质粒名称: | LCV2_mClover3_AAVS1_sgRNA_2 |
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出品公司: | 爱迪基因 |
目录编号: | 155099 |
存储实验室: | Jason Moffat |
载体骨架基本信息 |
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载体名称: | LCV2_mClover3 |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 14753 bp |
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: | 嘌呤霉素 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 30 ℃ |
插入基因信息 |
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插入基因名称: | AAVS1_sgRNA_2 |
物种来源: | H. sapiens (human) |
插入大小: | 20 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | U6 promoter |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | BsmBI/Esp3I(在克隆过程中被破坏) |
3' 克隆位点: | BsmBI/Esp3I(在克隆过程中被破坏) |
5' 测序引物 | ACTATCATATGCTTACCGTAAC |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | LCV2_mClover3_AAVS1_sgRNA_2 was a gift from Jason Moffat (Addgene plasmid # 155099 ; http://n2t.net/addgene:155099 ; RRID:Addgene_155099) |
参考文献部分: | Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism. Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan KA, Rahman M, Brown KR, Ross C, Usaj M, Nedyalkova L, Sizova O, Habsid A, Pawling J, Lin ZY, Abdouni H, Wong C, Weiss A, Mero P, Dennis JW, Gingras A, Myers CL, Andrews BJ, Boone C, Moffat J. Nat Metab (2020) 10.1038/s42255-020-0211-z |
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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 LCV2_mClover3_AAVS1_sgRNA_2 13724 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION lentiviral plasmid expressing mClover3, Cas9 and a gRNA targeting the AAVS1 safe harbor locus. ACCESSION . VERSION . KEYWORDS LCV2_mClover3_AAVS1_sgRNA_2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13724) AUTHORS Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan KA, Rahman M, Brown KR, Ross C, Usaj M, Nedyalkova L, Sizova O, Habsid A, Pawling J, Lin ZY, Abdouni H, Wong C, Weiss A, Mero P, Dennis JW, Gingras A, Myers CL, Andrews BJ, Boone C, Moffat J TITLE Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism JOURNAL Nat Metab (2020) REFERENCE 2 (bases 1 to 13724) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13724 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2608..2848 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2608..2628 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2779..2798 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2878..2953 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 3015..3226 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 3245..3254 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3251..7354 /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 7355..7402 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7403..7426 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7436..7489 /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 7496..8209 /codon_start=1 /product="bright green-yellow fluorescent protein derived from mClover2, with improved photostability (Bajar et al., 2003)" /label=mClover3 /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" primer_bind complement(7538..7559) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 8146..8167 /label=EGFP-C /note="EGFP, forward primer" CDS 8219..8275 /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 8276..8872 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 8769..8789 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 8888..9476 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8941..8961) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9359..9370) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 9548..9781 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(9807..9824) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9813..10037 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 10083..10511 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(10170..10189) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10380..10401 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10520..10540) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10564..10774 /label=SV40 promoter /note="SV40 early promoter" promoter 10586..10782 /label=SV40 promoter /note="SV40 early promoter" rep_origin 10633..10768 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10695..10714 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10830..10877 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10896..11270 /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 11400..11521 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11437..11456) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11491..11510 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11570..11586) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11570..11586) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11583..11605) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11594..11610 /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(11618..11648) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11663..11684 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11801..11818) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11972..12560) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12052..12071) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12731..13591) /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 13354..13373 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13592..13696) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg 2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 2821 ttggctttat atatcttgtg gaaaggacga aacaccggtc accaatcctg tccctaggtt 2881 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 2941 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc 3001 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg 3061 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt 3121 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt 3181 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag 3241 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg 3301 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa 3361 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga 3421 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa 3481 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt 3541 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc 3601 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca 3661 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc 3721 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga 3781 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga 3841 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag 3901 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct 3961 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga 4021 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa 4081 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc 4141 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct 4201 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc 4261 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa 4321 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa 4381 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga 4441 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg 4501 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg 4561 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag 4621 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa 4681 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa 4741 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta 4801 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc 4861 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg 4921 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc 4981 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct 5041 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga 5101 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa 5161 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac 5221 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa 5281 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat 5341 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg 5401 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat 5461 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga 5521 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag 5581 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa 5641 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca 5701 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga 5761 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct 5821 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa 5881 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt 5941 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat 6001 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc 6061 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac 6121 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga 6181 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct 6241 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga 6301 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt 6361 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat 6421 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg 6481 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa 6541 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga 6601 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc 6661 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact 6721 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa 6781 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa 6841 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc 6901 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta 6961 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct 7021 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc 7081 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca 7141 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa 7201 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac 7261 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga 7321 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc 7381 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggca gtggagaggg 7441 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac tcgaggtgag 7501 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 7561 aaacggccac aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct 7621 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 7681 caccttcggc tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga 7741 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga 7801 cgacggtacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 7861 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 7921 gtacaacttc aacagccact acgtctatat cacggccgac aagcagaaga actgcatcaa 7981 ggctaacttc aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta 8041 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 8101 ccatcagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 8161 gttcgtgacc gccgccggga ttacacatgg catggacgag ctgtacaagg gatccggcgc 8221 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga 8281 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct 8341 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat 8401 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa 8461 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga 8521 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct 8581 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg 8641 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt 8701 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc 8761 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga 8821 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta 8881 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 8941 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 9001 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 9061 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 9121 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 9181 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 9241 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 9301 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 9361 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 9421 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg 9481 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag 9541 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact 9601 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 9661 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 9721 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 9781 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 9841 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 9901 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 9961 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 10021 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 10081 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 10141 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 10201 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 10261 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 10321 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 10381 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 10441 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 10501 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 10561 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 10621 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 10681 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 10741 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt 10801 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg 10861 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc 10921 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 10981 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 11041 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 11101 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 11161 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 11221 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac 11281 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 11341 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca 11401 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 11461 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 11521 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 11581 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 11641 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 11701 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 11761 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 11821 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 11881 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 11941 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 12001 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 12061 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 12121 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 12181 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 12241 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 12301 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 12361 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 12421 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 12481 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 12541 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 12601 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 12661 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 12721 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 12781 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 12841 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 12901 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 12961 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 13021 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 13081 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 13141 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 13201 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 13261 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 13321 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 13381 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 13441 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 13501 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 13561 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 13621 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 13681 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac //
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