LOCUS Exported 7157 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION A CMV driven inverted SaCas9. SaCas9 is floxed to render the system cre-dependent.. ACCESSION . VERSION . KEYWORDS pJEP313-pAAV-CMV-SaCas9-DIO-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7157) AUTHORS Kumar N, Stanford W, Aradhana CS, Abraham ND, Dao TJ, Thaseen S, Sairavi A, Gonzalez CU, Ploski JE. TITLE The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase. JOURNAL Front. Mol. Neurosci. (2018) 13 REFERENCE 2 (bases 1 to 7157) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7157 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 190..209 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 371..511 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 382..511 /label=AAV2 ITR primer_bind 550..569 /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind 622..655 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 706..739 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." regulatory 761..770 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 773..793 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 818..3973 /codon_start=1 /product="Cas9 endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system" /label=SaCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKG" CDS 3974..4021 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" protein_bind complement(4072..4105) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4156..4189) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." primer_bind complement(4249..4273) /label=LNCX /note="Human CMV promoter, forward primer" promoter complement(4270..4473) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(4299..4319) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 4474..4777 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" repeat_region 4801..4930 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(4992..5580) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5072..5091) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5751..6611) /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 6374..6393 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6612..6716) /gene="bla" /label=AmpR promoter primer_bind 6784..6802 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6840..6862) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6962..6981 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(6998..296) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7135..7156) /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 61 actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa 121 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 181 gagaaaggaa gggaagaaag cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt 241 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta 301 tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 361 atcaggcgcc cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag 421 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag 481 ggagtggcca actccatcac taggggttcc tgcggccgct cggtccagtg aaaaaaatgc 541 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 601 ctcgagacta gtatcgaatt cataacttcg tatagcatac attatacgaa gttattcttt 661 gcaccattct aaagaataac agtgataatt tctgggttaa ggcaaataac ttcgtatagg 721 atactttata cgaagttatg ctagcgccac catgaccggt gccaccatgg ccccaaagaa 781 gaagcggaag gtcggtatcc acggagtccc agcagccaag cggaactaca tcctgggcct 841 ggacatcggc atcaccagcg tgggctacgg catcatcgac tacgagacac gggacgtgat 901 cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa aacaacgagg gcaggcggag 961 caagagaggc gccagaaggc tgaagcggcg gaggcggcat agaatccaga gagtgaagaa 1021 gctgctgttc gactacaacc tgctgaccga ccacagcgag ctgagcggca tcaaccccta 1081 cgaggccaga gtgaagggcc tgagccagaa gctgagcgag gaagagttct ctgccgccct 1141 gctgcacctg gccaagagaa gaggcgtgca caacgtgaac gaggtggaag aggacaccgg 1201 caacgagctg tccaccaaag agcagatcag ccggaacagc aaggccctgg aagagaaata 1261 cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa 1321 cagattcaag accagcgact acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc 1381 ctaccaccag ctggaccaga gcttcatcga cacctacatc gacctgctgg aaacccggcg 1441 gacctactat gagggacctg gcgagggcag ccccttcggc tggaaggaca tcaaagaatg 1501 gtacgagatg ctgatgggcc actgcaccta cttccccgag gaactgcgga gcgtgaagta 1561 cgcctacaac gccgacctgt acaacgccct gaacgacctg aacaatctcg tgatcaccag 1621 ggacgagaac gagaagctgg aatattacga gaagttccag atcatcgaga acgtgttcaa 1681 gcagaagaag aagcccaccc tgaagcagat cgccaaagaa atcctcgtga acgaagagga 1741 tattaagggc tacagagtga ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta 1801 ccacgacatc aaggacatta ccgcccggaa agagattatt gagaacgccg agctgctgga 1861 tcagattgcc aagatcctga ccatctacca gagcagcgag gacatccagg aagaactgac 1921 caatctgaac tccgagctga cccaggaaga gatcgagcag atctctaatc tgaagggcta 1981 taccggcacc cacaacctga gcctgaaggc catcaacctg atcctggacg agctgtggca 2041 caccaacgac aaccagatcg ctatcttcaa ccggctgaag ctggtgccca agaaggtgga 2101 cctgtcccag cagaaagaga tccccaccac cctggtggac gacttcatcc tgagccccgt 2161 cgtgaagaga agcttcatcc agagcatcaa agtgatcaac gccatcatca agaagtacgg 2221 cctgcccaac gacatcatta tcgagctggc ccgcgagaag aactccaagg acgcccagaa 2281 aatgatcaac gagatgcaga agcggaaccg gcagaccaac gagcggatcg aggaaatcat 2341 ccggaccacc ggcaaagaga acgccaagta cctgatcgag aagatcaagc tgcacgacat 2401 gcaggaaggc aagtgcctgt acagcctgga agccatccct ctggaagatc tgctgaacaa 2461 ccccttcaac tatgaggtgg accacatcat ccccagaagc gtgtccttcg acaacagctt 2521 caacaacaag gtgctcgtga agcaggaaga aaacagcaag aagggcaacc ggaccccatt 2581 ccagtacctg agcagcagcg acagcaagat cagctacgaa accttcaaga agcacatcct 2641 gaatctggcc aagggcaagg gcagaatcag caagaccaag aaagagtatc tgctggaaga 2701 acgggacatc aacaggttct ccgtgcagaa agacttcatc aaccggaacc tggtggatac 2761 cagatacgcc accagaggcc tgatgaacct gctgcggagc tacttcagag tgaacaacct 2821 ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa 2881 gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc gaggacgccc tgatcattgc 2941 caacgccgat ttcatcttca aagagtggaa gaaactggac aaggccaaaa aagtgatgga 3001 aaaccagatg ttcgaggaaa agcaggccga gagcatgccc gagatcgaaa ccgagcagga 3061 gtacaaagag atcttcatca ccccccacca gatcaagcac attaaggact tcaaggacta 3121 caagtacagc caccgggtgg acaagaagcc taatagagag ctgattaacg acaccctgta 3181 ctccacccgg aaggacgaca agggcaacac cctgatcgtg aacaatctga acggcctgta 3241 cgacaaggac aatgacaagc tgaaaaagct gatcaacaag agccccgaaa agctgctgat 3301 gtaccaccac gacccccaga cctaccagaa actgaagctg attatggaac agtacggcga 3361 cgagaagaat cccctgtaca agtactacga ggaaaccggg aactacctga ccaagtactc 3421 caaaaaggac aacggccccg tgatcaagaa gattaagtat tacggcaaca aactgaacgc 3481 ccatctggac atcaccgacg actaccccaa cagcagaaac aaggtcgtga agctgtccct 3541 gaagccctac agattcgacg tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa 3601 gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg aatagcaagt gctatgagga 3661 agctaagaag ctgaagaaga tcagcaacca ggccgagttt atcgcctcct tctacaacaa 3721 cgatctgatc aagatcaacg gcgagctgta tagagtgatc ggcgtgaaca acgacctgct 3781 gaaccggatc gaagtgaaca tgatcgacat cacctaccgc gagtacctgg aaaacatgaa 3841 cgacaagagg ccccccagga tcattaagac aatcgcctcc aagacccaga gcattaagaa 3901 gtacagcaca gacattctgg gcaacctgta tgaagtgaaa tctaagaagc accctcagat 3961 catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 4021 gggatccgat atctagatag ctggcctagg aattcacaag taaggcgcgc cataacttcg 4081 tataatgtat gctatacgaa gttattaaga ggtttcatat tgctaatagc agctacaatc 4141 cagctaccat tctgcataac ttcgtataaa gtatcctata cgaagttatt ccggagtcga 4201 ccggtgtctt ctatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt 4261 cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt 4321 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa 4381 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc 4441 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta 4501 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg 4561 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac 4621 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat 4681 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg 4741 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccacgc gtgcggccgc 4801 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4861 ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc 4921 tgcctgcagg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4981 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 5041 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 5101 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 5161 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 5221 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 5281 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 5341 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 5401 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 5461 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5521 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5581 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5641 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5701 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 5881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 6001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 6061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 6121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 6181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 6241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 6301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 6361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 6421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 6481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 6541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 6601 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6661 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 6721 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 6781 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac 6841 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 6901 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat 6961 gcggcatcag agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg 7021 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc 7081 ggcaaaatcc cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt 7141 tggaacaaga gtccact //