LOCUS       sequence-214112-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cyan color bright luminescent calcium indicator with intermediate 
            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+)_1.2u-pcDNA3
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        718..736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(769..771,772..774,775..1485)
                     /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(814..835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1075..1094)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1422..1443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2146..2223
                     /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(2608..2626)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2609..2626)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2646..2663)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2652..2876
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2922..3350
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3009..3028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3219..3240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3359..3379)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3364..3693
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3544..3679
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3606..3625
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3760..4554
                     /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(3814..3833)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4424..4443
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4728..4849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4765..4784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4819..4838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4898..4914)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4898..4914)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4911..4933)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4922..4938
                     /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(4946..4952,4953..4970,4971..4976))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4991..5012
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5129..5146)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5300..5888)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5380..5399)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6059..6850,6851..6919))
                     /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     6682..6701
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6920..7024)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7099..7118)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
       61 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      721 tacgactcac tatagggaga cccaagcttg gtaccgagct cggatccgat ggtgagcaag
      781 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      841 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      901 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      961 ctgtcctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1021 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1081 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1141 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1201 aactacttta gcgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1261 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag
     1321 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1381 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1441 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtac cgtcttcaca
     1501 ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt
     1561 gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa
     1621 aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat
     1681 gaaggtccat ggatgcatga ccaactgaca gaagagcaga ttgcagagtt caaagaagcc
     1741 ttctcattat tcgacaagga tggggacggc accatcacca caaaggaact tggcaccgtt
     1801 atgaggtcgc ttggacaaaa cccaacggaa gcagaattgc aggatatgat caatgaagtc
     1861 gatgctgatg gcaatggaac gatttacttt cctgattttc ttactatgat ggctagaaaa
     1921 atgaaggaca cagacagcga agaggaaatc cgagaagcat tccgtgtttt tgacaaggat
     1981 gggaacggct acatcagcgc tgctgattta cgtcacgtca tgacaaacct cggggagaag
     2041 ttaacagatg aagaagttga tgaaatgata agggaagcag atatcgatgg tgaaggccaa
     2101 gtaaactatg aagagtttgt acaaatgatg acagcaaagg gggggaagag gcgctggaag
     2161 aaaaacttca ttgccgtcag cgctgccaac cggttcaaga agatctccag ctccggggca
     2221 ctggagctcc tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac
     2281 cctgtggatg atcatcactt taaggtgatc ctgcactatg gcacactggt aatcgacggg
     2341 gttacgccga acatgatcga ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac
     2401 ggcaaaaaga tcactgtaac agggaccctg tggaacggca acaaaattat cgacgagcgc
     2461 ctgatcaacc ccgacggctc cctgctgttc cgagtaacca tcaacggagt gaccggctgg
     2521 cggctgtgcg aacgcattct ggcgtaagaa ttctgcagat atccatcaca ctggcggccg
     2581 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     2641 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2701 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2761 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2821 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2881 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2941 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3001 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3061 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3121 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3181 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3241 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3301 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3361 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3421 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3481 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3541 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3601 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3661 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3721 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3781 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3841 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3901 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3961 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4021 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4081 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4141 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4201 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4261 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4321 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4381 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4441 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4501 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4561 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4621 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4681 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4741 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4801 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4861 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4921 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4981 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5041 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5101 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5161 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5221 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5281 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5341 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5401 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5461 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5521 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5641 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5701 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5761 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5821 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5881 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5941 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6001 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6061 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6121 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6181 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6241 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6301 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6361 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6421 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6481 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6541 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6601 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6661 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6721 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6781 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6841 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6901 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6961 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7021 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     7081 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7141 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
//
