质粒名称: | pCI152-pLenti-SiT-Cas12a |
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出品公司: | Addgene |
目录编号: | 128405 |
存储实验室: | Randall Platt |
载体骨架基本信息 |
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载体名称: | pLenti |
载体出品公司: | -- |
空载体大小: | 4800 bp |
完整质粒大小: | 13260 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达,慢病毒 |
筛选标记: | 嘌呤霉素 |
载体抗性: | 氨苄青霉素,100μg/mL |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | -- |
插入基因信息 |
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插入基因名称: | AsCas12a-Triplex |
物种来源: | -- |
插入大小: | 4300 bp |
基因突变: | -- |
克隆方法: | -- |
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: | pCI152-pLenti-SiT-Cas12a was a gift from Randall Platt (Addgene plasmid # 128405 ; http://n2t.net/addgene:128405 ; RRID:Addgene_128405) |
参考文献部分: | Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Campa CC, Weisbach NR, Santinha AJ, Incarnato D, Platt RJ. Nat Methods. 2019 Aug 12. pii: 10.1038/s41592-019-0508-6. doi: 10.1038/s41592-019-0508-6. 10.1038/s41592-019-0508-6 PubMed 31406383 |
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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 pCI152-pLenti-SiT-Cas12a 13286 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express both AsCas12a and CRISPR arrays on single transcripts. ACCESSION . VERSION . KEYWORDS pCI152-pLenti-SiT-Cas12a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13286) AUTHORS Campa CC, Weisbach NR, Santinha AJ, Incarnato D, Platt RJ TITLE Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. JOURNAL Nat Methods. 2019 Aug 12. pii: 10.1038/s41592-019-0508-6. doi: 10.1038/s41592-019-0508-6. PUBMED 31406383 REFERENCE 2 (bases 1 to 13286) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..13286 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(53..72) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 244..623 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 625..823 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 841..1021 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1068..1193 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1686..1919 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2104..2148 /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 2446..2563 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" polyA_signal 2613..2661 /note="synthetic polyadenylation signal" misc_feature 2680..3268 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 2786..2797 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 3195..3215 /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3567..3647) /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS complement(3654..4247) /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 complement(3734..3754) /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS complement(4248..4304) /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 complement(4320..4367) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS complement(4368..8288) /codon_start=1 /product="CRISPR-associated protein from Acidaminococcus sp. BV3L6 (Zetsche et al., 2015)" /label=AsCpf1 /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES KDLKLQNGISNQDWLAYIQELRN" regulatory 4639..4648 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind complement(8335..8354) /label=T7 /note="T7 promoter, forward primer" promoter complement(8336..8354) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter complement(8399..8602) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" enhancer 8603..8906 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" LTR 9044..9277 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(9303..9320) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9309..9533 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9579..10007 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9666..9685) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9876..9897 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10016..10036) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10021..10350 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10201..10336 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10263..10282 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10398..10445 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10464..10838 /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 10968..11089 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11005..11024) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11059..11078 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11138..11154) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11138..11154) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11151..11173) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11162..11178 /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(11186..11216) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11231..11252 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11369..11386) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11540..12128) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11620..11639) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12299..13159) /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 12922..12941 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13160..13264) /gene="bla" /label=AmpR promoter ORIGIN 1 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct 61 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 121 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 181 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatataccc 241 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 301 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 361 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 421 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 481 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 541 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 601 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 661 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 721 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 781 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact 841 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 901 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 961 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 1021 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 1081 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 1141 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 1201 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 1261 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 1321 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 1381 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 1441 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 1501 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca 2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg 2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca 2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga 2581 cagcagagat ccagtttggt taattaaggt accacacaaa aaaccaacac acagatctaa 2641 tgaaaataaa gatcttttat tacgcgtcat ggattcgagg cggggaggcg gcccaaaggg 2701 agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca 2761 ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag aaggacgtcc 2821 cgcgcagaat ccaggtggca acacaggcga gcagccaagg aaaggacgat gatttccccg 2881 acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc 2941 aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc ccggaaagga 3001 gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac acagtgcaca 3061 ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc aaccaggatt 3121 tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta 3181 aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt 3241 cacaaatttt gtaatccaga ggttgattgt cgacttaacg cgatctatgt ctagaatcta 3301 caagagtaga aattagagac gagaaatcat gaaagcttgt gaaatagctg ttgaagctgg 3361 attagaattt gcgtctccat ctacaagagt agaaatttcg cagaccaata ccaggatctt 3421 gccatcctat ggaactgcct cggtgagttt tgcttttttt tttttaaagc tagggaaagg 3481 ccaaaaagca aaacctgaga aaacaaaagg ttgttttctc aggaaaagaa aaacctttac 3541 aaccctactg acgaatcgaa ttcttaggca tagtcgggga catcatatgg gtatgcataa 3601 tcaggcacgt cgtagggata agcgtaatct ggaacatcgt atgggtagga tccggcaccg 3661 ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg 3721 gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg 3781 ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc 3841 ttgccctggt ggtcgggcga gactccgacg gtggccagga accacgcggg ctccttgggc 3901 cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac 3961 tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg 4021 gtccagaccg ccacggcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg 4081 acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg 4141 acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg 4201 gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggttgg cccgggattc 4261 tcttcgacat cccctgcttg tttcaacagg gagaagttag tggctccgct tccggatccc 4321 tttttctttt ttgcctggcc ggcctttttc gtggccgccg gccttttgtt gcgcagctcc 4381 tggatgtagg ccagccagtc ctgattggag atgccgttct gcagcttcag atccttgctc 4441 tccttcaggt gattcagcag cagctggccc ttcagggcga tgtggtaggc gccattggca 4501 tcggcgtcca tgggccactc tgggttctga aaccgggagt cgaagcacac gccattcaga 4561 tcgcgcacgg ggctgttgat atagtcctcg cctgtggcgg cattggagtt ccgcatctgc 4621 agcacgctgc ggatcagggc caccatggtg tcgatggcgt gagaatcgtc attctccagc 4681 agctttggca ggatgttgga gccatccctg aacacgatgc ccttctcctc cagcagggcg 4741 atcagctcgt tggcaggata caggtcccgg tatctgccgg tgaatctgtg attctcgatc 4801 actggcacga ttctcttgcc ggcgatgaaa ggggtgccct tggcgtcaaa ctgtgtctcg 4861 ttcttctcga acacgatatc ccatgcaggc ataaagccgg gcaggcccct ctggaaggac 4921 agatttctgt tcatcttaaa gtgcaggatg aagtcgccgg ttttcacgtc gtagtgcaga 4981 aagtcgaagc cctccaggaa gtgcttgcgg ctctcgtgat tcttgatggt tttccacacg 5041 aaggggtcca cgaagccggt caggggatcg atcttagatg tatatggggc aggcacgtaa 5101 aacaggaagc cagactgggt gcccatcttg gcaaaggagg tgaactggtc tgtcagctgg 5161 tatgggttca gcacgcctcc cactttctct gctggatagt ccttcagcac caggcaattc 5221 agcttatcga tcagcatctt ctcgaactgc tggtacacgg ccttctcggc gatgccggtc 5281 ctcttgctct taaagccgaa attcaggttc tccagcacca ccacggcctg gtagtggatc 5341 atcaggtcca cgatctcgtg gatgacctgg ctcagatagc cctgcttcag atccttgatt 5401 gtgcccacca cagaccaggc ctgccttgct gccaccctct ccttctccct gttgtccagc 5461 ttcttctggt aatcaaactg ctggatggtg ttcaggctcc gctgctccag gatcttgccg 5521 gtggagtcga tcactgtgat atagatcagg tttctctcgc cccgatcgat gccgatgata 5581 ggtgtctcgg ggtgctcctt caggtaggca ttcaccctct ggttgaactt agatggggaa 5641 ttggcggcct gatagttcag tgtgataggc acgtggaaaa agaacttgtc gctggtaaag 5701 cgcctatcct tgatgatctc gtgagacacc tccttggtga tcacgttggg cagcagggcc 5761 ctggcctcat cagacaggtc gtgggacagt ctgtgattca catagtcgta cagctcctgg 5821 tacagggtgt cggggattgg ggttttctga tccttcagct tcttgttcag catcttctct 5881 cccagccggt gtgccatcct cttcatcctg gacttagggc ggtagaacag ctcggcctgg 5941 ccattcagct tgatgcttgt cttggccagg ttctctggag aaaacaggcc ggtccaatac 6001 agtgtgtgca gattaggctt gccgtggtgg cccttggcaa agtccttgtt atagatctgg 6061 aacaggtaca gcttgcctgt ctccacggca tccatgatct ccttctcggc gattctctgg 6121 aagctgatgt ggtacagcag gggattcagc tcggcatagt actcgcccag gtccttatac 6181 tgagaggatg gccgcaggct agacagatcg atagaggttg tcttggtata cttggacaga 6241 aaatcccttg tgaagtcgat ccacttgcac agggcctctc tgtagccctt ctggtcgccg 6301 gttttcttgg cgtaggctgt ctgaaacttc tttggctcct tctcaggatt gttcaggtcg 6361 tagatctcct ttgtgatctc cagaggctcg atgaaattgt tggacagcag gatgggggtt 6421 gtgtgggtct gaaagtgggc tgtcacggcc ttcagctggg tgctgcactt tgggatcatc 6481 ttggcggcat cagggaagta gtcatagtac atcttatcaa agccctcgct ggttttctct 6541 gtgggctcga agctcagggc cttatacctg cccttctgct ttggcatgat gcccagatag 6601 tacaggccgt tcttcacaaa caggatggcg ccattgttct tctccttatt cacgtcccag 6661 ccagaggcca gtgtaggcat ctgaaagttc agcttgaact tctccacgga gtagggcttc 6721 ttggtggcat aatttctggc cttgttgtag aagctcagag aaggctccat ctccagcttg 6781 atgccggtca gccgggcaga gaactcgggg tccacctcgt tggactcatc cacggcaaac 6841 cagtccagca ggtggtacag gcccagcagg ctgtccagct gagacttcag gatctccttc 6901 tcctcctgct tcttcagggt tgtaggcagt ggctgatcca gggcggcgtg tgcgtgggac 6961 aggatctcgc tggttttctg cttgaaggcc tcgctcagct ccttgcctgc ggcagagatg 7021 atctcctgca ggttgatatc ctcgtgcttc aggctgcgct gcaccttctc cttggcagac 7081 ttggtgatct tgcctgtcag ctcggagatt ctccgctcat acagggcatt cctcagtgta 7141 tcccagtggt cgcacagggc gctgctgatt gtctccagct tcttgtggct gatgaagatg 7201 tgtgtcaggt cgatgctgtt cagctcgtta aacagggcct cggctgtctc cagcacgttc 7261 tcgtttctca gcagtgtctt gtacttgcag aaggactgga tcacttcctc gtcgctctta 7321 aactcctcca ggatgaaaga cagggtgttc ctatcggaca ggatctgctt aaacaggggg 7381 atgaatctgt gtggcaggga ggcgatgatg tgggctgtct catcattctt ctggatggcc 7441 agattcagca cctcgttcag gcccttgatc ttctcggtgc ctgcctcccg agagattcct 7501 cccagcagct ggttatacag gtcgatctgg gtctgtgtca gcagctggtt ataaaaaggg 7561 aaggaaaaca cctcctcgat ggaggtgctc acgaagatgc cgatggcctt cttcacgttc 7621 tcaaagtgct cccgcaggct gggcacggcg gtgatcaggc gtgtgaagat gtgacaattc 7681 tccttaaact tggggaagtt gtcctgcacg atgcggtgtg ggatggctgt gctgatatcc 7741 tcggcgctga acacgttctt cctgttctca taaaagccgg agaagtaggt tgtaaacttg 7801 tcgaagctcc gcagcagggc gttctcgtgc tcggttgtgg tcacggtgcc cagctgcttc 7861 agcaccttgc cattaaacag ctcggccttg aacaggccct tgtagatctc ggcgtgtctc 7921 ttattgatgg catcggtcag gttgtctgtc cggccgatga agtagtcgtg gatggcattg 7981 cgatatgtgg cctgctcctc gatcagggcg ttccttgtct cctcggtttt ctcctttcta 8041 taggagtcga tggcggcgct caggttctcc caatccagct gcaccagctg caggcactgg 8101 tcggcatagg tcttgtagat ccgatcgatg atgggcttca gctccttgta gtgatcattg 8161 cgggccttgt cctcctcgat gaagccctgc tcctggatgt gcttcagggt cttgccctgt 8221 gggatcagct caaaccgcag tgtcttgctc acctgataca ggttggtaaa gccctcgaac 8281 tgtgtcatgg tggcggtacc aagcttaagt ttaaacgcta gccagcttgg gtctccctat 8341 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag 8401 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg 8461 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga 8521 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga 8581 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc 8641 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt 8701 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg 8761 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta 8821 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg 8881 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc 8941 ccgtaattga ttactattaa taactagtca cgctcgagtt taagaccaat gacttacaag 9001 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac 9061 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc 9121 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 9181 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 9241 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc 9301 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 9361 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 9421 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 9481 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 9541 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 9601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 9661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 9721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 9781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 9841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 9901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 9961 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 10021 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 10081 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 10141 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 10201 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 10261 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga 10321 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc 10381 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat 10441 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg 10501 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact 10561 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg 10621 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg 10681 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg 10741 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg 10801 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt 10861 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 10921 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 10981 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 11041 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 11101 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 11161 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 11221 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 11281 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 11341 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 11401 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 11461 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 11521 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 11581 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 11641 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 11701 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 11761 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 11821 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 11881 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 11941 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 12001 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 12061 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 12121 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 12181 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 12241 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 12301 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 12361 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 12421 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 12481 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 12541 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 12601 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 12661 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 12721 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 12781 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 12841 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 12901 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 12961 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 13021 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 13081 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 13141 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 13201 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 13261 cgcgcacatt tccccgaaaa gtgcca //
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