| 质粒名称: | pcDNA-dCas9-p300 Core |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 61357 |
| 存储实验室: | Charles Gersbach |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 载体出品公司: | Invitrogen; Life Technologies |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 11475 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，CRISPR，合成生物学 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | S.pyogenes dCas9 with c-terminal human p300 Core effector fusion (aa 1048-1664 of human p300) |
| 物种来源: | H. sapiens (human), Synthetic; S. pyogenes |
| 插入大小: | 6138 bp |
| 基因突变: | D10A; H840A in S.pyogenes Cas9 |
| GenBank编号 | WP_010922251.1 NP_001420.2 |
| 启动子 | CMV |
| 标签/融合蛋白: |
标志（插入时N端子）
SV40 NLS（N端子插入）
SV40 NLS（C端子插入）
HA（插入的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AscI(未破坏) |
| 3' 克隆位点: | PmeI (未破坏) |
| 5' 测序引物 | CMV F |
| 3' 测序引物 | Bgh R |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357 ; http://n2t.net/addgene:61357 ; RRID:Addgene_61357) |
| 参考文献部分: | Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA. Nat Biotechnol. 2015 May;33(5):510-7. doi: 10.1038/nbt.3199. Epub 2015 Apr 6. 10.1038/nbt.3199 PubMed 25849900 |
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 pcDNA-dCas9-p300 Core 11478 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION encodes S. pyogenes dCas9 with c-terminal fusion of human p300 HAT
core (aa 1048-1664) driven by CMV promoter.
ACCESSION .
VERSION .
KEYWORDS pcDNA-dCas9-p300 Core
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11478)
AUTHORS Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy
TE, Gersbach CA
TITLE Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates
genes from promoters and enhancers.
JOURNAL Nat Biotechnol. 2015 May;33(5):510-7. doi: 10.1038/nbt.3199. Epub
2015 Apr 6.
PUBMED 25849900
REFERENCE 2 (bases 1 to 11478)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11478
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 629..648
/label=T7
/note="T7 promoter, forward primer"
promoter 629..647
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 673..738
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 745..765
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 775..4878
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4903..4923
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4933..6783
/codon_start=1
/gene="EP300"
/product="catalytic histone acetyltransferase core of the
human E1A-associated protein p300 (Delvecchio et al.,
2013)"
/label=p300 core
/translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI
VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ
EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG
ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG
FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA
SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP
PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY
IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE
GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS
RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL
MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD"
regulatory 6547..6556
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 6784..6810
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
primer_bind complement(6835..6852)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 6841..7065
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 7111..7539
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7198..7217)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7408..7429
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(7548..7568)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 7553..7882
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 7733..7868
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 7795..7814
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 7949..8743
/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(8003..8022)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 8613..8632
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 8917..9038
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8954..8973)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9008..9027
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(9087..9103)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9087..9103)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9100..9122)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9111..9127
/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(9135..9165)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9180..9201
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9318..9335)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9489..10077)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(9569..9588)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10248..11108)
/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 10871..10890
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11109..11213)
/gene="bla"
/label=AmpR promoter
primer_bind complement(11288..11307)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
661 gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga catcgattac
721 aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg cggaatggat
781 aagaaatact caataggctt agctatcggc acaaatagcg tcggatgggc ggtgatcact
841 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
901 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
961 cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
1021 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
1081 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
1141 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
1201 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
1261 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
1321 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
1381 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
1441 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
1501 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
1561 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
1621 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
1681 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
1741 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
1801 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
1861 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
1921 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
1981 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
2041 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
2101 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
2161 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
2221 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
2281 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
2341 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
2401 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
2461 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
2521 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
2581 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
2641 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
2701 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
2761 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
2821 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
2881 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
2941 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
3001 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
3061 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
3121 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
3181 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
3241 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tgccattgtt
3301 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
3361 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
3421 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
3481 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
3541 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
3601 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
3661 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
3721 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
3781 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
3841 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
3901 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
3961 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
4021 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
4081 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
4141 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
4201 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
4261 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
4321 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
4381 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
4441 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
4501 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
4561 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
4621 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
4681 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
4741 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
4801 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
4861 agtcagctag gaggtgaccc aattgccgga tccaaggcta gcccgaaaaa gaaacgcaaa
4921 gttgggcgcg ccattttcaa accagaagaa ctacgacagg cactgatgcc aactttggag
4981 gcactttacc gtcaggatcc agaatccctt ccctttcgtc aacctgtgga ccctcagctt
5041 ttaggaatcc ctgattactt tgatattgtg aagagcccca tggatctttc taccattaag
5101 aggaagttag acactggaca gtatcaggag ccctggcagt atgtcgatga tatttggctt
5161 atgttcaata atgcctggtt atataaccgg aaaacatcac gggtatacaa atactgctcc
5221 aagctctctg aggtctttga acaagaaatt gacccagtga tgcaaagcct tggatactgt
5281 tgtggcagaa agttggagtt ctctccacag acactgtgtt gctacggcaa acagttgtgc
5341 acaatacctc gtgatgccac ttattacagt taccagaaca ggtatcattt ctgtgagaag
5401 tgtttcaatg agatccaagg ggagagcgtt tctttggggg atgacccttc ccagcctcaa
5461 actacaataa ataaagaaca attttccaag agaaaaaatg acacactgga tcctgaactg
5521 tttgttgaat gtacagagtg cggaagaaag atgcatcaga tctgtgtcct tcaccatgag
5581 atcatctggc ctgctggatt cgtctgtgat ggctgtttaa agaaaagtgc acgaactagg
5641 aaagaaaata agttttctgc taaaaggttg ccatctacca gacttggcac ctttctagag
5701 aatcgtgtga atgactttct gaggcgacag aatcaccctg agtcaggaga ggtcactgtt
5761 agagtagttc atgcttctga caaaaccgtg gaagtaaaac caggcatgaa agcaaggttt
5821 gtggacagtg gagagatggc agaatccttt ccataccgaa ccaaagccct ctttgccttt
5881 gaagaaattg atggtgttga cctgtgcttc tttggcatgc atgttcaaga gtatggctct
5941 gactgccctc cacccaacca gaggagagta tacatatctt acctcgatag tgttcatttc
6001 ttccgtccta aatgcttgag gactgcagtc tatcatgaaa tcctaattgg atatttagaa
6061 tatgtcaaga aattaggtta cacaacaggg catatttggg catgtccacc aagtgaggga
6121 gatgattata tcttccattg ccatcctcct gaccagaaga tacccaagcc caagcgactg
6181 caggaatggt acaaaaaaat gcttgacaag gctgtatcag agcgtattgt ccatgactac
6241 aaggatattt ttaaacaagc tactgaagat agattaacaa gtgcaaagga attgccttat
6301 ttcgagggtg atttctggcc caatgttctg gaagaaagca ttaaggaact ggaacaggag
6361 gaagaagaga gaaaacgaga ggaaaacacc agcaatgaaa gcacagatgt gaccaaggga
6421 gacagcaaaa atgctaaaaa gaagaataat aagaaaacca gcaaaaataa gagcagcctg
6481 agtaggggca acaagaagaa acccgggatg cccaatgtat ctaacgacct ctcacagaaa
6541 ctatatgcca ccatggagaa gcataaagag gtcttctttg tgatccgcct cattgctggc
6601 cctgctgcca actccctgcc tcccattgtt gatcctgatc ctctcatccc ctgcgatctg
6661 atggatggtc gggatgcgtt tctcacgctg gcaagggaca agcacctgga gttctcttca
6721 ctccgaagag cccagtggtc caccatgtgc atgctggtgg agctgcacac gcagagccag
6781 gactacccgt acgacgttcc ggactacgct tcttgagttt aaaccgctga tcagcctcga
6841 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
6901 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
6961 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
7021 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
7081 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
7141 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
7201 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
7261 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
7321 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
7381 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
7441 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
7501 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
7561 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
7621 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
7681 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
7741 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
7801 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
7861 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
7921 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
7981 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
8041 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
8101 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
8161 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
8221 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
8281 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
8341 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
8401 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
8461 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
8521 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
8581 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
8641 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
8701 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
8761 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
8821 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
8881 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
8941 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
9001 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
9061 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
9121 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
9181 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
9241 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
9301 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
9361 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
9421 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
9481 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
9541 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
9601 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
9661 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
9721 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
9781 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
9841 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
9901 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
9961 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
10021 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
10081 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
10141 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
10201 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
10261 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
10321 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
10381 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
10441 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
10501 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
10561 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
10621 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
10681 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
10741 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
10801 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
10861 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
10921 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
10981 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
11041 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
11101 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
11161 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
11221 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
11281 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
11341 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
11401 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
11461 gggccagata tacgcgtt
//
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