pcDNA3.1-hAsCpf1(TYCV) (pY210)

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联系方式:I47-825O-882O

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载体基本信息

质粒名称: pcDNA3.1-hAsCpf1(TYCV) (pY210) 
出品公司: Addgene
目录编号: 89351
存储实验室: Feng Zhang

载体骨架基本信息
载体名称: pcDNA3.1
载体抗性: 氨苄青霉素
空载体大小: 5402 bp
完整质粒大小: 9509 bp
载体类型: 哺乳细胞表达,CRISPR
生长条件: 37 ℃

插入基因信息
插入基因名称: Acidaminococcus sp. Cpf1 (RR variant)
物种来源: Synthetic
插入大小: 4107 bp
基因突变: S542R/K607R
启动子 CMV
标签/融合蛋白:
NLS(插入件上的N端子)
NLS(插入件上的C端子)
3xHA(插入件上的C端子)
克隆方法: Gibson克隆
5' 测序引物 CGCAAATGGGCGGTAGGCGTG

文献引用方法
材料和方法部分: pcDNA3.1-hAsCpf1(TYCV) (pY210) was a gift from Feng Zhang (Addgene plasmid # 89351 ; http://n2t.net/addgene:89351 ; RRID:Addgene_89351)
参考文献部分: Engineered Cpf1 variants with altered PAM specificities. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Nat Biotechnol. 2017 Jun 5. doi: 10.1038/nbt.3900. 10.1038/nbt.3900 PubMed 28581492

载体图谱质粒图谱和多克隆位点信息

pcDNA3.1-hAsCpf1(TYCV) (pY210)质粒图谱

载体简介

载体序列

LOCUS       pcDNA3.1-hAsCpf1(TYCV) (pY210)                9509 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expresses humanized AsCpf1 variant that recognizes TYCV PAMs.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-hAsCpf1(TYCV) (pY210)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9509)
  AUTHORS   Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, 
            Nishimasu H, Nureki O, Crosetto N, Zhang F
  TITLE     Engineered Cpf1 variants with altered PAM specificities.
  JOURNAL   Nat Biotechnol. 2017 Jun 5. doi: 10.1038/nbt.3900.
  PUBMED    28581492
REFERENCE   2  (bases 1 to 9509)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9509
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     213..233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(242..670)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(352..373)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     564..583
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    716..940
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     929..946
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1020..1100)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(1107..1154)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1155..5072)
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLARGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      1426..1435
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(5097..5117)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      5120..5129
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(5164..5183)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5165..5183)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5228..5431)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5257..5277)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5432..5811
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5983..6002
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6077..6181
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6182..7042
                     /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(6400..6419)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7213..7801
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7702..7721
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7955..7972
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8089..8110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8125..8155
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8163..8179
                     /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     8168..8190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8187..8203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8187..8203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    8252..8373
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8263..8282)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8317..8336
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(8547..9341)
                     /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(8658..8677)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     9268..9287
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(9408..228)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9422..48
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9476..9495)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
       61 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
      181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat
      241 taattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
      301 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
      361 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
      421 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
      481 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
      541 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
      601 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
      661 cagggcgcgt ggggataccc cctagagccc cagctggttc tttccgcctc agaagccata
      721 gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc
      781 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
      841 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
      901 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt
      961 taaacgggcc ctctagactc gagcggccgc cactgtgctg gatatctgca gaattcttag
     1021 gcatagtcgg ggacatcata tgggtatgca taatcaggca cgtcgtaggg ataagcgtaa
     1081 tctggaacat cgtatgggta ggatcccttt ttcttttttg cctggccggc ctttttcgtg
     1141 gccgccggcc ttttgttgcg cagctcctgg atgtaggcca gccagtcctg attggagatg
     1201 ccgttctgca gcttcagatc cttgctctcc ttcaggtgat tcagcagcag ctggcccttc
     1261 agggcgatgt ggtaggcgcc attggcatcg gcgtccatgg gccactctgg gttctgaaac
     1321 cgggagtcga agcacacgcc attcagatcg cgcacggggc tgttgatata gtcctcgcct
     1381 gtggcggcat tggagttccg catctgcagc acgctgcgga tcagggccac catggtgtcg
     1441 atggcgtgag aatcgtcatt ctccagcagc tttggcagga tgttggagcc atccctgaac
     1501 acgatgccct tctcctccag cagggcgatc agctcgttgg caggatacag gtcccggtat
     1561 ctgccggtga atctgtgatt ctcgatcact ggcacgattc tcttgccggc gatgaaaggg
     1621 gtgcccttgg cgtcaaactg tgtctcgttc ttctcgaaca cgatatccca tgcaggcata
     1681 aagccgggca ggcccctctg gaaggacaga tttctgttca tcttaaagtg caggatgaag
     1741 tcgccggttt tcacgtcgta gtgcagaaag tcgaagccct ccaggaagtg cttgcggctc
     1801 tcgtgattct tgatggtttt ccacacgaag gggtccacga agccggtcag gggatcgatc
     1861 ttagatgtat atggggcagg cacgtaaaac aggaagccag actgggtgcc catcttggca
     1921 aaggaggtga actggtctgt cagctggtat gggttcagca cgcctcccac tttctctgct
     1981 ggatagtcct tcagcaccag gcaattcagc ttatcgatca gcatcttctc gaactgctgg
     2041 tacacggcct tctcggcgat gccggtcctc ttgctcttaa agccgaaatt caggttctcc
     2101 agcaccacca cggcctggta gtggatcatc aggtccacga tctcgtggat gacctggctc
     2161 agatagccct gcttcagatc cttgattgtg cccaccacag accaggcctg ccttgctgcc
     2221 accctctcct tctccctgtt gtccagcttc ttctggtaat caaactgctg gatggtgttc
     2281 aggctccgct gctccaggat cttgccggtg gagtcgatca ctgtgatata gatcaggttt
     2341 ctctcgcccc gatcgatgcc gatgataggt gtctcggggt gctccttcag gtaggcattc
     2401 accctctggt tgaacttaga tggggaattg gcggcctgat agttcagtgt gataggcacg
     2461 tggaaaaaga acttgtcgct ggtaaagcgc ctatccttga tgatctcgtg agacacctcc
     2521 ttggtgatca cgttgggcag cagggccctg gcctcatcag acaggtcgtg ggacagtctg
     2581 tgattcacat agtcgtacag ctcctggtac agggtgtcgg ggattggggt tttctgatcc
     2641 ttcagcttct tgttcagcat cttctctccc agccggtgtg ccatcctctt catcctggac
     2701 ttagggcggt agaacagctc ggcctggcca ttcagcttga tgcttgtctt ggccaggttc
     2761 tctggagaaa acagaccggt ccaatacagt gtgtgcagat taggcttgcc gtggtggccc
     2821 ttggcaaagt ccttgttata gatctggaac aggtacagct tgcctgtctc cacggcatcc
     2881 atgatctcct tctcggcgat tctctggaag ctgatgtggt acagcagggg attcagctcg
     2941 gcatagtact cgcccaggtc cttatactga gaggatggcc gcaggctaga cagatcgata
     3001 gaggttgtct tggtatactt ggacagaaaa tcccttgtga agtcgatcca cttgcacagg
     3061 gcctctctgt agcccttctg gtcgccggtt ttcttggcgt acgctgtctg aaacttcttt
     3121 ggctccttct caggattgtt caggtcgtag atctcctttg tgatctccag aggctcgatg
     3181 aaattgttgg acagcaggat gggggttgtg tgggtctgaa agtgggcggt caccgccttc
     3241 agctgggtgc tgcaccgtgg gatcatcttg gcggcatccg gaaagtagtc atagtacatc
     3301 ttatcaaagc cctcgctggt tttctctgtg ggctcgaagc tgagggcctt atacctgccc
     3361 ttctgctttg gcatgatgcc cagatagtac aggccgttct tcacaaacag gatggcgcca
     3421 ttgttcttct ccttattcac gtcccagccc cgggccagtg taggcatctg aaagttcagc
     3481 ttgaacttct ccacggagta gggcttcttg gtggcataat ttctggcctt gttgtagaag
     3541 ctcagagaag gctccatctc cagcttgatg ccggtcagcc gggcagagaa ctcggggtcc
     3601 acctcgttgg actcatccac ggcaaaccag tccagcaggt ggtacaggcc cagcaggctg
     3661 tccagctgag acttcaggat ctccttctcc tcctgcttct tcagggttgt aggcagtggc
     3721 tgatccaggg cggcgtgtgc gtgggacagg atctcgctgg ttttctgctt gaaggcctcg
     3781 ctcagctcct tgcctgcggc agagatgatc tcctgcaggt tgatatcctc gtgcttcagg
     3841 ctgcgctgca ccttctcctt ggcagacttg gtgatcttgc ctgtcagctc ggagattctc
     3901 cgctcataca gggcattcct cagtgtatcc cagtggtcgc acagggcgct gctgattgtc
     3961 tccagcttct tgtggctgat gaagatgtgt gtcaggtcga tgctgttcag ctcgttaaac
     4021 agggcctcgg ctgtctccag cacgttctcg tttctcagca gtgtcttgta cttgcagaag
     4081 gactggatca cttcctcgtc gctcttaaac tcctccagga tgaaagacag ggtgttccta
     4141 tcggacagga tctgcttaaa cagggggatg aatctgtgtg gcagggaggc gatgatgtgg
     4201 gctgtctcat cattcttctg gatggccaga ttcagcacct cgttcaggcc cttgatcttc
     4261 tcggtgcctg cctcccgaga gattcctccc agcagctggt tatacaggtc gatctgggtc
     4321 tgtgtcagca gctggttata aaaagggaag gaaaacacct cctcgatgga ggtgctcacg
     4381 aagatgccga tggccttctt cacgttctca aagtgctccc gcaggctggg cacggcggtg
     4441 atcaggcgtg tgaagatgtg acaattctcc ttaaacttgg ggaagttgtc ctgcacgatg
     4501 cggtgtggga tggctgtgct gatatcctcg gcgctgaaca cgttcttcct gttctcataa
     4561 aagccggaga agtaggttgt aaacttgtcg aagctccgca gcagggcgtt ctcgtgctcg
     4621 gttgtggtca cggtgcccag ctgcttcagc accttgccat taaacagctc ggccttgaac
     4681 aggcccttgt agatctcggc gtgtctctta ttgatggcat cggtcaggtt gtctgtccgg
     4741 ccgatgaagt agtcgtggat ggcattgcga tatgtggcct gctcctcgat cagggcgttc
     4801 cttgtctcct cggttttctc ctttctatag gagtcgatgg cggcgctcag gttctcccaa
     4861 tccagctgca ccagctgcag gcactggtcg gcataggtct tgtagatccg atcgatgatg
     4921 ggcttcagct ccttgtagtg atcattgcgg gccttgtcct cctcgatgaa gccctgctcc
     4981 tggatgtgct tcagggtctt gccctgtggg atcagctcaa accgcagtgt cttgctcacc
     5041 tgatacaggt tggtaaagcc ctcgaactgt gtggctgctg ggactccgtg gataccgacc
     5101 ttccgcttct tctttggggc catggtggca agcttaagtt taaacgctag ccagcttggg
     5161 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     5221 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     5281 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     5341 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     5401 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     5461 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     5521 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     5581 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     5641 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     5701 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     5761 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     5821 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     5881 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     5941 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     6001 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     6061 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6121 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6181 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6241 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6301 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6361 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6421 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6481 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6541 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6601 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6661 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6721 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6781 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6841 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6901 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6961 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7021 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7081 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7141 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7201 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7261 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7321 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7381 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7441 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7501 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7561 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7621 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7681 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7741 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7801 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7861 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7921 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7981 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     8041 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     8101 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     8161 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     8221 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     8281 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     8341 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     8401 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     8461 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     8521 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     8581 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     8641 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     8701 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     8761 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     8821 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     8881 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     8941 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     9001 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     9061 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     9121 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     9181 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     9241 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     9301 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     9361 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     9421 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     9481 gcctctgcat aaataaaaaa aattagtca
//

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