LOCUS Exported 6895 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector with hSynapsin promoter, Cre-OFF hCAR (for efficient CAV-2 infection), Cre-ON ChETA-mRuby2 (for optogenetic activation), and W3SL regulatory cassette (for maximize cloning capacity). ACCESSION . VERSION . KEYWORDS pAAV-hSyn-DIO {hCAR}off-{ChETA-mRuby2}on-W3SL SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6895) AUTHORS Li SJ, Vaughan A, Sturgill JF, Kepecs A TITLE A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons. JOURNAL Neuron. 2018 Jun 6;98(5):905-917.e5. doi: 10.1016/j.neuron.2018.05.028. PUBMED 29879392 REFERENCE 2 (bases 1 to 6895) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6895 /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 660..693 /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 744..777 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." regulatory 783..792 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1884..1913 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS complement(1928..2638) /codon_start=1 /product="monomeric red fluorescent protein derived from mRuby, with improved photophysical properties (Lam et al., 2012)" /label=mRuby2 /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV AKFAGLGGGMDELYK" regulatory 2635..2644 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2654..3580) /codon_start=1 /product="H134R mutant of an N-terminal fragment of channelrhodopsin-2 from the alga Chlamydomonas reinhardtii (Nagel et al., 2005)" /label=ChR2(H134R) /note="blue light-activated cation channel" /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN PSMLYLATGHRVQWLRYATWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI RKTTKLNIGGTEIEVETLVEDEAEAGAV" regulatory 3577..3586 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3591..3624) /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(3675..3708) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(3778..3798) /label=WPRE-R /note="WPRE, reverse primer" repeat_region 4158..4298 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4158..4287 /label=AAV2 ITR rep_origin 4373..4828 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4460..4479) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4670..4691 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4845..4864) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4964..4986 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5024..5042) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5110..5214 /gene="bla" /label=AmpR promoter CDS 5215..6075 /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(5433..5452) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6246..6834 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6735..6754 /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 agtcgagaag gtaccggatc ctctagagtc gactccggaa 661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc 721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc 781 gcgccaccat ggcgctcctg ctgtgcttcg tgctcctgtg cggagtagtg gatttcgcca 841 gaagtttgag tatcactact cctgaagaga tgattgaaaa agccaaaggg gaaactgcct 901 atctgccgtg caaatttacg cttagtcccg aagaccaggg accgctggac atcgagtggc 961 tgatatcacc agctgataat cagaaggtgg atcaagtgat tattttatat tctggagaca 1021 aaatttatga tgactactat ccagatctga aaggccgagt acattttacg agtaatgatc 1081 tcaaatctgg tgatgcatca ataaatgtaa cgaatttaca actgtcagat attggcacat 1141 atcagtgcaa agtgaaaaaa gctcctggtg ttgcaaataa gaagattcat ctggtagttc 1201 ttgttaagcc ttcaggtgcg agatgttacg ttgatggatc tgaagaaatt ggaagtgact 1261 ttaagataaa atgtgaacca aaagaaggtt cacttccatt acagtatgag tggcaaaaat 1321 tgtctgactc acagaaaatg cccacttcat ggttagcaga aatgacttca tctgttatat 1381 ctgtaaaaaa tgcctcttct gagtactctg ggacatacag ctgtacagtc agaaacagag 1441 tgggctctga tcagtgcctg ttgcgtctaa acgttgtccc tccttcaaat aaagctggac 1501 taattgcagg agccattata ggaactttgc ttgctctagc gctcattggt cttatcatct 1561 tttgctgtcg taaaaagcgc agagaagaaa aatatgaaaa ggaagttcat cacgatatca 1621 gggaagatgt gccacctcca aagagccgta cgtccactgc cagaagctac atcggcagta 1681 atcattcatc cctggggtcc atgtctcctt ccaacatgga aggatattcc aagactcagt 1741 ataaccaagt accaagtgaa gactttgaac gcactcctca gagtccgact ctcccacctg 1801 ctaaggtagc tgcccctaat ctaagtcgaa tgggtgcgat tcctgtgatg attccagcac 1861 agagcaagga tgggtctata gtagagcaga agctgatcag cgaggaggac ctgtaattaa 1921 ttaattactt gtacagctcg tccatcccac caccaagccc ggcgaacttg gcaactgcgt 1981 gttcgcgttg tactacgaac atttcattgt cactttcctc taacctttcc aggcggtgat 2041 caacggcatg gataccgggc atcttgatgt tcccgacggt cttttttgac ctgtaagttg 2101 ttacgaaaga gcaagacaga tggccaccac catcaacttt cagtgccata tgagtgtatc 2161 ccctcagacc accatctgct ggatacatca tctctgtatt aggctcccaa cccttggtct 2221 tcttctgcat cacgggacca ttggagggaa agtttacccc tctgacttgg acgtggtaaa 2281 cgagacagcc atcctcaagg ctggtgtcct gcatgacggt gacgactcca ccatcttcgt 2341 atctcgtaac tctttcccaa gtaaaaccct caggaaagga ctgtttaaag aaatcaggaa 2401 tgcctttcgg gtacttgata aaagtacggc tgccatacat gaacgacgtg gcaagaatgt 2461 caaaggcaaa tggcaggggt cctccctcga tgactttgat cctcatggtt tgagttccca 2521 tgtacggatt gccttctcct tcacctgtgc atttgaattg gtggccgttg accgaacctt 2581 ccatgaccac cttcatacgc atattttcct tgatcagctc ttcgccctta gacaccatgg 2641 tggcggccgc tggcacggct ccggcctcgg cttcgtcttc gacgagagtc tcgacctcga 2701 tctccgttcc gccgatgttc agtttggtgg ttttgcggat atctccgtga atcaatatgt 2761 gctcgtggat caggacgcgc aggtagtgtc ccaacaaccc ccaacaattt ttactcatca 2821 gatcaataat cgtgtgacct acggtggagc catagacgct caggacgcca aaaccttcgg 2881 gccccaaaat gaagagaatt gggaacatac cccagctcac gaaaaacagc catgccatgc 2941 cggtcacgac ctggcggcac cgaccctttg gcacagtatg ataaccctcg atatatgctt 3001 tggcggcgtg aaaaaatgtg ttcgcgccat agcacaatcc aagacaaaag aagatgactt 3061 taacatagcc ggttgccatg gcgctggtag ccccccacac gatagtcccg atgtctgaga 3121 caaggagtcc catggttctc ctgctgtagt cgttgctcag gccggtgagg ttgctcaggc 3181 ggataaggat gacaggacaa gtgagcagcc aggttgcata gcgcagccac tgcacccggt 3241 gtcctgtggc aaggtagagc atagagggat tcttaaactc aaaaaagaac tcgagaatca 3301 ccttaaccat ttcaatggcg cacacataga tctcctccca gccgcatgta gatttccagg 3361 tttggtaggc atagaacatc agcagcaaaa tgctgaatcc tgctgcaagc cactgcagga 3421 catttgacgc ggtctgagcg ccgttcgtgc cgcgagattc aatccatccg gcacagtaac 3481 attgatcctc agggaccagg acggacccgt tcaccaccac aggattagta acgaacaaaa 3541 gttcgcgtcc gacggcagac aaagcgccgc catagtccat ggtggctagc ataacttcgt 3601 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta 3661 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attccgctcg 3721 agataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 3781 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 3841 ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga 3901 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 3961 ttccgtggtg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct 4021 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 4081 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg 4141 ttttttaaag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 4201 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 4261 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 4321 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 4381 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4441 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4501 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4561 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 4621 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 4681 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt 4741 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 4801 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 4861 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4921 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 4981 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 5041 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5161 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5401 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6061 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6121 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6361 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 6421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //