LOCUS Exported 5798 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AAV expression of ChETA (hChR2 with E123T/H134R mutations) fused to HA tag driven by hSynapsin promoter in Cre-ON manner for optogenetic activation. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-DIO {ChETA}on-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5798) 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 5798) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5798 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 2..142 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 13..142 /label=AAV2 ITR promoter 187..634 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" protein_bind 672..705 /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 756..789 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(802..828) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS complement(847..1773) /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 1770..1779 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1784..1817) /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(1868..1901) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 1926..2514 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1979..1999) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2397..2408) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2546..3022 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2745..2764) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3062..3202 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3062..3191 /label=AAV2 ITR rep_origin 3277..3732 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3364..3383) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3574..3595 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3749..3768) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3868..3890 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3928..3946) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4014..4118 /gene="bla" /label=AmpR promoter CDS 4119..4979 /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(4337..4356) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5150..5738 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5639..5658 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 61 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 121 aactccatca ctaggggttc ctgcggccgc acgcgtgtgt ctagactgca gagggccctg 181 cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac 241 gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct 301 atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc 361 accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga 421 aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg 481 cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca 541 cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg 601 cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga tcctctagag 661 tcgactccgg aataacttcg tataggatac tttatacgaa gttatgcaga atggtagctg 721 gattgtagct gctattagca atatgaaacc tcttaataac ttcgtatagc atacattata 781 cgaagttatg gcgcgtaatt aggcgtagtc gggcacgtcg taggggtaca tggtggcggc 841 cgctggcacg gctccggcct cggcttcgtc ttcgacgaga gtctcgacct cgatctccgt 901 tccgccgatg ttcagtttgg tggttttgcg gatatctccg tgaatcaata tgtgctcgtg 961 gatcaggacg cgcaggtagt gtcccaacaa cccccaacaa tttttactca tcagatcaat 1021 aatcgtgtga cctacggtgg agccatagac gctcaggacg ccaaaacctt cgggccccaa 1081 aatgaagaga attgggaaca taccccagct cacgaaaaac agccatgcca tgccggtcac 1141 gacctggcgg caccgaccct ttggcacagt atgataaccc tcgatatatg ctttggcggc 1201 gtgaaaaaat gtgttcgcgc catagcacaa tccaagacaa aagaagatga ctttaacata 1261 gccggttgcc atggcgctgg tagcccccca cacgatagtc ccgatgtctg agacaaggag 1321 tcccatggtt ctcctgctgt agtcgttgct caggccggtg aggttgctca ggcggataag 1381 gatgacagga caagtgagca gccaggttgc atagcgcagc cactgcaccc ggtgtcctgt 1441 ggcaaggtag agcatagagg gattcttaaa ctcaaaaaag aactcgagaa tcaccttaac 1501 catttcaatg gcgcacacat agatctcctc ccagccgcat gtagatttcc aggtttggta 1561 ggcatagaac atcagcagca aaatgctgaa tcctgctgca agccactgca ggacatttga 1621 cgcggtctga gcgccgttcg tgccgcgaga ttcaatccat ccggcacagt aacattgatc 1681 ctcagggacc aggacggacc cgttcaccac cacaggatta gtaacgaaca aaagttcgcg 1741 tccgacggca gacaaagcgc cgccatagtc catggtggct agcataactt cgtataaagt 1801 atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt 1861 gcaaagaata acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta 1921 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 1981 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 2041 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 2101 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 2161 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 2221 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 2281 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc 2341 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 2401 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 2461 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat 2521 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct 2581 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg 2641 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg 2701 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag 2761 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat 2821 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta 2881 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc 2941 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac 3001 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg 3061 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 3121 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 3181 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc 3241 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 3301 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 3361 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3421 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3481 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 3541 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 3601 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 3661 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3721 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 3781 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 3841 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 3901 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 3961 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 4021 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4081 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4141 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4201 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4261 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4321 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 4381 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 4441 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 4501 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 4561 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4621 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4681 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4741 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4801 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 4861 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 4921 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 4981 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5041 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5101 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5161 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5221 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5281 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5341 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5401 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 5461 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5521 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 5581 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5641 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5701 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5761 tacggttcct ggccttttgc tggccttttg ctcacatg //