质粒名称: | Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP |
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出品公司: | 爱迪基因 |
目录编号: | 118156 |
存储实验室: | Jorge Ferrer |
载体骨架基本信息 |
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载体名称: | lentiCRISPRv2 |
载体出品公司: | Feng Zhang (Addgene #52961) |
载体抗性: | 氨苄青霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 哺乳动物表达,慢病毒,CRISPR |
筛选标记: | -- |
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: | KRAB-dCas9-P2A-EGFP |
物种来源: | H. sapiens (human) |
插入大小: | 4907 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | EF1a core and U6 |
标签/融合蛋白: | HA(插入件上的C端子) |
克隆方法: | Gibson克隆 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | TAAGAAGTACGGCGGCTTCG |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP was a gift from Jorge Ferrer (Addgene plasmid # 118156 ; http://n2t.net/addgene:118156 ; RRID:Addgene_118156) |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP 15284 bp ds-DNA circular SYN 29-MAR-2019 DEFINITION Catalytically inactive Cas9 from S. pyogenes with P2A-EGFP under the EF1a core promoter, and cloning backbone for sgRNA. Contains BsmBI sites for insertion of spacer sequences.. ACCESSION . VERSION . KEYWORDS Lenti-(BB)-EF1a-KRAB-dCas9-P2A-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15284) TITLE Ferrer Lab Plasmids REFERENCE 2 (bases 1 to 15284) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 29, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15284 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(176..280) /gene="bla" /label=AmpR promoter primer_bind complement(355..374) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 546..925 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 927..1125 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1080..1100 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 1143..1323 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1370..1495 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1988..2221 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 2748..2865 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2916..3156 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2916..2936 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3087..3106 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 5046..5121 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 5183..5394 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 5407..5416 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5443..5628 /codon_start=1 /gene="ZNF10" /gene_synonym="KOX1" /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" CDS 5650..7368 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 9757..9783 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 9802..9822 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9829..9849 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9892..9945 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 9946..10659 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(9988..10009) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(10249..10268) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 10596..10617 /label=EGFP-C /note="EGFP, forward primer" misc_feature 10684..11272 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(10737..10757) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(11155..11166) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 11344..11577 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(11603..11620) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 11609..11833 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11879..12307 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11966..11985) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 12176..12197 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(12316..12336) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 12321..12650 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 12501..12636 /label=SV40 ori /note="SV40 origin of replication" primer_bind 12563..12582 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 12698..12745 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 12764..13138 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 13268..13389 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(13305..13324) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 13359..13378 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(13438..13454) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(13438..13454) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(13451..13473) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 13462..13478 /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)." promoter complement(13486..13516) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13531..13552 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(13669..13686) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13840..14428) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13920..13939) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(14599..175) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 15222..15241 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 61 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 121 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 181 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 241 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 301 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat 361 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc 421 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat 481 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac 541 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 601 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 661 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 721 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 781 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 841 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 901 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 961 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1021 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 1081 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta 1141 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 1201 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 1261 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 1321 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg 1381 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac 1441 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat 1501 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa 1561 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa 1621 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc 1681 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg 1741 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga 1801 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag 1861 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac 1921 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag 1981 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag 2041 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga 2101 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca 2161 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc 2221 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 2281 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca 2341 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc 2401 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt 2461 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg 2521 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt 2581 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa 2641 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg 2701 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa 2761 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata 2821 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg 2881 gacagcagag atccagtttg gttaattaag gtaccgaggg cctatttccc atgattcctt 2941 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa 3001 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 3061 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 3121 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac ggttgtaaat 3181 gagcacacaa aatacacatg ctaaaatatt atattctatg acctttataa aatcaaccaa 3241 aatcttcttt ttaataactt tagtatcaat aattagaatt tttatgttcc tttttgcaaa 3301 cttttaataa aaatgagcaa aataaaaaaa cgctagtttt agtaactcgc gttgttttct 3361 tcacctttaa taatagctac tccaccactt gttcctaagc ggtcagctcc tgcttcaatc 3421 attttttgag catcttcaaa tgttctaact ccaccagctg ctttaactaa agcattgtct 3481 ttaacaactg acttcattag tttaacatct tcaaatgttg cacctgattt tgaaaatcct 3541 gttgatgttt taacaaattc taatccagct tcaacagcta tttcacaagc tttcatgatt 3601 tcttcttttg ttaataaaca attttccata atacatttaa caacatgtga tccagctgct 3661 ttttttacag ctttcatgtc ttctaaaact aattcataat ttttgtcttt taatgcacca 3721 atatttaata ccatatcaat ttctgttgca ccatctttaa ttgcttcaga aacttcgaat 3781 gcttttgtag ctgttgtgca tgcacctaga ggaaaaccta caacatttgt tattcctaca 3841 tttgtgcctt ttaataattc tttacaatag cttgttcaat atgaattaac acaaactgtt 3901 gcaaaatcaa attcaattgc ttcatcacat aattgtttaa tttcagcttt cgtagcatct 3961 tgttttaata atgtgtgatc tatatatttg tttagtttca ttttttctcc tatatattca 4021 tttttaattt taattcttta ataatttcgt ctactttaac tttagcgttt tgaacagatt 4081 caccaacacc tataaaataa atttttagtt taggttcagt tccacttggg cgaacagcaa 4141 atcatgactt atcttctaaa taaaatttta gtaagtcttg tcctggcata ttatacattc 4201 catcgatgta gtcttcaaca ttaacaactt taagtccagc aatttgagtt aagggtgttg 4261 ctctcaatga tttcattaat ggttcaattt ttaatttctt ttcttctggt ttaaaattca 4321 agtttaaagt gaaagtgtaa tatgcaccca tttctttaaa taaatcttct aaatagtcta 4381 ctaatgtttt attttgtttt ttataaaatc aagcagcctc tgctattaat atagaagctt 4441 gtattccatc tttatctcta gctgagtcat caattacata tccataactt tcttcataag 4501 caaaaacaaa atttaatccg ttatcttctt ctttagcaat ttctctaccc attcatttaa 4561 atccagttaa agtttttaca atattaactc catatttttc atgagcgatt ctatcaccca 4621 aatcacttgt tacaaaactt gaatatagag ccggattttt tggaatgcta tttaagcgtt 4681 ttagatttga taattttcaa tcaattaaaa ttggtcctgt ttgatttcca tctaatctta 4741 caaaatgacc atcatgtttt attgccattc caaatctgtc agcatctggg tcattcataa 4801 taataatatc tgcatcatgt ttaataccat attcaagcgg tatttttcat gcaggatcaa 4861 attctggatt tggatttaca acatttttaa atgtttcatc ttcaaatgca tgctcttcaa 4921 cctcaataac gttatatcct gattcacgta atatttttgg ggtaaattta gttcctgttc 4981 cattaactgc gctaaaaata atttttaaat cttttttagc ttcttgctct tttttgtacg 5041 tctctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 5101 aaagtggcac cgagtcggtg cttttttgaa ttcgctagct aggtcttgaa aggagtggga 5161 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg 5221 gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa 5281 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt 5341 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg 5401 tgatccgcca ccatggatgc taagtcacta actgcctggt cccggacact ggtgaccttc 5461 aaggatgtat ttgtggactt caccagggag gagtggaagc tgctggacac tgctcagcag 5521 atcgtgtaca gaaatgtgat gctggagaac tataagaacc tggtttcctt gggttatcag 5581 cttactaagc cagatgtgat cctccggttg gagaagggag aagagcccgg cggttccggc 5641 ggagggtcga tggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc 5701 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac 5761 accgaccggc acagcatcaa gaagaacctg atcggcgccc tgctgttcga cagcggagaa 5821 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac 5881 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc 5941 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc 6001 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac 6061 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc 6121 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac 6181 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag 6241 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc 6301 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg 6361 ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac 6421 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac 6481 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc 6541 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg 6601 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct 6661 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac 6721 ggctacgccg gctacatcga tggcggagcc agccaggaag agttctacaa gttcatcaag 6781 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac 6841 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga 6901 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg 6961 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg 7021 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac 7081 ttcgaggaag tggtggacaa gggcgccagc gcccagagct tcatcgagcg gatgaccaac 7141 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac 7201 ttcaccgtgt acaacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc 7261 gccttcctga gcggcgagca gaaaaaagcc atcgtggacc tgctgttcaa gaccaaccgg 7321 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc 7381 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg 7441 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa 7501 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa 7561 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc 7621 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag 7681 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc 7741 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc 7801 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc 7861 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag 7921 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc 7981 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa 8041 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag 8101 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat 8161 gtggacgcta tcgtgcctca gagctttctg aaggacgact ccatcgataa caaagtgctg 8221 actcggagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag 8281 aagatgaaga actactggcg ccagctgctg aatgccaagc tgattaccca gaggaagttc 8341 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc 8401 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc 8461 cggatgaaca ctaagtacga cgagaacgac aaactgatcc gggaagtgaa agtgatcacc 8521 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag 8581 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg 8641 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac 8701 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc 8761 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc 8821 cggaagcggc ctctgatcga gacaaacggc gaaacaggcg agatcgtgtg ggataagggc 8881 cgggactttg ccaccgtgcg gaaagtgctg tctatgcccc aagtgaatat cgtgaaaaag 8941 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgac 9001 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc 9061 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg 9121 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat 9181 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag 9241 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc 9301 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac 9361 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg 9421 tttgtggaac agcacaaaca ctacctggac gagatcatcg agcagatcag cgagttctcc 9481 aagagagtga tcctggccga cgctaatctg gacaaggtgc tgagcgccta caacaagcac 9541 agagacaagc ctatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccagt 9601 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc 9661 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag 9721 acacggatcg acctgtctca gctgggaggc gacgcctatc cctatgacgt gcccgattat 9781 gccagcctgg gcagcggctc ccccaagaaa aaacgcaagg tggaagatcc taagaaaaag 9841 cggaaagtgg acggcattgg tagtgggagc aacggcagca gcggggatcc cgagggcaga 9901 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccagtgag caagggcgag 9961 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 10021 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 10081 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 10141 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 10201 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 10261 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 10321 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 10381 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 10441 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 10501 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 10561 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 10621 gccgccggga tcactctcgg catggacgag ctgtacaagg aattctaaac gcgttaagtc 10681 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 10741 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 10801 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 10861 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 10921 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 10981 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 11041 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 11101 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 11161 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 11221 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt 11281 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 11341 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 11401 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 11461 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 11521 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg 11581 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 11641 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 11701 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 11761 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 11821 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 11881 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 11941 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 12001 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 12061 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 12121 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 12181 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 12241 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 12301 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 12361 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 12421 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 12481 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 12541 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 12601 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg 12661 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata 12721 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag 12781 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg 12841 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt 12901 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt 12961 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt 13021 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt 13081 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca 13141 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 13201 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 13261 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 13321 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 13381 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc 13441 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 13501 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 13561 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 13621 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 13681 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 13741 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 13801 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 13861 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 13921 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 13981 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 14041 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 14101 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 14161 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 14221 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 14281 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 14341 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 14401 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 14461 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 14521 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 14581 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 14641 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 14701 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 14761 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 14821 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 14881 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 14941 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 15001 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 15061 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 15121 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 15181 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 15241 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggc //
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