LOCUS Exported 7345 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gi-coupled hM4D-IRES-mCitrine under the control of human synapsin promote. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-HA-hM4D(Gi)-IRES-mCitrine SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7345) AUTHORS Bryan Roth TITLE Roth lab DREADDs JOURNAL Unpublished REFERENCE 2 (bases 1 to 7345) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7345 /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)" CDS 663..689 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 2145..2718 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2312..2329) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2539..2558 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 2719..2728 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2725..3441 /codon_start=1 /product="enhanced variant of YFP (Heikal et al., 2001)" /label=Citrine /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2770..2791) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3031..3050) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3378..3399 /label=EGFP-C /note="EGFP, forward primer" misc_feature 3472..4060 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3525..3545) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3943..3954) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4092..4568 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4291..4310) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4608..4748 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4608..4737 /label=AAV2 ITR rep_origin 4823..5278 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4910..4929) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5120..5141 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5295..5314) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5414..5436 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5474..5492) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5560..5664 /gene="bla" /label=AmpR promoter CDS 5665..6525 /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(5883..5902) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6696..7284 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7185..7204 /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 gacgccacca 661 tgtacccata cgatgttcca gattacgcta tggccaactt cacacctgtc aatggcagct 721 cgggcaatca gtccgtgcgc ctggtcacgt catcatccca caatcgctat gagacggtgg 781 aaatggtctt cattgccaca gtgacaggct ccctgagcct ggtgactgtc gtgggcaaca 841 tcctggtgat gctgtccatc aaggtcaaca ggcagctgca gacagtcaac aactacttcc 901 tcttcagcct ggcgtgtgct gatctcatca taggcgcctt ctccatgaac ctctacaccg 961 tgtacatcat caagggctac tggcccctgg gcgccgtggt ctgcgacctg tggctggccc 1021 tggactgcgt ggtgagcaac gcctccgtca tgaaccttct catcatcagc tttgaccgct 1081 acttctgcgt caccaagcct ctcacctacc ctgcccggcg caccaccaag atggcaggcc 1141 tcatgattgc tgctgcctgg gtactgtcct tcgtgctctg ggcgcctgcc atcttgttct 1201 ggcagtttgt ggtgggtaag cggacggtgc ccgacaacca gtgcttcatc cagttcctgt 1261 ccaacccagc agtgaccttt ggcacagcca ttgctggctt ctacctgcct gtggtcatca 1321 tgacggtgct gtacatccac atctccctgg ccagtcgcag ccgagtccac aagcaccggc 1381 ccgagggccc gaaggagaag aaagccaaga cgctggcctt cctcaagagc ccactaatga 1441 agcagagcgt caagaagccc ccgcccgggg aggccgcccg ggaggagctg cgcaatggca 1501 agctggagga ggcccccccg ccagcgctgc caccgccacc gcgccccgtg gctgataagg 1561 acacttccaa tgagtccagc tcaggcagtg ccacccagaa caccaaggaa cgcccagcca 1621 cagagctgtc caccacagag gccaccacgc ccgccatgcc cgcccctccc ctgcagccgc 1681 gggccctcaa cccagcctcc agatggtcca agatccagat tgtgacgaag cagacaggca 1741 atgagtgtgt gacagccatt gagattgtgc ctgccacgcc ggctggcatg cgccctgcgg 1801 ccaacgtggc ccgcaagttc gccagcatcg ctcgcaacca ggtgcgcaag aagcggcaga 1861 tggcggcccg ggagcgcaaa gtgacacgaa cgatctttgc cattctgctt gccttcatcc 1921 tcacctggac gccctacaac gtcatggtcc tggtgaacac cttctgccag agctgcatcc 1981 ctgacacggt gtggtccatt ggctactggc tctgctacgt caacagcacc atcaaccctg 2041 cctgctatgc tctgtgcaac gccaccttta aaaagacctt ccggcacctg ctgctgtgcc 2101 agtatcggaa catcggcact gccaggtagg cggccgcgat atcgcccctc tccctccccc 2161 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 2221 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 2281 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 2341 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 2401 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 2461 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 2521 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 2581 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 2641 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 2701 tgaaaaacac gatgataagc caccatggtg agcaagggcg aggagctgtt caccggggtg 2761 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 2821 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 2881 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct gatgtgcttc 2941 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 3001 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 3061 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 3121 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 3181 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 3241 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 3301 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccaaactgag caaagacccc 3361 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 3421 ggcatggacg agctgtacaa gtgataggaa ttcgatatca agcttatcga taatcaacct 3481 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 3541 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 3601 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 3661 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 3721 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 3781 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 3841 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt 3901 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 3961 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 4021 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc 4081 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag 4141 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg 4201 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag 4261 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg 4321 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc 4381 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt 4441 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga 4501 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc 4561 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt 4621 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa 4681 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg 4741 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 4801 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 4861 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct 4921 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 4981 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 5041 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 5101 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc 5161 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat 5221 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta 5281 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 5341 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 5401 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 5461 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 5521 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 5581 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 5641 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 5701 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 5761 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 5821 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 5881 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 5941 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 6001 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 6061 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 6121 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 6181 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 6241 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 6301 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 6361 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 6421 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 6481 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 6541 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 6601 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 6661 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 6721 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 6781 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 6841 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 6901 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 6961 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 7021 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 7081 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 7141 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 7201 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 7261 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 7321 ttttgctggc cttttgctca catgt //