LOCUS       sequence-174274-        6404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green fluorescent indicator for calcium imaging in the mitochondria.
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 CEPIA2mt
FEATURES             Location/Qualifiers
     source          1..6404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        377..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     531..551
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             623..697
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             719..793
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(821..832,833..850,851..853,854..886,887..889,890..913,
                     914..940,941..997,998..1003,1004..1273,1274..1291,
                     1292..1726,1727..1729,1730..1732,1733..2173)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRI
                     VLKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVLR
                     SLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(821..832,833..850,851..853,854..886,887..889,890..913,
                     914..940,941..997,998..1003,1004..1273,1274..1291,
                     1292..1726,1727..1729,1730..1732,1733..2173)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWN
                     KTGHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG
                     PVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGE
                     ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
                     TYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRI
                     VLKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVLR
                     SLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDG
                     NGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      920..929
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1210..1231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1337..1358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2183..2212
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2248..2265)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2254..2478
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2524..2952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2821..2842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2961..2981)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2966..3295
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3146..3281
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3208..3227
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3321..4115
                     /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(3375..3394)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3985..4004
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4289..4410
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4326..4345)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4380..4399
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4443..4460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4614..5202)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4694..4713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5373..6164,6165..6233))
                     /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     5996..6015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6234..6338)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6403..6404,1..375)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attgattatt gagtagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacggttagt
      361 catcgctatt accatagtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      541 cggtaggcgt gtacggtggg aggtctatat aagcagagct ttctggctaa ctagagaacc
      601 cactgcttac tggcacgtga ccatgtccgt cctgacgccg ctgctgctgc ggggcttgac
      661 aggctcggcc cggcggctcc cagtgccgcg cgccaaaatt cattcactgg gggaccccat
      721 gagcgtgctc accccactcc tgctgcgggg gctgaccggc agcgctaggc ggctgccagt
      781 cccgcgggcc aagatccaca gtctcggcga tcccggatcc atgcggggtt ctcatcatca
      841 tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga
      901 cgatgacgat aaggatctcg ccaccatggt cgactcatca cgtcgtaagt ggaataagac
      961 aggtcacgca gtcagagcta taggtcggct gagctcactc gagaacgtct atatcatggc
     1021 cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg
     1081 cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1141 gctgcccgac aaccactacc tgagcaccca gtccaaactt tcgaaagacc ccaacgagaa
     1201 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1261 cgagctgtac aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac
     1321 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1381 gtccggcgag ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1441 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1501 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1561 cgaaggctac atccaggagc gcaccatctt cttcaaggac gacggctact acaagacccg
     1621 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgtgctga agggcatcga
     1681 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacacgc gtgaccaact
     1741 gactgaagag cagatcgcag aatttaaaga ggctttctcc ctatttgaca aggacgggga
     1801 tgggacaata acaaccaagg agctggggac ggtgctgcgg tctctggggc agaaccccac
     1861 agaagcagag ctgcaggaca tgatcaatga agtagatgcc gacggtaatg gcacaatcga
     1921 cttccctgag ttcctgacaa tgatggcaag aaaaatgaaa gacacagaca gtgaagaaga
     1981 aattagagaa gcgttccgtg tgtttgataa ggatggcaat ggctacatca gtgcagcaga
     2041 gcttcgccac gtgatgacaa accttggaga gaagttaaca gatgaagagg ttgatgaaat
     2101 gatcagggaa gcagacatcg atggggatgg tcaggtaaac tacgaagagt ttgtacaaat
     2161 gatgacagcg aaggcggccg cagaacaaaa actcatctca gaagaggatc tgaatggggc
     2221 cgcatagtct agaagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2281 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2341 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2401 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2461 atggcccggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2521 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2581 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2701 ttagtgcttt acggcacctc tcccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2761 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2821 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2881 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2941 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3001 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3061 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3121 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3181 tccgccccta ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3241 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3301 ccccccccgg gaggtccaca atggttgaac aagatggatt gcacgcaggt tctccggccg
     3361 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3421 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3481 ccggtgccct gaatgaactc caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3541 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3601 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3661 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3721 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3781 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3841 tcaaggcgcg tatgcccgac ggcgaggatc tcgtcgtgac tcatggcgat gcctgcttgc
     3901 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3961 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4021 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4081 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4141 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4201 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4261 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4321 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4381 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga tctttccgct
     4441 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4501 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4561 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4621 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4681 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4741 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4801 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4861 ggctgtatgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4921 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4981 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5041 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5101 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5161 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5221 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5281 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5341 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5401 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5461 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5521 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5581 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5641 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5701 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5761 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5821 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5881 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5941 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6001 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6061 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgggcaccc
     6121 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6181 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6241 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6301 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6361 cctgacgtca gatcgacgga tcgggagatc ggtaccgaat tcac
//
