LOCUS Exported 5455 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV genome that expresses tTA from the hSyn1 promoter that contains a positive feedback loop for amplifed expression of the tTA. ACCESSION . VERSION . KEYWORDS pAAV-ihSyn1-tTA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5455) AUTHORS Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu WL, Sanchez-Guardado L, Lois C, Mazmanian SK, Deverman BE, Gradinaru V TITLE Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. JOURNAL Nat Neurosci. 2017 Aug;20(8):1172-1179. doi: 10.1038/nn.4593. Epub 2017 Jun 26. PUBMED 28671695 REFERENCE 2 (bases 1 to 5455) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5455 /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..622 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind 662..680 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 698..716 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 1438..1548 /codon_start=1 /product="3 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP48 /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML" misc_feature 1582..2170 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1635..1655) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2053..2064) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2202..2678 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2401..2420) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 2718..2858 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2718..2847 /label=AAV2 ITR rep_origin 2933..3388 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3020..3039) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3230..3251 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3405..3424) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3524..3546 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3584..3602) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3670..3774 /gene="bla" /label=AmpR promoter CDS 3775..4635 /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(3993..4012) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4806..5394 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5295..5314 /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 gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca 181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga 241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg 301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt 361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac 421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc 481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca 541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt 601 cgtgtcgtgc ctgagagcgc agtcgagaac agatctctaa aacaggtaag tcccattaat 661 ctccctatca gtgatagaga aggtctgaag agtttactcc ctatcagtga tagagattaa 721 ttcctctact aaccttgttc atcttttctt tttttttcta caggtcctgg gtgattaaca 781 gcttaagggc ccggatccgg taccgccacc atgagtcggc tggataaatc taaagtcata 841 aacggcgctc tggaattact caatgaagtc ggtatcgaag gcctgacgac aaggaaactc 901 gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc 961 ctgctcgatg ccctggccat cgagatgctg gacaggcatc atacccactt ctgccccctg 1021 gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc 1081 ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac 1141 gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca 1201 ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag 1261 catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg 1321 agacaagcaa ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg 1381 gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac 1441 gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt 1501 gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaagaa 1561 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact 1621 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 1681 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 1741 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 1801 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 1861 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 1921 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 1981 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc 2041 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 2101 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 2161 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc 2221 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc 2281 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt 2341 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg 2401 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct 2461 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca 2521 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca 2581 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg 2641 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt 2701 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 2761 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 2821 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 2881 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 2941 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 3001 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 3061 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 3121 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 3181 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 3241 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt 3301 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 3361 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 3421 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 3481 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 3541 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 3601 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 3661 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 3721 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 3781 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 3841 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 3901 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 3961 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 4021 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 4081 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 4141 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 4201 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 4261 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 4321 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 4381 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 4441 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 4501 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 4561 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 4621 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 4681 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 4741 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 4801 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 4861 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 4921 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 4981 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 5041 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 5101 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 5161 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 5221 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 5281 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 5341 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 5401 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //