LOCUS       sequence-222573-        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+)_19u.
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+)_19u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..33)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        205..584
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        585..788
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     739..759
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     833..852
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        833..851
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             885..959
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             981..1052
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(1074..1076,1077..1079,1080..1790)
                     /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(1119..1140)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1380..1399)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1727..1748
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2451..2528
                     /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(2880..2898)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2881..2898)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2918..2935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2924..3148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3194..3622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3281..3300)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3491..3512
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3631..3651)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3636..3965
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3816..3951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3878..3897
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4032..4826
                     /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(4086..4105)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4696..4715
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5000..5121
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5037..5056)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5091..5110
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5170..5186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5170..5186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5183..5205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5194..5210
                     /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(5218..5224,5225..5242,5243..5248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5263..5284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5401..5418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5572..6160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5652..5671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6331..7122,7123..7191))
                     /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     6954..6973
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7192..7296)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
       61 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      121 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      181 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      241 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      301 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      361 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      421 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      481 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      541 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      601 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      661 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      721 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      781 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      841 ctcactatag ggagacccaa gcttgaaccg tcagatccgc tagcatgtcc gtcctgacgc
      901 cgctgctgct gcggggcttg acaggctcgg cccggcggct cccagtgccg cgcgccaaga
      961 tccattcgtt gggggatctg tccgtcctga cgccgctgct gctgcggggc ttgacaggct
     1021 cggcccggcg gctcccagtg ccgcgcgcca agatccattc gttgggggat ccgatggtga
     1081 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1141 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1201 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1261 ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     1321 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1381 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1441 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1501 agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag aacggcatca
     1561 aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact
     1621 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1681 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1741 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggtaccgtct
     1801 tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac ctggaccaag
     1861 tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc gtaactccga
     1921 tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc
     1981 cgtatgaagg tccatggatg catgaccaac tgacagaaga gcagattgca gagttcaaag
     2041 aagccttctc attattcgac aaggatgggg acggcaccat caccacaaag gaacttggca
     2101 ccgttatgag gtcgcttgga caaaacccaa cggaagcaga attgcaggat atgatcaatg
     2161 aagtcgatgc tgatggcaat ggaacgattt actttcctga ttttcttact atgatggcta
     2221 gaaaaatgaa ggacacagac agcgaagagg aaatccgaga agcattccgt gtttgggaca
     2281 aggatgggaa cggctacatc agcgctgctg atttacgtca cgtcatgaca aacctcgggg
     2341 agaagttaac agatgaagaa gttgatgaaa tgataaggga agcagatatc gatggtgaag
     2401 gccaagtaaa ctatgaagag tttgtacaaa tgatgacagc aaaggggggg aagaggcgct
     2461 ggaagaaaaa cttcattgcc gtcagcgctg ccaaccggtt caagaagatc tccagctccg
     2521 gggcactgga gctcctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     2581 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     2641 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     2701 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     2761 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     2821 gctggcggct gtgcgaacgc attctggcgt aactcgagca tgcatctaga gggccctatt
     2881 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     2941 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3001 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3061 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3121 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3181 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3241 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3301 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3361 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3421 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3481 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3541 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3601 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3661 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3721 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3901 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3961 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4021 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4081 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4141 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4201 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4261 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4321 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4381 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4441 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4501 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4561 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4621 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4681 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4741 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4801 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4861 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4921 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4981 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5041 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5101 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5161 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5221 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5281 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5341 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5401 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5461 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5521 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5581 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5641 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5701 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5761 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5821 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5881 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5941 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6001 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6061 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6121 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6181 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6241 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6301 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6361 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6421 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6481 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6541 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6601 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6661 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6721 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6781 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6841 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6901 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6961 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7021 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7081 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7141 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7201 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7261 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7321 tgacgtcgac ggatcgggag atctcccgat cccctat
//
