LOCUS Exported 6404 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Green fluorescent indicator for calcium imaging in the mitochondria (moderately low affinity variant). ACCESSION . VERSION . KEYWORDS pCMV CEPIA3mt SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6404) AUTHORS Suzuki J, Kanemaru K, Ishii K, Ohkura M, Okubo Y, Iino M TITLE Imaging intraorganellar Ca(2+) at subcellular resolution using CEPIA. JOURNAL Nat Commun. 2014 Jun 13;5:4153. doi: 10.1038/ncomms5153. PUBMED 24923787 REFERENCE 2 (bases 1 to 6404) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6404 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 12..388 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 390..593 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 544..564 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 636..710 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="MSVLTPLLLRGLTGSARRLPVPRAK" CDS 732..806 /codon_start=1 /product="mitochondrial presequence of human cytochrome c oxidase subunit VIII (Rizutto et al., 1989)" /label=COX8 presequence /translation="MSVLTPLLLRGLTGSARRLPVPRAK" CDS 846..863 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 867..899 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 903..926 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" regulatory 933..942 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 954..1010 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" primer_bind 1223..1244 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1350..1371) /label=EGFP-N /note="EGFP, reverse primer" CDS 1746..2186 /codon_start=1 /product="calcium-binding protein" /label=calmodulin /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLRSLGQNPTEA ELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" CDS 2196..2225 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" primer_bind complement(2261..2278) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2267..2491 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2537..2965 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2624..2643) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2834..2855 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2974..2994) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2979..3308 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3159..3294 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3221..3240 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3334..4128 /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="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3388..3407) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3998..4017 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4302..4423 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4339..4358) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4393..4412 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4456..4473) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4627..5215) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4707..4726) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5386..6246) /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 6009..6028 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6247..6351) /gene="bla" /label=AmpR promoter ORIGIN 1 gtaccgaatt cacattgatt attgagtagt tattaatagt aatcaattac ggggtcatta 61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 361 atctacggtt agtcatcgct attaccatag tgatgcggtt ttggcagtac atcaatgggc 421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctttctggc 601 taactagaga acccactgct tactggcacg tgaccatgtc cgtcctgacg ccgctgctgc 661 tgcggggctt gacaggctcg gcccggcggc tcccagtgcc gcgcgccaaa attcattcac 721 tgggggaccc catgagcgtg ctcaccccac tcctgctgcg ggggctgacc ggcagcgcta 781 ggcggctgcc agtcccgcgg gccaagatcc acagtctcgg cgatcccgga tccatgcggg 841 gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag caaatgggtc 901 gggatctgta cgacgatgac gataaggatc tcgccaccat ggtcgactca tcacgtcgta 961 agtggaataa gacaggtcac gcagtcagag ctataggtcg gctgagctca ctcgagaacg 1021 tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag atccgccaca 1081 acatcgagga cggcggcgtg cagctcgcct accactacca gcagaacacc cccatcggcg 1141 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaaa ctttcgaaag 1201 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 1261 ctctcggcat ggacgagctg tacaagggcg gtaccggagg gagcatggtg agcaagggcg 1321 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1381 acaagttcag cgtgtccggc gagggtgagg gcgatgccac ctacggcaag ctgaccctga 1441 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1501 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1561 agtccgccat gcccgaaggc tacatccagg agcgcaccat cttcttcaag gacgacggct 1621 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgtgc 1681 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaaca 1741 cgcgtgacca actgactgaa gagcagatcg cagaatttaa agaggctttc tccctatttg 1801 acaaggacgg ggatgggaca ataacaacca aggatctggg gacggtgctg cggtctctgg 1861 ggcagaaccc cacagaagca gagctccagg acatgatcaa tgaagtagat gccgacggta 1921 atggcacaat cgacttccct gagttcctga caatgatggc aagaaaaatg aaagacacag 1981 acagtgaaga agaaattaga gaagcgttcc gtgtgtttga taaggatggc aatggctaca 2041 tcagtgcagc agagcttcgc cacgtgatga caaaccttgg agagaagtta acagatgaag 2101 aggttgatga aatgatcagg gaagcagaca tcgatgggga tggtcaggta aactacgaag 2161 agtttgtaca aatgatgaca gcgaaggcgg ccgcagaaca aaaactcatc tcagaagagg 2221 atctgaatgg ggccgcatag tctagaagct cgctgatcag cctcgactgt gccttctagt 2281 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 2341 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 2401 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2461 aggcatgctg gggatggccc gggctctatg gcttctgagg cggaaagaac cagctggggc 2521 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2581 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2641 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2701 ttagggttcc gatttagtgc tttacggcac ctctccccca aaaaacttga ttagggtgat 2761 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2821 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 2881 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 2941 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa 3001 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3061 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 3121 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 3181 gttccgccca ttctccgccc ctaggctgac taattttttt tatttatgca gaggccgagg 3241 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 3301 tttgcaaaaa gctccccccc cgggaggtcc acaatggttg aacaagatgg attgcacgca 3361 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc 3421 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc 3481 aagaccgacc tgtccggtgc cctgaatgaa ctccaggacg aggcagcgcg gctatcgtgg 3541 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg 3601 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct 3661 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct 3721 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa 3781 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa 3841 ctgttcgcca ggctcaaggc gcgtatgccc gacggcgagg atctcgtcgt gactcatggc 3901 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt 3961 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct 4021 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc 4081 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg 4141 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg 4201 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 4261 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt 4321 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 4381 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt 4441 cgatctttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4501 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4561 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4621 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4681 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4741 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4801 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4861 tcgctccaag ctgggctgta tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4921 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4981 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5041 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5101 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5161 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5221 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5281 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5341 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5401 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 5461 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 5521 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 5581 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5641 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5701 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5761 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 5821 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 5881 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5941 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 6001 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 6061 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 6121 cactcgggca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 6181 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 6241 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 6301 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6361 ccgaaaagtg ccacctgacg tcagatcgac ggatcgggag atcg //