga atcagactgc ttcttcatag cgtctgaatt agccttaatc tcttcaggtg 40500
taggtttagc tgcttcctct gcacgagctt tcttagtagc taaagcttct tcggtagttg 40560
ggtctaacca gaactcctct acatctgtat aaccagcgtt ttcagtaatc ttagttaata 40620
gattgtaaat cttctgttca ttggttaaga tacctagacc accattaccc acaatgcttt 40680
gtgtcaattc aaacatacgc atcagatgca taagttgttg gtctttatcc ttagtaccgt 40740
tacctacaag aactttcata tccacttggt cacgccaagt agctgggtca attgttacgt 40800
acttaccgtt aagacggaag ctcatctgct catcttggtg tttcattgca agttgcttaa 40860
tagcaataaa caaatcacga acacctgtct cagcaaagat acgagctact aatttcaatc 40920
gtttctcggc ggtagacata actcggttaa cttgaccaga gccagtatga gagtttagaa 40980
ccttgtcatc aagaccttgt gtacgaggag aaacaccagt acggtttgtc ttaatctcat 41040
cgtagtaagc aaccatatcg taagtacctt taggtaactc aggctgtact agaggtcgta 41100
atgcaccttg catcttctca cgaacgacac cacctagctt gttgttttgc atatcagcta 41160
agttaacctg accttcaacg gcagcataac gaccattgtt aatcaagtac atgttgtcta 41220
gttggttacg aagaagtgct gtcttaactt cttgaacatc atgaaccatg tcataaatag 41280
attgaccgta gaactgatgt tgaatagggt taggagttaa tgaagaaaaa ggtacaaagt 41340
cccagtcatc atcactcaga atcgtattac cacacatgat gattcgacgt agctcattaa 41400
aaccatcgcc atccacatcc atacgaacat acgcttcaat aatccaatac ttgtcgttca 41460
ttgggtcatt accgtaatcg tcataatcgt gtgtctgagc accgttatac atctcacgga 41520
ttagtcgtag ttcagaatca gatgggtcat cttcatcgta gttatttaat tcttcaattt 41580
gttcggctgt ataacccatt tcatataggt cagccttagt aaccttacgt tgatgaccaa 41640
caaatgtagc gtctttaaca cacttagcat ttcggtcaat caaaaactct tctggcggaa 41700
caggttctac tttgatgtaa cgtttagttt cttctacaga aatcttaata tcaaacgtac 41760
catcttcatt ctcggcaatc tctagtagct cagcaccatc ttttgcaaga actgctgtaa 41820
caccatcatt agataagcct gtgtagtatt ctgctgtttg gttagtttct tccgtccagt 41880
aatgcttaac aataccacgc ttaaacatta gagcatcttt aaaccattga tgcataatca 41940
tgaaaccttc gttcaggtta aagaatgtgt agttagcgta gttactcata tgttctgcac 42000
catctacatg ggcaggagta cgagcttcat attgcacagc attgtaatct gttgcaaaga 42060
tttccattaa atctggcatc atccagtcaa tggcatctga gacatctcgt gagattactt 42120
gagaacgacc ttcaacttca ttgcccatct tctcgccgta gtagtagcgt tgagcttgtt 42180
tccagttctc ttgtagtttc tcgtcttgat aatcacgagc atctcgaata tatttacgaa 42240
ggtaatgaac tttcttatcc ttctccatta ccttcaattc ttcttgtttt ggcattaaac 42300
tatacctcta ttatcctgta caaagtattc tgtgtatggg tctccaccag aacctacaac 42360
tgcgtatttc tctcgtgaca tgattgcata tcgtgaagca gagattgcat catccttaat 42420
aggaacaatc ttaccatctt gacggtggta ctcacggaac tcttgaagcc agttacgaca 42480
ggttgagaac actttcaagc gtccttgttc catcatctga tacatctcca agataccgac 42540
ttcaacacta ttactacctt tacctgttga tttctcaccc aaggcaatag ggttcttaaa 42600
gtgctctgct gtcatgttca caccttgatt tcggtattgt tgagctagtt ggatacctga 42660
acctttatca tgttgatagc catcgtgagg ccaaacaact ggaatccaac gaggtcgaga 42720
gttaattgct gctgcatgaa caactgctgt ttgtttgttc tgataatgtt catcgtaaat 42780
gtaaacagta tctgactctc tatcccatgc tacccagata gcagcagtag ggtggtcata 42840
accaaagtct aaaccacaaa tacgtggata atggtctggt aaaacaaaag gttctaccac 42900
aatttggtct tcagggaatg ggaatacctg accagaaccg aataccggaa tacctcttgc 42960
acgcatctct cgttcgtgtg gagagtaaac tgcaagtagc tgttgcttgg tgtgctcatc 43020
aatgtgtgga gcatcatccc aagaagcttg aatcaggaac tgacccggtt taagttcgta 43080
cataaatgca cgtaccgttt ctgtcatacc attctctggt gtaaatgtca ttgacacaat 43140
accttttgta gtagctgtac gtgtaataca ctgtgtaaag atattgtgtg gtggttcctc 43200
atctagccaa ataaagtcta gtgctgaacc catgaatttg tgttcgccca tttcatagga 43260
cttaaagccc aatcgagaga cgccacccga cacatgtctg atagtggcgc tctggagagc 43320
atttgggaca cctggtaatc gtgtggtgtc aataatatct tcagcaggaa ttgctcctgt 43380
acctagtgca gtagggtcat ctggttgtcc tagcaactct agctgtagaa tgtcacgggt 43440
tgttacgtta gatacgccac atgcccatgc attaattggt ctatcaaagc gatgtccttc 43500
ccaatcctct gggtatttac cagttaggtg atatgccatt tcagcagcac ctgtaaaggt 43560
tttaccgata cggttagcac acatcgctag tttctgtctg ttgtctttgg aagcgtacat 43620
aaactttttc tgccaatcat atggttcaaa gaacttaagt ttattctttc gtttccaatc 43680
ttcttcagca gcaattagtt tctttaaacg ttctaaatca tccatatact ctcccaagtt 43740
ttaattaagc aaaagtttcc aatgaaatct tgctatccga agcaaccgat actttaaaat 43800
taacagcagg agtttcggga actaggatga tgtcgtctgt gattaccgga ctaggattcc 43860
aaatagcacc tcccggtttt tcattacctg attctaaacc ctgtagaatg aagtaattac 43920
ctaatgcacg ctgagcagca atatcttctg gtaggtatcc gataaagaca agcaagtctc 43980
caatattatc gttagttacc tgtaagaagt tacgatttgg tgaagcccaa acacgagcat 44040
attcaccatc accaatgtta ccacctttag tgacaacacc ctgtactcta tttaagttaa 44100
agcccattat gtatctgctc ctttatcttg tttctggttc tgatgtgtca taggagctga 44160
catccaaaac gcccgtttct tattacgctg tacgtaattc tgattatttc ccaatcgctg 44220
gggtcttact cgctgtacca tttgttctcc atgaacccat gttgtcagta gagccaattg 44280
ccggagcgga taatactcgc ttcatcggga atgcacactc gtcacatggt cgtgtatgaa 44340
cgtcatcttc tacaatcttt attgtggaga atccacagtt atcgcataag aagtctcttg 44400
attttctcat aaagataatc tccctgcctc agacgcttgg tgtctaccac cttctggtct 44460
gtagcttaat agttcgtcac ccttcatatc tcggtagata attgttccat cttttagctg 44520
ttctacctca gagatagggt tgtctggatt tgctaagtta taagatagtg ctcttcgttt 44580
agccaatccc ttaacagact gtccaccaac gtttgctgtg tctaacagtt gtcttgatac 44640
ttcgtcatac tcgccttcca taagagcttt gtttagctta ggaaagtaac tattattaag 44700
tacctgttca cccatattat atgatgcatc tatcaacatt gcctgaagtt caggtggtgc 44760
ttcgttgaag cccttacgca taccccactg acgattaagc agtgctaggt actcccgtgc 44820
tacctcttca tcaggcatat catctggtgc attaactgct tggcgagcta ccgaagtaac 44880
accaagatta ccagttggaa tgtcagtaac attatcacca ctaccctcta atacggctag 44940
atggttaaat actacctctg ttgggtcaaa ctgttgagtt tcagcagcct gttctacttg 45000
ttcgatgcct ctctgtttct gattagcttt agcattttct aaagggtcaa actcagaaag 45060
acgcattcta acatttggta agttaacttt tggtaggctt acctcttgta aagtaacctc 45120
tgggattaga gcctgacggt tataaatatc agtaagtaag ccatctgcca ttagttgtac 45180
ctcgccatca attcttgaat ttctctttgt cgttcctctg gagtcaattc atcagcctta 45240
gtatcctcaa taaccagcgt atctttctga atacctgaat tcttaagaat catctcaaga 45300
gctttaagac gtgtattcca aggaacagtc tcatctgtag ctgcggtaag aagtagtttc 45360
tgtgctaacg ctgaagtctg ccccattcta cgcttaacca ccgccatcac atggtcgttg 45420
tttttatcta aatacttttt agcgttttga gcagcagttt tctcactaaa accagcagca 45480
atgtaggctt gaatatggtc accaccattg tctgcatagg cttctgagaa cttttctaag 45540
cgttccttct caccgatacc acgtttaatt tcggttacgt tgctcattaa ttgctgtctc 45600
catgtcttta gcagagcctt taagttcttc gttttcacgt tctaaaaact ctactcgttg 45660
ttctaagtaa agtacgtatt cttgtactaa tttgcttttg ttgtacatat tgctctccta 45720
tatttggccg agggtatggg attcgaaccc atgcatcctt tcggacgtcg cattagcagt 45780
gcgatacatt aaaccactct gtcaaccctc tataactggc ggaagaacag ggattcgaac 45840
cctgacgtca catcactgta accttcggtt ttcaagaccg ctgccgctac ccagaatctt 45900
tcggcttatc cttcctttaa tttggtactt ctggttggaa tcgaaccaac gtctttcggt 45960
tatcagccga atgtaagagc ccctctacca cagaagtatt taatttggtc tgtgtggcag 46020
gatttgaacc tacgacccta agcttccaaa gctaacactc taccagactg agctacacac 46080
agatattttc cttattattc cttttgtcct cctaactact gtacgtttat acagtaattg 46140
ttgaaacatg tcctatatta tacactaata ttccgtcact gtcaagtata gaaatactaa 46200
atttatcatt ttcttccaaa taatttcaaa ataaagccac caaacccact aaatagtgaa 46260
gaaaggtagt ctaacgggga tggaatgaac ataccaataa tcacccactg ccaccactct 46320
aagctgttgt tgataactgt attacctgaa ctggtaacag cattcttaac atcatccatt 46380
gtatttgtaa ctgattcaat tggaactggt ggaactgtag tagaacaact ggtaagaaat 46440
agaactggaa ctatagtata tagtagtata gtcttactat aggttaatct catacttatt 46500
ccttatttgc tttagattta tcttaatctc tttatttgct ttaggtaaga gtaataatta 46560
gtaagtaata atgattaata attaataatc ttaataacta ttatctagtc cttatataag 46620
gataacataa aaaagtgtcc ctgtcaagta tagttttaca ttaattagtt attttatacc 46680
agttatagta aaatactaca gttttcctcc cccaaaattt taggcatagg ctttacagta 46740
caagggttca atggcatagt gtaaccttca ggggtagttt aagtaaaagt ccccctccgg 46800
tatatggatt caacgtatgc cctatcgcaa gaaacaaaaa tggggtatgg gggtgatata 46860
tgggggagaa attgcatagg tatatataca tactggataa aactacaggt gtatattttt 46920
acataggtgt gagtgtgcat aggggaaggt ttattcaggg tatttgtggc atagtaattg 46980
catagtatta atctataact gtctattcat acagtattca accagttgtt aatctaacgg 47040
tagcacataa cgggtatcag ttaacggtat ggggtagggt attaatcgct atacataatc 47100
tatagcatag ctaggctaag aaatatcatc tataagttat tgattaataa ggataaataa 47160
aaataattat aataaataca aataaaagct tgcaatcatt aggctgttag tattagtatt 47220
tagtcaacgg caagggaaac caaaccgata gctagaaaag gtaattaata tcttgacagc 47280
taaaagataa ggcattaagc tttatcacat gttctttaat aattagtatt tactttgatt 47340
ggattgctaa tcaatgtgct catataaccc atagcagtta tgggaactat cactgataag 47400
tcatcaataa acctttatta aggcggcaag aaaattaaaa taaatactaa attgttattg 47460
acaactaaaa cctagttaga ttaaactagc tagcaagtca gggataacgt agtgatacgg 47520
cgtctaagta gcaagtcata agcatacgaa attatgacac tgacaaaggt gcaataaata 47580
acgataaaaa gttattgaca attagaaacg aatcattata atggttcaca agttaggcaa 47640
gtgttggagg ataagccaat gcagtaatta acagttagac gggcgattgt gcctagactg 47700
gaattcaaag cctcgcatta atcaatacac tgatgcggtt ggtggagggc aaaggtaaaa 47760
caatacaggt tagacgaccg tgctactgta tagtgcaaat tacgtctggg atgctgaggc 47820
tatgtcccat gaaagaagat agccgagcgg ttagtctgcg tctccggtag tgtcgagagg 47880
aatagactgg gtaaaaccat attaaaccta gtagataaag tggctaggat atagatggca 47940
gctttacccc tcattacttg ctatgagtat aattattcat gtggagtatt tataccaatg 48000
gcacgtaaac aaaatcattt gaagaaagtt caaaagcgtt ttaaaaatgc tacacgtaac 48060
aaacagttta ttagaactga gagttatgaa tcacaaaaag gttttggtaa agttgtgcgt 48120
gataaagact cgcatgaata caaaaccatt caatccacca aaggtaaaca aaaagtttac 48180
agaacatcta aaccgttaat ttgaggacta tattatgact aatacagttg caattcttga 48240
accaaaaaca ctaaccgtta aacaaatcga cgatatgatt gtaaatatca agtcccgtgg 48300
taagaaactg gattcagata ttcaagtttg cggtttggct attcttaaac atgtaatgtt 48360
gcatggcgat attactaaag ccaatgctct atacaatgca ctacctaaag gctcacgtcg 48420
cactgcacta gcagcgtggt ttgttgagtt tggtatgctc aagtacagcg agaaaaagga 48480
taaaaaagag aatccacttg cttacaacaa gaaagctacc actaacctac aaggtgcaat 48540
gaataaacct tggtgggaat gtaaaggtac taatgaagat ggcggtcgtc cgtttgactt 48600
agaatctcaa cttaaatcac tacttactcg catggaaaaa gcccgtgaag atggtaagga 48660
agtaaaaact ggtaatgtta atgttgatga gctgaaaaag cttattgcgc aggttaaata 48720
aaaatgaaag ctactcagaa agttgttcgc cagtcacagt tcccgcaagg cggctggcgc 48780
aagcgtgacg ctactctata ttcatcgttt tatagctcta aacaagcccg taacagcgtt 48840
aaacagcgca agcaaggcgc tagcaaggct ttagcatcaa aagctcttag cgaggctttg 48900
agagcgttgt gaacattcga gtagtaaaac cgtgtggtgg cgaaattgtc atcacacgta 48960
acccaattat gaatacttgc ggtcagatga ctgtatctta tcagcgtaaa tggcacatcg 49020
taactaatgg agttatcaca ttatgaatct tgacagctta gcggcaatgg ctgcaaatca 49080
cacccttgtc gatatgatta ctaaaaaggg ctattttgat atttgcactt tagataaagc 49140
attgagaact atgggtattc aagcacaatg tggtaaaagt tacagtatct tgagttctct 49200
acactgtatg gattacaaag atatgccaaa cgaattgcgt cgtagtatcc caatgctagt 49260
gcaaaatgta gtcggtattg atgcagaata ctatgaagct gaacctgaac ctaaaccaga 49320
gcctaaacct aaacgcatgg ctctatcatt cttcaagtaa ggaaatacta tgattaaatt 49380
gagcgattta gtatctgtta acaatccaaa acttaaaaca tttaaccaag atggttttgt 49440
agttggtgta agctttggtg gcggtaatct aataaaagtg aagatgttaa acggtggaac 49500
acacgtattt aaacgtgaaa atcttactag aactaaggtt tggcgcatct ctcactacat 49560
gtaggggtaa atatgatagg tttacttggt ctatttctgc tcgctgtagt tattgttgtg 49620
gcaatcatgg tcttgtttga gaaaaacaag ttcaaatctg ctgtaattac agctctcgtt 49680
ggtctgacag cttatttcgg gctgtcagcc tatatcatgg cacctatgtt tgaggcatta 49740
atgaaatgaa tacaaacatg acactgtatg agttgaactc tcaattcatc ataaaacacg 49800
ctattaagtc tttgaagata gcatatctac agttaaatgc tctaaatgtg ttaaatattg 49860
agccatctgt cgaaaaacaa ggttacttca atgtaacatt gggctgtaat accactgaaa 49920
agacttatat catcactgtt tccatgaaaa acagcttaga acaactgtta aacagcttaa 49980
atctggcatt ggaaacactc acaaaccgtt aagagggtaa aacaatgcgc aaattcttct 50040
atctattcat aatcgtctac gttatcttgt ctattttctt cacttacaag gctgtagctg 50100
ctgaaaatgg cgagtttaag gtaatgtttg caggtttttc gcatcatatt ggtgcgcctg 50160
attatgcaga ccataacgaa aagcacaatt tggtaggtat atcgttcgat aacactgaga 50220
ttttctttat gaagaactcg tttcacaacg attctgtagg gattgcgcat aattttactt 50280
attcttacag ccaatatttg gaatttggtt tccgtgttgg tgctgcaagt ggctacgata 50340
ttgatggaat atacaatatt ggcggtattg caccgtttgc acagccaaat gtgacggtaa 50400
aatatggtaa tttaggggtt gagttgggat atttaccaca aatcagccat tcaacgccgt 50460
atggagcggc aacattaacg tttaattgga gtttatagca tgaaaattca ataccttatc 50520
aagtttttcg acattcttaa ctggttttta gctcgtacta tcgagaaaga taacaaagaa 50580
atccagaaaa accgtcaaaa gcgcaaggat ttaaagacag ctatgcgtaa actggatgaa 50640
gc 50642
Claims (10)
1. A vibrio parahaemolyticus phage RDP-VP-19010 is characterized in that the phage plaque is 4mm-5mm in diameter, circular, transparent and halo-free through electron microscope observation, the head of the phage plaque is icosahedron, the length of the phage plaque is about 61nm, the transverse diameter of the phage plaque is about 67nm, the tail length of the phage plaque is about 112nm, the diameter of the phage plaque is about 10nm, the phage plaque is of the family Myoviridae with uroviruses, and the preservation number is CGMCC No. 19384.
2. The vibrio parahaemolyticus phage RDP-VP-19010 of claim 1, wherein the phage is a virulent phage which has strong lytic activity against vibrio parahaemolyticus with a lysis rate of 99%, providing a phage source for industrial, large-scale, programmed production of phage and treatment of bacterial diseases in aquaculture animals caused by vibrio parahaemolyticus.
3. The Vibrio parahaemolyticus phage RDP-VP-19010 of claim 1, wherein the phage RDP-VP-19010 is cultured at 37 ℃ for 1 hour in the range of 5.0-9.0, and the titer is maintained at 109pfu/mL or more, and the titer reaches 10 at pH 7.010pfu/mL or more, and is resistant to weak acid and weak base conditions.
4. Use of the Vibrio parahaemolyticus bacteriophage RDP-VP-19010 of claim 1 in the preparation of a formulation against Vibrio parahaemolyticus.
5. The use of the vibrio parahaemolyticus bacteriophage RDP-VP-19010 as defined in claim 1 in the preparation of a medicament against vibrio parahaemolyticus ZHBV-18001.
6. A bactericidal composition for the prevention and treatment of vibrio parahaemolyticus infection comprising an effective amount of the vibrio parahaemolyticus phage RDP-VP-19010 of claim 1.
7. The bactericidal composition for preventing and treating vibrio parahaemolyticus infection of claim 6, wherein the content of vibrio parahaemolyticus phage RDP-VP-19010 in the bactericidal composition is not less than 109PFU/mL。
8. The bactericidal composition for preventing and treating Vibrio parahaemolyticus infection according to claim 6, further comprising an antibiotic for Vibrio parahaemolyticus.
9. An aquatic feed additive comprising the vibrio parahaemolyticus phage RDP-VP-19010 of claim 1.
10. A detergent or disinfectant comprising the vibrio parahemolyticus phage RDP-VP-19010 of claim 1.
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| CN110214181A (en) * | 2016-12-21 | 2019-09-06 | 尹特荣生物科技株式会社 | Novel vibrio parahaemolyticus phage VIB-PAP-4 and its purposes for inhibiting vibrio parahaemolytious bacterial multiplication |
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| CN110214181A (en) * | 2016-12-21 | 2019-09-06 | 尹特荣生物科技株式会社 | Novel vibrio parahaemolyticus phage VIB-PAP-4 and its purposes for inhibiting vibrio parahaemolytious bacterial multiplication |
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| CN114958778A (en) * | 2022-01-28 | 2022-08-30 | 武汉格瑞农生物科技有限公司 | Vibrio parahaemolyticus lytic phage GVP-P20 and application thereof in prevention and treatment of vibrio parahaemolyticus in eriocheir sinensis |
| CN114958778B (en) * | 2022-01-28 | 2023-10-27 | 武汉格瑞农生物科技有限公司 | Vibrio parahaemolyticus lytic phage GVP-P20 and application thereof in preventing and treating vibrio parahaemolyticus in eriocheir sinensis |
| CN115197919A (en) * | 2022-07-26 | 2022-10-18 | 中国科学院深圳先进技术研究院 | Vibrio phage composition and preparation method and application thereof |
| CN115197919B (en) * | 2022-07-26 | 2024-05-17 | 中国科学院深圳先进技术研究院 | Vibrio phage composition and preparation method and application thereof |
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Denomination of invention: A strain of Vibrio parahaemolyticus bacteriophage RDP-VP-19010 and its application Granted publication date: 20220617 Pledgee: China Construction Bank Co.,Ltd. Jimo Branch Pledgor: QINGDAO RUNDA BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024370010065 |