LOCUS Exported 7315 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION High photocurrent, low-light sensitive channelrhodopsin (ChRger3) for optogenetic activation with systemic delivery or for low-light activation. Driven by the CAG promoter and double floxed.. ACCESSION . VERSION . KEYWORDS pAAV-CAG-DIO(ChRger3-TS-YFP) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7315) AUTHORS Bedbrook CN, Yang KK, Robinson JE, Mackey ED, Gradinaru V, Arnold FH TITLE Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics. JOURNAL Nat Methods. 2019 Oct 14. pii: 10.1038/s41592-019-0583-8. doi: 10.1038/s41592-019-0583-8. PUBMED 31611694 REFERENCE 2 (bases 1 to 7315) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7315 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 210..513 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 515..792 /label=chicken beta-actin promoter primer_bind 1122..1144 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1206..1225 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 1292..1325 /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 1376..1409 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1421..2137) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1466..1487) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1815..1834 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2074..2095 /label=EGFP-N /note="EGFP, reverse primer" protein_bind complement(3300..3333) /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(3384..3417) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3442..4030 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3495..3515) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3913..3924) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4062..4538 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4261..4280) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4578..4718 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4578..4707 /label=AAV2 ITR rep_origin 4793..5248 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4880..4899) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5090..5111 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5265..5284) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5384..5406 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5444..5462) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5530..5634 /gene="bla" /label=AmpR promoter CDS 5635..6495 /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(5853..5872) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6666..7254 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7155..7174 /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 gcgtaacact agttattaat agtaatcaat tacggggtca 181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc 541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg 661 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg 721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc 781 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc 841 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac 901 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg 961 tggctgcgtg aaagccttga ggggctccgg gagcgccggc aggaaggaaa tgggcgggga 1021 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc 1081 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt 1141 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct 1201 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgcggcccaa 1261 cggtaccgga tcctctagag tcgactccgg aataacttcg tataggatac tttatacgaa 1321 gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttaataac 1381 ttcgtatagc atacattata cgaagttatg gcgcgcccta ttacttgtac agctcgtcca 1441 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct 1501 cgttggggtc tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca 1561 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt 1621 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca 1681 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt 1741 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact 1801 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga 1861 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc 1921 gggcgaagca ctgcaggccg tagccgaagg tggtcacgag ggtgggccag ggcacgggca 1981 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc 2041 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg 2101 gcaccacccc ggtgaacagc tcctcgccct tgctcaccac gttgatgtcg atctggtcca 2161 gggggatgta ctcgccctcg ctggtgatcc tgctcttggc ggccgcgacc gcgccagcct 2221 cggcctcgtc ctccaccagc gtctcgacct caatctcagt gccaccaatg ttcaatttgg 2281 tggtcttgcg aatgtcgccg tggatgagga tatgctcgtg gattttgatg cgcaggaagt 2341 gcccgagcag tccccaaata ttcttggaca gcaggtcggc gatggtgtgg ccaatagtgg 2401 acccagccac gctcaggacg ccgaagccct cggggccgag gatgaacagg atggggaaca 2461 taccccatga tacgaagaag agccaagcca tgccagtcac cacctggcga caccggccct 2521 tcggcacggt gtggtacgcc tccacgtaga tgattcctgc gttgtagaat gtctgggtgc 2581 cgtacaccag gccgatacag taaaacagcc acttcaccca cccagtggcc agagcggctg 2641 tcacgcccat gcagatagtg cccaggtctg acaccagcag agccatggta cgcttattat 2701 atgcctcact cagaccggtg atgttgctca gatggataag aatgacaggg catgtcagca 2761 gccactcggc gtaacgaagc catggggtaa tgttgcctcc gtacaggaac agcatcgcag 2821 gactgtcaaa ctcatggaaa tactcgatga tgaacttgat catctcgatc gtggccacgt 2881 aaatctcctc ccagccgcaa gtagacttcc aggtctggta gccgtagaac atcaggcaga 2941 gcgctgaaag cgcaaaagta atccactgca gaatgttggc agccaacttc tcggcatttg 3001 ttccgttgga tttaagccaa gccaagcaga agcactggcc gttgttcggg atgcaaataa 3061 cagaaccatt gttctcaaga gtgtatgagg tttggaagag catgcgctcg tgggcacggt 3121 ggaaaacgta gtcggggcca tcctgagtcg cgaccggcac cgtcgcgtca ctgccagtcg 3181 acttggtggg cttgtaggcg tccaccatca cgatgtgggc ggctcctgcg ctgccggccg 3241 ccagcgccac tgccagcgct agggcaagaa gccatggcct ccgcgacatg gtggctagca 3301 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt 3361 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa 3421 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact 3481 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 3541 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 3601 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 3661 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 3721 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 3781 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 3841 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc 3901 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 3961 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 4021 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc 4081 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc 4141 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt 4201 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg 4261 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct 4321 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca 4381 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca 4441 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg 4501 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt 4561 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 4621 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 4681 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 4741 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 4801 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4861 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4921 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4981 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 5041 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 5101 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt 5161 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 5221 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 5281 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 5341 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 5401 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 5461 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5581 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5641 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5701 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5761 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5821 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5881 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5941 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 6001 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 6061 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 6121 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 6181 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 6241 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 6301 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 6361 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6421 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6481 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6541 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6601 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6661 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6721 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6781 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 6841 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6901 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6961 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 7021 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 7081 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 7141 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 7201 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 7261 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //