LOCUS Exported 7283 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Optogenetics. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-DIO-ChRmine-mScarlet-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7283) AUTHORS Marshel JH, Kim YS, Machado TA, Quirin S, Benson B, Kadmon J, Raja C, Chibukhchyan A, Ramakrishnan C, Inoue M, Shane JC, McKnight DJ, Yoshizawa S, Kato HE, Ganguli S, Deisseroth K TITLE Cortical layer-specific critical dynamics triggering perception. JOURNAL Science. 2019 Aug 9;365(6453). pii: science.aaw5202. doi: 10.1126/science.aaw5202. Epub 2019 Jul 18. PUBMED 31320556 REFERENCE 2 (bases 1 to 7283) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7283 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 56..196 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 67..196 /label=AAV2 ITR polyA_signal 236..712 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind 494..513 /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" misc_feature 744..1332 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 850..861 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 1259..1279 /label=WPRE-R /note="WPRE, reverse primer" protein_bind 1357..1390 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 1441..1474 /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)." regulatory 1479..1488 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2481..3173 /codon_start=1 /product="bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016)" /label=mScarlet /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS TGGMDELYK" primer_bind complement(2611..2629) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2879..2898 /label=mCherry-F /note="mCherry, forward primer" protein_bind complement(3209..3242) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(3293..3326) /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)." promoter complement(3333..4511) /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3342..4280 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind complement(3365..3385) /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" repeat_region 4612..4741 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(4803..5391) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4883..4902) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5562..6422) /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 6185..6204 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6423..6527) /gene="bla" /label=AmpR promoter primer_bind 6595..6613 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6651..6673) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6773..6792 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(6809..7264) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6946..6967) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7158..7177 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgcccctgc 61 aggcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 121 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 181 atcactaggg gttcctgcgg ccgctcggtc cgcacgtggt tacctacaaa atcagaagga 241 cagggaaggg agcagtggtt cacgcctgta atcccagcaa tttgggaggc caaggtgggt 301 agatcacctg agattaggag ttggagacca gcctggccaa tatggtgaaa ccccgtctct 361 accaaaaaaa caaaaattag ctgagcctgg tcatgcatgc ctggaatccc aacaactcgg 421 gaggctgagg caggagaatc gcttgaaccc aggaggcgga gattgcagtg agccaagatt 481 gtgccactgc actccagctt ggttcccaat agaccccgca ggccctacag gttgtcttcc 541 caacttgccc cttgctccat accacccccc tccaccccat aatattatag aaggacacct 601 agtcagacaa aatgatgcaa cttaatttta ttaggacaag gctggtgggc actggagtgg 661 caacttccag ggccaggaga ggcactgggg aggggtcaca gggatgccac ccgtagatct 721 ctcgagcagc gctcggtatc gatgcgggga ggcggcccaa agggagatcc gactcgtctg 781 agggcgaagg cgaagacgcg gaagaggccg cagagccggc agcaggccgc gggaaggaag 841 gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca gaatccaggt 901 ggcaacatag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca ccacggaatt 961 gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg cgatgagttc 1021 cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac aggtggtggc 1081 aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc cacgttgcct 1141 gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca aggaggagaa 1201 aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag cgtatccaca 1261 tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa ttttgtaatc 1321 cagaggttga ttatcgataa gcttgatatc gaattcataa cttcgtatag catacattat 1381 acgaagttat tctttgcacc attctaaaga ataacagtga taatttctgg gttaaggcaa 1441 ataacttcgt ataggatact ttatacgaag ttatgctagc caccatggca cacgcaccag 1501 gcaccgacca gatgttctac gtgggcacaa tggacggctg gtatctggat accaagctga 1561 actccgtggc catcggcgcc cactggtctt gctttatcgt gctgacaatc accacattct 1621 acctgggcta tgagtcctgg acatctcggg gccctagcaa gagaacctcc ttttacgccg 1681 gctatcagga ggagcagaac ctggccctgt ttgtgaattt ctttgccatg ctgagctact 1741 tcggcaagat cgtggccgac acactgggcc acaacttcgg cgatgtgggc cccttcatca 1801 tcggcttcgg caattacagg tatgccgact acatgctgac ctgcccaatg ctggtgtacg 1861 atctgctgta tcagctgagg gccccctatc gcgtgtcttg tagcgccatc atctttgcca 1921 tcctgatgtc tggcgtgctg gccgagttct acgcagaggg cgaccctagg ctgaggaatg 1981 gcgcctacgc ctggtatggc tttggctgtt tctggtttat cttcgcctac tctatcgtga 2041 tgagcatcgt ggccaagcag tatagccggc tggcccagct ggcccaggat acaggcgccg 2101 agcacagcct gcacgtgctg aagttcgccg tgtttacctt ctccatgctg tggattctgt 2161 ttcccctggt gtgggccatc tgccctagag gcttcggctg gatcgacgat aactggacag 2221 aggtggccca ctgcgtgtgc gacatcgtgg ccaagtcctg ttacggcttt gccctggccc 2281 ggttcagaaa gacctatgat gaggagctgt ttcggctgct ggagcagctg ggacacgacg 2341 aggatgagtt ccagaagctg gagctggata tgaggctgag cagcaatggc gagcgcctgc 2401 ggagactgtc tgcggccgcc aagagcagga tcaccagcga gggcgagtac atccccctgg 2461 accagatcga catcaacgtg gtgagcaagg gcgaggcagt gatcaaggag ttcatgcggt 2521 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg 2581 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc 2641 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcacca 2701 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg 2761 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg 2821 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc 2881 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg 2941 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc gggcgctacc 3001 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca 3061 acgtggaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt 3121 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagttctgct 3181 acgagaacga ggtgtaatag ggcgcgccat aacttcgtat aatgtatgct atacgaagtt 3241 attaagaggt ttcatattgc taatagcagc tacaatccag ctaccattct gcataacttc 3301 gtataaagta tcctatacga agttatggat cctcacgaca cctgaaatgg aagaaaaaaa 3361 ctttgaacca ctgtctgagg cttgagaatg aaccaagatc caaactcaaa aagggcaaat 3421 tccaaggaga attacatcaa gtgccaagct ggcctaactt cagtctccac ccactcagtg 3481 tggggaaact ccatcgcata aaacccctcc ccccaaccta aagacgacgt actccaaaag 3541 ctcgagaact aatcgaggtg cctggacggc gcccggtact ccgtggagtc acatgaagcg 3601 acggctgagg acggaaaggc ccttttcctt tgtgtgggtg actcacccgc ccgctctccc 3661 gagcgccgcg tcctccattt tgagctccct gcagcagggc cgggaagcgg ccatctttcc 3721 gctcacgcaa ctggtgccga ccgggccagc cttgccgccc agggcggggc gatacacggc 3781 ggcgcgaggc caggcaccag agcaggccgg ccagcttgag actacccccg tccgattctc 3841 ggtggccgcg ctcgcaggcc ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg 3901 tcgccgcccg cggccccaaa aaccgaaata ccagtgtgca gatcttggcc cgcatttaca 3961 agactatctt gccagaaaaa aagcgtcgca gcaggtcatc aaaaatttta aatggctaga 4021 gacttatcga aagcagcgag acaggcgcga aggtgccacc agattcgcac gcggcggccc 4081 cagcgcccag gccaggcctc aactcaagca cgaggcgaag gggctcctta agcgcaaggc 4141 ctcgaactct cccacccact tccaacccga agctcgggat caagaatcac gtactgcagc 4201 caggtggaag taattcaagg cacgcaaggg ccataacccg taaagaggcc aggcccgcgg 4261 gaaccacaca cggcacttac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc 4321 acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag 4381 tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg 4441 ccgacccctc cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg 4501 gcaccggagc caattcccac tcctttcaag acctagaagg tccattagct gcaaagattc 4561 ctctctgttt aaaactttat ccatctttgc aaagcttacg cgtgcggccg caggaacccc 4621 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 4681 caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag 4741 gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4801 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4861 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4921 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4981 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5041 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5101 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5161 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5221 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 5281 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5341 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5401 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5461 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5521 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5581 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5641 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5701 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5761 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5821 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5881 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5941 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6001 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6061 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6121 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6181 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6241 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6301 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6361 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6421 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6481 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6541 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 6601 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 6661 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 6721 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 6781 gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt gttaaaattc 6841 gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat cggcaaaatc 6901 ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt ttggaacaag 6961 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 7021 gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa 7081 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 7141 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc gctggcaagt 7201 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 7261 gcgtactatg gttgctttga cgt //