阅读说明：本技术 转基因水稻mfb-MH3301的外源插入片段全序列及其旁侧序列 (Complete sequence of exogenous insertion fragment of transgenic rice mfb-MH3301 and flanking sequence thereof ) 是由 苗朝华 董美 高进 宛煜嵩 刘卫晓 王迪 梅英婷 王�锋 金芜军 于 2020-12-16 设计创作，主要内容包括：本发明在专利CN103667476A的基础上公开了转基因水稻mfb-MH3301的外源插入片段全序列及其旁侧序列。本发明通过hiTAIL-PCR和LA-PCR扩增的方法获得外源插入片段和水稻序列连接区的3’端旁侧序列,其核苷酸序列为SEQ ID No.1所示；本发明进一步公开了外源插入片段全序列,其核苷酸为SEQ ID No.2所示；本发明提供的分子特征为转基因水稻mfb-MH3301及其衍生品种检测方法的建立提供了基础。(The invention discloses a complete sequence of an exogenous insertion fragment of transgenic rice mfb-MH3301 and a flanking sequence thereof on the basis of a patent CN 103667476A. The invention obtains the flanking sequence of the 3' end of the connecting region of the exogenous insert fragment and the rice sequence by means of hitAIL-PCR and LA-PCR amplification, and the nucleotide sequence is shown in SEQ ID No. 1; the invention further discloses a complete sequence of the exogenous insertion fragment, and the nucleotide of the complete sequence is shown in SEQ ID No. 2; the molecular characteristics provided by the invention provide a foundation for establishing a detection method of the transgenic rice mfb-MH3301 and derived varieties thereof.)

1. The 3' end flanking sequence of the connecting region of the exogenous insertion fragment of transgenic rice mfb-MH3301 and the rice sequence is characterized in that: the nucleotide sequence is shown in SEQ ID No. 1.

2. The complete sequence of the exogenous insertion fragment of the transgenic rice mfb-MH3301 is characterized in that: the nucleotide sequence is shown as SEQ ID No. 2.

3. The method for preparing the 3' side sequence of the connection region of the exogenous insert fragment and the rice sequence of claim 1 and the complete sequence of the exogenous insert fragment of claim 2 is obtained by amplification by means of hitAIL-PCR and LA-PCR with the genome DNA of transgenic rice mfb-MH3301 as a template based on patent CN 103667476A.

4. The method of claim 3, comprising:

(1) extracting DNA of the transgenic rice mfb-MH 3301;

(2) according to sequence information provided in patent CN103667476A, an exogenous insertion fragment is positioned on a rice chromosome 9, a nested specific primer is designed on the chromosome sequence, and a hitAIL-PCR and LA-PCR method is utilized to step towards an unknown sequence at the 5' end;

(3) according to sequence information provided in patent CN103667476A, nested specific primers are designed at the sequence end, and the method of hitAIL-PCR and LA-PCR is utilized to step towards the unknown sequence at the 3' end; finally, overlapping the sequences obtained in the two directions to obtain a 3' end flanking sequence and a complete sequence of an exogenous insert;

(4) the correctness of the exogenous insert and the position of the exogenous insert on the chromosome is further verified by using an LA-PCR amplification technology.

Technical Field

The invention relates to a flanking sequence of transgenic rice, in particular to transgenic rice and a flanking sequence thereof, belonging to the field of molecular characteristic detection of the transgenic rice.

Background

Since the approval of the first transgenic product in 1996, the use of transgenic technology has increased exponentially and the number of transgenic crops entering the market has continued to increase. International agricultural biotechnology organization (ISAAA) reported that the planting area of 28 countries planting biotech crops in 2014 was 1.815 hundred million hectares. With the development and application of transgenic technology, a plurality of transgenic rice lines have been released into the environment and applied for commercial planting.

The flanking sequence of the exogenous insertion segment of the transgenic plant is one of the most important molecular characteristics of the transgenic plant line, so that the flanking sequence of the exogenous insertion segment is an important technical data for establishing a specific detection method of the transgenic plant line. The determination of the information of the whole sequence, the insertion site, the flanking sequence and the like of the exogenous insertion fragment of the transgenic organism can provide the basis of the molecular characteristics for the safety evaluation of the transgenic organism and is also the basis for the comparative research among transformants and the qualitative and quantitative detection method.

Disclosure of Invention

One purpose of the invention is to obtain a 3' end flanking sequence of a connecting region of an exogenous insertion fragment of transgenic rice mfb-MH3301 and a rice sequence;

the second purpose of the invention is to obtain the complete sequence of the exogenous insertion fragment of the transgenic rice mfb-MH 3301;

the above object of the present invention is achieved by the following technical solutions:

according to sequence information provided in patent CN103667476A, an exogenous insertion fragment is positioned on a rice chromosome 9, a nested specific primer is designed on the chromosome sequence, and a hitAIL-PCR and LA-PCR method is utilized to step towards an unknown sequence at the 5' end; according to sequence information provided in patent CN103667476A, nested specific primers are designed at the sequence end, and the method of hitAIL-PCR and LA-PCR is utilized to step towards the unknown sequence at the 3' end; finally, overlapping the sequences obtained in the two directions to obtain a 3' end flanking sequence and a complete sequence of an exogenous insert; the correctness of the exogenous insert and the position of the exogenous insert on the chromosome is further verified by using an LA-PCR amplification technology.

Drawings

FIG. 13' end hitAIL-PCR amplification results: 1, 3, 5, 7, which is a second round amplification product; 2, 4, 6, 8 is the third round of amplification product.

FIG. 23' end flanking sequence verification amplification results: 1. mfb-MH 3301; 2. minghui 86.

FIG. 3 Long fragment amplification validation results: 1, 3, 5, Minghui 86; 2, 4, 6 and mfb-MH 3301.

Detailed Description

Example 1 obtaining of exogenous insertion fragment of transgenic Rice mfb-MH3301 and 3' -flanking sequence of junction region of Rice sequence

First, experimental material

1. Plant material: transgenic rice mfb-MH3301, Minghui 86

2. Enzymes and reagents

Ex Taq of Takara^TMVersion 2.0plus dye and LA Taq^TMVersion 2.0plus dye, plant genome DNA extraction kit of Tiangen Biochemical technology Ltd, primer was synthesized by Shanghai, agarose of BIOWEST corporation.

3. Laboratory apparatus

A spectrophotometer: thermo ND-1000;

a centrifuge: thermo Bioguge Primo R;

a PCR instrument: ABI Veriti;

gel imager: BioRad T2A;

other instruments include water baths, precision balances, fume hoods, biosafety cabinets, electrophoresis apparatus, etc.

Second, Experimental methods

1. Extraction and detection of rice DNA

The plant genome DNA extraction kit DP305 (Tiangen Biochemical technology Co., Ltd.) is adopted for extracting and purifying the plant material DNA, and the extraction method is as follows:

1) taking a 200mg rice powder sample;

2) add 800. mu.L of 65 ℃ pre-heated GP1 buffer, mix by rapid inversion, place the tube in a 65 ℃ water bath for 1h, invert the tube during the bath to mix the samples several times.

3) Adding equal volume of phenol: chloroform (1:1), mixed well, and centrifuged at 12000rpm for 10 min.

4) Transferring the supernatant to a new centrifuge tube, adding equal volume of chloroform, mixing well, and centrifuging at 12000rpm for 10 min.

5) The supernatant was taken, added with equal volume of GP2 and mixed well.

6) Transferring the mixed liquid into adsorption column CB3, centrifuging at 12000rpm for 30s, and discarding waste liquid (the volume of the adsorption column is 700 μ L, and centrifuging can be added in times).

7) Add 500. mu.L of buffer GD to adsorption column CB3, centrifuge at 12000rpm for 30s, discard waste, place adsorption column CB3 in the collection tube.

8) 600. mu.L of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12000rpm for 30s, and the waste liquid was discarded, and the adsorption column CB3 was put into the collection tube.

9) And repeating the step 8.

10) The adsorption column CB3 was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

11) The adsorption column CB3 was transferred to a clean centrifuge tube, 60. mu.L of 0.1 XTE buffer was added to the middle of the adsorption membrane, the membrane was left at room temperature for 5min, centrifuged at 12000rpm for 2min, and the solution was collected in the centrifuge tube.

When an ultraviolet spectrophotometer is used for detecting the concentration and the purity of the DNA, the DNA has a remarkable absorption peak at OD260, and the ratio of OD260/OD280 is 1.7-1.9.

2.3' amplification of flanking sequences

According to the sequence information provided in the patent CN103667476A, a flanking sequence 2000bp of the 5' end is obtained, the sequence comprises a rice genome sequence, the 7709771-7710270 site of the chromosome 9 (GeneBank ID: NC-008402.2) of rice has the nucleotide sequence which is completely identical with the nucleotide sequence of the 1-500 site, therefore, a nested specific primer is designed on the nucleotide sequence after the 7710270 site, and three rounds of amplification are carried out with a degenerate primer, and the primer sequence is shown in the table 1.

Primers used for obtaining sequences flanking the' end of Table 13

TABLE 2 HITAIL-PCR reaction System

TABLE 3 hi TAIL-PCR amplification procedure

PCR product assay

The second and third PCR amplification products were analyzed by agarose gel electrophoresis (FIG. 1), and the appropriate third PCR amplification product was selected for sequencing.

4.3' end flanking sequence verification

Designing an upstream primer on the sequencing result of the PCR product, designing a downstream primer on the nucleotide sequence behind 7710270 th site of the rice chromosome 9, and carrying out PCR amplification and sequencing. The PCR reaction system is as follows: the total volume was 25.0. mu.L, wherein 12.5. mu.L of Ex Taq Mix buffer, 1. mu.L of each of 10. mu. mol/L upstream and downstream primers, and 2. mu.L of 25 ng/. mu.L DNA template were supplemented to 25. mu.L with deionized water. The conditions for PCR amplification were: denaturation at 94 deg.C for 5 min; 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 30s, 35 cycles; extension at 72 ℃ for 7 min. The electrophoretogram is shown in FIG. 2.

3 and 4, splicing the sequencing results to form a complete 3' end flanking sequence, wherein the nucleotide sequence of the flanking sequence is shown in a sequence table SEQ ID No. 1.

Example 2 acquisition of the complete sequence of exogenous insertion fragment of mfb-MH3301 transgenic Rice

First, experimental material

Same as example 1

Second, Experimental methods

1. Extraction and detection of rice DNA

Same as example 1

2. Amplification of the complete sequence of the foreign insert

According to the T-DNA region structure schematic diagram of the transformation vector pCDMARUBb-Hyg provided in the patent CN103667476A, a construction specificity experiment is carried out, sequencing is carried out, the sequence is spliced with a known sequence to obtain a 5 '-3' end part sequence, the construction linearity is drawn as follows, and the sequences of the used primers are shown in a table 4.

TABLE 4 primers used in the construction of specificity experiments

On the basis of the splicing sequence, a nested specific primer is designed with the nucleotide sequence shown in SEQ ID No.1 in the patent, degenerate primers are combined, the HITAIL-PCR and LA-PCR amplification is carried out for multiple times from two directions, finally, the sequences obtained from the two directions are overlapped to obtain the complete sequence of the exogenous insert, and the sequence of the used primer is shown in a table 5.

The LA-PCR reaction system is as follows: the total volume is 25.0. mu.L, wherein, 12.5. mu.L LA Taq Mix buffer, 1. mu.L each of 10. mu. mol/L upstream and downstream primers, 2. mu.L DNA template of 25 ng/. mu.L, are supplemented to 25. mu.L with deionized water. The conditions for PCR amplification were: denaturation at 94 deg.C for 5 min; 94 ℃, 30s, 66 ℃, 1kb/min, 30 cycles; extension at 72 ℃ for 10 min.

TABLE 5 amplification of the complete sequence of the foreign insert

3. All sequencing results: all the connection region sequences comprise exogenous insertion fragments and rice chromosome sequences at two sides, the nucleotide sequences are shown in a sequence table SEQ ID No.2, and the construction linearity is drawn and shown in a figure.

4. Integration structure obtained by long fragment amplification verification

And (3) designing a long fragment amplification primer, and verifying the obtained integrated structure. The sequences of the primers used are shown in the table, and the amplification results are shown in FIG. 3.

TABLE 6 full sequence verification primers

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Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> exogenous insertion fragment complete sequence of transgenic rice mfb-MH3301 and flanking sequence thereof

<141> 2020-12-10

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1898

<212> DNA

<213> Artificial Sequence

<400> 1

tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag 60

ttcctcttcg ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg 120

agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc 180

caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga 240

gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc 300

ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc 360

atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat 420

catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt 480

taaatatagg ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc 540

cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat 600

tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct 660

aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag 720

aaaacagctt tttcaaagtt gttttcaaag ttggcgtata acatagtatc gacggagccg 780

attttgaaac cggtgatcac aggcagcaac gctctgtcat cgttacaatc aacatgctac 840

cctccgcgag atcatccgtg tttcaaaccc ggcagcttag ttgccgttct tccgaatagc 900

atcggtaaca tgagcaaagt ctgccgcctt acaacggctc tcccgctgac gccgtcccgg 960

actgatgggc tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt 1020

tggctgcctg ttctgttgcc taggcacttt gggaaactca tgcttccaag agagaaaatt 1080

gaaatcaaca tccatggttt cgttaagagc atggcagact tcttttcgga ttttctcatg 1140

acgcttcact ggatcctgat caccgccttc actggtatct cgtcgagaat ccagaatgca 1200

gagcttctta tcattcatgt tgaaagcata tagagtccag tggccatcag atgatacagg 1260

gattaatacc tatatatata tacacacatt tcagcaaaaa aaaaacaatg ttcagattca 1320

atttggtaat ttcctggttg cattttgtaa cttaaatttg atcgacagac caattggatg 1380

acaagagatt agcgagagtt ttttcagttc tacccaattt ggatagaaat agaaaaggat 1440

cataccagat gtgaacctga aggatcatac atagtagaca aacgggaata ctcgttcctc 1500

agattgttgg gatcattgat atattgctgt ggaacaagga acaaaaatag ttgttattat 1560

gacactagga gaagagtaga actaggaatt atatgccgcc aacaacatcc caaggcgcac 1620

tcacgcaaac ttaccttcca atttgaatca acataatgcc tccaacctag acattttgtg 1680

tttttaaaaa tttcagcatc ctgtcttgcc ataattctaa cagccaagtc catgcaattt 1740

atacccatga ctccttcagg ttttattgtt tcctggatat ctacaagctt taaattgatc 1800

ggcaggttgc ctgataccca aacagacctg caattcaatc aataataagg ctggtcatga 1860

ctgcagtaac aaaactgatt gacttatgac aaagctaa 1898

<210> 2

<211> 26002

<212> DNA

<213> Artificial Sequence

<400> 2

taggcaagac gaaaactata tcattagagc tgggccaatc atatcccaaa caaggtctta 60

ttggcatata tagtcggaaa actatgtatt taaaagttca tgtgtcctat aacaaaacac 120

aaaagaaaat gcaaactctg atgtgtctaa ccagggagct ttttgatcaa ctcagagaca 180

ttagtaggca ggactgaatg atcatccaaa ctggagagta tgtatgctag aaagttcttc 240

cgcaactcct ttgtttccta acaagatgat gcatatacaa aattaatgac gataatatat 300

tgaatatgta ttccaatcaa aattaactct ccaggaatta tttcttacag ttgaaaacca 360

ccgaatcagt cgatgaccat cccataatcg cataaaattg aatacaaaga atccacagtc 420

acaactagaa ataaatcata gggaaaaatg agttgatcac ttatcatgca gtaaaacata 480

tacccataat aaagacggga gatatattgt ggtgtaaaca aattgacgct tagacaactt 540

aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 600

tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 660

acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 720

ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgacggt 780

atcgataagc ttgatccatg cctcacatgt taatgtacta ccaatggagg gcttccatgc 840

ctcacatgtt catgtacaca tttatgatta ggaaactttt taatatattt tatagatttc 900

ttatccatca tataaaaata cataattaat catacgattt tggagataca tattctgacg 960

tatcaaattc taattaaatt ttaaaatatt ttagtgacgt atcaaattct aattaaattt 1020

taaaatattt tagagacgta ttttcgtaac aatttaaaat gtatattata gatcacattc 1080

ataggtcatt ttataattta aaatattatg gagatgcatc ttcgtttatt ttacggagat 1140

atattttcgt aatttatcat aatagaattg ttcatgctat attttgttta tgtttgctca 1200

gatgaagatt taaaccttac aagcaatgtg caaaaaatga cgtacataaa tttagatggt 1260

ccaaaaatgt tataaataaa agatcaagaa gtgtcaaaaa aagtcaaaaa caacgataga 1320

gtagtataat gtcaaaataa aataaaatcc atgacactac tactattata tattaatgca 1380

ctaatgtgta tgtctaacta catcgcctct gcctcctctg tcagttatgt ctcgtaggcc 1440

atcaatcccc cgtcctccga cgttgtctcc ggtacatcaa tgtcccatgt gcctacgtca 1500

tgatggcatt taggacatgt ctcacatcag ccagatcagc aagatacatt tgtcaatgtc 1560

tatctacgca atctccacaa tgcgacgaca tataggcaag acatcctcaa cataatttag 1620

ttgtgcatgc ttctcctcta gtatctcccg atgagttgat cgaattaatt cctgcagccc 1680

ttggcaagct gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1740

aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 1800

atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 1860

tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta 1920

cgaattcgag ctcggtaccc agcttctaga gatctagtaa catagatgac accgcgcgcg 1980

ataatttatc ctagtttgcg cgctatattt tgttttctat cgcgtattaa atgtataatt 2040

gcgggactct aatcataaaa acccatctca taaataacgt catgcattac atgttaatta 2100

ttacatgctt aacgtaattc aacagaaatt atatgataat catcgcaaga ccggcaacag 2160

gattcaatct taagaaactt tattgccaaa tgtttgaacg atctgcaggt cggggtaccg 2220

aattctcagt actcagcctc gaaggtaact tcggcaggca caaactcaat acggtcaatg 2280

tacacttcat tgccagaatt gaacacatga gcgctaaggg tgaaaacgct tgatccgtta 2340

gagaagttga aaggagtagt gaaaccgacg gttctgaagc tgccggattg caagttgctg 2400

ccgcttgaca tggttgcgga gaagttaccc tgattgatag gccttccgtc gatggaggtg 2460

tggaattgca agttagtggt agatgcgtaa cgaatcctga cacgatatct ttgagaaagt 2520

ggtgcagtga tgttaactct gagggtgcta atctggccag gagaagttct tctaagaata 2580

tcacctcctg tgaagcctgg tcctttcacg acagaagttc cagatccaag gttagtagac 2640

ttggtcaatg ggatttgggt gatttgagag gaaggaatga tattgttgaa ctcagcacta 2700

cgatgaatcc atgagaacat aggagctctg atgatgctca cggaactgtt gctgaatccg 2760

gaacggaaca tggacacgtg gctcaacctg tgggagaatc cttgcctggg tggcacattg 2820

ttgttctgtg gtgggatttc gtccaaggaa tcaacggttc cgctctttct gtaaacagcg 2880

gatggcaagt tagaagaggt tccataggcg aactctgttc cgtcaagaac ggaaagttgc 2940

tggttgttga taccgatatt gaagggtctt ctgtacaagg tggaagacaa ggttctgtag 3000

acaccctgac ctagttgagc aacgatacgt tgttgtggag cggcgtttcc catagttcca 3060

tagagaggaa aggtaaactc gggcccgctg aatccaactg gagaggccat gatctggtgt 3120

ccagaccagt aatactctcc tctgtgagca tcggtgtaga tagttatgct gttcaagatg 3180

tccatcaagt gtgggctcct gatggagcct tcgatacctt gggcagaacc acggaagcta 3240

ccgtcgaagt tctcaagaac tgggttagta tagatttctc tggtaagttg ggacactgta 3300

cggatagggt aggttctgga gtcatagttc gggaagagag acacaatgtc caaaactgtg 3360

agggtcaatt ctctcctgaa ctggttgtat ctaatccaat ctctagaatc aggaccccag 3420

acacgctcca agccagtgtt gtaccaacga acagcgtggt cggtgtagtt tccaatcagc 3480

ctagtaaggt cgttgtaacg gctattgatg gttgcagcat cgaatcccca cctttgccca 3540

aacacgctaa cgtctcgaag cacgctgagg tgaagattag ctgcttgaac gtacacggac 3600

aagagaggaa cttggtagtt ctggactgcg aacaatggga tagctgtggt caaggcgctg 3660

ttcatgtcgt tgaattgaat acgcatttcc tcgcggagag ctgggttagt aggatcggct 3720

tcccactctc tgaagctctc tgcatagatt tggtagagat tgctcaatcc ttccaaccta 3780

gagatggcct ggttcctggc gaactcttcg atcctctggt tgatcaactg ctcaatttgc 3840

accaggaatg catcccattg agatggacca aagatacccc agatgatgtc aactagtccg 3900

agaacgaacc cagcacctgg cacgaactcg ctgagcagaa actgtgtcaa ggacaaggag 3960

atgtcgatgg gagtgtaacc ggtttcaatg cgttctccac caagtacttc aacttctggg 4020

ttactcaagc agttgtatgg aatgcattcg ttgatgtttg ggttgttgtc catggatcct 4080

ctagagtcga cctgcagaag taacaccaaa caacagggtg agcatcgaca aaagaaacag 4140

taccaagcaa ataaatagcg tatgaaggca gggctaaaaa aatccacata tagctgctgc 4200

atatgccatc atccaagtat atcaagatca aaataattat aaaacatact tgtttattat 4260

aatagatagg tactcaaggt tagagcatat gaatagatgc tgcatatgcc atcatgtata 4320

tgcatcagta aaacccacat caacatgtat acctatccta gatcgatatt tccatccatc 4380

ttaaactcgt aactatgaag atgtatgaca cacacataca gttccaaaat taataaatac 4440

accaggtagt ttgaaacagt attctactcc gatctagaac gaatgaacga ccgcccaacc 4500

acaccacatc atcacaacca agcgaacaaa aagcatctct gtatatgcat cagtaaaacc 4560

cgcatcaaca tgtataccta tcctagatcg atatttccat ccatcatctt caattcgtaa 4620

ctatgaatat gtatggcaca cacatacaga tccaaaatta ataaatccac caggtagttt 4680

gaaacagaat ttctactccg atctagaacg accgcccaac cagaccacat catcacaacc 4740

aagacaaaaa aaagcatgaa aagatgaccc gacaaacaag tgcacggcat atattgaaat 4800

aaaggaaaag ggcaaaccaa accctatgca acgaaacaaa aaaaatcatg aaatcgatcc 4860

cgtctgcgga acggctagag ccatcccagg attccccaaa gagaaacact ggcaagttag 4920

caatcagaac gtgtctgacg tacaggtcgc atccgtgtac gaacgctagc agcacggatc 4980

taacacaaac acggatctaa cacaaacatg aacagaagta gaactaccgg gccctaacca 5040

tggaccggaa cgccgatcta gagaaggtag agaggggggg ggggggagga cgagcggcgt 5100

accttgaagc ggaggtgccg acgggtggat ttgggggaga tctggttgtg tgtgtgtgcg 5160

ctccgaacaa cacgaggttg gggaaagagg gtgtggaggg ggtgtctatt tattacggcg 5220

ggcgaggaag ggaaagcgaa ggagcggtgg gaaaggaatc ccccgtagct gccgtgccgt 5280

gagaggagga ggaggccgcc tgccgtgccg gctcacgtct gccgctccgc cacgcaattt 5340

ctggatgccg acagcggagc aagtccaacg gtggagcgga actctcgaga ggggtccaga 5400

ggcagcgaca gagatgccgt gccgtctgct tcgcttggcc cgacgcgacg ctgctggttc 5460

gctggttggt gtccgttaga ctcgtcgacg gcgtttaaca ggctggcatt atctactcga 5520

aacaagaaaa atgtttcctt agttttttta atttcttaaa gggtatttgt ttaattttta 5580

gtcactttat tttattctat tttatatcta aattattaaa taaaaaaact aaaatagagt 5640

tttagttttc ttaatttaga ggctaaaata gaataaaata gatgtactaa aaaaattagt 5700

ctataaaaac cattaaccct aaaccctaaa tggatgtact aataaaatgg atgaagtatt 5760

atataggtga agctatttgc aaaaaaaaag gaaaacacat gcaccctaaa aagataaaac 5820

tgtaaagtcc tgttgtcaaa atactcaatt gtcctttaaa ccatgtctaa ctgttcattt 5880

atatgattct ctaaaacact gatattattg tagtactata aattatatta ttcgtagagt 5940

aaagtttaaa tatatgtata aagatagata aactgcactt caaacaagtg tgacaaaaaa 6000

aatatgggga aattttttat aacttaaaca tgcaaggctc attatctcta gagaggggca 6060

cgaccgggtc acgctgcact gcaggcatgc aagctgggga tcctctagag tcgacctgca 6120

ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 6180

ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 6240

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgctagagca 6300

gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taattcgatc catgcctcac 6360

atgttaatgt actaccaatg gagggctgta cacatttatg attacgaaat tttttaatat 6420

attttataga tttcttatgc atcatacaaa aatacataat tattcgtaac attttggaga 6480

tacatattca gatgcatcaa attctaatta aacgttaaaa tattttggag acgtatcttc 6540

gtaacaattt aaaacctata ctatagatca cattcgaagg tcattttata atttaaaata 6600

ttatggagat gcatcttcgt ttatgtttgc tcagatgaag atttaaacct tacaaacaat 6660

atgtaaaaaa tgacgtacat aaattcagat agtccaaaag tgtcatatat aaataaagat 6720

caataagtgt caaaaaaagt caagaacaac gatagagtag cataatgtca aaataaaata 6780

aaatccatga cactactact attatatatt aatgcactaa tgtgtatgtc taactacatc 6840

gtctctgcct cctctgtcag ttatgtctcg taagccatca atcccccgtc ctccggcgtt 6900

gtctccggta tatcaatgtc cccatgtgcc tacgtcatga tggcatctag gacatgtctc 6960

acatcagaca cattaggaaa gatacatttg ccaatgtata tctgcgcaat ctccacaayg 7020

caacgacata taggcaagac atcctcaaca taatttagtt gtgcatgctt ctcctctagt 7080

atcttccgat gagttgatca agcttatcga aactatcagt gtttgacagg atatattggc 7140

gggtaaacct aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaa 7200

aaggtttatc cgttcgtcca cttgtatgtg catgccaacc acagggttcc cctcgggatc 7260

aaagtacttt gatccaaccc ctccgctgct atagtgcagt cggcttctga cgttcagtgc 7320

agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt tacgcgacag gcggccgccc 7380

tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg cataaagtag aatacttgcg 7440

actagaaccg gagacattac gccatgaaca agagcgccgc cgctggcctg ctgggctatg 7500

cccgcgtcag caccgacgac caggacttga ccaaccaacg ggccgaactg cacgcggccg 7560

gctgcaccaa gctgttttcc gagaagatca ccggcaccag gcgcgaccgc ccggagctgg 7620

ccaggatgct tgaccaccta cgccctggcg acgttgtgac agtgaccagg ctagaccgcc 7680

tggcccgcag cacccgcgac atactggaca ttgccgagcg catccaggag gccggcgcgg 7740

gcctgcgtag cctggcagag ccgtgggccg acaccaccac gccggccggc cgcatggtgt 7800

tgaccgtgtt cgccggcatt gccgagttcg agcgttccct aatcatcgac cgcacccgga 7860

gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg cccccgccct accctcaccc 7920

cggcacagat cgcgcacgcc cgcgagctga tcgaccagga aggccgcacc gtgaaagagg 7980

cggctgcact gcttggcgtg catcgctcga ccctgtaccg cgcacttgag cgcagcgagg 8040

aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg tgaggacgca ttgaccgagg 8100

ccgacgccct ggcggccgcc gagaatgaac gccaagagga acaagcatga aaccgcacca 8160

ggacggccag gacgaaccgt ttttcattac cgaagagatc gaggcggaga tgatcgcggc 8220

cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg cggctgcatg aaatcctggc 8280

cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg aagaaaccga 8340

gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc atgcggtcgc 8400

tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat gaaggttatc 8460

gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca tctagcccgc 8520

gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca gggcagtgcc 8580

cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat cgaccgcccg 8640

acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat cgacggagcg 8700

ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt gctgattccg 8760

gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct ggttaagcag 8820

cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg ggcgatcaaa 8880

ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct gcccattctt 8940

gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg cacaaccgtt 9000

cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc asgcgctggc cgctgaaatt 9060

aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc acaaacacgc 9120

taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc agcctggcag 9180

acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac accaagctga 9240

agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa tacatcgcgc 9300

agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc ggctaaagga 9360

ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc catgtgtgga 9420

ggaacgggcg gttggccagg cgtaagcggc tgggttgcct gccggccctg caatggcact 9480

ggaaccccca agcccgagga atcggcgtga gcggtcgcaa accatccggc ccggtacaaa 9540

tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc 9600

ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa 9660

tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg aagccgccca 9720

agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata 9780

gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg 9840

aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca 9900

tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca 9960

tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg 10020

cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag 10080

aaacctgcat tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga 10140

acggccgcct ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa 10200

agagcgaaac cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg 10260

agatcacaga aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg 10320

atcccggcat cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag 10380

ccagatggtt gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt 10440

tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg 10500

aggaggcggg gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg 10560

aagcatccgc cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg 10620

aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta cattgggaac ccaaagccgt 10680

acattgggaa ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac 10740

acatgtaagt gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa 10800

aacttattaa aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag 10860

ccgaagagct gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc cccgccgctt 10920

cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc tggcctacgg ccaggcaatc 10980

taccagggcg cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca catggggttt 11040

ggggagcaat ggaaccaaaa accaacgtaa gctctgaatc ccaccgtaaa ggggctttcc 11100

gatctccagc tcacccgcca ggcattactc tctgatcagt gcctcgatga tcgggcagat 11160

cgttccgccg tctcccgcat cacattgctg caccagttcg cctaacagtt gctgcatgac 11220

gaccaagtcg gccatcttct gctcgatcaa cgcgagcttg cgtgcagccc gtgctcgcgt 11280

ttcggcacag gcgcacgact catccagcgt cagcagtcca ccgacttccg agagcgtgaa 11340

acccagcgcc tgagcccgct tgatgaaacg cagtcgcttc accatgtcca ccggatagcg 11400

ccgatggcca cccaagggtt tggctggttc atccagcagc ccgcgtcgct ggtagtagcg 11460

gatcgtctcg acgttcaccc cggcggcgtc tgccagcttg ccaatggtca gctctgtggc 11520

catcactttc tccttgattc cgtactttgg tacggagttt agaatagcac ctaggcaatc 11580

aggctcagga gatgggaatg gggatgcaac tcaccgggaa aggctcgctg gtcgcgagtt 11640

cgctgaccgc catcggtgcg tcggtgtgct gtgtcgggcc actggtgctg ttggcactcg 11700

gtgtcggcgg tacgtgggtg ggcgctctga ccatgatgga gccactacgc cccctcttca 11760

tcgggttgac tctactgttc ctgggattgg cattccgcaa gctctacctg gtgccacagg 11820

tttgtacgcc aggtacaccc tgcgccgatc cgcgcacgct cgtgcgacag cgactcgtgt 11880

tctggatcgt cagcgtgctg ctgctcggcc tattggccgt gccgtggctc gccccgctgt 11940

tctactgaag gagattaacc atgcgcaaac tgctgatcgc cgtgcttttc gccttgccct 12000

tcgtggcgct ggcggctccc ccgaaaaccg tcacgctcga cgtgcagaac atgacgtgcg 12060

gactctgtcc gatcacggtc aagaagtcgc tggagaaggt gtccggcgtg agtgacgtcc 12120

aggtcaattt cgaccagaag acggcgaccg tcacctacga tcccgataag gcccagcccg 12180

aggcactgac tgaggcgacc gcgaacgcgg gatacccctc cacagtgcag aagtgaggtc 12240

acgatgagcg ccattgtcct tgagtccgtg ctgacttgcc cgcgctgcgg cttcgccaag 12300

ccggaaacca tgcccacgga cgcctgccag ttctattacg agtgcagcaa ctgcaaggcg 12360

ctgctgcgcc ccaacccagg ggattgctgc gttttctgtt cgttcggctc ggtgaagtgc 12420

ccgccgatcc agcagcagct tgggtgttgc tcatagcgtt tagggggatc acaggtcgtc 12480

gtctggatta cgcagtggcc gcagctcgcc gcgcgcaact tcttccggta ccgcaaagga 12540

ataccgcccg agcatgttga tgtgctcgta gatcagcggc gatagccggg ccaaatcctc 12600

ttccagcact ggatagccgt gctgcttgag ccgttccacg gccgccgtca tgtagagggt 12660

gttccacagc acgatgatgt tcaccaccag gcccagagca ccgagctggt cttcctggcc 12720

ttcgcggtag cgctggcgga gctcgccgcg tttgccgtgg aacacggcgc gggccaggct 12780

gtgccggcct tcgcctcggt tcaactgggt cagggtggcg cggcgcttgg actcgtcgtc 12840

gatataggtc aacgtgtgca gagtcttttc gatccgcccg aattcggcca gcgcctgggc 12900

cagccgggtg ggtctatctc ccgtttgcag cgtgcgcatg atgccagtcg ccggcacccg 12960

gccgagtttg agcgagccgg ccaggcgcag caggtcgtcc cagtgctcgg cgatcaggtc 13020

gagtttgacc gactggcggg ccagcccgtt gagcttgccg tagtccgcgt ccgggcgcgt 13080

gcgccagaag cgggtaccgc cgacatcggc cagccgcgga ctgaagtggt agccaagtag 13140

gcggaagagc ccgaacacca catcgctgta ggccccggtg tcggtcatga tttgcgtcgg 13200

ctgcaactcg gtctgctgtt ccagcacgac cgccagcaac accaggctgt cgcgcagcgt 13260

gccgggcacg gtgatggcgt tgaggccgga gaattggtcg gaaatcaggt tgtaccaggt 13320

gacaccccgg ccggtgccga aatacttcgg attggggccg gcatgcacgg tgcgcaccgg 13380

tacgacgaag cgcatgccat cggcggaggc gacctcgccg ccaccccaga cttgggccag 13440

ttccagttgg ctttgcgctc cgaccaggat ggcgttagcc gctgacaggg tgtcgtcgcg 13500

gatataattc tggctgaccc aggacagccg gtcacggcgc agcgccgggt tgtcggtgcg 13560

gatcaagggc tccaggccgg tgttgcaggc cccgcccaac agcaccgcgc agaggctggt 13620

gaccaggttg tcggcgcgtg cattgcgttc ggagacatgg gtgaaggcct cggaaaagcc 13680

agtgcgggcg gcgatttcca agaggatttc cggcagatcg acacgcggca tcaggtcaga 13740

cacggccgcc cgcagttgca gcaacgagca gggctcgtcc agcttgtcca gcgccccgag 13800

cgacagttcg gtcttgccct cggtgttctc gctcagttga atcgccgggt tgtcgggcag 13860

gcgcgcggct actgccagcc aggttgcatc cagctcgacg gacaaggcgt ccagggtggt 13920

tttggcgtcg atggtcaggc ccagtgaccg gcagatgatc ggtcgcgccg ccagccattc 13980

ggcaccgtcg agcaggccaa ggcgcgggtc ggcatagcgc caactgggcg agacgaagac 14040

atcgcggcgg cgcagggccg tgcgcagcgc atcgagcgtg cagaacacat aggcacccat 14100

gtcgagggag ccatcttcgc gggtgatgtg cttctgccaa gccttggcca cgatctcctg 14160

cggcgcgtca tcctctggct tccggcgcgg caggttcagt tgcagccact ccagactagc 14220

cgccacgccc ttgccggccg ggctgaagcc gaagcggatg tgcttgagca ggtcgggcag 14280

gaagcggcgc acgctgcggt agcgcgcttc caatgcaaga aaatagacgt catctaccgg 14340

gcggatcagc gcgttgacct cttccagggc cttttccagg gtggtcctcg gcaggtcgtt 14400

gaacagccgg gcgcgcacgt tgtcatcgct gatcgagctg tccagcacga ccttgcacgc 14460

ggcggcgagc gtcgcggccg accgatccag gtctttcagg ctgcgcatgc gggctttctt 14520

gtcggccttc tccgcgttgc tgaacaggtc gcgcagcaag gcctccagga cttccagtgc 14580

gtcgtcgtgc gcagtcgcct ccaggcagag tgcgaaggcc accagtgtcg ccatccgccg 14640

cgacgccggc agccgattaa tcgcggtgac cttggccgtg ttggcgaagc gggccagggc 14700

ggcgatacgg ctgggaggga tgtgcgccgc cgccggcaag gtgatgccga tgccgcgcac 14760

gtcatcgagc cggcgcagtg cccgaatcaa cgcggggcca ctgaccatga ccgggccgga 14820

gcgcaattga tccagccggg agctgcggtt gccttcggcc accgtcagca agtcttgcag 14880

ttgcaatcgc tgttcctcag tcacgctgcg gcccagcgta aaccagaggc gttcttcgac 14940

ccgactgcgc aactgggcga taaagcgctc tagttgagac acaccaggca ggaggacttt 15000

ctgtgtgaac agccacgagg tggctcgctc aaacagcact cccggccggt cggtgcccgt 15060

ccagcagagg gcatacagcc agcggctcaa gcgaaagccg atgcccggat cggtgaaatg 15120

acgatagcca aagcggttct gaatatcggt ggcatgtatc cagcggcgat gatcgctata 15180

gcgctggagg cagtcggggt ctggaatcgc cagttgtcgg caaagcacct gcaggacttc 15240

caccggcacg gcggcgggct tgtccggcag aacgccaacg aagcggacgg tggtcagcag 15300

aacggcataa cccagacggt tatggttacc ccgcagcacc tggatggctt cacggtcttc 15360

atcgctcagg tggaagtaac gttccagctc ttcacggctg ggcgaatcaa catagcggcc 15420

aaaaccgtcg cgttgctctt gagtcagaaa accgaccggc attgatcaca gcacctcgac 15480

gcagaacacg gcgggcgtga cgatccgcgt gcgttcgatg tgcccgcgtt gcagcaggcc 15540

ggcgcggcga tcaccggtta ccaggtagtc cgcatcgccc gccaaggcca tggccagcaa 15600

aaacgagtca tccggatcat cggcttcgac ctcgatggtc aggcgctcca gtaccacagc 15660

ccgttgcagg ttattgatca tggcgcccac cttggcgggc tgtaggatgg cctgaagctt 15720

gggatagcgg ctggctcgac gaatttcatc gagttgcatc cgcgaggtca ccacctcgaa 15780

acgcgccgcc cgccaggcac ggtagatcgc atcgggcgcg ccatgtggcg agatcagggc 15840

gctgaacagg atgttggtat ccaacacgac ccgcatcagc gcttacgtgc ccagtcgagc 15900

gcttcgtcaa ccgcgttggt caattctgcc tcactcagat gggcgttagc ggccttggcc 15960

tgctcagcgc tcagctccag gatgtgtgcc cgtaccgctt cttcgatgaa gcgcgacagg 16020

tcgcccttac ggccgccgcc ctggctggcc agaaacatcc gaagcgactg gtcggtgtcg 16080

gccgagacgg cgacattcca gcgaatggta ttcatagcgt tctccgctaa taggtgtttg 16140

tgtgtttata cgccaatcac aaagggcgat gcaatggttc gacaaaacga aggttttgcc 16200

gaactctatc gaggaatggc tcggttgggg gtcgtttgcg ggagggggcg gaatcctacg 16260

ctaaggcttt ggccagcgat attctccggt gagattgatg tgttcccatc cgagcggcga 16320

aacatgggcc aagagatcgg gcgatagcag ctttccatcg cgtttctggt ttgcaacgac 16380

ctcgccgagc ttcatggtgt tccagaagat gatgatggcg gcgagcagat tcatgccggc 16440

gatgcggtaa tgctggcctt cggcggaacg gtcgcggatt tcaccgcggc ggtggaagct 16500

gattgcccgc ttcagcgcat gatgagcttc gcctttgttg agcccgatct gggcacgccg 16560

ttggagttcg gcatccagaa tccagtcgat catgaacagg gtgcgctcga cgcgaccgac 16620

ttcccgcagg gctgtcgcga gctcgttctg ccgcggatag gaggcgagtt tccgcagaat 16680

ctggcttggc gcgacggtcc cggcagcaat ggtggcggcg atgcgcagga tgtcgggcca 16740

attgcgctcg atcatggctt ggttgacctt tccgccgatc aacgctcgca ggtgcgccgg 16800

ggcggccgac ggattgaacg cgtagagccg tttggatggc aggtcgcgga tgcgcggagc 16860

gaaccggtag ccgagaatgg cacatgcggc aaagacgtga tcggtgaagc cgcccgtgtc 16920

ggtgaactgc tcgcggatat ggcgtccagc atcgttcatc agcaggccat cgaggatgta 16980

aggcgcttcg cttgccgttg caggaatcac ctgggttgcg aacggcgcat attggtcgga 17040

gacgtggcta taggctttca ggcccggggt attgccatat ttcgcgttga ccaggttcat 17100

ggcctcacct tgctctgtag cgacgaagaa ctgtccgtcg ctcgaagccg acgtgcccat 17160

gccccagaac cgggccatgg gtaacgctgc ctgtgcctcg accaccatgg ccagcgcccg 17220

gtcataggct tcgccctcga catgccaccg tccaatgcgg atcaattccc agaaggtgtg 17280

ggtgtttgtc gcatccgcca ttttgcgcaa gccgaggttg atcccttccg ccaagataac 17340

gttcattagc ccgatccggt cagcgcaggg tgctcctgtg cgcagatggg tgaacgcttc 17400

ggtgaagccg gtcgccgcat ccacctccag caggagatcg gtgatgcgcg tgggcgggat 17460

ctgcttgtag agatcgagca ccagatcttc ggcgcctgtc ggcgcggcgg cttcgagttt 17520

ctcgatatgc agaacgccgt tttcaatcga cccgcccggg atcgtgcctg cgcgagcggc 17580

acggccaagc tcgcgcaacc gcatgtcgag gcgagcttgc cggtctgcca gccattcctc 17640

cggccgcaat ggcacagcga gacgaccgcc ttccgcgatg gattgtgccg gaacgagtgc 17700

gtgtttcaga tcgccatagc gccgggacct agtaagccag acatctccgg agcggaacgc 17760

atcgcgcaga tggaacagca ccgcgatctc ccataggcga gcgtcgccag ccctctgggc 17820

ccgaaggtgg cgatgccatt tcgagctggg ccgcaagaag ctggtcatcg cggcatcgtt 17880

caaaccggta cgaagggccg tcaccgcttc cagaagcggc agtgcaacgg gcgcagctcg 17940

cagatcgagc aggcgcaaca tgcgtggagc gtatcggcgg aagcggtgat aaccgtcgag 18000

cacatgattg agcggatcgt cggccatggt ggcggtcagc ctggttgcca ttgcaacaag 18060

ggtttttaag ccgtcccacc ctgacccact cgcgatgaca tcgcccagcg gctggccatc 18120

atcctgtgca tcgaccaggg cgcccccgat ctcggcgaag gatttcaggg tgtcacgcac 18180

cacccccgct tcgtctgcga cctttgcatg gcaaatacgc tccgaagcac ggtagagacg 18240

gccgacgatc cggtcgtggg tttcgaccac tgcgtcggcc aacatcgcct gccattccga 18300

gacgcaaaca gccaagatcg caagccgcct gtcctccggg agatcgcgca tgccgtcggc 18360

ataataccgt tcaccctgcc tgcgcagacg agtcacccga tgggcaggaa cgccggcaag 18420

cagatcctcg gggagatcga tgcgttgcag atattcgagc cggtcgagca gccggttggc 18480

cgacgaagag ttcgagccag gctcgaactg gcgcagccac acaaaacggg tcacccgatc 18540

atcagccgtc tcctcgagca atgccagcaa ctgttctcgg atcgacatag gcagccgact 18600

ggcgatcctc gtctcgatgc gtcgctcggc atcgacgaga gccgcggcac aaagccgctc 18660

gatcgtggat gtcgcgggaa ggacagtgcg ggtgcgtcgg cactcggcta cgaagcgacg 18720

ggcgatatcc tcgttcgaca ccgccatctc ggcttctcgg aacaaccatt ccttcagctc 18780

gctcgcacca cgtccggaga aggtgcggaa gccgtagagc ccccgtaact cggcaagatg 18840

ctcgtgccgt gtttcctcgc gggcagcata gtctacgaga tcgtcggcac ccaggccaag 18900

ctgcgctccg ataaattcga tgacctctgc agggatcagt tcgcctggag ccagcacccg 18960

gccgggatag cgcaggacac acaattgcag ggcgaagccg aacctgttgt gagcgcgccg 19020

acgcagcctg atatgcccaa ggtcttcatc actcagcgta tagtgcttga gcaaatccgt 19080

ctgtgaagtc ggcaagcgca acagcgcgtc tttctgccga tcggttagag tgacgcgacg 19140

cggcatacat gttccttttt caaaatctga tagcgttcaa gacgctttgt ttatgaagct 19200

ggttgagata catttccaga ggtcaatgca atcgtggccg aagcgccgcc tcaaaccaac 19260

gtttgtgata catgctgatc ggatatgccc gcgtctccaa agccgatggc tcgcagtctc 19320

tcgacctgca gcacgacgcc ttgcgcgccg cacgtgtcga acgggacaat atctatgatg 19380

atcttgcttc cggcggtcgt gatgatcgcc ctggcttgac tgcctgcctc aagtcattgc 19440

gtgacggcga tgtgctggtg gtctggaagc tcgatcgcct cggacgatcg cttgcccatc 19500

tggtcaacac ggtgaaggag ctgtcagacc gcaagatcgg cctgcgggtt ctgactggaa 19560

agggcgctca gatcgacacc acgactgcat ccggtcgcat ggtgttcgga atcttcgcca 19620

ccttggccga gttcgagcgg gatctgatcc gagagcgcac catggcgggt ctcgcctccg 19680

cgagagcgcg cggtcgcaag ggcggacgaa aattcgcgct caccaaagct caggtgcgtc 19740

tcgcgcaagc cgccatggcc cagcgcgata cttcagtttc cgatctctgc aaggaactcg 19800

gcatcgagcg cgtcactctc taccgatatg tcggtcccaa aggcgagctc agagaccatg 19860

gaaagcatgt tctcggactt acgtagcaac tcgtttcttt tcgcaggttg agccacctcc 19920

gcgcttcatc agaaaactga aggaacctcc attgaatcga actaatattt tttttggtga 19980

atcgcattct gactggttgc ctgtcagagg cggagaatct ggtgattttg tttttcgacg 20040

tggtgacggg catgccttcg cgaaaatcgc acctgcttcc cgccgcggtg agctcgctgg 20100

agagcgtgac cgcctcattt ggctcaaagg tcgaggtgtg gcttgccccg aggtgatcaa 20160

ctggcaggag gaacaggagg gtgcatgctt ggtgataacg gcaattccgg gagtaccggc 20220

ggctgatctg tctggagcgg atttgctcaa agcgtggccg tcaatggggc agcaacttgg 20280

cgctgttcac agcctatcgg ttgatcaatg tccgtttgag cgcaggctgt cgcgaatgtt 20340

cggacgcgcc gttgatgtgg tgtcccgcaa tgccgtcaat cccgacttct taccggacga 20400

ggacaagagt acgccgcagc tcgatctttt ggctcgtgtc gaacgagagc taccggtgcg 20460

gctcgaccaa gagcgcaccg atatggttgt ttgccatggt gatccctgca tgccgaactt 20520

catggtggac cctaaaactc ttcaatgcac gggtctgatc gaccttgggc ggctcggaac 20580

agcagatcgc tatgccgatt tggcactcat gattgctaac gccgaagaga actgggcagc 20640

gccagatgaa gcagagcgcg ccttcgctgt cctattcaat gtattgggga tcgaagcccc 20700

cgaccgcgaa cgccttgcct tctatctgcg attggaccct ctgacttggg gttgatgttc 20760

atgccgcctg tttttcctgc tcattggcac gtttcgcaac ctgttctcat tgcggacacc 20820

ttttccagcc tcgtttggaa agtttcattg ccagacggga ctcctgcaat cgtcaaggga 20880

ttgaaaccta tagaagacat tgctgatgaa ctgcgcgggg ccgactatct ggtatggcgc 20940

aatgggaggg gagcagtccg gttgctcggt cgtgagaaca atctgatgtt gctcgaatat 21000

gccggggagc gaatgctctc tcacatcgtt gccgagcacg gcgactacca ggcgaccgaa 21060

attgcagcgg aactaatggc gaagctgtat gccgcatctg aggaacccct gccttctgcc 21120

cttctcccga tccgggatcg ctttgcagct ttgtttcagc gggcgcgcga tgatcaaaac 21180

gcaggttgtc aaactgacta cgtccacgcg gcgattatag ccgatcaaat gatgagcaat 21240

gcctcggaac tgcgtgggct acatggcgat ctgcatcatg aaaacatcat gttctccagt 21300

cgcggctggc tggtgataga tcccgtcggt ctggtcggtg aagtgggctt tggcgccgcc 21360

aatatgttct acgatccggc tgacagagac gacctttgtc tcgatcctag acgcattgca 21420

cagatggcgg acgcattctc tcgtgcgctg gacgtcgatc cgcgtcgcct gctcgaccag 21480

gcgtacgctt atgggtgcct ttccgcagct tggaacgcgg atggagaaga ggagcaacgc 21540

gatctagcta tcgcggccgc gatcaagcag gtgcgacaga cgtcatacta gatatcaagc 21600

gacttctcct atcccctggg aacacatcaa tcttaccgga gaatatcgtt ggccaaagcc 21660

ttagcgtagg atttcgccct ctcccgcaaa cgaccccggt tcgttaagct ccccgcgcta 21720

cggttcaaat aactggtacg cttaatcaaa gttcaactat tactaacgta gtcgagtaaa 21780

cagtactttt gatgaagatc ggttatgcgc gagtgagcac tcgggatcag aaagccgacc 21840

tacaagtcga tgccctgaaa caggccgggt gcgaacgcat ctaccaagac atcgccagcg 21900

gcgcgaaaag cgcccggccg gagttggaca aactgctggc caacgtgcgg ccgggtgatg 21960

ccgtggtgat ctggaagctg gatcgccttg ggcgttccct caagcacctg gtcgagttgg 22020

tcggcgagct ggcagagcgc aaggtcggct tacagagcct gaatgacccc atcgacacca 22080

cccacgccca aggccgcctg gtgttcaacc tgttcgcctc gctggcggag ttcgagcgcg 22140

agctgatccg cgagcggact caggcgggtc tgtcggccgc acgggcgcgt ggccggatcg 22200

gtggccgtcc caagggcctg ccagccaagg ctgaggccac cgccatggcg gccgaaaccc 22260

tctaccgcga aggtcgcctg agcgtcagcg cgatcggcga gaagctgcac atctccaaga 22320

gcacgctgta cagctacctg cgccaccgtg gtgtcgagat cggcgcgtac cagaagagcg 22380

ccaggtcacg cgaccagcag ccttcggccg cgtcgccggc agagccgccc gccgccgagc 22440

gggtggccac cgtcaccctg cgcctcgcgg tggtgaataa cagcaagttc gtgcgcggcc 22500

ggaagcgggc cacggagaac attgagcgct actgcctgga gccctatggc atgaagcggc 22560

tggatgccgg ccactatgag ttgaccattc cgtatcggag cgacgatgag ctggacaaga 22620

gcgtgcatga cctgctgacc gagatcagcc aggaggccga catgcgcaac tgttttgtcg 22680

agatgggcgc ctgggaagaa gacaccgaaa agcgttggta gggcttacgt tggtttttgg 22740

ttccattgct ccccaaaccc ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac 22800

ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat 22860

gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca 22920

gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag 22980

agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga 23040

gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 23100

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 23160

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 23220

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 23280

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 23340

cccctggaag ctccctcgtg cgctctcctg ttccgagcct gccgcttacc ggatacctgt 23400

ccgcctttct ccctttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 23460

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 23520

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 23580

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 23640

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 23700

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 23760

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 23820

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 23880

aactcacgtt aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa 23940

atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg 24000

acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac 24060

cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc 24120

acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt 24180

tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag 24240

ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg 24300

ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg 24360

cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag 24420

caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag 24480

tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt 24540

tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca 24600

ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag 24660

cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat 24720

ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga 24780

actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa 24840

agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg aaaccggtga 24900

tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg cgagatcatc 24960

cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt aacatgagca 25020

aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat gggctgcctg 25080

tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg cctgttctgt 25140

tgcctaggca ctttgggaaa ctcatgcttc caagagagaa aattgaaatc aacatccatg 25200

gtttcgttaa gagcatggca gacttctttt cggattttct catgacgctt cactggatcc 25260

tgatcaccgc cttcactggt atctcgtcga gaatccagaa tgcagagctt cttatcattc 25320

atgttgaaag catatagagt ccagtggcca tcagatgata cagggattaa tacctatata 25380

tatatacaca catttcagca aaaaaaaaac aatgttcaga ttcaatttgg taatttcctg 25440

gttgcatttt gtaacttaaa tttgatcgac agaccaattg gatgacaaga gattagcgag 25500

agttttttca gttctaccca atttggatag aaatagaaaa ggatcatacc agatgtgaac 25560

ctgaaggatc atacatagta gacaaacggg aatactcgtt cctcagattg ttgggatcat 25620

tgatatattg ctgtggaaca aggaacaaaa atagttgtta ttatgacact aggagaagag 25680

tagaactagg aattatatgc cgccaacaac atcccaaggc gcactcacgc aaacttacct 25740

tccaatttga atcaacataa tgcctccaac ctagacattt tgtgttttta aaaatttcag 25800

catcctgtct tgccataatt ctaacagcca agtccatgca atttataccc atgactcctt 25860

caggttttat tgtttcctgg atatctacaa gctttaaatt gatcggcagg ttgcctgata 25920

cccaaacaga cctgcaattc aatcaataat aaggctggtc atgactgcag taacaaaact 25980

gattgactta tgacaaagct aa 26002