LOCUS       sequence-214013-        7357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cyan color luminescent indicator for calcium signaling.Mitochondrial
            targeting signals of subunit VIII of human cytochrome c oxidase 
            (CoxVIII) were fused at the N-terminus of CeNL(Ca2+)_1.2u .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7357)
  AUTHORS   Hossain MN, Suzuki K, Iwano M, Matsuda T, Nagai T
  TITLE     Bioluminescent Low-Affinity Ca(2+) Indicator for ER with Multicolor 
            Calcium Imaging in Single Living Cells.
  JOURNAL   ACS Chem Biol. 2018 Mar 9. doi: 10.1021/acschembio.7b01014.
  PUBMED    29494125
REFERENCE   2  (bases 1 to 7357)
  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: "ACS Chem 
            Biol. 2018 Mar 9. doi: 10.1021/acschembio.7b01014."
COMMENT     Sequence Label: CoxVIIIx2-CeNL(Ca2+)_1.2u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     755..774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        755..773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             807..881
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             903..974
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(996..998,999..1001,1002..1712)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1041..1062)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1302..1321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1649..1670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2373..2450
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     promoter        complement(2802..2820)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2803..2820)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2840..2857)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2846..3070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3116..3544
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3203..3222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3413..3434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3553..3573)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3558..3887
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3738..3873
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3800..3819
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3954..4748
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4008..4027)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4618..4637
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4922..5043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4959..4978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5013..5032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5092..5108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5092..5108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5105..5127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5116..5132
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5140..5146,5147..5164,5165..5170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5323..5340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6253..7044,7045..7113))
                     /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     6876..6895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7114..7218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7293..7312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
       61 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      121 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      721 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      781 aagcttgaac cgtcagatcc gctagcatgt ccgtcctgac gccgctgctg ctgcggggct
      841 tgacaggctc ggcccggcgg ctcccagtgc cgcgcgccaa gatccattcg ttgggggatc
      901 tgtccgtcct gacgccgctg ctgctgcggg gcttgacagg ctcggcccgg cggctcccag
      961 tgccgcgcgc caagatccat tcgttggggg atccgatggt gagcaagggc gaggagctgt
     1021 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1081 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1141 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg tcctggggcg
     1201 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1261 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1321 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1381 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tactttagcg
     1441 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     1501 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1561 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga
     1621 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1681 ggatcactct cggcatggac gagctgtaca agggtaccgt cttcacactc gaagatttcg
     1741 ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg
     1801 tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga
     1861 gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtccatgga
     1921 tgcatgacca actgacagaa gagcagattg cagagttcaa agaagccttc tcattattcg
     1981 acaaggatgg ggacggcacc atcaccacaa aggaacttgg caccgttatg aggtcgcttg
     2041 gacaaaaccc aacggaagca gaattgcagg atatgatcaa tgaagtcgat gctgatggca
     2101 atggaacgat ttactttcct gattttctta ctatgatggc tagaaaaatg aaggacacag
     2161 acagcgaaga ggaaatccga gaagcattcc gtgtttttga caaggatggg aacggctaca
     2221 tcagcgctgc tgatttacgt cacgtcatga caaacctcgg ggagaagtta acagatgaag
     2281 aagttgatga aatgataagg gaagcagata tcgatggtga aggccaagta aactatgaag
     2341 agtttgtaca aatgatgaca gcaaaggggg ggaagaggcg ctggaagaaa aacttcattg
     2401 ccgtcagcgc tgccaaccgg ttcaagaaga tctccagctc cggggcactg gagctcctga
     2461 gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc
     2521 atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca
     2581 tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca
     2641 ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg
     2701 acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac
     2761 gcattctggc gtaactcgag catgcatcta gagggcccta ttctatagtg tcacctaaat
     2821 gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2881 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2941 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3001 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3061 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3121 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3181 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3241 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3301 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3361 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3421 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3481 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3541 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3601 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3661 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3721 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3781 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3841 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3901 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3961 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4021 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4081 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4141 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4201 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4261 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4321 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4381 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4441 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4501 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4561 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4621 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4681 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4741 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4801 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4861 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4921 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4981 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5041 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6361 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6781 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6841 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7021 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7081 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7141 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7201 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7261 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7321 agccagtatc tgctccctgc ttgtgtgttg gaggtcg
//
