LOCUS Exported 7291 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo. Cre inducible expression (double floxed inversed ORF). ACCESSION . VERSION . KEYWORDS pAAV EF1a DIO iChloC 2A dsRed SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7291) TITLE An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo. Cre inducible expression (double floxed inverse ORF) JOURNAL Unpublished REFERENCE 2 (bases 1 to 7291) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7291 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 242..1419 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 473..1410 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1367..1387 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1449..1482 /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 1533..1566 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1637..1660) /label=DsRed1-C /note="DsRed1, forward primer" primer_bind complement(1835..1854) /label=mCherry-F /note="mCherry, forward primer" primer_bind 2052..2072 /label=DsRed1-N /note="DsRed1, reverse primer" primer_bind 2104..2122 /label=mCherry-R /note="mCherry, reverse primer" regulatory 2248..2257 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 2252..2261 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2277..2330) /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" regulatory 3263..3272 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3277..3310) /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(3361..3394) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3419..4006 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3471..3491) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3889..3900) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4038..4514 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4237..4256) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4554..4694 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4554..4683 /label=AAV2 ITR rep_origin 4769..5224 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4856..4875) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5066..5087 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5241..5260) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5360..5382 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5420..5438) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5506..5610 /gene="bla" /label=AmpR promoter CDS 5611..6471 /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(5829..5848) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6642..7230 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7131..7150 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt 181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat 241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat 541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtacggatcc tctagagtcg 1441 actccggaat aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat 1501 tgtagctgct attagcaata tgaaacctct taataacttc gtatagcata cattatacga 1561 agttatggcg cgccctacag gaacaggtgg tggcggccct cggagcgctc gtactgttcc 1621 acgatggtgt agtcctcgtt gtgggaggtg atgtccagct tggtgtccac gtagtagtag 1681 ccgggcagct gcacgggctt cttggccatg tagatggtct tgaactccac caggtagtgg 1741 ccgccgtcct tcagcttcag ggcctggtgg atctcgccct tcagcacgcc gtcgcggggg 1801 tacaggcgct cggtggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg 1861 ggggggaagt tggtgccgcg gaacttcacc ttgtagatca gcgtgccgtc ctgcagggag 1921 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg 1981 aagccctcgg ggaaggacag cttcttgtag tcggggatgt cggcggggtg cttcacgtac 2041 gccttggagc cgtactggaa ctggggggac aggatgtccc aggcgaaggg cagggggccg 2101 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg 2161 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgcgcacctt gaagcgcatg 2221 aactctttga tgacgtcctc ggaggaggcc accatggcgg ccgcggatcc cggtgcaggg 2281 ccgggattct cctccacgtc accgcatgtt agaagacttc ctctgccctc ggccgcgggt 2341 accgcgccag cctcggcctc gtcctccacc agcgtctcga cctcaatctc agtgccacca 2401 atgttcaatt tggtggtctt gcgaatgtcg ccgtggatga ggatatgctc gtggatcagc 2461 acgcgcaggt agtggccgag cagaccccag cagttcttcg acatcaggtc aatgatggtg 2521 tggccgacgg tggagccgta cacgctcagg acgccgaagc cctcggggcc gaggatgaac 2581 aggatgggga acatacccca tgatacgaag aagagccaag ccatgccagt caccacctgg 2641 cgacaccggc ccttcggcac ggtgtggtaa ccctcgatgt aggccttggc agcgtgaaag 2701 aacgtgttag caccataaca cagacccagg cagaagaaga tgaccttgac gtatccggtg 2761 gccatggcgg aagtggcgcc ccacacaata cagccaatat tagacacaag cagacccatg 2821 gtgcgcctgc tgtagtcgtt ggacaagccc gtcaggtttg acaggtgaat gagaatgacc 2881 gggcaggtga gaagccactc ggcgtaacgc aaccactgga cgcggtggcc tgtggctaga 2941 tacagcatgg acgggttctt aaacgagaag aagaactcga gaatcacctt gaccatcctg 3001 atagcgcaca catagatctg ctcccagccg caggttgact tccatgtttg gtaggcgtaa 3061 aacataagca gtaggatgga gaagccagca gcaagccatt gcagcacgtt cgacgccgtt 3121 tgggcaccgt ttgtgccacg cgactcaatc cagcccgcgc agtaacactg gtcctcaggc 3181 acaagtacag agccattgac gactactggg ttcgttacaa atagcagctc gcgcccaacg 3241 gcactcaggg cgcctccata atccatggtg gctagcataa cttcgtataa agtatcctat 3301 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga 3361 ataacttcgt ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa 3421 tcaacctctg gatacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct 3481 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg 3541 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg 3601 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt 3661 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt 3721 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg 3781 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc 3841 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat 3901 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc 3961 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg 4021 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc 4081 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt 4141 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg 4201 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg 4261 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc 4321 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt 4381 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc 4441 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc 4501 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc 4561 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 4621 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 4681 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 4741 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 4801 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 4861 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 4921 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 4981 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 5041 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 5101 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 5161 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 5221 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 5281 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 5341 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 5401 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 5461 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 5521 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 5581 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 5641 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 5701 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 5761 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 5821 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 5881 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 5941 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 6001 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 6061 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 6121 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 6181 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 6241 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 6301 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 6361 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 6421 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 6481 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 6541 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 6601 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 6661 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 6721 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 6781 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 6841 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 6901 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 6961 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 7021 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 7081 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 7141 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 7201 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 7261 ctggcctttt gctggccttt tgctcacatg t //