LOCUS       sequence-296258-        6407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Red 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 6407)
  AUTHORS   Kanemaru K, Suzuki J, Taiko I, Iino M
  TITLE     Red fluorescent CEPIA indicators for visualization of Ca(2+) 
            dynamics in mitochondria.
  JOURNAL   Sci Rep. 2020 Feb 18;10(1):2835. doi: 10.1038/s41598-020-59707-8.
  PUBMED    32071363
REFERENCE   2  (bases 1 to 6407)
  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: "Sci Rep."; 
            date: "2020-02-18"; volume: "10(1):2835. doi"; pages: " 
            10.1038/s41598-020-59707-8"
COMMENT     Sequence Label: pCMV R-CEPIA3mt
FEATURES             Location/Qualifiers
     source          1..6407
                     /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             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             join(903..911,912..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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             954..1010
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     1706..1725
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2199..2228
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2264..2281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2270..2494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2540..2968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2627..2646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2837..2858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2977..2997)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2982..3311
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3162..3297
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3224..3243
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3337..4131
                     /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(3391..3410)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4001..4020
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4305..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4342..4361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4396..4415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4459..4476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4630..5218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4710..4729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5389..6180,6181..6249))
                     /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     6012..6031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6250..6354)
                     /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 ggtcgactct tcacgtcgta
      961 agtggaataa ggcaggtcac gcagtcagag ctataggtcg gctgagctca cccgtggttt
     1021 ccgagcggat gtaccccgag gacggcgccc tgaagagcga gatcaagaag gggctgaggc
     1081 tgaaggacgg cggccactac gccgccgagg tcaagaccac ctacaaggcc aagaagcccg
     1141 tgcagctgcc cggcgcctac atcgtcgaca tcaagttgga catcgtgtcc cacaacgagg
     1201 actacaccat cgtggaacag tgcgaacgcg ccgagggccg ccactccacc ggcggcatgg
     1261 acgagctgta caagggaggt acaggcggga gtctggtgag caagggcgag gaggataaca
     1321 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     1381 acgagttcga gatcgagggc gagggcgagg gccgccccta cgaggccttt cagaccgcta
     1441 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1501 tcatgtacgg ctccaaggcc tacattaagc acccagccga catccccgac tacttcaagc
     1561 tgtccttccc cgagggcttc aggtgggagc gcgtgatgaa cttcgaggac ggcggcatta
     1621 ttcacgttaa ccaggactcc tccctgcagg acggcgtatt catctacaag gtgaagctgc
     1681 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1741 ctacgcgtga ccaactgact gaagagcaga tcgcagaatt taaagaggct ttctccctat
     1801 ttgacaagga cggggatggg acgataacaa ccaaggatct ggggacggtg atgcggtctc
     1861 tggggcagaa ccccacagaa gcagagctcc aggacatgat caatgaagta gatgccgacg
     1921 gtgacggcac attcgacttc cctgagttcc tgacgatgat ggcaagaaaa atgaatgaca
     1981 cagacagtga agaggaaatt agagaagcgt tccgcgtgtt tgataaggac ggcaatggct
     2041 acatcggcgc agcagagctt cgccacgtga tgacagacct tggagagaag ttaacagatg
     2101 aggaggttga tgaaatgatc agggtagcag acatcgatgg ggatggtcag gtaaactacg
     2161 aagagtttgt acaaatgatg acagcgaagg cggccgcaga acaaaaactc atctcagaag
     2221 aggatctgaa tggggccgca tagtctagaa gctcgctgat cagcctcgac tgtgccttct
     2281 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2341 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2401 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2461 agcaggcatg ctggggatgg cccgggctct atggcttctg aggcggaaag aaccagctgg
     2521 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2581 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2641 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2701 cctttagggt tccgatttag tgctttacgg cacctctccc ccaaaaaact tgattagggt
     2761 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2821 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2881 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2941 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3001 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3061 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3121 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3181 ccagttccgc ccattctccg cccctaggct gactaatttt ttttatttat gcagaggccg
     3241 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3301 gcttttgcaa aaagctcccc ccccgggagg tccacaatgg ttgaacaaga tggattgcac
     3361 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3421 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3481 gtcaagaccg acctgtccgg tgccctgaat gaactccagg acgaggcagc gcggctatcg
     3541 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3601 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3661 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3721 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3781 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3841 gaactgttcg ccaggctcaa ggcgcgtatg cccgacggcg aggatctcgt cgtgactcat
     3901 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3961 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4021 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4081 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4141 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4201 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4261 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4441 cgtcgatctt tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4501 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4561 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4861 cgttcgctcc aagctgggct gtatgcacga accccccgtt cagcccgacc gctgcgcctt
     4921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5041 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6121 acccactcgg gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6241 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6301 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6361 tccccgaaaa gtgccacctg acgtcagatc gacggatcgg gagatcg
//
