LOCUS       sequence-214295-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cyan color bright luminescent calcium indicator with low affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  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 7200)
  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: CeNL(Ca2+)_110u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        92..471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        472..675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     626..646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     720..739
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        720..738
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(771..773,774..776,777..1487)
                     /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(816..837)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1077..1096)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1424..1445
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2148..2225
                     /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(2610..2628)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2611..2628)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2648..2665)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2654..2878
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2924..3352
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3011..3030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3221..3242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3361..3381)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3366..3695
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3546..3681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3608..3627
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3762..4556
                     /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(3816..3835)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4426..4445
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4730..4851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4767..4786)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4821..4840
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4900..4916)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4900..4916)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4913..4935)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4924..4940
                     /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(4948..4954,4955..4972,4973..4978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4993..5014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5131..5148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5302..5890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5382..5401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6061..6852,6853..6921))
                     /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     6684..6703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6922..7026)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7101..7120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
       61 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      661 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
      721 aatacgactc actataggga gacccaagct tggtaccgag ctcggatccg atggtgagca
      781 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      841 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      901 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      961 ccctgtcctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     1021 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1081 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1141 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1201 acaactactt tagcgacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     1261 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc
     1321 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1381 cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1441 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggt accgtcttca
     1501 cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg gaccaagtcc
     1561 ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta actccgatcc
     1621 aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc atcatcccgt
     1681 atgaaggtcc atggatgcat gaccaactga cagaagagca gattgcagag ttcaaagaag
     1741 ccttctcatt attcgacaag gatggggacg gcaccatcac cacaaaggat cttggcaccg
     1801 ttatgaggtc gcttggacaa aacccaacgg aagcagaatt gcaggatatg atcaatgaag
     1861 tcgatgctga tggcaatgga acgatttact ttcctgaatt tcttactatg atggctagaa
     1921 aaatgaagga cacagacagc gaagaggaaa tccgagaagc attccgtgtt tgggacaagg
     1981 atgggaacgg ctacatcagc gctgctgatt tacgtcacgt catgacaaac ctcggggaga
     2041 agttaacaga tgaagaagtt gatgaaatga taagggaagc agatatcgat ggtgaaggcc
     2101 aagtaaacta tgaagagttt gtacaaatga tgacagcaaa gggggggaag aggcgctgga
     2161 agaaaaactt cattgccgtc agcgctgcca accggttcaa gaagatctcc agctccgggg
     2221 cactggagct cctgagcggc gaccaaatgg gccagatcga aaaaattttt aaggtggtgt
     2281 accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg gtaatcgacg
     2341 gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc gccgtgttcg
     2401 acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt atcgacgagc
     2461 gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga gtgaccggct
     2521 ggcggctgtg cgaacgcatt ctggcgtaag aattctgcag atatccatca cactggcggc
     2581 cgctcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc
     2641 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2701 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2761 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2821 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2881 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2941 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3001 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3061 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3121 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3181 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3241 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3301 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3361 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3421 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3481 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3541 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3601 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3661 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3721 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3781 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3841 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3901 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3961 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4021 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4081 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4141 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4201 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4261 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4321 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4381 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4441 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4501 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4561 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4621 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4681 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4741 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4801 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4861 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4921 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4981 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5041 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5101 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5161 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5221 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5281 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5341 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5401 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5461 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5521 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5581 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5641 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5701 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5761 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5821 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5881 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5941 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6001 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6061 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6121 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6181 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6241 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6301 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6361 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6421 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6481 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6541 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6601 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6661 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6721 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6781 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6841 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6901 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6961 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7021 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     7081 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     7141 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
//
