LOCUS Exported 7684 bp ds-DNA circular SYN 05-JAN-2023 DEFINITION Cation channelrhodopsin ChroME2s targeted to the neuronal soma and proximal dendrites and separated from nuclear mRuby3 by P2A in a viral vector. ACCESSION . VERSION . KEYWORDS pAAV-CAG-DIO-ChroME2s-ST-P2A-H2B-mRuby3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7684) AUTHORS Sridharan S, Gajowa MA, Ogando MB, Jagadisan UK, Abdeladim L, Sadahiro M, Bounds HA, Hendricks WD, Turney TS, Tayler I, Gopakumar K, Oldenburg IA, Brohawn SG, Adesnik H TITLE High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks. JOURNAL Neuron. 2022 Jan 27. pii: S0896-6273(22)00008-3. doi: 10.1016/j.neuron.2022.01.008. PUBMED 35120626 REFERENCE 2 (bases 1 to 7684) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 5, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7684 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 94..111 /label=L4440 /note="L4440 vector, forward primer" repeat_region 379..508 /label=AAV2 ITR promoter 822..1099 /label=chicken beta-actin promoter primer_bind 1378..1400 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1462..1481 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 1532..1565 /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 1616..1649 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1661..2371) /codon_start=1 /product="monomeric red fluorescent protein derived from mRuby2, with improved brightness and photostability (Bajar et al., 2016)" /label=mRuby3 /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV AKYSNLGGGMDELYK" CDS complement(2381..2758) /codon_start=1 /gene="HIST1H2BJ" /product="human histone H2B" /label=H2B /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP GELAKHAVSEGTKAITKYTSAK" CDS complement(2765..2821) /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS complement(3032..3055) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" protein_bind complement(4049..4082) /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(4133..4166) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 4191..4779 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4244..4264) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4662..4673) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(4782..4798) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4783..4799) /label=pBluescriptKS /note="For pBluescript vector" polyA_signal 4828..4949 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4839..4858) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 4893..4912 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 5111..5240 /label=AAV2 ITR rep_origin 5419..5874 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5506..5525) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5716..5737 /label=F1ori-F /note="F1 origin, forward primer" promoter 5900..6004 /gene="bla" /label=AmpR promoter CDS 6005..6865 /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(6223..6242) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7036..7624 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7525..7544 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgc 181 gcgctcgctc gctcamykrg gscgcccggg caaagcncgg gcrwckggyg wsmkttrgyt 241 cgykyagcgg tcacgctssg cktamcnacc acacccgccg cgcttaatgc gccgctacag 301 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 361 tcttcgctat tacgccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 421 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 481 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta 541 tctacgtagc catgctctag gaagatctgt accattgacg tcaataatga cgtatgttcc 601 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 661 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 721 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 781 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt 841 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt 901 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggsgcgcgc crgggggggg 961 gggggggggg ggggggsggg gsggggsgag gcggagaggt gcggcggcag ccaatcagag 1021 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa 1081 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc 1141 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg 1201 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc 1261 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag 1321 cggctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt 1381 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct 1441 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca 1501 aagaattgga tcctctagag tcgactccgg aataacttcg tataggatac tttatacgaa 1561 gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttaataac 1621 ttcgtatagc atacattata cgaagttatg gcgcgcctca cttgtacagc tcgtccatgc 1681 caccaccaag gttgctgtat ttggcaactg ccacttctct ttgcactacg taggtttcat 1741 tgtcactctc ctcgatcctt tccaggcggt gatcaacggc atggacaccg ggcatcttga 1801 tgttcccgac ggtctttttt gacctgtaag ttgtcacgaa gttgcagtgc agatggccac 1861 caccatcaac tttcagtgcg atgtcagtgt atcctctcag accaccatct gctggataca 1921 tcatctctgt attaggctcc caacccttgg tcttcttctg catcacggga ccattggagg 1981 gaaagtttac ccctctgacc ttgacgttgt agacgagctc gccatcctca aggctggtgt 2041 cctgcgtgac ggtgacgact ccaccatctt cgtatctcgt aactctttcc caagtaaaac 2101 cctcaggaaa ggactgttta aagaaatcag ggatgtcggc cgggtacttg ataaaggtac 2161 ggctgccata catgaacgac gtggcaagaa tgtcaaaggc aaatggcagg ggtcctccct 2221 cgatgacttt gatcctcatg gtttgcactc cctcgtacgg tctgccttct ccttcacctg 2281 tgcatttgaa ttggtggccg ttgaccgaac cttccatgac caccttcata cgcatatttt 2341 ccttgatcag ctcttcgccc ttagacacca tctcgagtgc cttagcgctg gtgtacttgg 2401 tgatggcctt agtaccctcg gacacggcgt gcttggccaa ctccccaggc agcagcaggc 2461 gcacggccgt ctggatctcc ctggaggtga tggtcgagcg cttgttgtaa tgcgccaggc 2521 gggaagcctc acctgcgatg cgctcgaaaa tgtcgttcac aaacgaattc atgatgccca 2581 tggccttgga cgaaatgccg gtgtcagggt ggacctgctt cagaaccttg tacacataga 2641 tggaatagct ctccttgcgg ctgcgcttgc gcttcttgcc gcctttcttc tgcgccttag 2701 tcaccgcctt cttggagccc tttttcgggg cgggagcaga cttcgctggc tctggcatac 2761 tagtaggtcc agggttctcc tccacgtctc cagcctgctt cagcaggctg aagttagtag 2821 ctccgcttcc tgccgcgaat ctggtggctt cagggaagtc cgtggcacag ctgatgaagc 2881 tgtcaatgct ggacatgctc cgcatgtcga tgacgccctc cgtgcgtgcg ggcagagggg 2941 ccacggggct gggcatgctg ctcatctcca gctcctctgg aggcttgctc tgcggggcca 3001 tctccttggt gttgaggatg ggctgggact gcttatcgtc atcgtctttg taatctaccg 3061 gtgccactcc tccctcctcc tcctcgtcga cgaaggtctc gatctccatg ttctcgccgg 3121 ccacgttgat ggtggttgtc ttccggatgt cgccgtggat caggatgtgc tcgtggatct 3181 tcactctcag gtggtggccc aggaagcccc aagtgttctt ggcgatcaga tccaggatca 3241 ggtggccgat tccgctggtg taaggtgtga tcaggcccag tccctcgtgt ccggcgatga 3301 acatcacggg gaacatcagc caagagcaga agaagacgta ggccatgatc ttgcaaatct 3361 tccggcagac gcctttgggc agggtgtggt agctctcgat atacaccttg gcgatctgga 3421 agaagcaagt cacgccgtag aacaggccga tcatgaagaa caggatcttc agggggccct 3481 ttgtaaaggc ggctgtgatc ccccacacga tgttgccgat gtcggtcacc aggatggtca 3541 tggtccgctt gctgtactct tcgtgcagtc cggtcaggtt gctcaggtgg atcaggatca 3601 cggggcaagt caggagccac tcgctgtacc gcagccaaat cacggctcct ccgttggtct 3661 ggtacactgt ggctgggctg tcgacctcgt ggaacagctc gatgaagcac ttgacgagct 3721 cgatcacgca gacgtagacc tcctcccagc cggttgtggc cttgaagctg tgccaaccgt 3781 agaagatgag ctgcacgata gccacgatca cggtgaacca ctggagtccc actgcgatct 3841 tgtgctgcag ctcggttccg tggttgatgt gggggaagca accgtgatcg gctccggctg 3901 ttgtggcgtt agaggtttct ccgtgagcgt ctgctgctgg tgtcaccact gctgcggcgc 3961 tcagcaggcc tctaattgtg gcttcggcgc taggcacggc tgagataaag gcgtgggtca 4021 ttgtggcggc tgtttccata ctgctagcat aacttcgtat aaagtatcct atacgaagtt 4081 atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc 4141 gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat aatcaacctc 4201 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 4261 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 4321 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 4381 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 4441 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 4501 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 4561 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 4621 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 4681 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 4741 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgac 4801 ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa 4861 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 4921 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 4981 aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta 5041 aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc 5101 attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5161 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5221 gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg anngaggcsg 5281 gkygmsyaww ggkcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 5341 cgcgcagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 5401 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 5461 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 5521 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 5581 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 5641 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 5701 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 5761 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 5821 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg 5881 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5941 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 6001 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 6061 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 6121 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 6181 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 6241 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 6301 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 6361 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 6421 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 6481 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 6541 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 6601 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 6661 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6721 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 6781 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 6841 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 6901 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 6961 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 7021 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 7081 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 7141 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 7201 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 7261 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 7321 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 7381 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 7441 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 7501 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 7561 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 7621 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 7681 catg //