LOCUS Exported 7700 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION SpCas9 expression AAV backbone plasmid under control of nEF promoter. ACCESSION . VERSION . KEYWORDS pAAV-nEFCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7700) AUTHORS Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X, Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang K, Callaway EM, Zhang K, Belmonte JC TITLE In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. JOURNAL Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub 2016 Nov 16. PUBMED 27851729 REFERENCE 2 (bases 1 to 7700) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7700 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 175..386 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 399..667 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" CDS 704..730 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 734..754 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 764..4864 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 4868..4888 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 4907..4955 /note="synthetic polyadenylation signal" repeat_region 4963..5103 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4963..5092 /label=AAV2 ITR rep_origin 5178..5633 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5265..5284) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5475..5496 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5650..5669) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5769..5791 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5829..5847) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5915..6019 /gene="bla" /label=AmpR promoter CDS 6020..6880 /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(6238..6257) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7051..7639 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7540..7559 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gaaaggatct gcgatcgctc cggtgcccgt cagtgggcag 181 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaacgggtg 241 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt 301 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc 361 gcaacgggtt tgccgccaga acacagctga agcttcgagg ggctcgcatc tctccttcac 421 gcgcccgccg ccctacctga ggccgccatc cacgccggtt gagtcgcgtt ctgccgcctc 481 ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt aagtttaaag ctcaggtcga 541 gaccgggcct ttgtccggcg ctcccttgga gcctacctag actcagccgg ctctccacgc 601 tttgcctgac cctgcttgct caactctacg tctttgtttc gttttctgtt ctgcgccgtt 661 acagatccaa gctgtgaccg gcgcctacac cggtgccacc atgtacccat acgatgttcc 721 agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga agtacagcat 781 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt 841 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct 901 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac 961 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag 1021 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt 1081 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc 1141 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga 1201 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca 1261 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca 1321 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga 1381 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc 1441 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg 1501 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag 1561 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc 1621 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag 1681 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga 1741 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta 1801 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc 1861 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga 1921 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa 1981 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga 2041 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg 2101 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag 2161 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc 2221 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt 2281 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt 2341 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc 2401 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga 2461 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt 2521 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct 2581 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga 2641 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt 2701 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat 2761 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg 2821 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga 2881 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct 2941 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct 3001 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa 3061 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg 3121 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca 3181 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga 3241 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct 3301 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag 3361 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct 3421 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg 3481 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat 3541 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga 3601 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg 3661 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc 3721 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga 3781 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca 3841 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa 3901 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg 3961 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct 4021 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa 4081 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga 4141 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc 4201 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac 4261 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta 4321 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga 4381 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc 4441 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg 4501 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga 4561 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct 4621 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga 4681 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt 4741 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct 4801 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg 4861 cgacagcccc aagaagaaga gaaaggtgga ggccagctaa gaattcaata aaagatcttt 4921 attttcatta gatctgtgtg ttggtttttt gtgtgcggcc gcaggaaccc ctagtgatgg 4981 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 5041 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 5101 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac 5161 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 5221 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt 5281 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 5341 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga 5401 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 5461 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg 5521 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct 5581 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg 5641 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 5701 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 5761 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 5821 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 5881 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 5941 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 6001 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 6061 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 6121 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 6181 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 6241 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 6301 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 6361 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 6421 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 6481 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 6541 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 6601 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 6661 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 6721 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc 6781 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 6841 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 6901 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 6961 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 7021 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 7081 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 7141 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 7201 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 7261 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 7321 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 7381 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 7441 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 7501 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 7561 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 7621 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 7681 ctggcctttt gctcacatgt //