LOCUS Exported 5729 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV genome with Cre-dependent expression of the tTA from the hSyn1 promoter. Contains a positive feedback loop for amplifed expression of the tTA. ACCESSION . VERSION . KEYWORDS pAAV-ihSyn1-DIO-tTA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5729) 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 5729) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5729 /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" protein_bind 829..862 /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)." protein_bind 913..946 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(964..1074) /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" protein_bind complement(1714..1747) /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)." protein_bind complement(1798..1831) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 1856..2444 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1909..1929) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2327..2338) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2476..2952 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2675..2694) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 2992..3132 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2992..3121 /label=AAV2 ITR rep_origin 3207..3662 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3294..3313) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3504..3525 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3679..3698) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3798..3820 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3858..3876) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3944..4048 /gene="bla" /label=AmpR promoter CDS 4049..4909 /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(4267..4286) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5080..5668 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5569..5588 /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 taccggatcc tctagagtcg actccggaat aacttcgtat 841 aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata 901 tgaaacctct taataacttc gtatagcata cattatacga agttatggcg cgccttaccc 961 ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa 1021 gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggccgg 1081 cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa 1141 aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtc tcagaagtgg 1201 gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc 1261 ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc 1321 cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt 1381 ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca 1441 gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca 1501 gaagtgggta tgatgcctgt ccagcatctc gatggccagg gcatcgagca gggcccgctt 1561 gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct 1621 tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcgccgt ttatgacttt 1681 agatttatcc agccgactca tggtggcgct agcataactt cgtataaagt atcctatacg 1741 aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata 1801 acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca 1861 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 1921 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 1981 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 2041 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 2101 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 2161 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 2221 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta 2281 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 2341 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 2401 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct 2461 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg 2521 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg 2581 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg 2641 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca 2701 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct 2761 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt 2821 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag 2881 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc 2941 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc 3001 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 3061 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3121 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3181 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 3241 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt 3301 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 3361 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 3421 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 3481 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc 3541 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa 3601 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 3661 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 3721 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 3781 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 3841 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 3901 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 3961 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4021 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4081 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4141 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4201 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 4261 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 4321 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 4381 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 4441 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 4501 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 4561 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 4621 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 4681 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 4741 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 4801 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 4861 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 4921 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 4981 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5041 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5101 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5161 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5221 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5281 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 5341 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5401 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 5461 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 5521 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5581 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 5641 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 5701 ggccttttgc tggccttttg ctcacatgt //