| 质粒名称: | hCas9-VPR |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 68497 |
| 存储实验室: | George Church |
|
载体骨架基本信息 |
|
| 载体名称: | hCas9 Plasmid #41815 |
| 完整质粒大小: | 11319bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SP-Cas9-VPR |
| 物种来源: | Synthetic |
| 插入大小: | 5799bp |
| 启动子: | CMV |
| 标签/融合蛋白: | VPR (C terminal on insert) |
| 克隆方法: | Gateway Cloning |
| 5’测序引物 | TCAAGTACTTCGACACCACCA |
| 3’测序引物 | AACACCCGTGCGTTTTATTC |
|
文献引用方法 |
|
| 材料和方法部分: | hCas9-VPR was a gift from George Church (Addgene plasmid # 68497 ; http://n2t.net/addgene:68497 ; RRID:Addgene_68497) |
| 参考文献部分: | Cas9 gRNA engineering for genome editing, activation and repression. Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR, Collins JJ, Weiss R, Church G. Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580. 10.1038/nmeth.3580 PubMed 26344044 |
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 hCas9-VPR 11319 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION nuclease competent SP-Cas9 fused to VPR.
ACCESSION .
VERSION .
KEYWORDS hCas9-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11319)
AUTHORS Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian
J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR,
Collins JJ, Weiss R, Church G
TITLE Cas9 gRNA engineering for genome editing, activation and repression.
JOURNAL Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580.
PUBMED 26344044
REFERENCE 2 (bases 1 to 11319)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..11319
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 50..429
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 430..633
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 584..604
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 630..654
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 742..751
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 748..4851
/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 4864..4884
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind 4921..4945
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4975..5124
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 5149..5169
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5599..5955
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 5974..6543
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
protein_bind complement(6548..6572)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(6622..6639)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(6705..6724)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 6749..6797
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 6999..7427
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7086..7105)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7296..7317
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(7436..7456)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 7441..7770
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 7621..7756
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 7683..7702
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 7837..8631
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(7891..7910)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 8501..8520
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 8807..8928
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8844..8863)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8898..8917
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(8977..8993)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8977..8993)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8990..9012)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9001..9017
/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(9025..9055)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9070..9091
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9208..9225)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9379..9967)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(9459..9478)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10138..10998)
/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 10761..10780
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(10999..11103)
/gene="bla"
/label=AmpR promoter
primer_bind complement(11178..11197)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgatatc
781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
1261 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
3241 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct
3301 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
4861 gaccccaaga agaagaggaa ggtgtcgcca gggatccgtc gacttgacgc gttaatatca
4921 acaagtttgt acaaaaaagc aggctacaaa gaggccagcg gttccggacg ggctgacgca
4981 ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac
5041 atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt
5101 gatgatttcg acctggacat gctgattaac tctagaagtt ccggatctcc gaaaaagaaa
5161 cgcaaagttg gtagccagta cctgcccgac accgacgacc ggcaccggat cgaggaaaag
5221 cggaagcgga cctacgagac attcaagagc atcatgaaga agtccccctt cagcggcccc
5281 accgacccta gacctccacc tagaagaatc gccgtgccca gcagatccag cgccagcgtg
5341 ccaaaacctg ccccccagcc ttaccccttc accagcagcc tgagcaccat caactacgac
5401 gagttcccta ccatggtgtt ccccagcggc cagatctctc aggcctctgc tctggctcca
5461 gcccctcctc aggtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct
5521 gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg
5581 gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg
5641 cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc
5701 gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct gaaccagggc
5761 atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga ggccatcacc
5821 cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct gggagcacca
5881 ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc cgatatggat
5941 ttctcagcct tgctgggctc tggcagcggc agccgggatt ccagggaagg gatgtttttg
6001 ccgaagcctg aggccggctc cgctattagt gacgtgtttg agggccgcga ggtgtgccag
6061 ccaaaacgaa tccggccatt tcatcctcca ggaagtccat gggccaaccg cccactcccc
6121 gccagcctcg caccaacacc aaccggtcca gtacatgagc cagtcgggtc actgaccccg
6181 gcaccagtcc ctcagccact ggatccagcg cccgcagtga ctcccgaggc cagtcacctg
6241 ttggaggatc ccgatgaaga gacgagccag gctgtcaaag cccttcggga gatggccgat
6301 actgtgattc cccagaagga agaggctgca atctgtggcc aaatggacct ttcccatccg
6361 cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg
6421 aacctggact cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac
6481 gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg
6541 ttttgaaacc cagctttctt gtacaaagtg gttgatatca tctagagcgg ccgcaggtac
6601 ctgaataact aaggccgctt ccctttagtg agggttaatg cttcgagcag acaaagggtt
6661 cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag
6721 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
6781 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
6841 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
6901 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
6961 agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
7021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
7081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
7141 agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
7201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
7261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
7321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc
7381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
7441 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
7501 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
7561 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
7621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
7681 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
7741 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
7801 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
7861 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
7921 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
7981 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
8041 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
8101 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
8161 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
8221 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
8281 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
8341 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
8401 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
8461 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
8521 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
8581 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
8641 tggggttcgc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
8701 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
8761 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
8821 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
8881 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
8941 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
9001 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
9061 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
9121 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
9181 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
9241 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
9301 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
9361 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
9421 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
9481 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
9541 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
9601 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
9661 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
9721 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
9781 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
9841 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
9901 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
9961 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
10021 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
10081 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
10141 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
10201 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
10261 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
10321 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
10381 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
10441 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
10501 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
10561 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
10621 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
10681 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
10741 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
10801 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
10861 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
10921 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
10981 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
11041 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
11101 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
11161 ctatggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
11221 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
11281 caaggcttga ccgacaattg catgaagaat ctgcttagg
//
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