质粒名称: | pDIRECT_22C |
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出品公司: | Addgene |
目录编号: | 91135 |
存储实验室: | Daniel Voytas |
载体骨架基本信息 |
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载体名称: | pTRANS_220d |
载体出品公司: | -- |
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: | 卡那霉素 |
载体抗性: | 卡那霉素 |
生长温度: | 37 ℃ |
宿主菌: | ccdB Survival |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Engineering Reagent: 35S:Csy4-P2A-AtCas9 + CmYLCV: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_22C was a gift from Daniel Voytas (Addgene plasmid # 91135 ; http://n2t.net/addgene:91135 ; RRID:Addgene_91135) |
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
pDIRECT22C载体基本信息
pDIRECT22C载体图谱质粒图谱和多克隆位点信息
pDIRECT22C载体简介
pDIRECT22C载体序列
LOCUS pDIRECT_22C 16058 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Direct Cloning, Type: T-DNA, Engineering Reagent: 35S:Csy4-P2A-AtCas9 + CmYLCV:gRNAs with Csy4 spacers , Plant Selection: 2x35S:npt II. ACCESSION . VERSION . KEYWORDS pDIRECT_22C SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16058) 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 16058) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16058 /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(1843..2640) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1954..1973) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 2564..2583 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(2703..3379) /label=CaMV 35S promoter (enhanced) /note="cauliflower mosaic virus 35S promoter with a duplicated enhancer region" protein_bind 3570..3591 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3606..3636 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3644..3660 /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 3649..3671 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3668..3684 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3668..3684 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 4214..4559 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" CDS 5151..5207 /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 5208..9311 /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 9324..9344 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind 10113..10127 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" misc_RNA 10128..10203 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind 10209..10223 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" CDS 10543..10848 /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 10727..10746 /label=ccdB-fwd /note="ccdB gene, forward primer" polyA_signal 10876..11050 /label=CaMV poly(A) signal /note="cauliflower mosaic virus polyadenylation signal" primer_bind complement(11069..11086) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(11069..11085) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11078..11100) /label=M13/pUC Forward /note="In lacZ gene" misc_feature 11288..11312 /label=RB T-DNA repeat /note="right border repeat from nopaline C58 T-DNA" CDS 12613..13242 /codon_start=1 /product="stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" /label=pVS1 StaA /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI" CDS 13671..14744 /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 14810..15004 /label=pVS1 oriV /note="origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)" primer_bind complement(15240..15262) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 15348..15488 /label=bom /note="basis of mobility region from pBR322" primer_bind 15403..15422 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(15503..15520) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(15674..204) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(15754..15773) /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 agcttgtcga 1801 tcgactctag ctagaggatc gatccgaacc ccagagtccc gctcagaaga actcgtcaag 1861 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa 1921 gcggtcagcc cattcgccgc caagttcttc agcaatatca cgggtagcca acgctatgtc 1981 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt 2041 ttccaccatg atattcggca agcaggcatc gccatgtgtc acgacgagat cctcgccgtc 2101 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgttcttc 2161 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg 2221 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat 2281 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg 2341 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac 2401 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctggag 2461 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga 2521 cagccgaaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa 2581 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatccccat 2641 ggtcgatcga cagatctgcg aaagctcgag agagatagat ttgtagagag agactggtga 2701 tttcagcgtg tcctctccaa atgaaatgaa cttccttata tagaggaagg gtcttgcgaa 2761 ggatagtggg attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc 2821 tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca 2881 tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga 2941 tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct 3001 gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc 3061 ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc 3121 accatgttca catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac 3181 gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac 3241 gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt 3301 ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac 3361 cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt 3421 ggagtagacg agagtgtcgt gctccaccat gttggcaagc tgctctagcc aatacgcaaa 3481 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3541 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 3601 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 3661 tttcacacag gaaacagcta tgacatgatt acgaattcga gctcggtacc cggggatcgg 3721 cgcgccagat ttgccttttc aatttcagaa agaatgctaa cccacagatg gttagagagg 3781 cttacgcagc aggtatcatc aagacgatct acccgagcaa taatctccag gaaatcaaat 3841 accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc atcaagaaca 3901 cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg attcaaggct 3961 tgcttcacaa accaaggcaa gtaatagaga ttggagtctc taaaaaggta gttcccactg 4021 aatcaaaggc catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga 4081 ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca 4141 acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag 4201 accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc 4261 attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca 4321 aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc 4381 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 4441 cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc 4501 actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac 4561 gggggactcc tgcaggaaaa atggatcatt atcttgatat tagacttaga cctgatccag 4621 aatttccacc agctcaactt atgtctgttc tttttggaaa acttcatcaa gctcttgttg 4681 ctcaaggagg agatagaatt ggagtttctt ttcctgatct tgatgaatca agatcaagac 4741 ttggagaaag acttagaatt catgcttctg ctgatgatct tagagctttg cttgctagac 4801 cttggcttga aggacttaga gatcatcttc aatttggaga accagctgtt gttccacatc 4861 caactcctta tagacaagtt tcaagagttc aagctaaatc taatccagaa agacttagaa 4921 gaagacttat gagaagacat gatctttctg aagaagaagc tagaaaaaga attcctgata 4981 ctgttgctag agctttggat ttgccttttg ttacacttag atcacaatct actggacaac 5041 attttagact ttttattaga catggaccac ttcaagttac tgctgaagaa ggaggattta 5101 cttgttatgg actttctaag ggaggttttg ttccttggtt tggatctgga gctactaatt 5161 tttctcttct taagcaagct ggagatgttg aagaaaatcc tggacccatg gataagaagt 5221 actctatcgg actcgatatc ggaactaact ctgtgggatg ggctgtgatc accgatgagt 5281 acaaggtgcc atctaagaag ttcaaggttc tcggaaacac cgataggcac tctatcaaga 5341 aaaaccttat cggtgctctc ctcttcgatt ctggtgaaac tgctgaggct accagactca 5401 agagaaccgc tagaagaagg tacaccagaa gaaagaacag gatctgctac ctccaagaga 5461 tcttctctaa cgagatggct aaagtggatg attcattctt ccacaggctc gaagagtcat 5521 tcctcgtgga agaagataag aagcacgaga ggcaccctat cttcggaaac atcgttgatg 5581 aggtggcata ccacgagaag taccctacta tctaccacct cagaaagaag ctcgttgatt 5641 ctactgataa ggctgatctc aggctcatct acctcgctct cgctcacatg atcaagttca 5701 gaggacactt cctcatcgag ggtgatctca accctgataa ctctgatgtg gataagttgt 5761 tcatccagct cgtgcagacc tacaaccagc ttttcgaaga gaaccctatc aacgcttcag 5821 gtgtggatgc taaggctatc ctctctgcta ggctctctaa gtcaagaagg cttgagaacc 5881 tcattgctca gctccctggt gagaagaaga acggactttt cggaaacttg atcgctctct 5941 ctctcggact cacccctaac ttcaagtcta acttcgatct cgctgaggat gcaaagctcc 6001 agctctcaaa ggatacctac gatgatgatc tcgataacct cctcgctcag atcggagatc 6061 agtacgctga tttgttcctc gctgctaaga acctctctga tgctatcctc ctcagtgata 6121 tcctcagagt gaacaccgag atcaccaagg ctccactctc agcttctatg atcaagagat 6181 acgatgagca ccaccaggat ctcacacttc tcaaggctct tgttagacag cagctcccag 6241 agaagtacaa agagattttc ttcgatcagt ctaagaacgg atacgctggt tacatcgatg 6301 gtggtgcatc tcaagaagag ttctacaagt tcatcaagcc tatcctcgag aagatggatg 6361 gaaccgagga actcctcgtg aagctcaata gagaggatct tctcagaaag cagaggacct 6421 tcgataacgg atctatccct catcagatcc acctcggaga gttgcacgct atccttagaa 6481 ggcaagagga tttctaccca ttcctcaagg ataacaggga aaagattgag aagattctca 6541 ccttcagaat cccttactac gtgggacctc tcgctagagg aaactcaaga ttcgcttgga 6601 tgaccagaaa gtctgaggaa accatcaccc cttggaactt cgaagaggtg gtggataagg 6661 gtgctagtgc tcagtctttc atcgagagga tgaccaactt cgataagaac cttccaaacg 6721 agaaggtgct ccctaagcac tctttgctct acgagtactt caccgtgtac aacgagttga 6781 ccaaggttaa gtacgtgacc gagggaatga ggaagcctgc ttttttgtca ggtgagcaaa 6841 agaaggctat cgttgatctc ttgttcaaga ccaacagaaa ggtgaccgtg aagcagctca 6901 aagaggatta cttcaagaaa atcgagtgct tcgattcagt tgagatttct ggtgttgagg 6961 ataggttcaa cgcatctctc ggaacctacc acgatctcct caagatcatt aaggataagg 7021 atttcttgga taacgaggaa aacgaggata tcttggagga tatcgttctt accctcaccc 7081 tctttgaaga tagagagatg attgaagaaa ggctcaagac ctacgctcat ctcttcgatg 7141 ataaggtgat gaagcagttg aagagaagaa gatacactgg ttggggaagg ctctcaagaa 7201 agctcattaa cggaatcagg gataagcagt ctggaaagac aatccttgat ttcctcaagt 7261 ctgatggatt cgctaacaga aacttcatgc agctcatcca cgatgattct ctcaccttta 7321 aagaggatat ccagaaggct caggtttcag gacagggtga tagtctccat gagcatatcg 7381 ctaacctcgc tggatctcct gcaatcaaga agggaatcct ccagactgtg aaggttgtgg 7441 atgagttggt gaaggtgatg ggaaggcata agcctgagaa catcgtgatc gaaatggcta 7501 gagagaacca gaccactcag aagggacaga agaactctag ggaaaggatg aagaggatcg 7561 aggaaggtat caaagagctt ggatctcaga tcctcaaaga gcaccctgtt gagaacactc 7621 agctccagaa tgagaagctc tacctctact acctccagaa cggaagggat atgtatgtgg 7681 atcaagagtt ggatatcaac aggctctctg attacgatgt tgatcatatc gtgccacagt 7741 cattcttgaa ggatgattct atcgataaca aggtgctcac caggtctgat aagaacaggg 7801 gtaagagtga taacgtgcca agtgaagagg ttgtgaagaa aatgaagaac tattggaggc 7861 agctcctcaa cgctaagctc atcactcaga gaaagttcga taacttgact aaggctgaga 7921 ggggaggact ctctgaattg gataaggcag gattcatcaa gaggcagctt gtggaaacca 7981 ggcagatcac taagcacgtt gcacagatcc tcgattctag gatgaacacc aagtacgatg 8041 agaacgataa gttgatcagg gaagtgaagg ttatcaccct caagtcaaag ctcgtgtctg 8101 atttcagaaa ggatttccaa ttctacaagg tgagggaaat caacaactac caccacgctc 8161 acgatgctta ccttaacgct gttgttggaa ccgctctcat caagaagtat cctaagctcg 8221 agtcagagtt cgtgtacggt gattacaagg tgtacgatgt gaggaagatg atcgctaagt 8281 ctgagcaaga gatcggaaag gctaccgcta agtatttctt ctactctaac atcatgaatt 8341 tcttcaagac cgagattacc ctcgctaacg gtgagatcag aaagaggcca ctcatcgaga 8401 caaacggtga aacaggtgag atcgtgtggg ataagggaag ggatttcgct accgttagaa 8461 aggtgctctc tatgccacag gtgaacatcg ttaagaaaac cgaggtgcag accggtggat 8521 tctctaaaga gtctatcctc cctaagagga actctgataa gctcattgct aggaagaagg 8581 attgggaccc taagaaatac ggtggtttcg attctcctac cgtggcttac tctgttctcg 8641 ttgtggctaa ggttgagaag ggaaagagta agaagctcaa gtctgttaag gaacttctcg 8701 gaatcactat catggaaagg tcatctttcg agaagaaccc aatcgatttc ctcgaggcta 8761 agggatacaa agaggttaag aaggatctca tcatcaagct cccaaagtac tcactcttcg 8821 aactcgagaa cggtagaaag aggatgctcg cttctgctgg tgagcttcaa aagggaaacg 8881 agcttgctct cccatctaag tacgttaact ttctttacct cgcttctcac tacgagaagt 8941 tgaagggatc tccagaagat aacgagcaga agcaactttt cgttgagcag cacaagcact 9001 acttggatga gatcatcgag cagatctctg agttctctaa aagggtgatc ctcgctgatg 9061 caaacctcga taaggtgttg tctgcttaca acaagcacag agataagcct atcagggaac 9121 aggcagagaa catcatccat ctcttcaccc ttaccaacct cggtgctcct gctgctttca 9181 agtacttcga tacaaccatc gataggaaga gatacacctc taccaaagaa gtgctcgatg 9241 ctaccctcat ccatcagtct atcactggac tctacgagac taggatcgat ctctcacagc 9301 tcggtggtga ttcaagggct gatcctaaga agaagaggaa ggtttgactc gagatatgaa 9361 gatgaagatg aaatatttgg tgtgtcaaat aaaaagcttg tgtgcttaag tttgtgtttt 9421 tttcttggct tgttgtgtta tgaatttgtg gctttttcta atattaaatg aatgtaagat 9481 cacattataa tgaataaaca aatgtttcta taatccattg tgaatgtttt gttggatctc 9541 ttctgcagca tataactact gtatgtgcta tggtatggac tatggaatat gattaaagat 9601 aaggagctcc ggtgacggac ggcgcgccga agagctggca gacatactgt cccacaaatg 9661 aagatggaat ctgtaaaaga aaacgcgtga aataatgcgt ctgacaaagg ttaggtcggc 9721 tgcctttaat caataccaaa gtggtcccta ccacgatgga aaaactgtgc agtcggtttg 9781 gctttttctg acgaacaaat aagattcgtg gccgacaggt gggggtccac catgtgaagg 9841 catcttcaga ctccaataat ggagcaatga cgtaagggct tacgaaataa gtaagggtag 9901 tttgggaaat gtccactcac ccgtcagtct ataaatactt agcccctccc tcattgttaa 9961 gggagcaaaa tctcagagag atagtcctag agagagaaag agagcaagta gcctagaagt 10021 agtcaaggcg gcgaagtatt caggcacgtg gccaggaaga agaaaagcca agacgacgaa 10081 aacaggtaag agctaagctt cctgcaggtt cactgccgta taggcaggtt ttagagctag 10141 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 10201 tgcgttcact gccgtatagg cagaagccag ataacagtat gcgtatttgc gcgctgattt 10261 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaag gaggtatgct 10321 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat 10381 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc 10441 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg 10501 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac 10561 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac 10621 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc 10681 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc 10741 gatatggcca gtgtgccggt ttccgttatc ggggaagaag tggctgatct cagccaccgc 10801 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataagc tcttcagtcg 10861 atcgacaagc tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag 10921 ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt 10981 atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa 11041 aatccagatc ccccgaatta ccggtggcac tggccgtcgt tttacaacgt cgtgactggg 11101 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 11161 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 11221 aatgctagag cagcttgagc ttggatcaga ttgtcgtttc ccgccttcag tttaaactat 11281 cagtgtttga caggatatat tggcgggtaa acctaagaga aaagagcgtt tattagaata 11341 atcggatatt taaaagggcg tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc 11401 aaccacaggg ttcccctcgg gatcaaagta ctttgatcca acccctccgc tgctatagtg 11461 cagtcggctt ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct 11521 aagttacgcg acaggctgcc gccctgccct tttcctggcg ttttcttgtc gcgtgtttta 11581 gtcgcataaa gtagaatact tgcgactaga accggagaca ttacgccatg aacaagagcg 11641 ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc 11701 aacgggccga actgcacgcg gccggctgca ccaagctgtt ttccgagaag atcaccggca 11761 ccaggcgcga ccgcccggag ctggccagga tgcttgacca cctacgccct ggcgacgttg 11821 tgacagtgac caggctagac cgcctggccc gcagcacccg cgacctactg gacattgccg 11881 agcgcatcca ggaggccggc gcgggcctgc gtagcctggc agagccgtgg gccgacacca 11941 ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt 12001 ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt 12061 ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag ctgatcgacc 12121 aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt 12181 accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct 12241 tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat gaacgccaag 12301 aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca ttaccgaaga 12361 gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc acggctcaac 12421 cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg cctggccggc 12481 cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta 12541 aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata aacaaatacg 12601 caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc aggcaagacg 12661 accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt tctgttagtc 12721 gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta 12781 accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat cggccggcgc 12841 gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc cgcgatcaag 12901 gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg ggccaccgcc 12961 gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct acaagcggcc 13021 tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg 13081 gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc 13141 actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag 13201 gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga ggtaaagaga 13261 aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg 13321 ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac tttcagttgc 13381 cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag accattaccg 13441 agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga ataaatgagt 13501 agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc aggcaccgac 13561 gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag cggctgggtt 13621 gcctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc gtgagcggtc 13681 gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt 13741 tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat 13801 cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg 13861 cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc 13921 tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt 13981 cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag 14041 aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg 14101 cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg 14161 gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg 14221 gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca 14281 tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct 14341 tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg 14401 agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg 14461 ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac 14521 gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg 14581 gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg 14641 acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc 14701 gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc 14761 tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg cctctttcct gtggatagca 14821 cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa 14881 agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg 14941 atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg 15001 cataactgtc tggccagcgc acagcccaag agctgcaaaa agcgcctacc cttcggtcgc 15061 tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa 15121 tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg 15181 accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac 15241 ctctgacaca tgcagctccc ggaaacggtc acagcttgtc tgtaagcgga tgccgggagc 15301 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 15361 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 15421 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 15481 gcatcaggcc ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 15541 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 15601 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 15661 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 15721 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 15781 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 15841 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 15901 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 15961 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 16021 cagcagccac tggtaacagg attagcagag cgaggtat //
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