LOCUS       sequence-214007-        7282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Orange 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 
            OeNL(Ca2+)_18u.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7282)
  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 7282)
  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-OeNL(Ca2+)_18u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        95..474
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..678
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     629..649
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     723..742
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        723..741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             775..849
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             871..942
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             964..1617
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2266..2343
                     /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(2695..2713)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2696..2713)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2733..2750)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2739..2963
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3009..3437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3446..3466)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3451..3780
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3631..3766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3693..3712
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3847..4641
                     /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(3901..3920)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4511..4530
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4815..4936
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4852..4871)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4906..4925
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4985..5001)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4985..5001)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4998..5020)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5009..5025
                     /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(5033..5039,5040..5057,5058..5063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5078..5099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5216..5233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5387..5975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5467..5486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6146..6937,6938..7006))
                     /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     6769..6788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7007..7111)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7186..7205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      121 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      241 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      301 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      361 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      421 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      481 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      541 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      601 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      661 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
      721 attaatacga ctcactatag ggagacccaa gcttgaaccg tcagatccgc tagcatgtcc
      781 gtcctgacgc cgctgctgct gcggggcttg acaggctcgg cccggcggct cccagtgccg
      841 cgcgccaaga tccattcgtt gggggatctg tccgtcctga cgccgctgct gctgcggggc
      901 ttgacaggct cggcccggcg gctcccagtg ccgcgcgcca agatccattc gttgggggat
      961 ccgatggtga gcgtgatcaa gcccgagatg aagatgaggt actacatgga cggctccgtc
     1021 aatgggcatg agttcacaat cgagggtgag ggcacaggca gaccttacga gggacatcag
     1081 gagatgacac tgcgcgtcac aatggccgag ggcgggccaa tgcctttcgc cttcgacctg
     1141 gtgtcccacg tgttctgtta cggccacaga gtttttacca agtacccaga agagatccca
     1201 gactatttca agcaggcctt tcctgagggc ctgtcctggg agaggtccct ggagttcgag
     1261 gacggcggct ccgcctccgt gagcgcccac atcagcctga ggggcaacac cttctaccac
     1321 aagtccaagt tcaccggcgt gaacttcccc gccgacggcc ccatcatgca gaaccagagc
     1381 gtggactggg agccctccac cgagaagatc accgccagcg acggcgtgct gaagggcgac
     1441 gtgaccatgt acctgaagct ggagggcggc ggcaaccaca agtgccagtt caagaccacc
     1501 tacaaggccg ccaaggagat cctggagatg cccggcgacc actacatcgg ccacaggctg
     1561 gtgaggaaga ccgagggcaa catcaccgag caggtggagg acgccgtggc ccactccggt
     1621 accgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt
     1681 gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa
     1741 aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat
     1801 gaaggtccat ggatgcatga ccaactgaca gaagagcaga ttgcagagtt caaagaagcc
     1861 ttctcattat tcgacaagga tggggacggc accatcacca caaaggaact tggcaccgtt
     1921 atgaggtcgc ttggacaaaa cccaacggaa gcagaattgc aggatatgat caatgaagtc
     1981 gatgctgatg gcaatggaac gatttacttt cctgattttc ttactatgat ggctagaaaa
     2041 atgaaggaca cagacagcga agaggaaatc cgagaagcat tccgtgtttt tgacaaggat
     2101 gggaacggct acatcagcgc tgctgattta cgtcacgtca tgacaaacct cggggagaag
     2161 ttaacagatg aagaagttga tgaaatgata agggaagcag atatcgatgg tgaaggccaa
     2221 gtaaactatg aagagtttgt acaaatgatg acagcaaagg gggggaagag gcgctggaag
     2281 aaaaacttca ttgccgtcag cgctgccaac cggttcaaga agatctccag ctccggggca
     2341 ctggagctcc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac
     2401 cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt aatcgacggg
     2461 gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac
     2521 ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat cgacgagcgc
     2581 ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt gaccggctgg
     2641 cggctgtgcg aacgcattct ggcgtaactc gagcatgcat ctagagggcc ctattctata
     2701 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2761 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2821 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2881 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2941 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3001 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3361 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3541 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3601 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3781 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3841 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3901 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3961 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4021 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4081 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4141 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4201 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4261 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4321 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4381 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4441 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4501 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4561 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4621 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4681 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4741 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4801 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4861 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4921 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4981 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5041 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5101 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5161 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5221 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5281 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5341 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5401 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5461 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5521 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5581 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5641 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5701 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5761 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5821 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5881 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5941 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6001 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6061 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6121 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6181 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6241 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6301 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6361 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6421 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6481 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6541 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6601 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6661 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6721 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6781 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6841 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6901 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6961 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7021 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7081 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7141 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     7201 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     7261 cgcgagcaaa atttaagcta ca
//
