LOCUS Exported 7281 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette. ACCESSION . VERSION . KEYWORDS Nme2Cas9_AAV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7281) AUTHORS Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ TITLE A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. JOURNAL Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 10.1016/j.molcel.2018.12.003. PUBMED 30581144 REFERENCE 2 (bases 1 to 7281) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7281 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 7..54 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 55..144 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" CDS 151..177 /codon_start=1 /product="nuclear localization signal (Makkerh et al., 1996)" /label=NLS /translation="PAAKKKKLD" primer_bind complement(183..202) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 248..303 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" repeat_region 316..456 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 316..445 /label=AAV2 ITR rep_origin 531..986 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(618..637) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 828..849 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(1003..1022) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1122..1144 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1182..1200) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1268..1372 /gene="bla" /label=AmpR promoter CDS 1373..2233 /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(1591..1610) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2404..2992 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2893..2912 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 3054..3194 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3065..3194 /label=AAV2 ITR misc_RNA complement(3222..3314) /label=Nm tracrRNA /note="trans-activating CRISPR RNA for the Neisseria meningitidis CRISPR/Cas9 system (Hou et al., 2013)" promoter complement(3373..3613) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(3423..3442) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(3593..3613) /label=hU6-F /note="Human U6 promoter, forward primer" regulatory 3949..3958 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3961..3981 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3988..4035 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" ORIGIN 1 gaggataagc ggcccgcagc aaccaagaag gcaggacagg ccaagaagaa gaagtacccc 61 tatgacgtgc ctgactacgc cgggtatccc tacgacgtgc ctgattacgc cgggtcctat 121 ccctacgacg tgccagatta cgctgcagct ccagcagcga agaaaaagaa gctggattaa 181 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 241 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 301 tcactcggcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 361 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 421 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc 481 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 541 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 601 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 661 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 721 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 781 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 841 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt 901 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 961 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 1021 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 1081 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 1141 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 1201 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 1261 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 1321 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 1381 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 1441 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1501 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1561 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 1621 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1681 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1741 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1801 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1861 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1921 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1981 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 2041 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat 2101 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 2161 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 2221 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 2281 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 2341 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2401 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2461 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2521 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 2581 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2641 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2701 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2761 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2821 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2881 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2941 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 3001 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag 3061 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac 3121 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat 3181 cactaggggt tcctgcggcc tctagagttt aaacaaaaaa ataaacgatg ccccttaaag 3241 cagaagcttt aaggggcaga gcgttgcggc acatcttttc agacggcctt attgtagcaa 3301 cggttctcat ttcgtttccg aaatgagaaa gggagctaca actgaagagc tggaatgctc 3361 ttccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag 3421 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa 3481 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat 3541 taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg 3601 gaaataggcc ctcttcctgc ccgaccttga cgtcgactct agaatggagg cggtactatg 3661 tagatgagaa ttcaggagca aactgggaaa agcaactgct tccaaatatt tgtgattttt 3721 acagtgtagt tttggaaaaa ctcttagcct accaattctt ctaagtgttt taaaatgtgg 3781 gagccagtac acatgaagtt atagagtgtt ttaatgaggc ttaaatattt accgtaacta 3841 tgaaatgcta cgcatatcat gctgttcagg ctccgtggcc acgcaactca tacttaagca 3901 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaacaccgc caccatggtg 3961 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa 4021 gccaagaaaa agaaaatggc cgccttcaag cctaacccaa tcaattacat cctgggactg 4081 gacatcggaa tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct 4141 atccggctga tcgatctggg cgtgagagtg tttgagaggg ccgaggtgcc aaagaccggc 4201 gattctctgg ctatggcccg gagactggca cggagcgtga ggcgcctgac acggagaagg 4261 gcacacaggc tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca 4321 gacttcgatg agaatggcct gatcaagagc ctgccaaaca ccccctggca gctgagagca 4381 gccgccctgg acaggaagct gacaccactg gagtggtctg ccgtgctgct gcacctgatc 4441 aagcaccgcg gctacctgag ccagcggaag aacgagggag agacagcaga caaggagctg 4501 ggcgccctgc tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg 4561 acacctgccg agctggccct gaataagttt gagaaggagt ccggccacat cagaaaccag 4621 aggggcgact atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg 4681 ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc 4741 gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg 4801 ggacactgca ccttcgagcc tgcagagcca aaggccgcca agaacaccta cacagccgag 4861 cggtttatct ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg 4921 ccactgaccg acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg 4981 acatatgccc aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg 5041 agatacggca aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc 5101 atctctcgcg ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc 5161 tccgagctgc aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc 5221 acaggccgcc tgaaggacag ggtgcagcct gagatcctgg aggccctgct gaagcacatc 5281 tctttcgata agtttgtgca gatcagcctg aaggccctga gaaggatcgt gccactgatg 5341 gagcagggca agcggtacga cgaggcctgc gccgagatct acggcgatca ctatggcaag 5401 aagaacacag aggagaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct 5461 gtggtgctga gggccctgtc ccaggcaaga aaagtgatca acggagtggt gcgccggtac 5521 ggatctccag cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac 5581 cggaaggaga tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc 5641 aagtttaggg agtacttccc taactttgtg ggcgagccaa agtctaagga catcctgaag 5701 ctgcgcctgt acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg 5761 gtgcggctga acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc 5821 tgggacgatt cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc 5881 aatcagacac catacgagta tttcaatggc aaggacaact ccagggagtg gcaggagttc 5941 aaggcccgcg tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag 6001 aagttcgacg aggatggctt taaggagtgc aacctgaatg acaccagata cgtgaaccgg 6061 ttcctgtgcc agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg 6121 ttcgcctcta atggccagat cacaaacctg ctgaggggat tttggggact gaggaaggtg 6181 cgggcagaga atgacagaca ccacgcactg gatgcagtgg tggtggcatg cagcaccgtg 6241 gcaatgcagc agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc 6301 aagaccatcg ataaggagac aggcaaggtg ctgcaccaga agacccactt cccccagcct 6361 tgggagttct ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct 6421 gagtttgagg aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct 6481 agcaggccag aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat 6541 cggaagatgt ctggcgccca caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac 6601 aacgagaaga tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag 6661 aacatggtga attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg 6721 ctggaggcct acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat 6781 aagaagggag gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg 6841 ctgctgaata agaagaacgc ctacacaatc gccgacaatg gcgatatggt gagagtggac 6901 gtgttctgta aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc 6961 tggcaggtgg ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat 7021 agctatacat tctgtttttc cctgcacaag tatgacctga tcgccttcca gaaggatgag 7081 aagtccaagg tggagtttgc ctactatatc aattgcgact cctctaacgg caggttctac 7141 ctggcctggc acgataaggg cagcaaggag cagcagtttc gcatctccac ccagaatctg 7201 gtgctgatcc agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg 7261 aagaagcgcc cacccgtgcg g //