LOCUS       sequence-296222-        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-CEPIA4mt
FEATURES             Location/Qualifiers
     source          1..6407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        379..582
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     533..553
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             625..699
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             721..795
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             835..852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             856..888
                     /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(892..900,901..915)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      922..931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             943..999
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     1695..1714
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2188..2217
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2253..2270)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2259..2483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2529..2957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2616..2635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2826..2847
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2966..2986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2971..3300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3151..3286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3213..3232
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3326..4120
                     /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(3380..3399)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3990..4009
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4294..4415
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4331..4350)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4385..4404
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4448..4465)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4619..5207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4699..4718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5378..6169,6170..6238))
                     /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     6001..6020
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6239..6343)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acattgatta ttgagtagtt attaatagta atcaattacg gggtcattag ttcatagccc
       61 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      181 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacggtta
      361 gtcatcgcta ttaccatagt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      421 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      481 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      541 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctttctggct aactagagaa
      601 cccactgctt actggcacgt gaccatgtcc gtcctgacgc cgctgctgct gcggggcttg
      661 acaggctcgg cccggcggct cccagtgccg cgcgccaaaa ttcattcact gggggacccc
      721 atgagcgtgc tcaccccact cctgctgcgg gggctgaccg gcagcgctag gcggctgcca
      781 gtcccgcggg ccaagatcca cagtctcggc gatcccggat ccatgcgggg ttctcatcat
      841 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac
      901 gacgatgacg ataaggatct cgccaccatg gtcgactctt cacgtcgtaa gtggaataag
      961 gcaggtcacg cagtcagagc tataggtcgg ctgagctcac ccgtggtttc cgagcggatg
     1021 taccccgagg acggcgccct gaagagcgag atcaagaagg ggctgaggct gaaggacggc
     1081 ggccactacg ccgccgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     1141 ggcgcctaca tcgtcgacat caagttggac atcgtgtccc acaacgagga ctacaccatc
     1201 gtggaacagt gcgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     1261 aagggaggta caggcgggag tctggtgagc aagggcgagg aggataacat ggccatcatc
     1321 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1381 atcgagggcg agggcgaggg ccgcccctac gaggcctttc agaccgctaa gctgaaggtg
     1441 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     1501 tccaaggcct acattaagca cccagccgac atccccgact acttcaagct gtccttcccc
     1561 gagggcttca ggtgggagcg cgtgatgaac ttcgaggacg gcggcattat tcacgttaac
     1621 caggactcct ccctgcagga cggcgtattc atctacaagg tgaagctgcg cggcaccaac
     1681 ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc tacgcgtgac
     1741 caactgactg aagagcagat cgcagaattt aaagaggctt tctccctatt tgacaaggac
     1801 ggggatggga cgataacaac caaggatctg gggacggtga tgcggtctct ggggcagaac
     1861 cccacagaag cagagctcca ggacatgatc aatgaagtag atgccgacgg tgacggcaca
     1921 ttcgacttcc ctgagttcct gacgatgatg gcaagaaaaa tgaatgacac agacagtgaa
     1981 gaggaaattc gcgaagcgtt ccgtgtgtgg gataaggatg gcaatggcta catcggcgca
     2041 gcagagcttc gccacgtgat gacagacctt ggagagaagt taacagatga ggaggttgat
     2101 gaaatgatca gggtagcaga catcgatgga gaaggtcagg taaactacga agagtttgta
     2161 caaatgatga cagcgaaggc ggccgcagaa caaaaactca tctcagaaga ggatctgaat
     2221 ggggccgcat agtctagaag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2281 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2341 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2401 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2461 tggggatggc ccgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2521 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2581 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2641 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2701 ccgatttagt gctttacggc acctctcccc caaaaaactt gattagggtg atggttcacg
     2761 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2821 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2881 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2941 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3001 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3061 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3121 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3181 cattctccgc ccctaggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3241 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3301 aagctccccc cccgggaggt ccacaatggt tgaacaagat ggattgcacg caggttctcc
     3361 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3421 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3481 cctgtccggt gccctgaatg aactccagga cgaggcagcg cggctatcgt ggctggccac
     3541 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3601 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3661 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3721 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3781 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3841 caggctcaag gcgcgtatgc ccgacggcga ggatctcgtc gtgactcatg gcgatgcctg
     3901 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3961 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4021 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4081 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4141 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4201 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4261 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4321 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4381 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgatcttt
     4441 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4501 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4561 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4621 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4681 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4741 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4801 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4861 agctgggctg tatgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4921 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4981 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5041 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5101 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5161 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5221 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5281 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5341 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5401 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5461 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5521 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5581 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5641 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5701 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5761 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5821 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5881 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5941 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6001 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6061 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcggg
     6121 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6181 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6241 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6301 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6361 tgccacctga cgtcagatcg acggatcggg agatcggtac cgaattc
//
