质粒名称: | pDIRECT_26H |
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出品公司: | Addgene |
目录编号: | 91150 |
存储实验室: | Daniel Voytas |
载体骨架基本信息 |
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载体名称: | pTRANS_260d |
载体出品公司: | -- |
载体抗性: | 卡那霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 植物表达,CRISPR |
筛选标记: | Basta |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Engineering Reagent: ZmUbi:Csy4-P2A-TaCas9 + PvUbi1:gRNAs with Csy4 spacers |
物种来源: | Synthetic |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | -- |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pDIRECT_26H was a gift from Daniel Voytas (Addgene plasmid # 91150 ; http://n2t.net/addgene:91150 ; RRID:Addgene_91150) |
参考文献部分: | A multi-purpose toolkit to enable advanced genome engineering in plants. Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF. Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922. 10.1105/tpc.16.00922 PubMed 28522548 |
买家导航
pDIRECT26H载体基本信息
pDIRECT26H载体图谱质粒图谱和多克隆位点信息
pDIRECT26H载体简介
pDIRECT26H载体序列
LOCUS pDIRECT_26H 19461 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Direct Cloning, Type: T-DNA, Engineering Reagent: ZmUbi:Csy4-P2A-TaCas9 + PvUbi1:gRNAs with Csy4 spacers, Plant Selection: PvUbi2:bar. ACCESSION . VERSION . KEYWORDS pDIRECT_26H SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 19461) AUTHORS Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF TITLE A multi-purpose toolkit to enable advanced genome engineering in plants. JOURNAL Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922. PUBMED 28522548 REFERENCE 2 (bases 1 to 19461) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..19461 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(291..1085) /codon_start=1 /gene="aphA-3" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF" misc_feature 1510..1534 /label=LB T-DNA repeat /note="left border repeat from nopaline C58 T-DNA" polyA_signal 1612..1786 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" CDS complement(1793..2344) /codon_start=1 /gene="Streptomyces hygroscopicus bar" /product="phosphinothricin acetyltransferase" /label=BlpR /note="confers resistance to bialophos or phosphinothricin" /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF WQLDFSLPVPPRPVLPVTEI" protein_bind 4284..4305 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4320..4350 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4358..4374 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 4363..4385 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4382..4398 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4382..4398 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 7015..7071 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7072..11175 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 11176..11223 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS complement(12225..12236) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" protein_bind 13508..13522 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" misc_RNA 13523..13598 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind 13604..13618 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" CDS 13938..14243 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 14122..14141 /label=ccdB-fwd /note="ccdB gene, forward primer" polyA_signal 14271..14445 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" primer_bind complement(14472..14489) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(14472..14488) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14481..14503) /label=M13/pUC Forward /note="In lacZ gene" misc_feature 14691..14715 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS 16016..16645 /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" CDS 17074..18147 /codon_start=1 /product="replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 RepA /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI LVMRYRNLIEGEASAGS" rep_origin 18213..18407 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" primer_bind complement(18643..18665) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 18751..18891 /label=bom /note="basis of mobility region from pBR322" primer_bind 18806..18825 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(18906..18923) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(19077..204) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(19157..19176) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 61 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 241 cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat 301 tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca 361 aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag 421 gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact 481 ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc 541 tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg 601 tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat 661 tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga 721 aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca 781 tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca 841 tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg 901 tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa 961 tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta 1021 tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta 1081 gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt 1141 ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa 1201 cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt 1261 tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc 1321 tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat 1381 cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac 1441 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg 1501 gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa 1561 cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat 1621 tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa 1681 tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac 1741 cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agtcaaatct 1801 cggtgacggg caggaccgga cggggcggta ccggcaggct gaagtccagc tgccagaaac 1861 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat 1921 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca 1981 cgctcttgaa gccctgtgcc tccagggact tcagcagatg ggtgtagagc gtggagccca 2041 gtcccgtccg ctggtggcgg gggctgacgt acacggtgga ctcggccgtc cagtcgtagg 2101 cgttgcgtgc cttccaggga cccgcgtagg cgatgccggc gacctcgccg tccacctcgg 2161 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt 2221 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc 2281 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc 2341 tcataagctt ctgcaaaaga gaaccagaca acagggtaag tgcctagcag taaacaaaca 2401 gaactcatca caagcaaaca gcaacatcat attcatacca acaggtcatg tgtgttcatc 2461 acatcattag tactaagcat gccatcatcc aagtatatca aagtaagggc aaagagcatg 2521 catgatcatc aggtgcacaa aagaatcatc aaattgtagc agtacatatc ttcatctatc 2581 atgcatatct atccataaca ggacgatgca tgttgaccag gtaaaagcta caggatccta 2641 taggaacagc agcgtatata tcttcaccaa tcttagcatc ttaatcatgt ggcacatgca 2701 gtttcaattt aagcacatga gctagttgat tatgaggtac cagagaatca tcaaactgat 2761 gtagcagcat atatcctcat ctatcatgca atctaatcta atctaactaa acaggaaagg 2821 tgtgctattc agttaaaagc taccgcatca tatacaaacg gcagcataag aaaaagcata 2881 atcatcttaa tcatgcaaca aacgcagatt cataataagc ccaagagcta gcttgtgatg 2941 atcttattct actctgatct acagcaatca gataacgacc taaccttgca catggcaaca 3001 aaacaatcga tcggacgaat cagttgtttg ttcctagcta gcaccatcga accagataat 3061 agatgcacgt acagatcccg aaaacgaacc caaaaacagg gcagacctag ctgaacctag 3121 gcagcgaccc agcagatcgt gagaacgatc tcatctacga acagcctaga agcaacccca 3181 cgattcccgg acaaacgacc taaaatcccc acaaatcaca tgagcatgac aggataaaca 3241 gcggaaccga tcagatctac acgaaaaccc cacctcccag ccacccacga tcaggaaaca 3301 cgcggatcta gcatgatttc gtcaacgcct cagcctagtt cctagccaca gaccaagcag 3361 aaccaccaaa ccacgccgag cgaggagatg gggcaagagg acgggggaga cgatcgccgt 3421 accttgaagc gggggaagga tcgccgaggg tcgcgaggag agcaatttgg atttggaacc 3481 cgggggttgt gcgctccgaa cgatgagacg atgtgagatt gtgggaagag gcgcggaggg 3541 ccctgtattt atgggctgcg acggggggag gagaggtggg gagggttggg gaaggaatcc 3601 cccacccgtg ccgtgacggt tccgggccgt gtgagaggag cccgctcgtc tccgccacgc 3661 aatttccgcg atcggagcgg agctttcgag aggcggctgg atggttggtg gccgttagat 3721 ttgtagacgc cgttaacgcc tcgcctccac cgggaagagt tttgagcagc cgcttatgac 3781 aatggcttaa cgacgttaga cggagcgtta gtggcaggcc atgatagaat agacgtattg 3841 caatgggatt ataattaaat aaataagaat ataataagat atggcaagtc ggcactcatg 3901 acatggtctt cgaaatgata gtgctcactt tcttagccga gaaagttgac gcgactgatt 3961 tagaagttaa gatattattt ctctcttctt ttctttcctc gtcatataag gatgaaataa 4021 actttagaga ttgccggtag tgattttgga tttcggcgat caggcttggt ttgccggttt 4081 cggacggtgt gccttaggcc acccgcagtg tatcttgtaa tgttcaaccg ataagcaagg 4141 gtggggctca agcaagtagt aaacaactat gtcaaatgtc accatggtta tggtcttgtt 4201 tagttggctt cttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 4261 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 4321 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 4381 acaggaaaca gctatgacat gattacgaat tcgagctcgg tacccgggga tctgcaggcg 4441 cgcctgcagt gcagcgtgac ccggtcgtgc ccctctctag agataatgag cattgcatgt 4501 ctaagttata aaaaattacc acatattttt tttgtcacac ttgtttgaag tgcagtttat 4561 ctatctttat acatatattt aaactttact ctacgaataa tataatctat agtactacaa 4621 taatatcagt gttttagaga atcatataaa tgaacagtta gacatggtct aaaggacaat 4681 tgagtatttt gacaacagga ctctacagtt ttatcttttt agtgtgcatg tgttctcctt 4741 tttttttgca aatagcttca cctatataat acttcatcca ttttattagt acatccattt 4801 agggtttagg gttaatggtt tttatagact aattttttta gtacatctat tttattctat 4861 tttagcctct aaattaagaa aactaaaact ctattttagt ttttttattt aataatttag 4921 atataaaata gaataaaata aagtgactaa aaattaaaca aatacccttt aagaaattaa 4981 aaaaactaag gaaacatttt tcttgtttcg agtagataat gccagcctgt taaacgccgt 5041 cgacgagtct aacggacacc aaccagcgaa ccagcagcgt cgcgtcgggc caagcgaagc 5101 agacggcacg gcatctctgt cgctgcctct ggacccctct cgagagttcc gctccaccgt 5161 tggacttgct ccgctgtcgg catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg 5221 cacggcaggc ggcctcctcc tcctctcacg gcaccggcag ctacggggga ttcctttccc 5281 accgctcctt cgctttccct tcctcgcccg ccgtaataaa tagacacccc ctccacaccc 5341 tctttcccca acctcgtgtt gttcggagcg cacacacaca caaccagatc tcccccaaat 5401 ccacccgtcg gcacctccgc ttcaaggtac gccgctcgtc ctcccccccc ccctctctac 5461 cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 5521 tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 5581 cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 5641 ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 5701 tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 5761 catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 5821 ctagatcgga gtagaattaa ttctgtttca aactacctgg tggatttatt aattttggat 5881 ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga tggaaatatc 5941 gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca gagatgcttt 6001 ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg ttctagatcg 6061 gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact gtatgtgtgt 6121 gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct aggataggta 6181 tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag catctattca 6241 tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta taattatttt 6301 gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt ttttagccct 6361 gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc accctgttgt 6421 ttggtgttac ttcctgcagg aagcatggac cactacctcg acatcaggct caggccagac 6481 ccagagttcc caccagccca gctcatgtcc gtcctcttcg gcaagctcca ccaggccctc 6541 gtggcccagg gcggcgacag gatcggcgtg tccttcccag acctcgacga gtccaggtcc 6601 aggctcggcg agaggctccg catccacgcc tccgccgacg acctcagggc cctcctcgcc 6661 aggccgtggc tggagggcct cagggaccac ctccagttcg gcgagccagc cgtggtgcca 6721 cacccaaccc catacaggca agtgtccagg gtgcaagcca agtccaaccc agagaggctc 6781 aggaggaggc tcatgaggag gcacgacctc tccgaggaag aggccaggaa gcgcatccca 6841 gacaccgtgg ccagggccct cgacctccca ttcgtgaccc tcaggtccca gtccaccggc 6901 cagcacttcc gcctcttcat caggcacggc ccactccagg tgaccgccga ggagggcggc 6961 tttacctgct acggcctctc caagggcggc ttcgtgccgt ggttcggctc cggcgccacc 7021 aacttctccc tcctcaagca agccggcgac gtggaggaga acccaggccc aatggacaag 7081 aagtactcga tcggcctcga catcgggacg aactcagttg gctgggccgt gatcaccgac 7141 gagtacaagg tgccctctaa gaagttcaag gtcctgggga acaccgaccg ccattccatc 7201 aagaagaacc tcatcggcgc tctcctgttc gacagcgggg agaccgctga ggctacgagg 7261 ctcaagagaa ccgctaggcg ccggtacacg agaaggaaga acaggatctg ctacctccaa 7321 gagattttct ccaacgagat ggccaaggtt gacgattcat tcttccaccg cctggaggag 7381 tctttcctcg tggaggagga taagaagcac gagcggcatc ccatcttcgg caacatcgtg 7441 gacgaggttg cctaccacga gaagtaccct acgatctacc atctgcggaa gaagctcgtg 7501 gactccaccg ataaggcgga cctcagactg atctacctcg ctctggccca catgatcaag 7561 ttccgcggcc atttcctgat cgagggggat ctcaacccag acaacagcga tgttgacaag 7621 ctgttcatcc aactcgtgca gacctacaac caactcttcg aggagaaccc gatcaacgcc 7681 tctggcgtgg acgcgaaggc tatcctgtcc gcgaggctct cgaagtccag gaggctggag 7741 aacctgatcg ctcagctccc aggcgagaag aagaacggcc tgttcgggaa cctcatcgct 7801 ctcagcctgg ggctcacccc gaacttcaag tcgaacttcg atctcgctga ggacgccaag 7861 ctgcaactct ccaaggacac ctacgacgat gacctcgata acctcctggc ccagatcggc 7921 gatcaatacg cggacctgtt cctcgctgcc aagaacctgt cggacgccat cctcctgtca 7981 gatatcctcc gcgtgaacac cgagatcacg aaggctccac tctctgcctc catgatcaag 8041 cgctacgacg agcaccatca ggatctgacc ctcctgaagg cgctggtccg ccaacagctc 8101 ccggagaagt acaaggagat tttcttcgat cagtcgaaga acggctacgc tgggtacatc 8161 gacggcgggg cctcacaaga ggagttctac aagttcatca agccaatcct ggagaagatg 8221 gacggcacgg aggagctcct ggtgaagctc aacagggagg acctcctgcg gaagcagaga 8281 accttcgata acggcagcat cccccaccaa atccatctcg gggagctgca cgccatcctg 8341 agaaggcaag aggacttcta ccctttcctc aaggataacc gggagaagat cgagaagatc 8401 ctgaccttca gaatcccata ctacgtcggc cctctcgcgc gggggaactc aagattcgct 8461 tggatgaccc gcaagtctga ggagaccatc acgccgtgga acttcgagga ggtggtggac 8521 aagggcgcta gcgctcagtc gttcatcgag aggatgacca acttcgacaa gaacctgccc 8581 aacgagaagg tgctccctaa gcactcgctc ctgtacgagt acttcaccgt ctacaacgag 8641 ctcacgaagg tgaagtacgt caccgagggc atgcgcaagc cagcgttcct gtccggggag 8701 cagaagaagg ctatcgtgga cctcctgttc aagaccaacc ggaaggtcac ggttaagcaa 8761 ctcaaggagg actacttcaa gaagatcgag tgcttcgatt cggtcgagat cagcggcgtt 8821 gaggaccgct tcaacgccag cctcgggacc taccacgatc tcctgaagat catcaaggat 8881 aaggacttcc tggacaacga ggagaacgag gatatcctgg aggacatcgt gctgaccctc 8941 acgctgttcg aggacaggga gatgatcgag gagcgcctga agacgtacgc ccatctcttc 9001 gatgacaagg tcatgaagca actcaagcgc cggagataca ccggctgggg gaggctgtcc 9061 cgcaagctca tcaacggcat ccgggacaag cagtccggga agaccatcct cgacttcctc 9121 aagagcgatg gcttcgccaa caggaacttc atgcaactga tccacgatga cagcctcacc 9181 ttcaaggagg atatccaaaa ggctcaagtg agcggccagg gggactcgct gcacgagcat 9241 atcgcgaacc tcgctggctc ccccgcgatc aagaagggca tcctccagac cgtgaaggtt 9301 gtggacgagc tcgtgaaggt catgggccgg cacaagcctg agaacatcgt catcgagatg 9361 gccagagaga accaaaccac gcagaagggg caaaagaact ctagggagcg catgaagcgc 9421 atcgaggagg gcatcaagga gctggggtcc caaatcctca aggagcaccc agtggagaac 9481 acccaactgc agaacgagaa gctctacctg tactacctcc agaacggcag ggatatgtac 9541 gtggaccaag agctggatat caaccgcctc agcgattacg acgtcgatca tatcgttccc 9601 cagtctttcc tgaaggatga ctccatcgac aacaaggtcc tcaccaggtc ggacaagaac 9661 cgcggcaagt cagataacgt tccatctgag gaggtcgtta agaagatgaa gaactactgg 9721 aggcagctcc tgaacgccaa gctgatcacg caaaggaagt tcgacaacct caccaaggct 9781 gagagaggcg ggctctcaga gctggacaag gccggcttca tcaagcggca gctggtcgag 9841 accagacaaa tcacgaagca cgttgcgcaa atcctcgact ctcggatgaa cacgaagtac 9901 gatgagaacg acaagctgat cagggaggtt aaggtgatca ccctgaagtc taagctcgtc 9961 tccgacttca ggaaggattt ccagttctac aaggttcgcg agatcaacaa ctaccaccat 10021 gcccatgacg cttacctcaa cgctgtggtc ggcaccgctc tgatcaagaa gtacccaaag 10081 ctggagtccg agttcgtgta cggggactac aaggtttacg atgtgcgcaa gatgatcgcc 10141 aagtcggagc aagagatcgg caaggctacc gccaagtact tcttctactc aaacatcatg 10201 aacttcttca agaccgagat cacgctggcc aacggcgaga tccggaagag accgctcatc 10261 gagaccaacg gcgagacggg ggagatcgtg tgggacaagg gcagggattt cgcgaccgtc 10321 cgcaaggttc tctccatgcc ccaggtgaac atcgtcaaga agaccgaggt ccaaacgggc 10381 gggttctcaa aggagtctat cctgcctaag cggaacagcg acaagctcat cgccagaaag 10441 aaggactggg acccaaagaa gtacggcggg ttcgacagcc ctaccgtggc ctactcggtc 10501 ctggttgtgg cgaaggttga gaagggcaag tccaagaagc tcaagagcgt gaaggagctc 10561 ctggggatca ccatcatgga gaggtccagc ttcgagaaga acccaatcga cttcctggag 10621 gccaagggct acaaggaggt gaagaaggac ctgatcatca agctcccgaa gtactctctc 10681 ttcgagctgg agaacggcag gaagagaatg ctggcttccg ctggcgagct ccagaagggg 10741 aacgagctcg cgctgccaag caagtacgtg aacttcctct acctggcttc ccactacgag 10801 aagctcaagg gcagcccgga ggacaacgag caaaagcagc tgttcgtcga gcagcacaag 10861 cattacctcg acgagatcat cgagcaaatc tccgagttca gcaagcgcgt gatcctcgcc 10921 gacgcgaacc tggataaggt cctctccgcc tacaacaagc accgggacaa gcccatcaga 10981 gagcaagcgg agaacatcat ccatctcttc accctgacga acctcggcgc tcctgctgct 11041 ttcaagtact tcgacaccac gatcgatcgg aagagataca cctccacgaa ggaggtcctg 11101 gacgcgaccc tcatccacca gtcgatcacc ggcctgtacg agacgaggat cgacctctca 11161 caactcggcg gggataagag acccgcagca accaagaagg cagggcaagc aaagaagaag 11221 aagtgactcg agtatatgaa gatgaagatg aaatatttgg tgtgtcaaat aaaaagcttg 11281 tgtgcttaag tttgtgtttt tttcttggct tgttgtgtta tgaatttgtg gctttttcta 11341 atattaaatg aatgtaagat cacattataa tgaataaaca aatgtttcta taatccattg 11401 tgaatgtttt gttggatctc ttctgcagca tataactact gtatgtgcta tggtatggac 11461 tatggaatat gattaaagat aaggagctcg ttaaccggtg acggacggcg cgccgaagag 11521 cacgtcagtg tttggtttcc actagcacga gtagcgcaat cagaaaattt tcaatgcatg 11581 aagtactaaa cgaagtttat ttagaaattt ttttaagaaa tgagtgtaat tttttgcgac 11641 gaatttaatg acaataatta atcgatgatt gcctacagta atgctacagt aaccaacctc 11701 taatcatgcg tcgaatgcgt cattagattc gtctcgcaaa atagcacaag aattatgaaa 11761 ttaattttac aaactatttt tatttaatac taataattaa ctgtcaaagt ttgtgctact 11821 cgcaagagta gcgcgaacca aacacggcct ggaggagcac ggtaacggcg tcgacaaact 11881 aacggccacc acccgccaac gcaaaggaga cggatgagag ttgacttctt gacggttctc 11941 cacccctctg tctctctgtc actgggccct gggtccccct ctcgaaagtt cctctggccg 12001 aaattgcgcg gcggagacga ggcgggcgga accgtcacgg cagaggattc cttccccacc 12061 ctgcctggcc cggccatata taaacagcca ccgcccctcc ccgttcccca tcgcgtctcg 12121 tctcgtgttg ttcccagaac acaaccaaaa tccaaatcct cctcctcctc ccgagcctcg 12181 tcgatccctc acccgcttca aggtacggcg atcctcctct cccttctccc ctcgatcgat 12241 tatgcgtgtt ccgtttccgt ttccgatcga gcgaatcgat ggttaggacc catgggggac 12301 ccatggggtg tcgtgtggtg gtctggtttg atccgcgata tttctccgtt cgtagtgtag 12361 atctgatcga atccctggtg aaatcgttga tcgtgctatt cgtgtgaggg ttcttaggtt 12421 tggagttgtg gaggtagttc tgatcggttt gtaggtgaga ttttccccat gattttgctt 12481 ggctcgtttg tcttggttag attagatctg cccgcatttt gttcgatatt tctgatgcag 12541 atatgatgaa taatttcgtc cttgtatccc gcgtccgtat gtgtattaag tttgcaggtc 12601 ctagttaggt ttttcctact gatttgtctt atccattctg tttagcttgc aaggtttggt 12661 aatggtccgg catgtttgtc tctatagatt agagtagaat aagattatct caacaagctg 12721 ttggcttatc aattttggat ctgcatgtgt ttcgcatcta tatctttgca attaagatgg 12781 tagatggaca tatgctcctg ttgagttgat gttgtacctt ttacctgagg tctgaggaac 12841 atgcatcctc ctgctacttt gtgcttatac agatcatcaa gattatgtag ctaatattcg 12901 atcagtttct agtatctaca tggtaaactt gcatgcactt gctacttatt tttgatatac 12961 ttggatgata acatatgctg ctggttgatt cctacctaca tgatgaacat tttacaggcc 13021 attagtgtct gtctgtatgt gttgttcctg tttgcttcag tctatttctg tttcattcct 13081 agtttattgg ttctctgcta gatacttacc ctgctgggct tagttatcat cttatctcga 13141 atgcattttc atgtttatag atgaatatac actcagatag gtgtagatgt atgctactgt 13201 ttctctacgt tgctgtaggt tttacctgtg gcaactgcat actcctgttg cttcgctaga 13261 tatgtatgtg cttatataga ttaagatatg tgtgatggtt ctttagtata tctgatgatc 13321 atgtatgctc ttttaacttc ttgctacact tggtaacatg ctgtgatgct gtttgttgat 13381 tctgtagcac taccaatgat gaccttatct ctctttgtat atgatgtttc tgtttgtttg 13441 aggcttgtgt tactgctagt tacttaccct gttgcctggc taatcttctg cagatcctgc 13501 aggttcactg ccgtataggc aggttttaga gctagaaata gcaagttaaa ataaggctag 13561 tccgttatca acttgaaaaa gtggcaccga gtcggtgcgt tcactgccgt ataggcagaa 13621 gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat 13681 atgtataccc gaagtatgtc aaaaggaggt atgctatgaa gcagcgtatt acagtgacag 13741 ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt 13801 aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa 13861 tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga 13921 gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc 13981 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc 14041 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc 14101 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtttccg 14161 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta 14221 acctgatgtt ctggggaata taagctcttc agtcgatcga caagctcgag tttctccata 14281 ataatgtgtg agtagttccc agataaggga attagggttc ctatagggtt tcgctcatgt 14341 gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 14401 aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatcccccg aattagagct 14461 ctaccggtgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 14521 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 14581 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcta gagcagcttg 14641 agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgacaggata 14701 tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg 14761 gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct 14821 cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt 14881 tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct 14941 gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat 15001 acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg 15061 ggctatgccc gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac 15121 gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg 15181 gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta 15241 gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc 15301 ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc 15361 atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc 15421 acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc 15481 ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg 15541 aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc 15601 agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg 15661 accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac 15721 cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga 15781 tcgcggccgg gtacgtgttc gagccgcccg cgcacggctc aaccgtgcgg ctgcatgaaa 15841 tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag 15901 aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg 15961 cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa 16021 ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct 16081 agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg 16141 cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga 16201 ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga 16261 cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct 16321 gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt 16381 taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc 16441 gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc 16501 cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac 16561 aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc 16621 tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca 16681 aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc 16741 ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc 16801 aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac 16861 atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc 16921 taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat 16981 gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgcctgcc ggccctgcaa 17041 tggcactgga acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg 17101 gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc 17161 gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct 17221 gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag 17281 ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc 17341 cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga 17401 gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg 17461 gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc 17521 gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca 17581 cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac 17641 ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag 17701 gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag 17761 atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg 17821 taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt 17881 ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag 17941 gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc 18001 aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat 18061 ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc 18121 gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta 18181 gcaggggaaa aaggtcgaaa aggcctcttt cctgtggata gcacgtacat tgggaaccca 18241 aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac 18301 cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac 18361 tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag 18421 cgcacagccc aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc 18481 gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca 18541 ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat 18601 caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 18661 cccggaaacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 18721 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 18781 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 18841 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gccctcttcc 18901 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 18961 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 19021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 19081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 19141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 19201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 19261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 19321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 19381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 19441 aggattagca gagcgaggta t //
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