质粒名称: | pL-CRISPR.EFS.GFP |
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出品公司: | Addgene |
目录编号: | 57818 |
存储实验室: | Benjamin Ebert |
载体骨架基本信息 |
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载体名称: | pLKO.005 |
载体出品公司: | -- |
空载体大小: | -- |
完整质粒大小: | 11707 bp |
载体修饰: | -- |
载体类型: | 慢病毒,CRISPR |
筛选标记: | -- |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | Stbl3 |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | SpCas9 |
物种来源: | S. pyogenes |
插入大小: | 4300 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子: | EFS |
标签/融合蛋白: | FLAG(插入件上的N端子) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | BamHI(未破坏) |
3’克隆位点: | Nhei(未破坏) |
5’测序引物 | ATAAGTGCAGTAGTCGCCGTG |
3’测序引物 | AAAGCAGCGTATCCACATAGC |
文献引用方法 |
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材料和方法部分: | pL-CRISPR.EFS.GFP was a gift from Benjamin Ebert (Addgene plasmid # 57818 ; http://n2t.net/addgene:57818 ; RRID:Addgene_57818) |
参考文献部分: | Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951. 10.1038/nbt.2951 PubMed 24952903 |
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载体简介
载体序列
LOCUS pL-CRISPR.EFS.GFP 11707 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses eGFP via P2A cleavage site. EFS Promoter driven. ACCESSION . VERSION . KEYWORDS pL-CRISPR.EFS.GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11707) AUTHORS Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL TITLE Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. JOURNAL Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951. PUBMED 24952903 REFERENCE 2 (bases 1 to 11707) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11707 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 5..231 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 232..412 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 459..584 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1077..1310 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1495..1539 /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" promoter 1717..1957 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1717..1737 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1888..1907 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1984..2059 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2107..2224 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2293..2504 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2524..2533 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2533..2556 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2563..2583 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2608..6708 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6709..6756 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6772..6828 /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 6829..7545 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6874..6895) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(7135..7154) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7482..7503 /label=EGFP-C /note="EGFP, forward primer" misc_feature 7564..8152 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(7617..7637) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8035..8046) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 8224..8457 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 8535..8656 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8572..8591) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 8626..8645 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 8696..8831 /label=SV40 ori /note="SV40 origin of replication" primer_bind 8758..8777 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(8851..8870) /label=T7 /note="T7 promoter, forward primer" promoter complement(8852..8870) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(8880..8897) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(8880..8896) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8889..8911) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 9038..9493 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9125..9144) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9335..9356 /label=F1ori-F /note="F1 origin, forward primer" promoter 9519..9623 /gene="bla" /label=AmpR promoter CDS 9624..10484 /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 complement(9842..9861) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 10655..11243 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 11144..11163 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 11397..11414 /label=L4440 /note="L4440 vector, forward primer" protein_bind 11531..11552 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 11567..11597 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11605..11621 /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 11610..11632 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 11629..11645 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 11629..11645 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 11664..11684 /label=T3 /note="T3 promoter, forward primer" promoter 11666..11684 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct 1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta 1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgtgaccgtc 1981 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 2041 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat 2101 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 2161 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 2221 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg 2281 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 2341 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 2401 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 2461 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga 2521 tccgccacca tggattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag 2581 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc 2641 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc 2701 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg 2761 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac 2821 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag 2881 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag 2941 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac 3001 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg 3061 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc 3121 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac 3181 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg 3241 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag 3301 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc 3361 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac 3421 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc 3481 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc 3541 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg 3601 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc 3661 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc 3721 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag 3781 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac 3841 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc 3901 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg 3961 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc 4021 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc 4081 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc 4141 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag 4201 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg 4261 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc 4321 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc 4381 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac 4441 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc 4501 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag 4561 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac 4621 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac 4681 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag 4741 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc 4801 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc 4861 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag 4921 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc 4981 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac 5041 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg 5101 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc 5161 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc 5221 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt 5281 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat 5341 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca 5401 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa 5461 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt 5521 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc 5581 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac 5641 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct 5701 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg 5761 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc 5821 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg 5881 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc 5941 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc 6001 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc 6061 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc 6121 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag 6181 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga 6241 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat 6301 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat 6361 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag 6421 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc 6481 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg 6541 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac 6601 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc 6661 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgtcctgct 6721 gctactaaga aagctggtca agctaagaaa aagaaagcta gcggcagcgg cgccaccaac 6781 ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat ggtgtctaag 6841 ggcgaagagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 6901 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 6961 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 7021 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 7081 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 7141 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 7201 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 7261 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 7321 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 7381 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 7441 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 7501 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaac gcgttaagtc 7561 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 7621 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 7681 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 7741 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 7801 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 7861 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 7921 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 7981 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 8041 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 8101 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt 8161 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 8221 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 8281 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 8341 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 8401 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 8461 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 8521 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 8581 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 8641 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 8701 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 8761 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 8821 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 8881 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 8941 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 9001 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 9061 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 9121 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 9181 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 9241 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 9301 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 9361 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 9421 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 9481 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 9541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 9601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 9661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 9721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 9781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 9841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 9901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 9961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 10021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 10081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 10141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 10201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 10261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 10321 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 10381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 10441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 10501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 10561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 10621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 10681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 10741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 10801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 10861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 10921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 10981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 11041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 11101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 11161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 11221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 11281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 11341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 11401 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 11461 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 11521 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 11581 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 11641 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc 11701 tgcaagc //
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