LOCUS Exported 9118 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of dmBACCS2VL, a modified Drosophila melanogaster blue light-activated Ca2+ channel switch, along with dOrai and mCherry. ACCESSION . VERSION . KEYWORDS dmBACCS2VL-IRES-dOrai-IRES-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9118) AUTHORS Ishii T, Sato K, Kakumoto T, Miura S, Touhara K, Takeuchi S, Nakata T TITLE Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS. JOURNAL Nat Commun. 2015 Aug 18;6:8021. doi: 10.1038/ncomms9021. PUBMED 26282514 REFERENCE 2 (bases 1 to 9118) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9118 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 61..364 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 365..568 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 519..539 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 603..612 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 2186..2759 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 2209..2761 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" regulatory 3281..3290 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 3843..4416 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 3866..4418 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 4418..5128 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(4563..4581) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4831..4850 /label=mCherry-F /note="mCherry, forward primer" misc_feature 5173..5761 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5226..5246) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5644..5655) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 5906..6027 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5943..5962) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5997..6016 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(6034..6489) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6171..6192) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6383..6402 /label=F1ori-R /note="F1 origin, reverse primer" promoter 6516..6620 /gene="bla" /label=AmpR promoter promoter 6622..6979 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(6645..6665) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 6830..6965 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6892..6911 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 7014..7808 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7068..7087) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 7678..7697 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(7996..8015) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 8040..8087 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 8416..9004 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8905..8924 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcctcg 601 aggccaccat gggaccggtc ggaggaagcg gtggatcttt ggctactaca cttgaacgta 661 ttgagaagaa ctttctcatt actgacccaa gattgccaga taatcccatt atattcgcgt 721 ccgatagttt cttgcagttg acagaatata gccgtgaaga aattttggga agaaactgca 781 ggtttctaca aggtcctgaa actgatcgcg cgacagtgag aaaaattaga gatgccatag 841 ataaccaaac agaggtcact gttcagctga ttaattatac aaagagtggt aaaaagttct 901 ggaacctctt tcacttgcag cctatgcgag atcagaaggg agatgtccag tactttattg 961 gggttcagtt ggatggaact gagcatgtcc gagatgctgc cgagagagag ggagtcatgc 1021 tgattaagaa aactgcagaa aatctgcaat catggcttca atacacatat gaactagaaa 1081 gtaagaatca tcagaagaag cgcacgtcgg ctgagaagca gctacagtcg gccagagagg 1141 cttgtgagaa attgcgtaag aaacggtcaa gtttggtggg tgcgttcgtt tccacgcacg 1201 gaaagagtat tgatgatgtg gatcggtcga ttgttgaggc acggaatgcc ctcggagatg 1261 taacaaacga gctgcaagaa cgactgcatc gctggaagca aatcgagacg tgccttggct 1321 taaacattgt gaacaacaat ggtctgcccg gaggatccgg aggaagtgga ggaggtatcc 1381 tgcagtctcg gggtggcagt ggtggttcag gcggttctgg atctttggct actacacttg 1441 aacgtattga gaagaacttt ctcattactg acccaagatt gccagataat cccattatat 1501 tcgcgtccga tagtttcttg cagttgacag aatatagccg tgaagaaatt ttgggaagaa 1561 actgcaggtt tctacaaggt cctgaaactg atcgcgcgac agtgagaaaa attagagatg 1621 ccatagataa ccaaacagag gtcactgttc agctgattaa ttatacaaag agtggtaaaa 1681 agttctggaa cctctttcac ttgcagccta tgcgagatca gaagggagat gtccagtact 1741 ttattggggt tcagttggat ggaactgagc atgtccgaga tgctgccgag agagagggag 1801 tcatgctgat taagaaaact gcagaaaatc tgcaatcatg gcttcaatac acatatgaac 1861 tagaaagtaa gaatcatcag aagaagcgca cgtcggctga gaagcagcta cagtcggcca 1921 gagaggcttg tgagaaattg cgtaagaaac ggtcaagttt ggtgggtgcg ttcgtttcca 1981 cgcacggaaa gagtattgat gatgtggatc ggtcgattgt tgaggcacgg aatgccctcg 2041 gagatgtaac aaacgagctg caagaacgac tgcatcgctg gaagcaaatc gagacgtgcc 2101 ttggcttaaa cattgtgaac aacaatggtc tgcccggcgg ctctggaggt agcggactag 2161 tttgagtcga gccgcggcaa ttccgcccct ctccctcccc cccccctaac gttactggcc 2221 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc 2281 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta 2341 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag 2401 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga 2461 accccccacc tggcaacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg 2521 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 2581 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 2641 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa 2701 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat 2761 atgtctgtgt ggaccacggc caacaattcg ggcctagaga cgccaacaaa gtcgccgatc 2821 acgtcgtcgg ttccgcgggc atcgagaagt tctgcggtga tcaccactgg aaatcaccag 2881 cagcaccact tccagcacgt tgtggccgcc gccgttgcag ccgccacttc agtggccacc 2941 ggtcatcagt tccagcagca gttcccgctg cacgcacatc cgcatccgca tccacagcac 3001 cacagcaaca gtcccacggg tagcggcagc aacagcaaca acagcgcagg tttccagcgc 3061 accagcatca gcaactcgct gctgcagttc ccgccgcccc cgcccccttc ctcacagaac 3121 caggcgaagc cgcgcggaca ccatcgcacc gccagcagca gcatgtcgca atccggcgag 3181 gatctccact cgcccaccta cctgtcctgg cggaagctcc agcttagtcg ggccaagcta 3241 aaggcatcca gcaagacgtc cgccctgctc tccggattcg ccatggtggc gatggtggag 3301 gtgcaactgg atcacgatac gaatgtacca ccgggcatgc tgatagcctt cgccatctgc 3361 accaccctcc tggtggcggt gcacatgctg gccctgatga tcagcacctg tatcctgcca 3421 aatattgaga cggtgtgtaa tttgcatagt atttccctgg tccacgaatc gccacacgag 3481 cgtctccatt ggtacattga aacggcgtgg gcattctcca cgctgctggg actcatactc 3541 tttctactgg agatagccat cctgtgctgg gtgaaattct atgacctaag tccgccggcg 3601 gcttggtcag cgtgtgtggt cctcataccg gtgatgatca tctttatggc cttcgccatt 3661 cacttctatc gctccctggt gtcgcacaaa tatgaggtga cagtctcggg catccgtgag 3721 ctggagatgc tcaaggagca gatggagcag gatcatttgg agcatcacaa taacatacga 3781 aataatggca tgaactatgg cgcatccggg gacattgtct aggtcgagcc gcggcaattc 3841 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 3901 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 3961 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 4021 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 4081 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg caacaggtgc 4141 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 4201 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 4261 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 4321 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 4381 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gtgagcaagg gcgaggagga 4441 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa 4501 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac 4561 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc 4621 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt 4681 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg 4741 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa 4801 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg 4861 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca 4921 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc 4981 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc 5041 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac 5101 cggcggcatg gacgagctgt acaagtaacc gcggtcgacg gtaccgcggg cccgggatct 5161 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 5221 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 5281 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 5341 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 5401 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 5461 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 5521 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 5581 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 5641 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 5701 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 5761 catcgatacc gtggatcgat ccaccggatc tagataactg atcataatca gccataccac 5821 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 5881 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 5941 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 6001 tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa 6061 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 6121 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 6181 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 6241 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 6301 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 6361 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 6421 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 6481 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 6541 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 6601 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt 6661 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 6721 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6781 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 6841 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 6901 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 6961 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg 7021 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 7081 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 7141 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 7201 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 7261 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 7321 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 7381 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 7441 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 7501 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 7561 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 7621 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 7681 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 7741 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 7801 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 7861 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 7921 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc 7981 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac 8041 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt 8101 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc 8161 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc 8221 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat 8281 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 8341 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 8401 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 8461 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 8521 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 8581 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 8641 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 8701 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 8761 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 8821 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 8881 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 8941 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 9001 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 9061 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat //