LOCUS Exported 7131 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Anion channelrhodopsin from Geminigera cryophila (CCMP2564) for expression in mammalian cells. ACCESSION . VERSION . KEYWORDS pcDNA3.1-GcACR_457-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7131) AUTHORS Govorunova EG, Sineshchekov OA, Rodarte EM, Janz R, Morelle O, Melkonian M, Wong GK, Spudich JL TITLE The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity. JOURNAL Sci Rep. 2017 Mar 3;7:43358. doi: 10.1038/srep43358. PUBMED 28256618 REFERENCE 2 (bases 1 to 7131) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7131 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 135..152 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS complement(175..192) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(202..243) /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS complement(273..989) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(318..339) /label=EGFP-C /note="EGFP, forward primer" primer_bind 667..686 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 926..947 /label=EGFP-N /note="EGFP, reverse primer" regulatory 1448..1457 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind complement(1990..2009) /label=T7 /note="T7 promoter, forward primer" promoter complement(1991..2009) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter complement(2054..2257) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(2083..2103) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 2258..2637 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" primer_bind 2809..2828 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 2903..3007 /gene="bla" /label=AmpR promoter CDS 3008..3868 /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(3226..3245) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4039..4627 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4528..4547 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4781..4798 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4915..4936 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4951..4981 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4989..5005 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 4994..5016 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5013..5029 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5013..5029 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" polyA_signal 5078..5199 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5089..5108) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5143..5162 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(5375..6169) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5486..5505) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 6096..6115 /label=Neo-R /note="Neomycin resistance gene, reverse primer" promoter complement(6236..6565) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6250..6385 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(6304..6323) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 6550..6570 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin complement(6579..7007) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6689..6710) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6901..6920 /label=F1ori-R /note="F1 origin, reverse primer" polyA_signal 7053..146 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" ORIGIN 1 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga 61 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac 121 agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa ctcaatggtg 181 atggtgatga tgaccggtac gcgtagaatc gagaccgagg agagggttag ggataggctt 241 accttcgaac cgcgggccct ctagagcata tgttacttgt acagctcgtc catgccgaga 301 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg 361 tctttgctca gggcggactg gtagctcagg tagtggttgt cgggcagcag cacggggccg 421 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg 481 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag 541 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg 601 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg 661 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct 721 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag 781 cactgcaggc cgtagccgaa ggtggtcacg agggtgggcc agggcacggg cagcttgccg 841 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg 901 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc 961 ccggtgaaca gctcctcgcc cttgctcacg gcggccgcca gcttcacgtc gaagttctct 1021 ttgggaggct cgatcacgcc ctgcttctca tcggtctcca tgagcatgcc ggcttcgtcc 1081 ctgttcctgg catactctct gtcccacttg ccgttcagca ggccccaggt ggtgctccac 1141 agcagcaggc cgtagcagtt cttgcaggag atatcggcca ccaggtagag cagagcgcta 1201 gcgttctcgt cgatcacgca catgtaggtg ctgctgatga tccacaggat gggaaagcag 1261 ctccaggtgc cgaagaacac catcctcagc aggtacagcc tcttcaccac tttcatggcc 1321 agagggctgc caatctgctg gaagtcgtcg atggtcaggg cgaagatggc gaaggcgcag 1381 gacatctcga aaatgaagca cacgaaggcg aagaaaaagt gggtccagat cacgtcgttg 1441 gtttcggcca tggtggcgga gatgccgaac actgtcctga tgatggaggc ggcgatcatc 1501 atggggttca ggggctggct cttgtacttg atgagggcca tgttggacac caggcccagc 1561 ataatgggac aggtgcacag ccaggcggcc attctgcacc agggcagcac tttgccattg 1621 gccagcctca catatccctg gccgaagaag gccaggctgt agctgatcat ctcggtggcg 1681 gggagataga tggcctccca gggcactctg ggggctctgc tgatcatgaa caggacctgg 1741 caggcgctca tcacggccat acacagctgg gtgatcttca cgaaaggcat ggtgaccacg 1801 ctcatggcga ggcactcggc gttgtccctg gcgtagctgc catagatctc agggctcagg 1861 tcgatgacct tctgcaggta ctcggccctc ttgtcggcag cggacatgtt gccgtatctg 1921 ggtgtcctgg gggggcaggc ggacatggat ccgagctcgg taccaagctt aactagccag 1981 cttgggtctc cctatagtga gtcgtattaa tttcgataag ccagtaagca gtgggttctc 2041 tagttagcca gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt 2101 gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa 2161 acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct 2221 atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata 2281 cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg 2341 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat 2401 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc 2461 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg 2521 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc 2581 tatgaactaa tgaccccgta attgattact attaataact agtcaataat caatgtcaac 2641 gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct 2701 tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact 2761 cagcgacctc caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc 2821 agattgtact gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg 2881 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 2941 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 3001 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 3061 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 3121 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 3181 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 3241 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 3301 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 3361 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 3421 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 3481 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 3541 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 3601 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 3661 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 3721 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 3781 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 3841 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 3901 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 3961 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 4021 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 4081 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 4141 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 4201 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 4261 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 4321 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 4381 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 4441 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 4501 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 4561 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 4621 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 4681 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 4741 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 4801 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 4861 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 4921 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 4981 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 5041 caagctctag ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga 5101 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 5161 gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag 5221 catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc 5281 caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc 5341 ttggtcggtc atttcgcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat 5401 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag 5461 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc 5521 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca 5581 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc 5641 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat 5701 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg 5761 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag 5821 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca 5881 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc 5941 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca 6001 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga 6061 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct 6121 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc 6181 ctcatcctgt ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc 6241 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg 6301 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg 6361 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact 6421 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc 6481 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg 6541 actttccaca ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta 6601 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 6661 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 6721 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 6781 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 6841 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 6901 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 6961 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct 7021 agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg 7081 cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac c //