LOCUS       sequence-89562-e        6404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green fluorescent indicator for calcium imaging in the mitochondria 
            (low affinity variant).
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2014-06-13"; volume: "5:4153. doi"; pages: " 
            10.1038/ncomms5153"
COMMENT     Sequence Label: pCMV CEPIA4mt
FEATURES             Location/Qualifiers
     source          1..6404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(834..845,846..863,864..866,867..899,900..902,903..926,
                     927..953,954..1010,1011..1016,1017..1286,1287..1304,
                     1305..1739,1740..1742,1743..1745,1746..2186)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRI
                     VLKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLR
                     SLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVWDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGEGQVNYEEFVQMMTAK"
     CDS             join(834..845,846..863,864..866,867..899,900..902,903..926,
                     927..953,954..1010,1011..1016,1017..1286,1287..1304,
                     1305..1739,1740..1742,1743..1745,1746..2186)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRI
                     VLKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLR
                     SLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVWDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGEGQVNYEEFVQMMTAK"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1223..1244
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1350..1371)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     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(join(5386..6177,6178..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 agaaattcgc gaagcgttcc gtgtgtggga taaggatggc aatggctaca
     2041 tcagtgcagc agagcttcgc cacgtgatga caaaccttgg agagaagtta acagatgaag
     2101 aggttgatga aatgatcagg gaagcagata tcgatggaga aggtcaggta 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
//
