CN112359048B - Preparation method of strychnos ignatii C - Google Patents

Preparation method of strychnos ignatii C Download PDF

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CN112359048B
CN112359048B CN202011409396.7A CN202011409396A CN112359048B CN 112359048 B CN112359048 B CN 112359048B CN 202011409396 A CN202011409396 A CN 202011409396A CN 112359048 B CN112359048 B CN 112359048B
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luz27
strychnine
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CN112359048A (en
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杜艺岭
施鑫杰
黄立明
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The invention discloses a preparation method of strychnine ignatide C, and belongs to the field of biological pharmacy. The preparation method comprises the following steps: actinomyces igniarius DSM43766 is taken as a starting strain, and a acetylase gene luz27 is inactivated through a gene inactivation technology, wherein the nucleotide sequence of the acetylase gene luz27 is shown as SEQ ID No. 2. And then inoculating the constructed mutant strain of the actinomyces lusononi madura in a fermentation culture medium, performing fermentation culture, and separating and purifying a fermentation product to obtain the strychnine ignatide C. The invention inactivates the acetylase gene luz27 of the actinomyces ignatii madura by means of genetic engineering to block the acetylation modification process of the actinomycins ignatii C, thereby specifically accumulating the component of the actinomycins ignatii C with higher biological activity. The Ignin C is produced specifically by a microbial fermentation method, and the method is green, economic and efficient and has an industrial production prospect.

Description

Preparation method of strychnos ignatii C
Technical Field
The invention relates to the field of biological pharmacy, in particular to a method for preparing an antiviral active molecule Ignin C in a green, high-efficiency and specific manner.
Background
Iguratin pepetimin (luzopeptin) is a kind of double-embedded family non-ribosomal peptide compound produced by Actinomadura sp.DSM 43766. The strain was isolated in 1980 by Japanese scientists from soil samples from the Philippine Igscht island. The Ignin has good antitumor activity and antibacterial activity, and is a high-efficiency HIV reverse transcriptase inhibitor.
The actinomyces ignasoren madura can produce A, B, C and other components under different fermentation conditions. Structural general formulas of the Ignin A-C are shown as formula (I),
Figure BDA0002817174200000011
ignin A and R1＝R2＝CH3CO; lvrong peptide B: R1＝H,R2＝CH3CO; lvrong peptide C: R1＝R2＝H。
The strychnine peptin C contains two 4-hydroxy-2, 3,4, 5-tetrahydropyridazine-3-carboxylic acid structural units, wherein the 4-hydroxy can be subjected to acetylation modification. The Ignin A and the Ignin B are respectively a diacetyl and a monoacetyl compound of the Ignin C. The Iguratin C has the best water solubility and the strongest transmembrane capacity, more importantly, the Iguratin C has the strongest HIV reverse transcriptase activity, and inhibits the IC of HIV-1 and HIV-2 reverse transcriptase50Respectively less than 7nM and 68nM, and the Iguratin C has no obvious toxicity to HIV host cells.
However, the original starting strain of the actinomadura ignatii strain is not produced with the strychnine peptide C under different culture conditions, or coexists with other components, the yield is low, and the separation and purification are difficult to be influenced by other components.
Meanwhile, the chemical synthesis of the strychnine hydrochloride C is very difficult. Complete synthesis of strychnine peptaibol C was reported by Dale l. boger and Delphine Valognes in 1999 and 2001, respectively. However, the strychnine peptide C has a complex structure, and both total synthesis routes require about 20 steps, so that the cost is high and the yield is low.
Therefore, the development of a method for preparing strychnine C with low cost and high efficiency is a problem to be solved by those skilled in the art. In the breeding work of microorganisms, the modification of the kind or yield of metabolites by inactivation or high expression of specific genes has been widely used. It is a feasible scheme to modify the actinomyces ignasoren madura by genetic engineering means to enable the actinomyces ignason madura to be capable of producing the ignason peptidoglycin C specifically.
Disclosure of Invention
The invention aims to provide a method for efficiently preparing strychnine hydrochloride C in a green way by microbial fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme:
sequencing the whole genome of the Igschuna actinomycetes (Actinomarda sp.DSM43766) to identify the biosynthesis gene cluster of Igschatacin, wherein the gene cluster comprises 5 related genes which participate in amino acid synthesis and post-modification, wherein the gene cluster comprises a gene luz27 for coding acetylase, and the nucleotide sequence is shown as SEQ ID NO. 2. The function of the gene is destroyed by the strategies of gene knockout or gene inactivation and the like so as to block the acetylation modification of the strychnine ignatide C, and further realize the accumulation of the strychnine ignatide C component.
The invention provides a mutant strain of actinomyces lusonoides, and a construction method of the mutant strain comprises the following steps: actinomyces igniarius DSM43766 is taken as a starting strain, and a acetylase gene luz27 is inactivated through a gene inactivation technology, wherein the nucleotide sequence of the acetylase gene luz27 is shown as SEQ ID No. 2.
The actinomyces ignasonii DSM43766 is available from German culture Collection of microorganisms and cell cultures (DSMZ) and has the number DSM NO. 43766.
The gene inactivation technology can realize luz27 deletion mutation by homologous recombination, CRISPR/Cas9 and other technologies; or inactivation of luz27 by transposon mutagenesis, insertion mutation, point mutation, etc.
Specifically, the method for knocking out the acetylase gene luz27 by using the homologous double-exchange method comprises the following steps:
(1) using genomic DNA of the actinomyces madurae of the Igscht as a template, carrying out PCR amplification on two upstream and downstream homologous arms of a target knockout region, and inserting the upstream and downstream homologous arms into a shuttle plasmid vector pSET152 to obtain a knockout vector pSET 152H-delta luz27 of luz 27;
(2) pSET 152H-delta luz27 was transformed into E.coli, and mixed with wild type Strongyloides obtusifolia DSM43766, and luz27 deletion mutant strain was selected.
Preferably, in step (1), the primers for amplifying the upstream homology arms are:
27LF：5’-agcagcAAGCTTatcacgtcgaggttgtgctcgatcacc-3’；
27LR：5’-agcagcTCTAGAaaccgctcagcacgaagaagaacga-3’；
the primers for amplifying the downstream homology arm are as follows:
27RF：5’-agcagcTCTAGAgtgaacctggccttcaaccagctcac-3’；
27RR：5’-agcagcGAATTCcagcagcatcgtcgtggtgatgtgac-3’。
in the step (2), the luz27 knockout vector is introduced into escherichia coli ET12567/pUZ8002 through transformation, then conjugal transfer is carried out and transferred into actinomycetes of Strobilanthus lugens, after resistance verification, a conjugant is subjected to relaxation culture and continuous passage, then a non-resistant colony is obtained through photocopy culture and verified through PCR, and the selected colony is proved to be an expected luz27 inactivated mutant.
The invention also provides a preparation method of the strychnine hydrochloride C, which comprises the following steps: and inoculating the constructed mutant strain of the actinomyces lusononi madura in a fermentation culture medium, performing fermentation culture, and separating and purifying a fermentation product to obtain the strychnine ignatii C.
The fermentation medium is an MS medium and comprises the following components in percentage by mass: 2% of D-mannitol, 2% of soybean meal, 2% of agar powder and the balance of water. The fermentation culture condition is that the culture is carried out for 11-13 days at 27-37 ℃. Preferably, the fermentation culture condition is culture at 28-32 ℃ for 11-13 days.
The separation and purification comprises the following steps: and after fermentation, soaking a culture medium containing the thalli in ethyl acetate, filtering a leaching solution, evaporating the ethyl acetate to dryness to obtain a crude extract, dissolving the crude extract with methanol, performing Sephadex LH20 gel chromatography on the dissolved crude extract, collecting a target fraction, and separating the target fraction by using a YMC-C18 chromatographic column to obtain a target product.
Preferably, the time for soaking in ethyl acetate is 10-12 h.
When Sephadex LH20 gel chromatography is performed, methanol is used as a mobile phase for elution, and fractions containing Iguratin C are collected. Further, purification was performed by using preparative C18 column, and isocratic elution was performed with 50% (acetonitrile + 0.05% formic acid)/(water + 0.05% formic acid) at a flow rate of 4mL/min, and the objective product was collected.
The invention has the following beneficial effects:
(1) the invention determines the gene luz27 of coding acetylase involved in the biosynthesis of Igscht actinomyces luscht through whole genome sequencing analysis of the Igscht actinomyces luscht, and further inactivates the acetylase gene luz27 of the Igscht actinomyces luscht (Actinomandura sp.DSM43766) through a genetic engineering means to block the acetylation modification process of the Igscht peptidmyces luscht C, thereby specifically accumulating the component Igscht C with higher bioactivity.
(2) The invention specifically produces the strychnine peperomin C by using the mutant strain of the actinomyces igniarius with inactivated acetylase gene luz27 as a fermentation strain and a microbial fermentation method, and the method is green, economic and efficient and has industrial production prospect.
Drawings
FIG. 1 shows the biosynthesis gene cluster of Ignin (luz 1-luz 30).
FIG. 2 is a schematic diagram of the construction of the mutant strain DS-1, wherein the right panel is a gel electrophoresis test.
FIG. 3 shows the measurement results of the leaching liquor components of the mutant strain DS-1 in example 1 by high performance liquid chromatography.
FIG. 4 shows the mass spectrometric results of Ignin C (MW 1342.5) (M/z 1/2[ M +2H ]]2+)
FIG. 5 shows Ignin C1H(600MHz,CDCl3) Spectra.
FIG. 6 shows Ignin C13C NMR(150MHz,CDCl3) Spectra.
Detailed Description
The invention is further illustrated by the following specific examples, but is not limited thereto.
The starting strain of mutant strain DS-1, Lvsonemadora, in the examples below (Actinomarda sp. DSM43766) was purchased from the German Collection of microorganisms and cell cultures (DSMZ).
The LB medium comprises 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and 2% agar powder; the MS culture medium comprises 2% of D-mannitol, 2% of soybean meal and 2% of agar powder.
Example 1
Firstly, constructing a mutant strain DS-1, comprising the following steps:
1. sequencing the whole genome of the Igschuna actinomycetes (Actinomardra sp.DSM43766) to identify the biosynthesis gene cluster of Igschitonin. The gene cluster is 48kb in length, shown as SEQ ID NO.1, and comprises 30 genes, shown as figure 1.
Further functional analysis was performed on 30 genes to determine a gene encoding acetylase, which had the nucleotide sequence shown in SEQ ID NO.2 and was named luz 27. The amino acid sequence of the acetylase encoded by the gene is shown in SEQ ID NO. 3.
2. The E.coli-Streptomyces based shuttle plasmid vector pSET152(Genbank SEQ ID NO: AJ414670.1) was designed for luz27 gene knock-out by the homologous double crossover method.
Using genomic DNA of the IgY madura actinomycete as a template, two homologous arms (the length is 2.2kb and 2.6kb respectively) at the upper and lower parts of a target knockout region are amplified through a polymerase chain reaction. Wherein the left homology arm of 2.6kb in length was amplified with primers 27LF (5 '→ 3': agcagcAAGCTTatcacgtcgaggttgtgctcgatcacc) and 27LR (5 '→ 3': agcagcTCTAGAaaccgctcagcacgaagaagaacga) and the right homology arm of 2.2kb in length was amplified with primers 27RF (5 '→ 3': agcagcTCTAGAgtgaacctggccttcaaccagctcac) and 27RR (5 '→ 3': agcagcGAATTCcagcagcatcgtcgtggtgatgtgac).
The left homology arm was inserted into pSET152 using two enzymatic cleavage sites HindIII and XbaI, and the right homology arm was inserted using two enzymatic cleavage sites EcoRI and XbaI, resulting in luz27 knock-out vector pSET152H- Δ luz 27.
After sequencing verification, the DNA was introduced into the competence of the E.coli strain ET12567/pUZ8002 by transformation. Picking single colony to be inoculated into a test tube culture medium for 12 hours, transferring the single colony into an LB culture medium according to the proportion of 1:50, shaking the single colony for 5 hours at 37 ℃ and 180rpm, centrifugally collecting escherichia coli thalli at 5000rpm and 8min, washing the escherichia coli thalli three times by LB, and collecting the thalli under the same centrifugal conditions each time.
Meanwhile, 1 ml of spore suspension of wild type Lignason madura actinomyces is taken, centrifuged at 5000rpm for 5min, then the upper glycerol is removed, 800 microliters of 2 XYT culture medium is added for resuspension, the mixture is placed into a water bath kettle at 45 ℃ for heat shock for 5min, then the washed escherichia coli is used for resuspension, and the two bacteria are mixed uniformly and then are spread on an MS (with the components of 2% D-mannitol, 2% soybean meal and 2% agar powder) plate containing 10mM magnesium chloride for culture at 30 ℃. After 18 hours, the cells were covered with apramycin at a final concentration of 50mg/L and nalidixic acid at a final concentration of 25 mg/L.
After 10 days, the zygotes were picked on MS plates containing apramycin to make spores. Spores were then collected and serially passaged on antibiotic-free MS plates until mutants were obtained. Streaking separation is carried out after 6 passages and every passage to obtain a single colony. Single colonies were picked and grown on MS plates without apramycin and MS plates without antibiotic, and single colonies that grew on antibiotic-free plates but did not grow on apramycin plates were picked for PCR identification with primers 28RF (5 '→ 3': agcagcTCTAGAattccggcaccgatgcgatgtccac) and 26LR (5 '→ 3': agcagcTCTAGAccactgtgcggatgtcgtggtcatg), respectively.
As shown in FIG. 2, the starting strain produced a target band of 1.6kb, while the mutant produced the expected band of 0.8 kb. The DS-1 mutant strain with luz27 deletion mutation is obtained by screening.
Fermentation of DS-1 mutant strain and identification of Ignin C
Collecting spores of the DS-1 mutant strain, coating the spores on an MS culture medium, culturing at 30 ℃ for 11-13 days, soaking the MS culture medium in ethyl acetate for 12h, evaporating the leaching solution to dryness, dissolving the leaching solution in methanol, and analyzing by high performance liquid chromatography (mobile phase ratio: 0-25 min, acetonitrile: water: 10: 90-80: 20; 25-26 min, acetonitrile: water: 80: 20-100: 0; 26-28 min, acetonitrile: water: 100: 0; 28-29 min, acetonitrile: water: 100: 0-10: 90; 29-30 min, acetonitrile: water: 10: 90. mobile phases all contain 0.05% formic acid, a chromatographic column is an EC-C18 analytical column, a detector is a diode array detector, and the flow rate is 1.2 mL/min).
Comparing with HPLC spectrogram of original strain and standard substance of Ignin A, C, as shown in FIG. 3, it is proved that the compound directionally accumulated by mutant strain engineering strain DS-1 is Ignin C, and no longer synthesizes Ignin A and other components.
Example 2
Preparation of Lv Song peptide rhzomorph C
The strain DS-1 of the Actinomadura sp is cultured for 12 days in MS culture medium (1L) in large scale, and then soaked in ethyl acetate for 3 times, the leaching liquor is combined and filtered, the ethyl acetate is evaporated to dryness to obtain crude extract, the crude extract is dissolved in 4ml of methanol and then is subjected to Sephadex LH20 gel chromatography (pure methanol mobile phase). The eluate was received in 5 ml aliquots per tube. 20 microliters of the components in each tube were analyzed by an EC-C18 chromatography (mobile phase ratio: 0-25 min, acetonitrile: water: 10: 90-80: 20; 25-26 min, acetonitrile: water: 80: 20-100: 0; 26-28 min, acetonitrile: water: 100: 0; 28-29 min, acetonitrile: water: 100: 0-10: 90; 29-30 min, acetonitrile: water: 10: 90; mobile phase contains 0.05% formic acid, detector is a diode array detector, flow rate is 1.2 mL/min). Fractions containing Iguratin C were found by comparison with standards of Iguratin C and separated by YMC-C18 chromatography column, isocratic washing with 50% (acetonitrile + 0.05% formic acid)/(water + 0.05% formic acid)Removing at flow rate of 4mL/min, and detecting at 254nm and 350nm ultraviolet to obtain Ignin C (63.2mg, t)R＝20.8min)。
The compound is further confirmed to be the Ignin C by mass spectrometry and nuclear magnetic resonance analysis, see FIGS. 4-6, and the nuclear magnetic resonance data are shown in Table 1.
TABLE 1
Figure BDA0002817174200000071
It should be noted that several variations and modifications can be made by those skilled in the art without departing from the principles of the present invention, such as fermentation with different media, luz27 deletion mutation by CRISPR/Cas9 and luz27 inactivation by transposon mutagenesis, insertion mutation, point mutation, etc. These should also be considered as the scope of protection of the present invention.
Sequence listing
<110> Zhejiang university
<120> preparation method of Ignin C
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 47885
<212> DNA
<213> LvSong Madura actinomycetes (Actinomarda sp.)
<400> 1
atgctcgacg gcttcgtccc gtggccggac cacctcgccg aggagtaccg ccgggccggt 60
taccggctcg ggcgcccgct cggcgacctg ctccacgagt cgtgcgcgcg gcacgccggc 120
cgggtggccg tcgtctgcgg ggagcgccgg atgacctacg ccgagctgtc gcgggcggcg 180
gaccggatgg cgtacggcct gctggagctg ggcgtcaagc cgctggaccg ggtcgtggtg 240
cagctgccga acgtgcccga gttcgtcgtg ctgctgttcg cgctgctgcg cgccggcgcc 300
atccccgtcc tggcgctgcc cgggcaccgg cgggccgaga tcgccggagt gcacgggcac 360
gcgggcgcgg tggcgtacgc gatcaaggac gagttcggcg gcttcgacta ccgccggctc 420
gcccgcgagc tgccctcgcc cgggcacgtg ctggtcagcg gcgacgcgca ggagttcacc 480
gcgctcgaca cgctggaggg gacggcgcgg gagctgccgg cggccgaccc gggcgacccg 540
gcgctgttcc tgctgtcggg cgggacgacc gggctgccca agctgatccc gcgcacgcac 600
gacgactacg gctactgcat gcgggcgacg gccgaggcga tgcgggccgg ctccggcctc 660
gtgtacctgg cggtcaaccc ggcggcgcac aacgcggcgc tcggctgtcc cggggtgctc 720
ggcagcctgc tgctcggcgg caaggcggtg ctcacctcca gcgtgcggcc cgacgaggtc 780
ttcccgctga tcgagcggga gggcgtcacc gtgaccacga tcgtgccggc ggtgctgcgg 840
ctgtgggccg acgcgggccc gcgcgggcac gacctgtcgc ggctcacgtt gcaggtcggc 900
agcgccccgc tggacccggc gctggcgcgc cgggcccgcg agggcctggg gtgcgggctg 960
acccggtggt tcggcctcag cgagggcctg ctcacccaca cccggctgga cgaccccgac 1020
gaggtgatcg acggcacgga ggggcggccg ctcgcggcgg ccgacgaggt cctgctcgtg 1080
gggccgtccg gggagccggt gcccgacggc gaggcgggcg agctgctggt acgcgggccg 1140
tacacgatcc gcggctactt ccgggcgccg gagcacaacg cgcgctcgtt cgcccccggc 1200
gggtacttcc ggaccgggga cctggtgcgg cggcggcctg acggcaacct cgtcgtcgtg 1260
ggccgggtca aggacgtgat caaccgggcg ggcgagaaga tctcccccga ggaggtggag 1320
cgctgcctgc tcacgcatcc ggcggtgcgg gacgcggccg tgctcggcgt gccggacggc 1380
gcgctcggcg agcgcacgta cgccttcctc gtgctggccg gcgacgtccg gccgccggcg 1440
gtcaaggcgt acctgcgcga gcgcgggctg gccacgtaca agatcccgga tcggatcgtg 1500
ccgatggcgg agctgccgcg cacccccatg ggcaagatcg acaagagggc gctgcgcgac 1560
ggcgtcaccg cccggcgcgc ctcctgacgc cggtcaggag gcgcgggaca gggcgcggat 1620
caggaggcgc ggatcaggag gcgcgggcgg ccctgcgggc gcggtcacgc agccggcggg 1680
cggccaggtg gatggtcgag aagtgccggg cggcattggc ccgcatcggc gcggggatgt 1740
cggcgagccc ccggtagagc acgcacgaca gctgcaggta gctcatgaac agcgcgagct 1800
cgtccaccag cgggtcgagc cgcccctcgg cggtcaccgc cagcggcccc cgccagccgt 1860
ccacgagctc cagcagggcc cgctgccgct cgaccaggac gggcagcacc tcgcccaccg 1920
ggtaggggcc cgagccgcgc atggggtgcg cggccagcac ggcggcgatg gcgtcggccg 1980
cctcggcgac cgcgtcggcg aggcggtgcc ggtcggtgtc gggtgcggtg gtcatggcgt 2040
gctcccttcc agggcccggc gcaggtaaag ggcggtgggc gagccggcga cgtcgagcag 2100
cccggcgggc gggccctggt agagcagccg gccgccccgg ctgccgccct cggggccgag 2160
ctcgatcagc cagtcggcgt gcgcgatcac gtcgaggttg tgctcgatca ccaccacggt 2220
ggcggcgtag gagtcgacca gccggtccag cgtgccgagc agccgctcca cgtcggccag 2280
gtgcagcccg ctggtgggct cgtcgaggac gtaggtggtg ctgcggctgc tcaggtgccc 2340
ggcgagcttg atgcgctggc actcgccgcc ggacaggctg gtgagcggct gcccgagccg 2400
caggtagtcg agcccgacgt cgccgatcgc ggtcagcacg ggcacgatct tgcggttgcc 2460
ggcgaagaac tcgcggcctt cggccacggt cagctccagc acgtcgctga tgtcgaggcc 2520
gcgcagccgg tagccgagca cctcgtcggt gaagcgtctg ccgttgcagg tctggcaggt 2580
gagggtcacg ccctccagcg aggcgaggtc ggtgaagacc acgccgaggc cgttgcaggt 2640
ggggcaggcg ccggaggagt tggcgctgaa cagggcgggg ctgacgtggt gggccttggc 2700
gaactcggcg cggatcgcgt cgccgatgcc ggtgtgggtg ctggtggtgg agcgccggtt 2760
ggcgtgcggc atcgactggt cgacgatcac cgcctccggg tgctgcgccc ggaaggcgcc 2820
gttgatcagg ctggacttgc ccgcgccggc cacgccgcag agcacggtga gcgcccgctc 2880
gggcagctcg accgtgaggt cgcgcaggtt gtgcatccgg gcgtgcgcca ccgtgagcat 2940
gcccgccggg gtgcgcacgc gctccttcag ccgggtggcg ccggtcatga acctgccggt 3000
gagcgtgccc gagccggcca gctcggcgac ctggccggcg aagaccacct tgccgcccgc 3060
cgcgcccgcg ccggggccga ggtcgatgac gtagtcggcg gcggcgatca cgtcgcggtc 3120
gtgctccacc accaggacgg tgttgccctg gtcgcgcagc gcgtgcagca ggttcgtcag 3180
ccggtgcacg tcgcgcgggt gcaggcccat gctgggctcg tcgaagacgt agagcatgtc 3240
gttgaggctg ccggacaggt gccggacgat cttcacccgc tgcgactcgc cgcccgacag 3300
cgtggcggtg cggcgctcca ggctgagata gccgaggccg atctccacca ggtggccgac 3360
gcgcgagatc agcgcctcga tcaccggctt ggcctgcggc aggtcgagcg cgcgcaggac 3420
gcccaccagc tcctcggcct ccatcgcggc gtagtcggcg atgttgcggc cgtcgatcct 3480
gcagtcgagg gcgagcgccg acagccgggt gccgccgcag accgggcagg gcaccgaggt 3540
gacgaaccgg gcggccatct gccgggtgcg ggcgccgagc tcgcggtccg gcttgacgaa 3600
gcggcggcgg aacttcacca ccgcgccctc gtaggccatg ttgatccgct gcccgccgac 3660
ctccagcggc accgtgccgt cggtgccgtg cagcagctcc tcgcgctcgg ccggggtgta 3720
gtcggcgacc ggcttgtcgg ggtcgaaccg gccggaggcg gccagcaggt accagtcgcg 3780
cgagccgacg tcgaagaccg gcgcgcgcag cgcgccctcg ttgagcgact tcgacgggtc 3840
gaggaaggcg tccatgtcga tgccgacgac ctcgcccagg ccctcgcact cggggcacat 3900
gccggccggc atgttgaagg agaaggcgtc ggcgtagccg acgaacggct cgcccgcgcg 3960
ggagaacagc agccgcagca gcggcgcgat gtcggtgatg gtgccgaccg tggagcgcgg 4020
cccgccggaa aggggccgct gatccaccgc gacggtggcc ggcagatgct cgatcagatc 4080
gaagtcgggg cgggccgaac tgggcaggaa attgcggacg aatgccggca gcgtctcatt 4140
ccattgccgt tgcgcctcga ccgcgacggt gtcgaaaacc agcgacgact ttcccgatcc 4200
ggatactccg gtgacgaccg tgagggtgcg tttggggatc tccgcgtcga cggtgtccag 4260
gttgtgctcc cgagcgccga ccacccggat ccattccggc accgatgcga tgtccacgcg 4320
ccgttcagca ttcacgactt cgaccctatc attgaagttt tacgttgaga gtgagccgcg 4380
ataagacccg gaaaacacgg gacgaccgga tgccaactct caaccggacc tgattgccgc 4440
cggtccggcc agggtagaca ggcggcgccc gccgctcaac accccggacg cgttgacggg 4500
cgaaagcacg gcgtgcgaac aatgaggcgc cgggccggtc cgggaaagcc gcggcgcctt 4560
ttcattcgcg ccgcccgccc cggaccgccg cctttccggc ggccgcgggc ggatgtcctg 4620
tcgccgggac acccaccgcc gggccgcgcg agcgagaggg gaaatcgcat gacctccacg 4680
accgtctcca gccgtttacc gtcgctgacc gggctgcgtt tcgtggccgc gttctgcgtg 4740
ctgctcgaac acctcaacat catcgcggtt ttcgtgccgc cggcgcgggt ctcggtgtcg 4800
ttcttcttcg tgctgagcgg tttcgtgctc acctggtcgt cccggccggg cgacacgtac 4860
cggtcgttct ggcggcgcag gttctggaag atcttcccca accacaccgt gagctgggcg 4920
ctgatgctgg tcatcctggc cgtcaccggc gtctcgctgg tgccgggggt actgccgcca 4980
gggccgacgc cgcccctgcc cgccgccgcc aacctgacgc tgctgcactc gtgggtgccc 5040
acgttcgact acatgttcag cgtcaacccg gtgagctggt cgctgagcag cgagatgctc 5100
ttctacctgc tgttccccgc gctgctgccg ctggtgctgc gcatcccggc gcggcggctg 5160
gcgctcgccg ccgcggggct ggtggcgctg gcctggctga tcccgctggt gtcgctgacc 5220
ttctccggcc cggcgctcat gcccgggctg ctgcagagcc agtactggtt cgcctacttc 5280
ttcccgcccg cccggctgcc ggagttcgtg ctcggcatgg tgctggcccg gatggtcatc 5340
cacggcttca ccccgcggct gggcgtggcg ctgaccgggg ccgccacgct ggtgttcctg 5400
ttcgtcgcga cggccgcggg gctgcccgcg ccgttcatgt tcgcggccgt gaccctcgtg 5460
cccgtggccc tggtggtgct gggcggcgcc tccatcgacg tccggcacgc gcgctcgttc 5520
tggggcaccc gcctcatggt cttcctcggc gagatctcct tcgcgctcta cctcatccac 5580
ccgctggtga acctggcctt caaccagctc acgcagggcc gggtccgggc ggcgctcggc 5640
gacgccggcg cgaacgtcat cgtcatcgcg ctctgcctgc tggccgcctg ggcgctgtac 5700
cgggccgtgg agcggccgct catgcggcgg ttcggctcct cccgcccggc acccgccccg 5760
gcgccggcgg cgcccacggc ccagggggcc tgaccctcct ccgcccatcg tccgtccacc 5820
tccgacgaca aggaccggca tgaccacgac atccgcacag tgggacaccg acgaacgcca 5880
gttctggctc cgcggcgagc agcccccggc ctgggtcgac ttcgacgagg ccatcggcgt 5940
ctggcgggtc ttcggctacc cggaggccct gcgcgccttc accgagatcg acaccttctc 6000
ctccaacacc gagcggctgg cgccgatgga ggtccagtac accgagggca acctcgtgga 6060
gatggacccg ccgcagcacg gcaagctgcg caagctggtc acgcacgcgt tcaccccgcg 6120
catggtcgcc aacctggagc cccgcgtccg ggagatcacg aacgagctgc tcgacgcgac 6180
cggcggggcc gggcgcatcg agctgatcaa cgacctggcc tacccgctgc cggtcatcgt 6240
catctgcgag ctgctgggca tccccgccga cgaccgccac ctgttcaagc gctgggtgga 6300
cgagatgatg gcgcagacgc acgagtactc cttctccgag cccaccgagc tgcaggaaag 6360
cagcatgcag cacgccatgg agcagatggg ccggctcgtc gactacatct gcgagcacgc 6420
cgaggaccgg cgccgcacac cccgcgagga cctgctcagc agcctcgtgc acgccgaggt 6480
ggacggccgc cggatgagca acaacgaggt cgccatcttc gccaactcgc tgctggtgaa 6540
cgggcacctg accaccacga tgctgctggg caactcggtg ctgtgcctgg acgcccaccc 6600
cgagcagttc gccgaggtgc gcgccgaccg gtcgaagatc cccggcgcga tcgaggaggc 6660
cctgcgcttc gtcacgcccg cgccggtgct cggccgcgcc acgctgcgcg agaccgagct 6720
cggcggccgc gtcatccccg cggaccagat catccaggtc tgggtgggcg ccgcgaaccg 6780
ggacccccgc cagttcgagc ggccggacgt ctacgacctc caccgcgacc ccaacccgca 6840
cctgaccttc gggcgcggca tccacttctg cgtcggcgcg gggctggcgc gcctggagag 6900
ccgggtggtg atggacctcc tgctcgaccg ctaccgcagc gtgcgcagcg acccggacga 6960
gccgcccgcg ttcctcacca acccgtacgt cacggccctg cgctcgttcc cgctgctggt 7020
cgcctgaagg agaagccgtc atgaccgtcc tcacggaccg atggaacgtc aatccgcggc 7080
acttctggct gcgcggcgag aagccggagc agccggtcgc cttcgacccc gcgctcggga 7140
tctggaacgt ctacggctac ccggaggcgg tggccgtcct cggcgacccg aagaccttct 7200
cctccgacac cacgcggctg gtggcggccg acctcgaccc ctcgctcaac gacggcgtgc 7260
tgttccagat ggacccgccg gcgcaccgca agctccgcaa cctggtcagc cacgccttct 7320
ccccgaagat cgtcgccggg ctggagccgc gcatcagcgg gctcaccgcc gagctgctgg 7380
acgaggccgc ggagcggggc cggctggagc tggtgggcga cctcgcctac ccgctgccgg 7440
tgatcgtcat cgccgagctg ctcggcgtcc ccgcgagcga ccgcgacctg ttcaagggct 7500
gggtggaccg gctgttcgag agcacgcacc agttctccat ggccgaggag ccgcaagggc 7560
tggagaccca cctggagatc accaaggcga tgaccggcta cctgcgcgag cacgccgtcg 7620
agcgccggcg gcggccgcgc gaggacctgc tgtcggagct ggtgctcgcg gaggtggacg 7680
gcgagcggct gaacgacaac cagatcgtca acttcgccaa catcctgctg atggccggtc 7740
acatcaccac gacgatgctg ctgggcaaca ccgtgctctg cctggacgcg cacccggagt 7800
ggttcgcggc ggtgcgcgcc gaccggtcgg tgctgccggc ggcgctggag gagacgctgc 7860
gttacttcag cccgttcacc gccgccgcgc gctccaccac ggccgcgacg gagctggccg 7920
gtcacaccgt ccccgccgac cagatgatca tggtgtgggt ggccgcggcc aaccgcgacg 7980
ggcggcagtt ccccgacccg gacgtcttcg acccgtcccg cgaccccaac ccgcacctgg 8040
gcttcggccg cggcgtgcac ttctgcctgg gcgcgccgct ggccaggatc gaggccagga 8100
tcgcgctgga ggtgctgctg gagcgccatc cggtgctgcg caccgacccc ggcgacccgc 8160
cgtcgttcgt gccggcgccg gacatgagcg gggtcgagcg cctgccgctg ctcacctcat 8220
gagcgctgtg acgagccccg gctccgcatc tgcgcgagga cggcgtcgcc gagcggcgtg 8280
ccgcggcggc cgaggtccag cggcaggtcg aagtggttgc gcccgcgggc gatgaaggag 8340
accagctccg gcgtccgcgg gcgggcggcg gcgacgaagt cgtggtgctg ccgggtgaag 8400
gccgccgtct cgttgtcgcc cacggcgacg accagcggcg gcagccggtc cggcagccgc 8460
cgcagggggc tgttgcgcgc cgcggccgcc gcgtccagcc cgagcgccgc gttgacgtag 8520
gtgcggcgca gctcgccgag ctcgtagacg ccgctgagca gcgccgcgcc ggcgaccggc 8580
ggcccgtcga gcagggccat ggccaccagg tgggcgcccg ccgagctgcc gctgaggtgg 8640
accggcccgt gggcgcgcgc ggccagccac cgcacgccct cccgcaccat ggcgacgatc 8700
tcgtcgagcc ggtagcgggg ggccagcccg tagccgagcg cggcgaaggc catgccctcg 8760
cgccggaagt cggccgcggc gaacgcggag tcctccctgc cgagctcctg ccagtagccg 8820
ccgtgcacgt agaccatgag aggcgcgtcc ggggagccgc ccgggaagtg gtcggcgacc 8880
tgctcgggcc gcgggccgta ccggagcggc cggtagccgg cggcccgcgc ggcccggctg 8940
cgccgctcgt actcctccag atgaacccgc aggtcggcga cgcgcgtgct gggcgagtag 9000
tcgcggtcga ggctcaccgc gcaccccttt ctccgagacg aatgctgatg atccgggagg 9060
cgaagtgtcc agagactcgg ccgaggtacg gttcgtccgc tggccggccc aggcggagct 9120
gcgcaaccgc tgcaggagcg aggggatccc gtgcctgctg gtggtggagg gcggcgcgcg 9180
gcccccggtg tgcagcgacc cgcgggagga ctgggtccgc gcgccgatct cccggccgga 9240
cctggaggcc cgcgtcgcgg cgctccggca gcgcgtccac gaccggcgca acccggtgct 9300
ggacgcgacc ggcacgttgt tgttcgacgc gcggtcggtc accgtctcca gcacgcacac 9360
cgagctgatg gcgttgttcg tggagcggtt ctgcgaggtc gtctaccgcg aggagctgat 9420
ggagcgcctg gccgcgtgcg ggccgccgcc gaccaggaac gcgctggacc tgcacatcat 9480
gcggctgcgc cgccgggtcg gcacggtggg cctggcgatc aggacggtgt ggggcctcgg 9540
ctacctgctg gagccctcac acgcggcgtg aggcgtcgca ctcggccacg aagtccagca 9600
gccgcgccac gccctcacgc agcacctcgg gcgcgacgca gaaggcgatc cgcacgagcc 9660
ggtgcgacca cggcgccggc tcgatcgagc ggacggcccc ggcgccgtcc aggccgggct 9720
ggagcgcgaa cccggtgccg ggcacgacgg ccacgcgccg ctcgcgcagc agccgggccg 9780
cgaacgtctc ggcgtcgagc cccgagcggg cgatgtccag catcgcgtac cagccgccgg 9840
acggcagccc gggcagcagg cccgcggcca ccaggccggg caggaccgcg tcccggttgc 9900
gccggaccag ctcgccgtgc gcgtccgcgg ccggcaggca ccgcatggcg gcgatcccgg 9960
cgtgctggac gggggtggac gggccgatga tgctggcctc ctggaccacg cgcagcgtgc 10020
tcgcggtccg ctcgtcggcc gtcaccacgt agccgagccg gtagccggtc atggccatgc 10080
tcttggagaa gctgaacgtg ctgtgcacga tgcgctcggc ctccggcagc ccgcgttcca 10140
gcgacgccgg ggagacgtgc tcgccgtcga agacgaagtg ctcgtacgcc tcgtcgctga 10200
ccacccgcca gccgcgggac ctggccgcgt gcagcagccg gcccacggcg gcggcgtcca 10260
tgaccacccc ggacgggttg gccggggagt tcagcagcag cacgcgggtg gccggcccgg 10320
cggcggccag gacggcctcg gggtcgggcc ggtagccggg gccgaggggg tagaacaccg 10380
gccgcagccc ggccagcagc gcctgctgga ggtggaccgg ccagtgcagc tcgggcagca 10440
ggatctccgc gcccggctcg gcgatcgcct tcagcagcga cgccaggccc tggcaggagc 10500
ccggggtgac gaacacgcgc gagggggcgg cgtcgatgcc gttggcggcg cggagcttgg 10560
ccacgatcag ctcgcgcagc tccagaaggc cgtccaccgg ggtgtagccg gtctcgccgc 10620
gccgcaccgc ggccacgagc gcgtcggcga cctccggcgg cgggtcgaag tacggctcgc 10680
ccacgtggag cttggccacc tgcccgccgg ggcctcgttc ctcctcggcg gccgcgtaga 10740
agatctcgtg cagccacgac agcgggatcg tcgaggcgat cggcacctgg ctcggcacgt 10800
ggctcggcac ctggctcagc atggtcatgg tctcctcctg ggctgtcatg tggtgcggcc 10860
cccgtccacc gggacgagcg caccggtgat ccatccggcg aggtccgggt cgagcagctg 10920
gcagacccac cgcgcgatct cctcgcccgt cccgaagcgg cccatggggg tggccgcgag 10980
caggctctcg cgcaggtccc gccggccctc cggggtcagc ccggcctcct cccacatcgg 11040
ggtgtccacc gggccgggca ggacggcgtt gacgcgtacc tcgggcgcga gctcccgggc 11100
cagggagcgg gtcaggctgt tgagcgcggc cttgctggcg ccgtagaagg agcggccggg 11160
catggacagc acgccgccga cggaggagac gttgaccacc gcgccgcggg cggcccgcag 11220
gtgcggcagc gcgcttctga tcagctcggc gggggcgcgc acgttgacgg cgagcatccg 11280
gtcgaactcc tcggggccgg cctcctccag gcgggcgaac cgggccagac cggcgttgtt 11340
caccacgccg tgcagcgcgc cgaaccgctc cacggcggcg cgcacgatcc ggccggccgc 11400
gcccggcgcg gccacgtcct ccgggacggc ctgcgccgcc ccgtccgtgg cgagctcctc 11460
cagcgccgcg cgccgccgcc cgacggcggt gacgagcgcg ccctccttca gcgcccgctc 11520
cgcgatggcc cggccgatgc cggtgcccgc cccggtgacc accaccgatc tgccctggag 11580
gctcataccg gcgtcacctg caccgtgtcg gcgatcagcc cggcgaggtc gtgcctgagg 11640
tggaagtggc cgccgggccg gacccgcagg gcgaagtccc cggtggtgac ccgcgcccac 11700
tcgggctgcg tcgcctcctc caccaggggg tcgccgtcgc cgtggtagca gcggaccgcg 11760
caggacagcg gcgccgtccc ggggcgcggc cggtaggtct cgctggcccg cacgtcggag 11820
cgcagcatgg gcagcaggac cgagcgcagc tcagtgttgc cggcgatgtc ggggtcgatg 11880
ccgccgagct cgcacagcgt cgtccagaac tcctcgtccg gccccaggtg ggtgcgcccg 11940
cccgcggcca gctcgggcgg cggcgagctg gagacgcaga ggcgcagcgg ctgcgacccc 12000
gcctcgcgca gcgccagcgc cgtctcgtag gcgaccagcg cgcccaggct gtggccgaac 12060
agcacgcagg gcgtctgcct cgcggtctcc agcagctcgg cggcgacgcc cgcggccatc 12120
gtccggacgt cgcccgcggg cggctcgcgg aacctgtcgc cccggccggg gtactggacg 12180
gccagcagcc gcacggcggg cggcagcgcg ggcacccact ccgcgaagaa cccggcggag 12240
ccccccgagt gcgggaagca caccacgctc agcccggggc gctccccccg ggtcagcggc 12300
cgcagccagc gccgttccca cgttcgcacc atgacctcct ccgtcgcggc cggccttcgc 12360
tgaccactgt tccgcccggg aggggcggcg gcggcgtccg cgaaagatgt gggcaagaga 12420
tcgggccgcc ggcggccgcc gcggcgacga tggcggggcg acgacggcag ggaaaggagc 12480
gcccacgtga gcccgtcact cgatctgacc gacccggccg cgttcgtcag gaccgacccc 12540
cacgcgttct ggcgcgaggt gcggagggag catccggtgt actggcacga gccgtccggc 12600
gggcggcccg gcttctgggt ggtctcccgg tacgccgacg tgctggcctg ctacgacgac 12660
tggaagcggc tcagctcggc ccgcggtacg gtcctcgacg tgctgctgcg cggcggcgac 12720
tcggcgggcg gcaagatgct cgccgtcacc gaccgcccgc gccaccggca cctgcgcaac 12780
ctgatgctcc aggcgttctc gccaaggcgg ctgggcgagg tcgccgcgaa ggtggaggcg 12840
cgcaccgccc gcctggtcgc ggccgtgacc gagctgggcc gcttcgactt cgccgccgag 12900
gtggccgagc acatcccgat gaacacgatc tgcgacctgc tgtccatccc ggagcaggac 12960
cgcaaggagc tgctgcagtg gagcaagtcg gcgctgtcgt cggagcgtcc cgacagcgcc 13020
gagctggact ccctggaggc caggaacgag atcgtgctct acctcatgga cctcgccttc 13080
gaacgccgcg cccgccccgg cgacgacgtg atcagcatga tcgccggcgc cgaggtggag 13140
ggccgcccgc tcacgcccga ggagatcgcg ctcaactgct acagcctgct gctcggcggc 13200
gacgagtcct cccggatgtc gggcatcgcc gcggtgctgg ccttcgcccg cgatcccggc 13260
gagtggcggc ggctgcgcga gggcgcggtc ggcctggaca ccgccgtgga ggagatcctg 13320
cggtgggcca cgccgggcat gcacttcgcc cgcacggcgc tgaccgatct cacgctcggc 13380
gggcagcggg tgcgcgcggg cgacatcgtc accctgtgga acacctcggc gaacaacgac 13440
gagacggtct tccccggccc gcgcgtcctc gacctgggac gccggccgaa caagcacctc 13500
accttcggcc acgggccgca tttctgcgtg ggcgcgttcc tcggccggac cgagctgcgg 13560
gccttgctgg agtcactggt ggcgtgcgtc gggacgatcg aactcgacgg cacgcccgcc 13620
cgcatctact cgaacttcct gtacggccac agcagcctgc cggtccggtt cctgccgcgc 13680
gcgcaggcgt agccccgggg ctcctcaggc gtagcgtgag cgcagcccgg gaccggccgc 13740
gggcccggcg cggaagacgt ggtcggcggt gtcgacgcgg cggccggtgc gccggtccac 13800
cacgacgggc tccacctcct gcccggtgcg ggcgtccacc aggatgaggg cgcgccggcc 13860
gggctccagg tcccggttgc cccaggcggc caggacggcg atgagcggcc gcagcgaccg 13920
gccgaggtcc gtcggccggt agccgtcgcg gtgcggggcg agcaggcccg ccgccacggc 13980
cgcgtccagc cgttcgcgca gcgctccggc ggtgaggccg aggtcgtccc gcatccgccg 14040
cacgtcgtgg acgccgttga gcgtctcgtg cagcagctcc agcgtccgcc agtcgccgat 14100
gcggtccacg gcccgcgcca gcgggcagtc ctcgatgcgt gcggccatgc tctcctccgg 14160
ggaacgggct ccccgggggc ggccccgggg agcgcctggg tcagttcggg tcggccgcca 14220
tggcctcgcg caggctgcgc gggcgcatgt ccgtccagtg ctgctcgacg tactccaggc 14280
aggccacccg gtcctcctcg ccgaacacga cccgccagcc ggccggcacc tcggcgaacg 14340
acggccacag cgagtgctgc tcctcgtcgt tgaccaggac gtagaaccgg ccgtcggcgt 14400
cgtcgaacgg gttcgtgctc atcatcggac tcctttgctc cggtgttcca ggatcgaggc 14460
gacgatctcg ccgctcctga ccgcgatgtt cgacagcagc gacgagctca gcccgtggct 14520
gtgctcggtg ccgccctgca ggtagatgcc gcagtccagg tccgggtccg tcaccatccg 14580
gtagtcgcgt tcgacgcggt agcggccggc cgcgtcgcgc aggcagtgcc ggtcgagctc 14640
gccgaggacg cggcccgggt ccatcgggtc gtagccggtg gcgaagacca ccaggtcgac 14700
ctcgaagtcg tcgtggtcgt cggtgagcag cgagtgcacc gagatcctgg tgtcgtcggc 14760
gacgcgcttg acgccccgca cccgggtcat gggcaggaac gacagccgcc tggtgccgcg 14820
cagctcctcg tcgtaggccc ggcggtgcag gccctggatg acctcgtcgt ccacgacgga 14880
gtagttggtg ttgccgtggt agcgccagat ggcctgcttg gacggctcgg agccgtcgaa 14940
gtagacgtcc accgcggccg ggtcgaagac ccggttggcg aagggcgtgt cgtcggcgac 15000
gcagtagccg taggagggca tgacggcgaa gatgtgcgcc cccggcacct cgtcgtagag 15060
gaacctggcg atctccgccg cgctctgccc ggcgccgacc accgccgcgc gcctgatcgc 15120
gccgccgtcc caggcgcgga agttgtccag gaactcggag ctgtgccaga cgcgctggtc 15180
gcgctcgacg cccggcggca gcttcggcac cagcccggtg gagatgacga cgttgcgggc 15240
caggatgcgc cactcgccgc ccgcgtgggc gtgcctgcgc agctccacct cgacgcagcc 15300
gcccggccgg tgcgccggcc ggcgcagcgc gaccacctcg gcgttgtaca ccggccgctg 15360
ggtgagccgc gactccgccc actccagata ggagtggaac tcctcccgcg tcgggaagaa 15420
gtcctggttg ttgacgaact gcggcagccg cccggccgcg tgcaggtagg agacgaagcc 15480
gaagcgcgag cgcgggttgc ggaaggtggc gaggtccttc aggaacgaga cctgcatctt 15540
cgccgagggc accagcatgc cgcggtgcca gccgagcgac ggctgccgtt cgaagaagac 15600
cgccgagagc tccgcggcct cctcctccag cgcgatggcg agggcgaggt tcgacggccc 15660
gaacccgatg cccaccacgt cgtaggtgtc gtagctggac ggcacggtca gtcctccccg 15720
agcgcgagat tgacgatccg gcagatctcg gccgcgtgct ccggccggta catctcctgg 15780
tgggtgcagc ggatgtcgtg ctgcctgagc tccccctcga cgtgcggccg ccacagcgcg 15840
gtgtagtcct cggggttcag cagcgcgttg aagaacagcg tggtcccgcg gtagacgggc 15900
gagcggaacc tcttcatgat cgcggtgtgc tcgaccatga gcgaggccga cgtctcgacg 15960
atgaggtcgt actcgtccat gtcgtcgagg tggcgcatgt acttctcgaa gaactccctg 16020
acctggccgg ggtcggggtc gtccggcgcg tgccgggtga agtagctcgc cggcgcgcag 16080
tccagcaggc ccagcagcgc cacctcgtgg ccgcggcgct gcagctcggc ggcggcggcg 16140
tgcgcgaccg tgccgccgag cgaccagccg agcaggtgga aggggccctc gggctggacg 16200
gcgaggagct gcgccaggta gtcgtcgatc atctcctcga tcgaggaggg ccgcgggtgc 16260
gcgcggtcga agccgcgggc ctgcacgccg tacacggggt ggtcctcgcg caggtgcgcg 16320
gcgaacccca tgtacggcca gcacagcccg ccgccggagt ggaaccacca cagcggcgcc 16380
cgccggccgc cggtcctgat cggcagcacc acggcgaacg ggtcctggcc ctcgggtggc 16440
tgggcggcgt ccaggtgcgc ggccagctcg gcgacggtgg gcgcctggaa gaccgtcctg 16500
atggggacct cgacgcccag ctcggcgcgg atgcgccagg tgaggcggat gacccgcagc 16560
gagtgcccgc ccatcgcgaa gaagtcgtcg tcgacgccga tccggtccag gccgagcacc 16620
tcggcgaaga gcctggccag cgtctcctcc cgctggtcgc gcggcgcccg gtagacgccg 16680
cccgcgaact cgggctcggg cagcgcggcc cggtcgagct tgccgttgcg ggtcagcggg 16740
agccggtcga gcaccacgaa cgcgctcggc atcatgtact cgggcagcct gccggtcacg 16800
cgttcgcgca gcccggccag cagcgccccg acgccgcggg cgacggtcgg ggtgttggcc 16860
agccgcccgc gcacgccgct gggccggaag acgccggtca cgcaggtccg cgccgacggg 16920
gtgagcaccg cctcgaagcc gtccgggtcg gtcgccgacc aggtgcagcg cacggtgagg 16980
ccgcgcgccg cgccccacgc gcacaacgcc tcggggtcga cgccgcccgc ggggccggcc 17040
tcgctgagca ggcgggcgtt gggcagcccg gtcagccgca ccgggccgcc gggacgcagg 17100
tccgcgagcg cgctgacgtc ctcgccccag gtgaggacgg gaacgccggc cgcgtccatg 17160
ggccgcaccg gctgcttgtg cagcacgacc tcgtagcggt gcctggtcag ctcgttgtgg 17220
tggccgccgc gcttgagctg cacgtccacc gcgccgacgg cggtgttgcg cgcggcccac 17280
gccgtgaagt actccgggtc gaggacgagc tccttgtcgg cgagcacggc ccgttcgacg 17340
gcggcccgca cggcggcggg cgggtcgtcg gggtgctggg cccggtgcac ggcggtgtgg 17400
aaggcccgca gcgtcgccag gttgcgcacg tcgcccacga cgatccgccc gcccggggcc 17460
agcaggccca tggcgaggtc gagcacctgc gccaggtagc cggcgtcggg gaagtactgc 17520
acgacggagt tcagcacgac ggtgtcgaag tggccggcgg gcaggccggc ggcgtcctcg 17580
gcgggctggc agcgcagggt gacgcgcccg ccccacggct gcccggccac ctggccgcgc 17640
agccggtcga tgacgggggc ggagaagtcg gtgccccagt actcctcgac gtgcggcgcc 17700
agcggggcca gcagcaggcc ggagccgacg cccagctcca ggacccggcg cggccggtgc 17760
gcgaggacgc gggccacggc ggcgtcgcgc caggcccgca tctcctccag cgggatcggg 17820
cggccggtgt aggagctgtt ccagccggtg aagtcggcgc cgagcgggcc gggccgcacc 17880
tcgccgccct cgtacatgga gtcgtagacc tgccgccact cgcggacgtg ctcggccgcc 17940
tcgtcgccgg aggccagggc ggcgccgggc accacgtagg cgaccagcct gcggtccccc 18000
gggcggtcct cgcgggcgat caccgcggcc cgggcgaccc ccgcgtcctc cagcagcacg 18060
gcctcgatct cggcgggctc gatgcggaac cccctgacct tgagctggtc gtccacgcgg 18120
cccaggtact ccagccggcc ggcggcgttc caccggacca ggtcgccggt gcggtacatg 18180
cgctcgcccg gccggccgtg gcggcaggcg acgaaccgct cggcggtgag cccggggcgg 18240
ccgacgtagc cgcgcccgag cccctccccc gccaggtaca gctcgcccgc cacgcccggc 18300
ggcgccagct ccaggccctc gtccagcacg tagagccgca tgccccgcat gggctcgccg 18360
atgggcacgc tctcggcgcc gggcggcagc tcgcggacgc ggtgccgggc ggcgaaggtg 18420
gtggtctcgg tgggcccgta gccgttcacc acggccaggc cgggccgcag cacgcgccgc 18480
acggcctcgg gcggcagcgc ctcgccgccg gcccagacct cctccacgcc ggcgaagcag 18540
cccgggtgct cctccgccat gacggcgaac aggccggccg tcagccacag gccggtgacc 18600
ccgccctgct cgacggcctc ggccatggcc gccaggtaga gcttgcccgg cggggccagc 18660
acgagggtgc cgccggaggt cagcggcgtc cacagctcgt aggtggaggc gtcgaaggtg 18720
tgcggggagt gcacgagcac ccgccggtgc gccggccccc agcagtcgtc gagcacgagg 18780
tccgcgacgt tgcggtgggt gatctccacg cctttgggcc gtccggtgga gccggaggtg 18840
tacatgacgt aggcggcgtt gtccggccgc acccgggccg cgggcggcgg gccgtcctgc 18900
ggctcgtccc ggaaccgcgc ggcccactcc tggtccagga cggccaccgg cccggcgtcg 18960
cgcagcatgt aggcgacgcg gtccggcggg aagtccgggt cgatcggcac gtacgccgcg 19020
ccgctcttca ccacgcccag gatggccgcg accagcagcg gcgagcggtc gagcagcagc 19080
gccacccgcc gctcagggcc cgcgccgcgg ctgatgaggt gccgggcgac gcggtcggac 19140
cagcggtcga gctcggcgta ggtccattcg tccccgccgc cggtcagggc cgcggcgtcc 19200
ggggtgcggc gcacctgggc ggcgaacagc tcgggcaggg tccggcgcgt gtcgcgggcc 19260
gtgcccgccc gcgccagcag ctcctcgcgc tcgcccggca gcagcacgtc gccaccgccg 19320
atggtgtcgc ccgcgtgggt cacgaggtgg tccacgaggc gggcgaagcg ggcggcgacg 19380
gcctcggcgg tgccgcggtc gaacaggtcc gtcgcgtact ccagcgtgcc ggcgccgtcg 19440
ggcgagaggt tgaggaacag gtcgaacttg gcccggcgcg tggtcaccgg ctccagccgg 19500
acccgcaggc ccggcatcgc gctcgcgctc ggggcgttgt tctgccaggc gaacatgacc 19560
tggaacagcg ggtggtaggc ggtggaccgc tccgggttca gcagctccac caggtcctcg 19620
aagggcacgt cctggttgtc gtaggcggcc agggccttgc ccttgacgcg gccgagcagc 19680
tcgccgaacg acggccgccc ggacaggtcc acccgcagca cccaggtgtt gacgaagaag 19740
ccgaccaggt cttccagcgc ctcgtcggtg cggccggcga tcggcgagcc gatcgtcacg 19800
tcgtcgccgg cgccgagccg ccacagcagc accgcgagcg cggcctgcag caccatcggg 19860
acggtcacgc cccgctccct ggccagctcc tcgaccctgg ccagcacggc ggggccgagc 19920
cggacggcga cggcgtcgcc gccgttgccg gccacggccg ggcggggacg gtcggtgggg 19980
agctggacgg gctgcggaac tccgtccagc tccccgcgcc agtagcgggt ctgcgccgag 20040
accaggctgt ccgggtcgtt ctcgtcgccc agcaggtcgc gctgccacag cgtgtagtcc 20100
gcgtactgga cgggcaggcc ggcccagcgc ggggccgtgc ccgccagccg ggccgcgtag 20160
gcgcgggaca ggtcgcgggc gagcggggcg gaggagccgc cgtcgccggc gatgtggtgg 20220
accagcagga cgagcacgtg ctcgcgcggc gacacccgga gcacgctcgc ccggaccggg 20280
atctccgcgg ccaggtcgaa ctcgtgcccg acggcgcgga cgaccgcctc ctccagctcg 20340
gcgggggcgg tgacgggcat gtcgagcacg acctcgccgg gcgggacgac gtgctggtac 20400
ggcatgccct cggcgtcctc gcggaagacg gtgcgcaggc tctcgtgcct ggtcaccacg 20460
tcgaggagcg cggcgcgcag cgccggcacg tcgagctcgc cggtcagccg cagcacgatg 20520
ggcaggttgt acgtcggcga cggcccctcg aagcggtgga tgaaccacag ccggcgctgc 20580
gcgaacgaca gcggcagccg gtccggccgc ggcatggggg cgagccgggg gcgcacccgg 20640
tggccgccgc cgagccggtc ggccagcccg gcgggcgtgg gcgtctcgaa cagggcgcgg 20700
atcggcagct cgacgccgag gacggtgcgg atgcggctca ccagccgggt ggccagcagc 20760
gagtggccgc ccaggtcgaa gaagccgtcg tcggcgccga cccggtccac gccgagcacc 20820
tcggcgaaca gcccgcacag cttctcctcg cgcggggtgc gcggggcgcg gccggtgccg 20880
gagccggcct ggccgggggc gggcagcgcg cggcggtcga gcttgccgtt ggccgtcagc 20940
ggcacccggc tgatcgcgac cacggtggcg ggcaccatgt actcgggcag ccgctcccgc 21000
gcgtgcgcgc gcagcagcgc gccgagcgcg ccgatctcgc gggcggcggc cggctcgttc 21060
gcccaggccc ggccgtcgcg ggcggcggcc ggggcgaagg tcccggcgca gacctggtcg 21120
tcgggcacgc cgtcgggcag cacgatcgcg tcgaagcact cggcccccgc cggggaccag 21180
gtgaccaggg cggtgccgcc ccgctcgcgg gcccaggcgg tcagctcgtg cgggtccagc 21240
gcgggcccgg cgggcggcgg gcccgccagc acggacagcg cgcgggccgt cgcggcctcc 21300
ccggcgagac gggcgttggg gatgccggtg acgcgcaccg gcccgtcgtc ggggacgtcg 21360
tccagcccgg tccacgggac cgtcccgacg tcgtcgagcc gcagcggccg cacgggctgc 21420
ttgtgcagca cgacctcgta gcggtgcctg gtcagctcgt tgtggcggcc gcccggtttg 21480
agccgcacgt cgagcgcgcc ggcctcgtgc gacgcggccc aggcggcgaa ccactcggga 21540
tccaccacca gttcccgttc gaccagcagc gcctgctcca ccagggcgcg gacggccggg 21600
gccggcgcct cgggccggcg tccccgctgg acggccgcct ggagcagcgg cagggtggcg 21660
gcccgccgga cgtcgccgac cacgatccgg ccgccggggg ccagcagccg ccaggcctgg 21720
cgcagcaccc ggtccaggta gccggtgccg gggaagtact gcgcgacgga gttgagcacg 21780
acggtgtcga agtgcccttc gggcaggccg tccgcgtcgt cggccggccg gcacagcagc 21840
cggacgttcg ggcccacctc gggccggagc cgctcgatga ccgaagggga gaagtcggtc 21900
gcccagtact cctgcgtgcc cggcgcgatc ctggacagca gcaggccgtt gcccgcgccg 21960
atctccagca cccgttgcgg cgaccaccgg gtgacgcccg ccacggccgc gtcccgccat 22020
tcccgcatgt cctccatggg gatcggctcg ccggtgtagc tggacgtcca gaccgagaag 22080
tcgtcggccc attccgccgc ggcggagtcc gcgtacgcct ggtcgtagac gtgctgccac 22140
tcctcgacct gggcggtggc gccctcgtcg gtgccgcgga cggccgggac cacgtaggcg 22200
accaggcgct cgtcgcgtgc cacgacgacg gcctgctcga cgtgggggtg cgcggcgagg 22260
gtggcttcga cctcgcccgg ctcgatgcgg tagccgcgga tcttgacctg gtcgtcgagc 22320
cgtcccaggt actccagttc gccgttctgg ttccagcggg ccaggtcgcc ggtgcggtac 22380
atgcgtcctg ggtggaaggg gtcggggacg aatcggtcgg cggtcagggc gggccggtgg 22440
tggtagccgc gggccaggcc ggtgccggcg atgtagagct cgccgggggt ctgggcgggg 22500
acgggttgca ggtaggggtc caggacgtag acccgggtgt tggcgatggg ccggccgatg 22560
gtcggcgggc cggtcacggt ggcggcggtg gaccagatgg tggtctcggt ggggccgtag 22620
aggttggtca gctcggccgc gtgctcgcgc agctgggtcg ccaggtgcgc gggcagcgcc 22680
tcgcctccgg tcagcaggcg cagaccggcc agccgggcgg ggtcgtgggc ggtcatcatc 22740
tgccacagcg acggggtggc ctgcatgagc gtgatgtcgt gccgggtgat caggtcggcc 22800
agcaggggcg ggtcggggtg ttcggcgagg atcacgtgac cgcccgtggt gagcgggccg 22860
aacagctcca gcgcggcgat gtcgaaggcg acggtggtga ccgccagcag ccggtcctga 22920
tcggacaggc ggcagcggcg gcgcatgtcg gtgaggaagt tgaccagcgc ctcgtggctg 22980
atcacgacgc ctttgggggt gccggtggag ccggaggtgt agatgacgta ggcggcgctg 23040
cccggcaacg ggcggcgggc ggggtcggtg tcggggaggc cggtcaggtc gggctcgccg 23100
gtcaggtgca gcagcggggc ggcgtcgtcg agcacgaact ccacccgggc cggcgggtag 23160
gccgggtcca gggggacgta ggcgccgccg gtcttggcca ccgccagcaa cgccaccacc 23220
agttccgccg agcgcggcag ccggaccgcg atccgccgct ccgggcccgc gccacgggcc 23280
agcagccagt gcgccagccg gttggcgcgc cggttcagct ccccgtaggt gagcgtccgg 23340
ccgccgcagg tcagcgccgg agcgccggga gcgcgcgcgg cctgccgctc gacgagaccg 23400
gcgaggtcgg ccggctcgac cggcgccgcc gtgcggttgt gctcgtgcag gagccgctgc 23460
cgctcctggc cggtcagcac ccgcaccgcc gcgatcggga cggccgggtc ggccgtgacc 23520
tggcggacga cgtccgcgaa ccgctcggcc agccgctccg ccgacgaccg gtcgaacagg 23580
tcggtggcgt actccagcgt ccccgtgccg tcgggcgcga ggttgaagaa caggtcgaac 23640
ttggccgacc ccggggagac ctgctccatc gtggcgtcga gccctggcag gtcgaggtgc 23700
ggccacgggg tgttctgcca ggcgaacatg acctggaaca gcggatggta ggcggtggag 23760
cgctcggggt tgagcagttc caccagccgt tcgaacggca cgtcctggtt gtcgtaggcg 23820
gcgatcgcct tacgccggac gtgggcgacg acgtcctcga acgacggccg cccggacagg 23880
tccacccgca ggacccaggt gttgaggaag agtccgacca ggtcggtcag ggcctcgtcg 23940
gtgcggccgg cgatgggcga gccgatcgcc acatccgtac ccgcacccaa ccgatgcagc 24000
aacaccgcca gcgcggcctg caacaccatc ggcaccgtcg ccccaccacg ccgcgccacc 24060
ccctccaccg ccgccagcaa ccccggctcc aaccggaacc ccaccgtccc gccccgataa 24120
ctcgccaccg ccggccgcgg ccgatccacc ggcaaccgca ccggctgcgg cacccccgcc 24180
aactccgcac gccaataccc cacctgcgca cccaccagcc gctcatcacc cagcaactcg 24240
tgctgccaca acgtgtaatc ggcgtactgc accggcaact ccgcccaccc cggcacccgc 24300
cccgccagcc gcgcccgata cgccaccgac agatcccgcg ccaacggcgc cgccgactcc 24360
ccatccgccg cgatgtgatg caacaacaac accagcacgt gctcacccgc ccccacccgc 24420
aacaagcacg cccgcaccgg gatctcccgc gacagatcga actcataggc caccgcccgc 24480
gccaccgccg cccccacctc caccggctcc gacaccggca gctccaccac cacctcaccc 24540
accggaacca cccgctgccc cggcaccccg tccgcgtcct ccaccagcac cgtccgcaaa 24600
ctctcgtgcc ggcccaccac atcaccgatc gccgcccgca acgccggcac atccagctca 24660
ccgcacaacc gcaacaccac cggcaggttg tacgtcgacg acggaccctc gaaccgatcg 24720
acgaaccaca accggcgctg cgcgaaggac agggggatca tgacggtcac ctttcggtca 24780
tcctgcggag cggcgctctg acggacgcgg acagctcctg ccggcgttgc gccagccggg 24840
ctacggacgg ggactcgaag agcatccgga tcggtacctc gacgcggagc gtcgcgcgga 24900
tgcggctgac gagctgggtg gcgagcagcg agtgcccgcc cagggcgaag aagtcgtcgt 24960
ccacgccgac ccgctccacc cccagcacct cggcgaacag cccgcacagg acctcctcgt 25020
cgctggtgga cgggggccgg taggccgacg agccgaattc gggcgcgggc agcgcggccc 25080
ggtccacctt gccgttcggc gtcagtggca gggcctggag ggcgatcacc agcggcacca 25140
tgtactccgg cagccgctcg cgcgcgaagc tccgcaacgc ctcgtcctgt acctggccca 25200
ccacgtaggc caccagccgg ccgtcgcgtg ccacgaccac ggcctgctcg acgtcggggt 25260
gcgcggccag gaccgcctcc acctcgcccg gctcgatccg gaagccgcgg atcttgacct 25320
ggtcgtccag tcgtcccagg tactccagtt cgccggctgg gttccaccgc acgagatctc 25380
ccgtgcggta catgcgtcct gggtggaagg ggtcggggac gaatcggtcg gcggtcaggg 25440
cgggccggtg gtggtagccg cgggccaggc cggtgccggc gatgtagagc tcgccggggg 25500
tctgggcggg gacgggttgc aggtaggggt ccaggacgta gacccgggtg ttggcgatgg 25560
gccggccgat ggtcggcggg ccggtcacgg tggcggcggt ggaccagatg gtggtctcgg 25620
tggggccgta gaggttggtc agctcggccg cgtgctcgcg cagctgggtc gccaggtgcg 25680
cgggcagcgc ctcgcctccg gtcagcaggc gcagaccggc cagccgggcg gggtcgtggg 25740
cggtcatcat ctgccacagc gacggggtgg cctgcatgag cgtgatgtcg tgccgggtga 25800
tcaggtcggc cagcaggggc gggtcggggt gttcggcgag gatcacgtga ccgcccgtgg 25860
tgagcgggcc gaacagctcc agcgcggcga tgtcgaaggc gacggtggtg accgccagca 25920
gccggtcctg atcggacagg cggcagcggc ggcgcatgtc ggtgaggaag ttgaccagcg 25980
cctcgtggct gatcacgacg cctttggggg tgccggtgga gccggaggtg tagatgacgt 26040
aggcggcgct gcccggcaac gggcggcggg cggggtcggt gtcggggagg ccggtcaggt 26100
cgggctcgcc ggtcaggtgc agcagcgggg cggcgtcgtc gagcacgaac tccacccggg 26160
ccggcgggta ggccgggtcc agggggacgt aggcgccgcc ggtcttggcc accgccagca 26220
acgccaccac cagttccgcc gagcgcggca gccggaccgc gatccgccgc tccgggcccg 26280
cgccacgggc cagcagccag tgcgccagcc ggttggcgcg ccggttcagc tccccgtagg 26340
tgagcgtccg gccgccgcag gtcagcgccg gagcgccggg agcgcgcgcg gcctgccgct 26400
ccaccaggtc ggccagggtc gtacgcggca gcggcgcggc ggtgtcgttg acctccgcca 26460
ccaggtgccg ccgttcgtcc gcggtcacca cgcgcaccgc tcccacggag acggccgggt 26520
cggccgtgac ctggcggacg acgtccgcga accggtcggc gagccgctgc gcggacccct 26580
cgtcgaacag gtccttcgcg tactccagcg tccccgtgcc gtcgggcgag acgctgaaga 26640
acaggtcgaa cttcgcggtg cccgtgccga ccggctcgta ggtcacccgc aggccgggga 26700
agtcggcgtc ggcctgggtg ttgttctgcc aggcgaacat gacctggaac agcggatggt 26760
aggcggtgga gcgctcgggg ttgagcagtt ccaccagccg ttcgaacggc acgtcctggt 26820
tgtcgtaggc gttcagcgcc ttgcgccgca cctcgtccac cacctgctcg aacgaggggt 26880
cgccggacag gtcgacgcgc agcacccagg tgttggcgaa cagcccgatg aggtccttga 26940
gcgcttcgtc ggtgcggccg gcgatgggcg agccgatcgc cacgtccgta cccgcgccca 27000
accggtgcag caacaccgcc agcgcggcct gcaacaccat cggcaccgtc gccccaccac 27060
gccgcgccac cccctccacc gccgccagca accccggctc caaccggaac cccaccgtcc 27120
cgccccgata actcgccacc gccggccgcg gccgatccac cggcaaccgc accggctgcg 27180
gcacccccgc caactccgca cgccaatacc ccacctgcgc acccaccagc cgctcatcac 27240
ccagcaactc gtgctgccac aacgtgtaat cggcgtactg caccggcaac tccgcccacc 27300
ccggcacccg ccccgccagc cgcgcccgat acgccaccga cagatcccgc gccaacggcg 27360
ccgccgactc cccatccgcc gcgatgtgat gcaacaacaa caccagcacg tgctcacccg 27420
cccccacccg caacaagcac gcccgcaccg ggatctcccg cgacagatcg aactcatagg 27480
ccaccgcccg cgccaccgcc gcccccacct ccaccggctc cgacaccggc agctccacca 27540
ccacctcacc caccggaacc acccgctgcc ccggcacccc gtccgcgtcc tccaccagca 27600
ccgtccgcaa actctcgtgc cggcccacca catcaccgat cgccgcccgc aacgccggca 27660
catccagctc accgcacaac cgcaacacca ccggcaggtt gtacgtcgac gacggaccct 27720
cgaaccgatc gacgaaccac aaccggcgct gcgcgaagga cagcggcagg cgggcgggcc 27780
gcgtggcgag cgcccgcagc gggggccgga ccgccaggcc gggcgtcagg tgaggggcca 27840
gttcggcgac cgtgggggct tggaagacgg tcctgatggg gacctcgacg cggagcgccg 27900
cgcggatgcg gctgaccagc cgggtggcga gcagcgagtg cccgcccagg gcgaagaagt 27960
cgtcgtccac gccgacccgc tccaccccca gcacctcggc gaacagcccg cacaggacct 28020
cctcgtcgct ggtggacggg gaccggtagg ccgacgagcc gaactcgggc gcgggcagcg 28080
cggcccggtc caccttgccg ctcggcgtca gcggcagcgc cgccgccacc acgaccgcgg 28140
agggcaccat gtactccggc agccggccct ggacgaagcc gcgcagggtc tcggcctcga 28200
cccggcccac cgcgtaggcc accagccggc cgtcgcgtgc cacgaccacg gcctgctcga 28260
cgtgggggtg cgcggccagg gccgcctcga cctcggccgg ctcgatccgg aagccccgga 28320
tcttcacctg gtcgtccagc cgccccaggt actccagctc gccggccggg ttccagcggg 28380
ccaggtcgcc ggtgcggtac atgcgcgagc cggccgggcc gaacgggtcg gcgacgaacc 28440
ggtcggcggt cagggcgggc cggccgtggt agccgcgccc catgcaggcg ccggtgacgt 28500
agagctcgcc cgcgacgccg ggcggcaccg gcctgagccc gggaccgagc acgtgcacgc 28560
gcatgttgcc gagcggcctg ccgatggggg cgccgccgct gccgctccgg gtggcggccg 28620
tgccgagcac gcaggtggtg gcgtagaagg tctccgtctg cccgtacgcg ttgacgatct 28680
tcgcgccggg cagcgccgtg cgggcctgcc ggacgagcga ggccggcagc gcctcgcccg 28740
cgaacacgac cgcgtcggcc gtgacccgcc cggcgagcgc gccggcgagc tcggcgaagg 28800
cggacgggac cgtgctgatc acgccgccgg tccagccgtc gcgttcgccg agcacgagca 28860
cgtccctgac gatctcggca gtcccgcccc ggcagagcgt gctgaacagc tcgaacaccg 28920
acacgtcgaa gctgaccgag gtgcccgcga gcatccgggc gcgcggcggc aggccgagca 28980
cgtcgttgag gcgcagcacg ccgttgacga cgttaccgtg ggtgatcgcg acgcccttcg 29040
gctcgccggt ggagccggag gtgtacatga ggtaggcggt gttgccgggc agcagcccgg 29100
tgcgcgggtc gtgcccgggc ccggagccga gctcgtcgag gtggacgcgc gggacgcgcg 29160
cgtcgcgcac cgcccgcccg gcggcggcgt cggtcagcag cagcgcggga cgggcgtcgg 29220
cgagcacgta ctccagccgc tcgctgggga accccgggtc gatcggcagg tacgccgcgc 29280
ccgacttcag gacgccgagc agggccacga ccagctccgc ggaccggggc agggccagcg 29340
ccaccacggt ctcggggccc gcgccgcggg cgatcaggtc gtgggcgagc cggttggcgc 29400
gggtgttcag ctcgcggtag gtgagggtac ggccgccgga ggtcacggcc ggggcgtcgg 29460
gcgtccgctc cgcctgccgt tcgaccaggc cggggacggt gagcggcggg gtcgcgacgg 29520
cggtgtcgtt gagctgccgc agccgttggt gttcgccggg ttcgagcagg tcgagcgtgg 29580
cgaggcgggc ctccgggtcg tcggccaggc gccgcaggac gcgcacgacg cgccgcgcca 29640
gccggtcggc gtcggcgccg gagagccggt cccgctggta ctgcacggtg agccgcaggc 29700
gagggtcggc ggcggccagc agcgcgaccg ggtagtgcgt cgcggtgtac ggccggacgc 29760
cggtgaccgc gaggccggcc gtgctgttgg cctcggcgag gccgatccgg tccaccgggt 29820
aggactcgaa cacgacgagc gtgtcgaaca gggtgctcag accggtgacc cgctggatgt 29880
cgccgagccc ggtgtggtgg tgttcgagca gcgccgcctg ccggtcctgc aggcgggtga 29940
gcacctgcgc gaacgtgtcg gcgggcgcgc acggcacgcg gaccggcacg gtgttgatga 30000
ggaggccgac caggtcctcg acgccgggca gcgcgggggt gcggccggag acggtggtgc 30060
cgaacagcac gtcccggcgg ccggtcagct cggccagcag cacgccccag gcgccctgca 30120
ccagcgtgtt gagggtgacg ccgagctcgg cggcgcggcg ggacagggcg gcggccacgt 30180
cgggcggcag cgggacgtcg gtctggccga tgcccggctc ggcggtacgg gcgggcaggc 30240
tcgccgccag cagcgtcggc tccgtcacgc cgtcgagctc gcgggcccag gcccgcagcg 30300
acgcctcctg gtcgcgggag cccagccagg cgaggaagtc gcggaacccg ccggccctgg 30360
ccaggccgtc cggctcgccg ccggccgcgt acagccgcag gaggtcgcgc atcagccgcg 30420
gcagcgacca gccgtcgagg agggcgtggt gggtgctgag gacgagctcg gcccggccgg 30480
cgccgagcgt gacgagcgtc agccgcagca gcggcgggac ggcggggtcg aagcggacgc 30540
cgcggtccgc ggccagcagc cgctccagct cgcgggcccg ctcggcctcg ccgagccggg 30600
acaggtcgtg ctcgcgccac ggcagccgga ccccgtccac cacgagctgg gcgaggtcgc 30660
cggcctcgtc ggcggcgaac gcgacgcgca ggttcgggtg ccggtcgagc agcgcctgcc 30720
cggcggcgcg cagccgcgcc gggtcgagcg gcccgtcgag gcggaggacg agctgcatct 30780
ggtacgcgtc cgcgcccgtc ccgccgagca tcgagtggaa gagcatgccg gactggaggg 30840
gcgtcagggg ccacacgtcg gacaggccgg gccaggaccg ctcccaccgt tcgagctggt 30900
ggcggctcac gccggcgcgc gtcaggtccg cgggcgtcag gccgccggcg tccggcgcgg 30960
cggcgtgccc ggcgagtgcc gtgacggcgg cggcccacag ctcggccagg tcccgcacct 31020
cgtcgcggga cagcacaccg tcggggaagg tgaacagggc cgtcagctcc tgctcgccgc 31080
tgatcagcgc gtcgacctgg agcgtggccg ggacgggctg gccggggccg ggcatggcga 31140
ccagttccgg ccattccggc gcgggccccc actcgccgcc ggtcaggtcc tcggcgagac 31200
gtccgaggta gttgaagacg atctggccct cggggtggcg gcccagctcg gccgcggtcc 31260
cgtcgtcgag gtggcggagc aggccgtacc cgatgccgtg cccgggcagg gagcgcagtt 31320
gttccttgac cgccttgatg gcggctccgg cggcggggcc gccggcgaac gcctgctcga 31380
cgtcgatgcc ggccaggtcg agccgggccg ggtggacgct ggtgaaccag ccgacggtgc 31440
gggacaggtc cgcgcccggc accacggcct cctcgcggcc gtgcccctcc agccggatca 31500
ccgcggcggt gcccggcacg ccctcgcggc ggcggcggcg cagcagcgcc agcatgagcg 31560
cggccagcag gccctcgtcc acgcccgcgc ggaaggcggc gggccagcgg gcgagcaggg 31620
tgcgggtgat cgccggcgtc agccggacgc gcacggtcga ggtggccgcg gcgacgtcgc 31680
gggcggggtc cagcgggcgg gcgccgagcc gcggctcgtc ggtggtgacg acgtcgagcc 31740
agaccggcag ctcgtgcccg cggcgggccg cctccgaccg cagcgcgtgc gcccagcggc 31800
gcatggaggt gcccacgggc ggcaggacgg ccgggcggcc gtcgcgcacg gccgcccagg 31860
cggcggcgag gtcgggcagc aggatgcgcc aggagacgcc gtccacgacg aggtggtgcg 31920
cgagaatcac cagccgcccg cccgcgacgg cgccggccga accggaggcg ccgccgccgg 31980
aaggaccgtc gccgccgccg gagggggcga gccagatgaa gcgcagcatc gagccgtgtt 32040
ccgggtccag ctcgcgctgg gcggcctcca gggcgggccg gacctcgtcc gggtgcccga 32100
cgcggcggat caggcgggcg accggcaccg agccggccgg ctccacgcgc aggtcgccgc 32160
cgtcgagccg ggagcgcagc acgtcgtggt ggtccaccac ggcggtgagc gtcgcgacca 32220
gcccggcgtg gtcgatcccc ggcggcaggg tcaccgccat ccactgcgcg aaccgcgcga 32280
acgcggggcc gcgcgcggcg gcctcgcggg ccaccggcag gagcggcatc gggccggtgc 32340
cgccgccgtc cagctcctcc agcgcgctcc cggggccggt cccggccgcg tcggcgaggt 32400
cggtgacggt gcggcgttcg aagacgtcgc gcgggctgag cgcgaggccg cgggcgcggg 32460
cccgggagac gacctggatg gcgcggatgc tgtcgccgcc cagggtgaag aagtcgtcct 32520
cggcgctgac ccggtccagg ccgagcactt cggcgtagac ctccgccagg acctcctcgg 32580
cgggcgtgcc gggagcgacg tagtcgccgc ccgcgaacac gggctcgggc agggcccgcc 32640
ggtccagctt gccgttggtg gtcagcggca gccggtcgag cagcacgaac gcggccggga 32700
ccatgtactt gggcagccgt cccgacacga acgcgcgcag ctctcccgcg tcggcgcccg 32760
cgcggaagtc gaccgcctcc ccgccgccga tgccgcccgc gtcccgcggc acgacgtagg 32820
cgacgagctg cctgccgccg gtcccgcggc cctcgcgggc gatcaccgcg gcctgcgaca 32880
cgcccgggtg ccgctccagg gcggcctcga tctcggcggg ctcgatgcgg aagccccgga 32940
tcttcacctg gtggtcggcc cgtcccatga actcgaggcg gccgtcggcg gcccagcgcg 33000
cgaggtcgcc gctgcggtac atgacggcgc cgggcgggcc gaacgggcag gcgacgaaac 33060
gctgggcggt gagtgcggcc cggccgtggt agccgcggcc gacgcccgcg ccggcgacgt 33120
acagctcgcc gatgacgccg ggcggcaccg gggcgaggcc gggcccgagc acgtacatcc 33180
gcatgttgcc gatcggcacc ccgatggggc tcgcgccgcg ctcggcgaac tccgccagca 33240
tctccaggta ggtgacgtgg acggtggtct cggtgatgcc gtacatgttg accagcaccg 33300
gcgcgccggg cgtcgcgccg gggtaccagc cgcgcagccg ggccgggtcc agggcctcgc 33360
cggcgaagac caccatgcgc agccgcagcc ccgaggtgtc ggcgggccgg ttggcggaca 33420
gctcgtagaa ggcggagggc gtctggctca ggaccgtcac gccgtggcgg agcagcagcc 33480
gccacagggc gtcgggggaa cgcacgacgt cccaggtggc caggacgatg cggccgccgt 33540
gcagcagggc gccgcacagc tcccacatgg agacgtcgaa cgcctgcgag tggcagcacg 33600
accacacgtc gtccgggccg aagcggcacc accgctcggt gccggcgaag agcgccacga 33660
ggttgcggtg ggtgatggcg acgcccttgg gcgtgcccgt ggagccggag gtgtacatga 33720
ggtaggcgac gttgccgggg cgggcccgcg cgggcgggtc gtcctcgccg ccctcctgcc 33780
gggtgacgtc gatgccgagg cgcggtgtcc cggccgcgcg cggcacggcc cgctcccccg 33840
cctcgtcggt caccaggacg tgcggccggg cgtcggacag cacgtagtcg agccggccgc 33900
tcgggaactc gggatcgatc ggcaggtaga cggcgccggc cttgtagacg ccgagcaggg 33960
ccacgacgag gtcggccgag cgggtcacgg cgaccgccac cacggactcg ggcgtgacgc 34020
cgcgcccggc cagggcgtgc gccagccggt tggcgcgggt gttcagctcc ccgtaggtca 34080
gcgtcacgtc gccggcgacc acggccacgg cgtccggccg ggccgccgcg tgccgttcga 34140
acagctcggc caccgtcgcg tcgggcagcg gcacggcggt gtcgttgaag tcgtcgagca 34200
gccgccgccg ctcgtggccg gtcaggagct cgatcgagcc ggccggggca ccggggtgcg 34260
cggtcagggc gcggacggtg gcgatcaggc gttcgctcag gccggccggg tcgtgctcgc 34320
cgtagaacgc cgccggggcg tcgacgcgca cgtggaggtc gctgccgggc cggccgtcga 34380
agtaggtgga gatcatcagc tcgtccaccg agccgaacga gccgccggcg aagtgcgccg 34440
tgctgccggc gaggtccagc ccttcgacga aggggatgat gttgacgatc gcgccgaacg 34500
ggctgcggaa ggctccggcg cggcccgtct cgcgctggat gtcggagacc tggtgcgtgg 34560
cgtgccggaa gacctcggtc ttggcgcgct gggtcgcgtc gaggacggcg ggcaggtcgg 34620
cggtgacggg cacgccgatc ctgatcggga cgaggttcga caggaccccg ggggtgcgcc 34680
gcgtggcgcc gaagcggttg ttgacggtga cggtgaacag cggctcgggc aggtcgcaca 34740
ggtggcggaa gaagacggcg gccgcggcca tcagcagggt cggcagggtg acgccgtacg 34800
agcgggccag ccgttcccag gcgtcggcct cctcgcgcgg cacgcgcgcg gtgtggctcc 34860
acacgccgct ggggttggcg cgggctgggg tgggggtgcc gggcaggcgg gcgggggcgg 34920
acgggtccga caggtagcgc cgccagaact cggcgtccct ggcgaaccgg tcggaggccc 34980
ggtagcgcgc gtcccgctcg tgcagcgccg cgggctcctc caccggccag tggggagcgg 35040
ggtcgccctt gagcgtcgcg gtgtagacct cggcgatccg ccgcgagacg agcgtgagca 35100
ggccgaaggc gtcggtcacg acgtggttgc agaccagcac gaggaacgac cgctgctccc 35160
ccagcgtgag cagaccggcc cggaacagca cgtcccgctc caggtcgaac ggctgggccg 35220
cgagctcggc cacatacgcc cgcgccgccg cctcggggtc ggccgccccg ctgaggtccg 35280
cggcgaaggg ccgccacgcg ccgaggtcgc gcggttcctg gcgcagccgc ccggcctcct 35340
ccctgaacgt gaccaggacg gtgcgcgcct cggtgagcac gacccgcagc gcggagttca 35400
gcaaggcact gtcgagctcg ccctggacct cccagacggc cgccacgttg ttgggcggct 35460
ccggccgcag ccgctgtgcc agccaaaggc tttcctgcgc cgccaccacg ccgatcgaag 35520
tcatccgggc caccctcctc aaaggtcttt tccagcctta ttcgccgcgg tcccgcgagt 35580
caattgccag cgccattgtc ctgccgaccg gacaccgtcc ccgcaggtcc gcggagtttt 35640
ccggcgagta atgcggagcg cgttccccgg tcaattcccg tcagttcgaa accgcccgcc 35700
gcgaacaatt cccgccattc gccggccgtc cgctcgcgcc cgccgagcag caccagcatg 35760
tgcaggtcga ggagcttttc cgggtggcgg gcgtcgtcct cgggaatcac ctgctcgacg 35820
acgagcagcg tgccgtgcgg cggcatggcg gcggccacct cgcgcaggat ccgggccgca 35880
gcgtcgtcgt cccagtcgtg caggatctgg gccagcacgt agacgtcgcc gccctcgggc 35940
acgccggtga agaagtcgcc gccgatgacg gcgcagcgct ccgccgggac gtcggcctcc 36000
cggaccacgt gcggcaggtc gaacagcacg ccgcgcaggt gcgggtggcc cgcgagcagc 36060
gtcctcagca gctcgccgtt gccgccgccg acgtccacga ccgtgcgggc gccgctccag 36120
tcccgctcga tcagcggttg catgcgcagg gtgaccaggc ccgcctgggc ggcgtcgaag 36180
gccgccgact cggcggggcg ggcgcgcagc caggcgaaca tgggctgccc gaacagctcg 36240
tcgaaggccg gtttcccggt gcgcagcgag tgcccggcga ggccccatgc ccggtagggc 36300
gcgccggcca tcagggtgac caggtgccgc agcgagccgg gccggtcggc gcgcaggtgc 36360
gcgccggtcg cggtgagggc gaaggtgccg cgcggcccct cggtgaacag gccctcggcg 36420
acgagcacgc gcaggaaccg ggtgagcgcg ccggcgtccg ccccggcgcg cgccgccagg 36480
ccggcggcct ccgcggggcc gtcggcgagc aggtcgggca cgccggcctc ggtgaccgcg 36540
aagatggcct gcgagacgat gtagcccatg atcgcgtgcc gcagcgcgcg gtagccgtcg 36600
tcgtgctcag gcatgcgggc ctcctggtga gagcgcgggg aacagcgggc gttcggccgc 36660
ctcccgcagg tagtcggcgc cggagctgcc gccggtgccc ggcacccggc cgatgatctt 36720
gagggtgatc ccccagtgcg tgcgtttcat cgcgtgccac gcctggtcca ggcccgtcat 36780
ggcggcggcc aggtcgtcca gtcgtggccc gccgcccgcc gccaggtact cctcctggag 36840
tgagcggcct ggccgcggcc gctcgtccag ccggagcagc tccatgggga gccgttcggt 36900
gggcaggggc gcgctcaggc gctccacctc gcggtaggcg cgggactgga tggcgctgcg 36960
gccgtcggtg gacgaccgga tgaccgcgaa cgactccttc ggcatggtgg tcagcacccg 37020
gtagaggctg ccggtggcgg ccacgcgggc cgtcgcgccg cgcacctcgg ccgccgcggc 37080
cgccgggtcc ccgccggcga gggccgtcac cgcccgttcc aggcagcgcg ccacctgccg 37140
gtagaccagc tcgaacacct ggatgctgcg gatgaacatc acctcgtcgt gcagcggcgt 37200
catgggcagc agggtgatct cggcggccag ccgcacggcg ggcggcacgg cctccagggc 37260
gagcagggcg agctcccccg cccggtcggc ggcgggcgcc ccggggaagc gggcggtcac 37320
cccggccgct ccgggcgcca gctcccccgc cctggccagg gcccgcaggc aggcccggac 37380
ccggacctgc tgcggggcgg agccgcacac ccggccggcg gcggcctcga aggccagcag 37440
gtcgcccagc agcgccacct gctgcaggtc gaggcccgtg tcggggcctc cggccgcggt 37500
cccggccacc tggtgcagca ggggcatcgc gccgtaggag tcgtagtcgc cgtcctcctg 37560
gtcgaaggtg cagggcagcc aggacgacag gatcgtcccg tgggccggcc tcccgccgcc 37620
gccgtgccgc cggcagccct gctcggccag cacgcgcagg gccttgacca gctcggcgtc 37680
cgcccggccg cggcccaccg cctggtagtg ccgcacgatg gggcggaacg ggaacgcggc 37740
cgggccggcc gtctcctgcc actccgcgag gctgttcagg gccgaggacg ggcacctcgc 37800
cgtctcgtcc agcgcccgcg ggctgaggcc gccgaccccg acgttcgtcg tggctcgcat 37860
ccgatgtcct tcccgctggc tcactggggg tcaggcacgt gccacagctc ggggaaggac 37920
ggcgtgaaga ccgtcgcgcg caggaactcc gcgccggtcg agccgccggt cccggccttg 37980
tcgccgatca cccgccgggc caggacggcg tgctggaagc gccactcctg gaaggcctgg 38040
tccaggtcgc gcagcccctc gcacacctgc gccgccaccg cgtcgtcccg ctccaccgtg 38100
cggagggcca gcggcacgtc gtgcggcggg tacccgcagg ccgccaggta ctccaggaag 38160
gaggcgaaca gccggggccg gcccgccctg tcctcgatgc gcgccctggc cgggctgccc 38220
gcggggaact gccgcgccac gtcgccgtcg caccggccca gcaccagctc gatctcgcgg 38280
aactgggcgg agtagaaacc gctgccgccc agccgctcgc ggaaggcgtc gaactgcccg 38340
gcggtgagcg tctccagcgc ccggagctgc gccgtcagca ccgcgaaccc ggcgccgagc 38400
cgccgcagcg tccgcagggc gtggccgctg tcggccgcgg cgagggccgc ctggaggccg 38460
cccagctcgt gcagcagctc cttgaaccac aactcgtgcg actggtggac gacgacgaac 38520
aacaattcgt cgggctgccc ggtattgggc ttctgcaggt ccagcaacgt gtccagagac 38580
agatattcgc ggtacgcgcg atatgcggaa agagcgctgg tcatacctct tccctaacac 38640
ggccgccggc ccctcgggca aaacctttca ccagtcattc acagcccgcc tcgcgaggca 38700
cgaaaaagcg ccccggaata tctccgaggc gcttttcgaa cggtcaggag ccggcttgca 38760
ccagcccttc gatatcggcc agggtggccg cctcgaaaat gcttctgatc ggcaccgaga 38820
caccgaactc ccggcgaatt ctgcgggcca gcagggtggc cagcaccgaa tggccgccga 38880
ggtcgaagaa gttgtcctca gggcgggcct ccggcacgcc caggatctcg gccgccaggc 38940
cgcacacgtc gtcgcccgcc gccgccggcg cgggggcccg gcgcagggcg gcgcggtcga 39000
tcttcccgtt gggcagggtg gggaagctct ccagcacgac gacggcggcg ggcaccatgt 39060
gcgcgggcag cgcggcggcg agatgggcgc gcaggtcggc tccggtgacc gggtcgccgg 39120
ggcgggtccg ggcgtaggcg accacgcggg cctcggcgag cccgccctcc acgaccgcgc 39180
aggccagctc gaccgccggg tgcgcgctca gccgggcctc gatctcgccc agctcgaccc 39240
ggaagccccg gatcttcacc tggtcgtcgg ctcgcccggc gaagaacagc tcgccgtcgg 39300
cccggcgccg gccgcggtcc ccggagcggt acatgcggga gccggcgggg ccgtacgggt 39360
cggcgacgaa ccgggcggcg gtctccgcgg gacggccgag gtacccccgg gcgaggccgg 39420
gcccggccag gtacagctcc ccctccgcgt cgggggccgc ctcgccgagg cgttcgtcca 39480
gcacgtggac ggcgctgccg agcagggggc ggccgatgct gatctcctcg cggccggtga 39540
tgggccccat ggcgatgccc acggtgacct cggtggggcc gtagccgttg aacatgcgcc 39600
ggccggcgga ccaggtcgtg gcgagcggcc gggtgacggc gtcgccggtg gacatgacga 39660
ccccgcccgg cagcagcccg gcggcgtcgg tgacgctcag cgcgaccggc ggcagggtcg 39720
cgtgggtgac cgcgttcctg cgcatggtgt cgtgcagcgg cggcccgggc agcaggtcgc 39780
cggcgggcgc gagcacgagc gccgcgccgg acagcagggc gaggcagatg tcccagaagg 39840
ccgcgtcgaa gctgatggag gcccactgga gcacccggtc gcccggcccg gcgccgagct 39900
ccgcggcctg actggccgcg agggcggcca cgccggagtg cgtcaccgcg acgcccttgg 39960
gccggcccgt cgagccggag gtgtagatca cgtacatcag gtggccggga cggagggggg 40020
cgcggcggtc ccggtccgtc aggtcgtggt gcggctgcgc cgcgcacgcg gcggcgacgg 40080
cgggatcgtc gatcagcagc tcgggaatgt ccgcgccggg cacgtgcgcc tccgacgagc 40140
gcacgagcag caccggccgc gcgtcccgca gcatgaacga cagccgctcc gccgggtagg 40200
cggggtcgag gggcaggtag gcgccgcccg ccttgagcac cgcgagcagc acggtcatca 40260
gcccggcggt gcgcgggacc gcgacggcga ccagcgtgtc gggcccgacg ccggccgcga 40320
cgagccgtct ggccaggcgg ttggcgtcgg cgttgagccg ggcgtagccg accgtcccgc 40380
cgggcgtgat cagggccggc cggtcgccca tgcgggccac ggtcgattcg aacaggtcat 40440
tgacggtgcg catgtccgct cgcgtttcct ggtcggcgct tgctacggct tgctacgacg 40500
ctctccggcc cggtccttcc tggaccgtcg ccgggtcgcg cggcgggggg acgggctcac 40560
cggcgaggcg gcccatgagc tcggcgacct ggcgggcgcc gcaggagggc cgcgccgcga 40620
aggaggcccg cacgcgttcc ctgatctccg gccgctgctc ctggctgagc ttgaacatgc 40680
cttcggcgcg ggtcacggcg atccggaacg cgcccactcc cggcaggatg cgccggaagt 40740
agccgagcga atccgtcatg tcccagcccg tcccgaacgc cgcctcgaag gcgcgcacgg 40800
tggcgcacac cacctccagc gtctcggcct cgccgcggat ggggctgagg gtgccgtgga 40860
cgtggacgga ggtgaagttc cacgtcggcg cggccgggct gacccggtaa acggccggcg 40920
agacgtaggc gtgcgggccg gtgaaggtca ccagcgccgg cccgcccggc tccagggcct 40980
tccagtgcgg gttcgcccgg ttcatgtgcc ccagcaacac ggcgcccgac aggtcggccg 41040
cccacggctc gggaccggcc gggtcgggga tgaccggcac gtgggtggcg taggggccgg 41100
cgtcggcggg cccgttggtg gccagcagcg ccagcgggtt gccgcggacc agctctgccg 41160
tccagccgcc ggccggctgg cggtagggac cggggacgaa catgcgatgc ggtcctttcc 41220
tcaggcggtc ttcctgtcag gagcgttccg gtcgccggac gcgtcgtcga tgatccgcac 41280
gatccagtcc atgaacggca tgtgggccgc ctccgacgcc tgcatccacc ggtcccggcg 41340
ctcggagtcc aggctcagcg tcacctgcac gcgggtcgcc gggtcggcct cgtcgagctt 41400
gcgcgcccgc ttgcgcagct cctgccggag ccggcgggcc gaccagctct cccgctcggc 41460
gcgggcgagc cagagatcct gctcggcctc cggcagcgag gcggcctcgg cgtggtgctg 41520
gaggctcagg ctgtcccgtc gccgggacgg cgggaacttc ctggccaccc aggcatagtt 41580
gcgcaacgtc tggtagctga gcgaggtctc ctcgcagacg acctggtagc ggccggggaa 41640
ctgctcctcg ccgtagacca gccagtcggc cagccaccag cacgaggagt cgctgatccg 41700
ccggatctgc tgcccgatcc ggcaccagga ctcgaagccg agcccgacgg ggatgcgcag 41760
ctcgacgcgg cccgcgtaga cccccgcgtc gatccccagc ctgccccgga cggcgccctg 41820
ctcgcccggg gctccggcgc gcacccctga ggcgagcccc ggacgggtct gtcggttgac 41880
ggcggcactc atcagaacga tcgcatcctt cctcctcgaa ggacgaagca actggtgacg 41940
cgatcgccag ctaaaaccgt ccggttggtt ttgtcaattc tgtcatcacc tgggatgttt 42000
attgagtaaa tccaggtcag cgaggttatc aaaaacagca cagaaggttt gttcggtgtg 42060
aaggtgaggg acttggcgat gatccaggaa cgcgcggaac tcacccgaaa caagatcatt 42120
cacggtgccg cggaagcttt cgccgaacgc ggtttcgccg gcacctcgct catggagatc 42180
gtgcagctca gcggcaccac caagggcgcg ctgtacttcc acttcggctc caaggaccgg 42240
ctcgccgagg agatccggga gcacgccgac gagatctgga ccggcatgga gcggctggcg 42300
tcctgccgtt gcgacggccg ggccgccgtc caggcgatga tcgacttcac ccacctgctc 42360
acccgcaacc tggccaggga cgtcgtcttc cgcgccggcc tccggctgga cggcgagggc 42420
ggcaccccgc gcgagcgctg gatcccgctg ctccggctcc tgctggcgga ggccgccccg 42480
gccccggaga gcgcggaggg cgtggaggcc atcctcgtgg cgttcgtgcc gggcctggag 42540
gcgctctccc gccgccgccc cgaccggctc gacgcgcgga cgatcaccgc cgtctggcgg 42600
acgctcctgc cggcgctgac cacggccccg tcggcctacc tgcccgccgg ccggccgcac 42660
ccgcaccccg gcgacccgct cggctgcggg cggcccgcgg cgcgcgccgc gggggcggcg 42720
gaggcggcta tccggcctgg gacatcagga tgacgctgcg cccctcgggc agcagcccgt 42780
tgagcagcac ctgccacatc gtgtcgagcc gctgcatgag gtcttcacgg cggctgtaga 42840
tgaacgacag gatctgcgtg ccgtagaagg cgctgttgat gaacctggcg cacgcctccg 42900
cgtcgaggtc gccgcggagc tcgccgcggt ccttggcctc ctcgagcagc ttgacgagca 42960
cgaggtccca gtcggtgaac gcctccagcg tggggctgcc gaaggtgccc tccgccacca 43020
gccgcacgcc ggccaggtgg atcgggttgt ccttgatgag ccgcgcgtag gcgtaggaca 43080
ggttgatcag ggtgcgcagc ggcgtggcgc cctgggcggc gacggtctcg gccagccggg 43140
cgccctcctc gcgatgggcg gcgacgacgg cctgcgccag ctcctccttg gaggagaagt 43200
ggaagtacac cgcgcccttg gtcaccgcgg tgcgggcgat gatgtccgcc atgcgcgcgc 43260
cacggaagcc gtccgcggcg aagacctcgg ccgcggcccg gagcacgacg gcgcgggtgc 43320
gttcggcgcg ctgctggagc cgtcgtgcca ttcgaccgcc aagtacagga aagtcttttg 43380
gccggtatct tagctggccg gtccggtagc tacaatgcgg gattttcctg ttcagagcgg 43440
ctgatatgac cgtaaagcta cagaaatctg gctgacggtg gtgagaaaca aggataccgg 43500
taggtttgtt ttcaccatcc tctcgggagg tgacatccgc atggggctca ccgggccgga 43560
tctgcgccac gaagagagcc tgttcgaaca gaccatcccc cgccggctcg tccaccggac 43620
cgcggtcacc gaggtgctga tcaccggctt ccgggcggac ggcccggacc gttatctgct 43680
gggcgcccag tggccgctct gccaccgctt ctaccggacc gtccacgggc ggcacgaccc 43740
cttcctgctc gtcgagagcg tgcgccaggc cgggctgctc atcgggcacg ccggctacgg 43800
cgtcccggcc gggcacgcct tcgtcatgga ccacctggcc ttcgagatcc cggccgacgc 43860
gctcgcctgc gagccgaccc acagcgacgt ggtgctgtcg gtgacctgcc gcccgaccgg 43920
ccgcggcggg cgcgcaccca agggcctcgg ctacaccatg gagctgcacc gcggcgcccg 43980
gcacgcgggc acggcgcggg cgcgggcccg ctgcatcagc ccggccgtgc tcgcccgcct 44040
gcgcgcgggc caggtcaggc gttgcggtca gccgttcgcc gccggcccgc cggtgcccgc 44100
cgagcgggtg ggcaaggcgc accccgagga cgtcgtcctg tccgccaccg gcgaggccga 44160
ggtctggacg ctgcgctgcc gcccgcaccc ggtcctgttc gaccacccca tggaccacgt 44220
gccgggcaac ctcatgatcg aggcgatgct ccaggcgacc cggtgccggc actggccgcg 44280
ccaggtcggc gtgcgcgccc tcagcgccaa cttccaccgc tacgccgacc tcgaccggcc 44340
cgtcaccgta cgggccgccg acccgatcgg cgacgacgag cccaccctgg tctccctgac 44400
acaggacggc gagcacctgg ccgacgggct gatcacaccg gccgccctga cctgaccaca 44460
cgggagacga ccatgaccga cagcgcgttc ctgaccggca aggtcatcct gatcaccggg 44520
gccagccggg gcatcggcgc ggccgccgcc cggctgttcg cccgcgaggg cgcggccgtc 44580
gtcctgaccg cgcgcggcgg gcgcgagctc ggcgacctcg tggacgagct gaccgggcag 44640
gggcacgagg cggtgcacgt cgcgggcgac gtgaccgtgc ccggcgacgc cgcccgcgtg 44700
gtctccgccg ccctcggccg gtacggcagg ctggacgcgg ccttcaacaa cgccggccgc 44760
ggcggcggcc ccgacgccat gcacctggcc gacgacgcgg ccctcgaccg ggtgatggcg 44820
gtgaacgcgg gcggcacctg gcactgcatg cgcgaggaga tccgggccat gctgccgcgc 44880
ggcggcggct cgatcgtcaa caacgcgagc gtcgccgggc tgctggccgg gcccgtgtcg 44940
gccgcctacg tggccgccaa gcacgccgtc atcggcctga ccaaggcggg cgcggccgag 45000
tacggcgcgc gcggcatccg cgtcaacgcc gtcgccaccg gcgtggcccg cacgcccatg 45060
agcgaggcgt ggatggcggc cagccccgcc gtggagccct cgctggccga gaccgccccg 45120
ctggccaggg tggcgggcgc cggggagatc gccgaggccg cggcgtggct gtgcagcgac 45180
cgggcctcct acgtcaccgg cgccacgctc accgtggacg gcgggctcag ctcatgtgtc 45240
ctgcaacagg cgcgccggtg agggcgggcc gccgtcgtgg gcgtccatgc tcaggtagcc 45300
gtgcagcacg gcggcgccct ccagcatcgc ggcggcggtg cgggtgagcg cgtgccgccg 45360
ctcctcgacc gcgacgcgca gggcctgcgt gctgccggtc ctgcgcagct cgtcgagcag 45420
ggccctgatc tgcggcagcg ggtgccggcc ttgccgcagg atgtggatca tccgggcgtc 45480
gcggacgtcg tcggggccgt accggcggta gccggtgacc cgctcgcgtt ccggtgcgag 45540
cagcccggcg gcctcccaca cccgcagcgc cgaggtgcgc agcccgaggt gccgggcgac 45600
ctccccgatc cgcgtcaggt tcctcgccgg gaccggggcg ggatcgtccg ccacggcctg 45660
cagggcctcg ctggtcgagc ggagcgcgag ccgccgggcg tgcaggccgg cgtgggactc 45720
gtcgatgagg gcgagcgcgc ccggcacgtc gcccgcgtgg atcgtcagca tgatcgaacg 45780
ggcggtgtcc cagccgaagc ccttcgccag cgaccggtag gcgagcaggg cggcccggtg 45840
ccgggcgtcg aaccggcggt agcccgaggg cgtgcgctcg gtgggcggca ggatgccggc 45900
gtcggcgtag ttgcggatct gctgggtgga gacgcccacc tcgcgcgcca ggtcaaccgg 45960
gcgcaacgcg ggagttgagg gtttcgcgac cacggagcca tcctatgggc gctcaacagc 46020
atgacaacgt tcaacgtaaa acttcaatga gagactttaa gtatggacat cacgcaccgg 46080
aaagccgcag aaccatcgga gaaaggccgg gccccttccg cgacccccgt gatccagacc 46140
cgcgacctgc gcatgcggta cgggacgacg gacgtgctgc acggcgtcga cctgcgtgtc 46200
caccagggag aggtggtcgc cctgctcggc ccgaacggcg cgggcaagac gaccaccatc 46260
gagatcctcg agggcttccg ccgccgctcc gctggcgagg tcgaggtcct cggcgccgac 46320
cccggccagg ccggcgacgc gtggctggcc cgcatcggca tcgtcctgca gtcgtggcgg 46380
gaccacggca ggtggcgcgc cggggagctg ctcgaccacc tcagccgctt ctacgtgcct 46440
ttccgcgagc cgtaccgggc cgacgagctg ctgcgcctgg tcgggctcgc cgaccgggcc 46500
ggcgcggtga tcggcaccct gtcgggcggc cagcgccgca ggctcgacgt ggccatcggc 46560
ctggtcggcc gccccgacct gctcttcctg gacgagccga cggtcggctt cgacccccag 46620
gcccggcgcg acttccacga cctcgtccag agcctgtcgc acggccagga caccgcgatc 46680
ctgctcacca cgcacgacct cgacgaggcc gagcggctcg cggaccgcat cgtgatcctc 46740
gcgggcgggc gcatcgtggc cgacggcacc gccgaggagc tggccgccca ggtgtcgagc 46800
gagtcggagg tccgctacac cctcgacggc gtcccgcaca ccgagtcggt cgcggacgcc 46860
accaggtacg tccgcgagct gttcgccacg cacggcgacg ccgtcggcaa cctccaggtg 46920
cgcgcggtca gcctggagga cgtctacgtg agcctggtcc aggagtacga gtccgggcgg 46980
cgcacccagc cgccggcgaa actgagggag gtggcccgat gaccacggcg acggacgccc 47040
gcacggcggc cgcgcgcgcc ggtctgcagc gcggctggat cgagttccgc cagaccatga 47100
ccagcggcca ggacctctgg tcggtgatgt tcttcccggt cgtgaccctg gtggtcatgt 47160
tcctgctgcg gcacaacacc gtgccgggca ccggcttctc cctcggctcg caggcccttc 47220
cgggcatcct cggcatgaac gtcgtgctca acggcatgat gatgctggcc ctgtcgctga 47280
cgatggaccg cgaggacggc acgctgctgc gcgcgaaggc caccccgaag gggatgaccg 47340
gctacctcgt cggcaaggtg accagccggg cgggcacggc cgtcatcggg ctgctggtgc 47400
cgctcgtccc ggcggcgttc ctgttcggcg ggctggacct cgggcgggtc tcggcctggg 47460
tcaccctcgg gtgggtggtg gcgctcgggc tcgtcgcggt gctgccgttc ggggcgatcc 47520
tcggctcgtt gttcggctcg gcgcagggcg tcggcttcgt caccttcccg atgatggtcc 47580
tgatggggat ctccggcgtc ttctacccga tctccggctt ccctgagtgg ctgcagtggg 47640
tcgggcaggt gttcccgctg tactggctgg cgctcggcct gcgctcggcg atgctgccgg 47700
acgcgatggc cgtcgccgag atcggcgagt cgtggcggca cctggagacg gcggtcgtgc 47760
tgggggtgtg ggcggccgcc gggttcctgc tcgctcccgt ggtgctgcgg cgcatggcgc 47820
ggcggcagtc cggctcggcc gtcgaggagc agcggcgcaa ggccatgcag cagcgcacga 47880
cgtga 47885
<210> 2
<211> 1125
<212> DNA
<213> LvSong Madura actinomycetes (Actinomarda sp.)
<400> 2
atgacctcca cgaccgtctc cagccgttta ccgtcgctga ccgggctgcg tttcgtggcc 60
gcgttctgcg tgctgctcga acacctcaac atcatcgcgg ttttcgtgcc gccggcgcgg 120
gtctcggtgt cgttcttctt cgtgctgagc ggtttcgtgc tcacctggtc gtcccggccg 180
ggcgacacgt accggtcgtt ctggcggcgc aggttctgga agatcttccc caaccacacc 240
gtgagctggg cgctgatgct ggtcatcctg gccgtcaccg gcgtctcgct ggtgccgggg 300
gtactgccgc cagggccgac gccgcccctg cccgccgccg ccaacctgac gctgctgcac 360
tcgtgggtgc ccacgttcga ctacatgttc agcgtcaacc cggtgagctg gtcgctgagc 420
agcgagatgc tcttctacct gctgttcccc gcgctgctgc cgctggtgct gcgcatcccg 480
gcgcggcggc tggcgctcgc cgccgcgggg ctggtggcgc tggcctggct gatcccgctg 540
gtgtcgctga ccttctccgg cccggcgctc atgcccgggc tgctgcagag ccagtactgg 600
ttcgcctact tcttcccgcc cgcccggctg ccggagttcg tgctcggcat ggtgctggcc 660
cggatggtca tccacggctt caccccgcgg ctgggcgtgg cgctgaccgg ggccgccacg 720
ctggtgttcc tgttcgtcgc gacggccgcg gggctgcccg cgccgttcat gttcgcggcc 780
gtgaccctcg tgcccgtggc cctggtggtg ctgggcggcg cctccatcga cgtccggcac 840
gcgcgctcgt tctggggcac ccgcctcatg gtcttcctcg gcgagatctc cttcgcgctc 900
tacctcatcc acccgctggt gaacctggcc ttcaaccagc tcacgcaggg ccgggtccgg 960
gcggcgctcg gcgacgccgg cgcgaacgtc atcgtcatcg cgctctgcct gctggccgcc 1020
tgggcgctgt accgggccgt ggagcggccg ctcatgcggc ggttcggctc ctcccgcccg 1080
gcacccgccc cggcgccggc ggcgcccacg gcccaggggg cctga 1125
<210> 3
<211> 374
<212> PRT
<213> LvSong Madura actinomycetes (Actinomarda sp.)
<400> 3
Met Thr Ser Thr Thr Val Ser Ser Arg Leu Pro Ser Leu Thr Gly Leu
1 5 10 15
Arg Phe Val Ala Ala Phe Cys Val Leu Leu Glu His Leu Asn Ile Ile
20 25 30
Ala Val Phe Val Pro Pro Ala Arg Val Ser Val Ser Phe Phe Phe Val
35 40 45
Leu Ser Gly Phe Val Leu Thr Trp Ser Ser Arg Pro Gly Asp Thr Tyr
50 55 60
Arg Ser Phe Trp Arg Arg Arg Phe Trp Lys Ile Phe Pro Asn His Thr
65 70 75 80
Val Ser Trp Ala Leu Met Leu Val Ile Leu Ala Val Thr Gly Val Ser
85 90 95
Leu Val Pro Gly Val Leu Pro Pro Gly Pro Thr Pro Pro Leu Pro Ala
100 105 110
Ala Ala Asn Leu Thr Leu Leu His Ser Trp Val Pro Thr Phe Asp Tyr
115 120 125
Met Phe Ser Val Asn Pro Val Ser Trp Ser Leu Ser Ser Glu Met Leu
130 135 140
Phe Tyr Leu Leu Phe Pro Ala Leu Leu Pro Leu Val Leu Arg Ile Pro
145 150 155 160
Ala Arg Arg Leu Ala Leu Ala Ala Ala Gly Leu Val Ala Leu Ala Trp
165 170 175
Leu Ile Pro Leu Val Ser Leu Thr Phe Ser Gly Pro Ala Leu Met Pro
180 185 190
Gly Leu Leu Gln Ser Gln Tyr Trp Phe Ala Tyr Phe Phe Pro Pro Ala
195 200 205
Arg Leu Pro Glu Phe Val Leu Gly Met Val Leu Ala Arg Met Val Ile
210 215 220
His Gly Phe Thr Pro Arg Leu Gly Val Ala Leu Thr Gly Ala Ala Thr
225 230 235 240
Leu Val Phe Leu Phe Val Ala Thr Ala Ala Gly Leu Pro Ala Pro Phe
245 250 255
Met Phe Ala Ala Val Thr Leu Val Pro Val Ala Leu Val Val Leu Gly
260 265 270
Gly Ala Ser Ile Asp Val Arg His Ala Arg Ser Phe Trp Gly Thr Arg
275 280 285
Leu Met Val Phe Leu Gly Glu Ile Ser Phe Ala Leu Tyr Leu Ile His
290 295 300
Pro Leu Val Asn Leu Ala Phe Asn Gln Leu Thr Gln Gly Arg Val Arg
305 310 315 320
Ala Ala Leu Gly Asp Ala Gly Ala Asn Val Ile Val Ile Ala Leu Cys
325 330 335
Leu Leu Ala Ala Trp Ala Leu Tyr Arg Ala Val Glu Arg Pro Leu Met
340 345 350
Arg Arg Phe Gly Ser Ser Arg Pro Ala Pro Ala Pro Ala Pro Ala Ala
355 360 365
Pro Thr Ala Gln Gly Ala
370
<210> 4
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agcagcaagc ttatcacgtc gaggttgtgc tcgatcacc 39
<210> 5
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agcagctcta gaaaccgctc agcacgaaga agaacga 37
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agcagctcta gagtgaacct ggccttcaac cagctcac 38
<210> 7
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
agcagcgaat tccagcagca tcgtcgtggt gatgtgac 38
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agcagctcta gaattccggc accgatgcga tgtccac 37
<210> 9
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
agcagctcta gaccactgtg cggatgtcgt ggtcatg 37

Claims (9)

1. An acetylase gene luz27, characterized in that its nucleotide sequence is shown in SEQ ID NO. 2.
2. The mutant strain of actinomyces lusonodes is characterized in that the construction method comprises the following steps: actinomyces igniarius DSM43766 is taken as a starting strain, and a acetylase gene luz27 is inactivated through a gene inactivation technology, wherein the nucleotide sequence of the acetylase gene luz27 is shown as SEQ ID No. 2.
3. The mutant strain of actinomadura ignettii according to claim 2, wherein the acetylase gene luz27 is knocked out by homologous double crossover comprising the steps of:
(1) using genomic DNA of the actinomyces madurae of the Igscht as a template, carrying out PCR amplification on two upstream and downstream homologous arms of a target knockout region, and inserting the upstream and downstream homologous arms into a shuttle plasmid vector pSET152 to obtain a knockout vector pSET 152H-delta luz27 of luz 27;
(2) pSET 152H-delta luz27 was transformed into E.coli, and mixed with wild type Strongyloides obtusifolia DSM43766, and luz27 deletion mutant strain was selected.
4. The mutant actinomadura ignettii strain according to claim 3, wherein in step (1), the primers for amplifying the upstream homology arms are:
27LF：5’-agcagcAAGCTTatcacgtcgaggttgtgctcgatcacc-3’；
27LR：5’-agcagcTCTAGAaaccgctcagcacgaagaagaacga-3’；
the primers for amplifying the downstream homology arm are as follows:
27RF：5’-agcagcTCTAGAgtgaacctggccttcaaccagctcac-3’；
27RR：5’-agcagcGAATTCcagcagcatcgtcgtggtgatgtgac-3’。
5. a method for preparing strychnine B-directional C is characterized by comprising the following steps: the mutant strain of actinomyces luschmanii of any one of claims 2 to 4 is inoculated into a fermentation medium, fermented and cultured, and the fermentation product is separated and purified to prepare the strychnine ignatide C.
6. The method for preparing strychnine peptin C according to claim 5, wherein the fermentation medium is MS medium, and comprises the following components by mass percent: 2% of D-mannitol, 2% of soybean meal, 2% of agar powder and the balance of water.
7. The method of producing strychnine in claim 5, wherein the fermentation is carried out at 28-32 ℃ for 11-13 days.
8. The method of producing strychnine in claim 5, wherein the isolation and purification comprises: and after fermentation, soaking a culture medium containing the thalli in ethyl acetate, filtering a leaching solution, evaporating the ethyl acetate to dryness to obtain a crude extract, dissolving the crude extract with methanol, performing Sephadex LH20 gel chromatography on the dissolved crude extract, collecting a target fraction, and separating the target fraction by using a YMC-C18 chromatographic column to obtain a target product.
9. The method of producing strychnine in claim 8, wherein the soaking time is 10-12 h.
CN202011409396.7A 2020-08-27 2020-12-04 Preparation method of strychnos ignatii C Active CN112359048B (en)

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