LOCUS       sequence-193343-        7590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of a Nano-lantern-based luminescent Ca2+ 
            indicator with Kd=300nM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7590)
  AUTHORS   Saito K, Chang YF, Horikawa K, Hatsugai N, Higuchi Y, Hashida M, 
            Yoshida Y, Matsuda T, Arai Y, Nagai T
  TITLE     Luminescent proteins for high-speed single-cell and whole-body 
            imaging.
  JOURNAL   Nat Commun. 2012;3:1262. doi: 10.1038/ncomms2248.
  PUBMED    23232392
REFERENCE   2  (bases 1 to 7590)
  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: "Nat 
            Commun."; date: "2012"; volume: "3:1262. doi"; pages: " 
            10.1038/ncomms2248"
COMMENT     Sequence Label: Nano-lantern(Ca2+)_300/pcDNA3
FEATURES             Location/Qualifiers
     source          1..7590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(78..97)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        269..648
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        649..852
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     803..823
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     897..916
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        897..915
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(948..950,951..953,954..1466)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(993..1014)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1254..1273)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1601..1622
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2781..2858
                     /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(3177..3195)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3178..3195)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3215..3232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3221..3445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3491..3919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3578..3597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3788..3809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3928..3948)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3933..4262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4113..4248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4175..4194
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4329..5123
                     /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(4383..4402)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4993..5012
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5297..5418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5334..5353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5388..5407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5467..5483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5467..5483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5480..5502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5491..5507
                     /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(5515..5521,5522..5539,5540..5545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5560..5581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5698..5715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5869..6457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5949..5968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6628..7419,7420..7488))
                     /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     7251..7270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7489..7590,1..3))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
       61 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      121 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      181 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      241 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      301 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      361 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      421 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      481 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      541 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      601 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      661 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      721 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      781 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      841 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      901 acgactcact atagggagac ccaagcttgg taccgagctc ggatccgatg gtgagcaagg
      961 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1021 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1081 tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1141 tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     1201 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1261 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1321 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1381 actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     1441 acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc
     1501 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc
     1561 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1621 tgaccgccgc cgggggtacc aaggtgtacg accccgagca gaggaagagg atgatcaccg
     1681 gcccccagtg gtgggccagg tgcaagcaga tgaacgtgct ggacagcttc atcaactact
     1741 acgacagcga gaagcacgcc gagaacgccg tgatcttcct gcacggcaac gccactagca
     1801 gctacctgtg gaggcacgtg gtgccccaca tcgagcccgt ggccaggtgc atcatccccg
     1861 atctgatcgg catgggcaag agcggcaaga gcggcaacgg cagctacagg ctgctggacc
     1921 actacaagta cctgaccgcc tggttcgagc ttctgaacct gcccaagaag atcatcttcg
     1981 tgggccacga ctggggcgcc gccctggcct tccactacgc ctacgagcac caggacagga
     2041 tcaaggccat cgtgcacatg gagagcgtgg tggacgtgat cgagagctgg gacgagtggc
     2101 cagacatcga ggaggacatc gccctgatca agagcgagga gggcgagaag atggtgctgg
     2161 agaacaactt cttcgtggag accgtgctgc ccagcaagat catgagaaag ctggagcccg
     2221 aggagttcgc cgcctacctg gagcccttca aggagaaggg cgaggtgaga agacccaccc
     2281 tgagctggcc cagagagatc cccctggtga agggcccatg gatgcatgac caactgacag
     2341 aagagcagat tgcagagttc aaagaagcct tctcattatt cgacaaggat ggggacggca
     2401 ccatcaccac aaaggaactt ggcaccgtta tgaggtcgct tggacaaaac ccaacggaag
     2461 cagaattgca ggatatgatc aatgaagtcg atgctgatgg caatggaacg atttactttc
     2521 ctgaatttct tactatgatg gctagaaaaa tgaaggacac agacagcgaa gaggaaatcc
     2581 gagaagcatt ccgtgttttt gacaaggatg ggaacggcta catcagcgct gctcaattac
     2641 gtcacgtcat gacaaacctc ggggagaagt taacagatga agaagttgat gaaatgataa
     2701 gggaagcaga tatcgatggt gatggccaag taaactatga agagtttgta caaatgatga
     2761 cagcaaaggg gggcgggtcc aagaggcgct ggaagaaaaa cttcattgcc gtcagcgctg
     2821 ccaaccggtt caagaagatc tccagctccg gggcactgga gctcggcaag cccgacgtgg
     2881 tgcagatcgt gagaaactac aacgcctacc tgagagccag cgacgacctg cccaagctgt
     2941 tcatcgaggg tgaccccggc ttcttcagca acgccatcgt ggagggcgcc aagaagttcc
     3001 ccaacaccga gttcgtgaag gtgaagggcc tgcacttcct ccaggaggac gcccccgacg
     3061 agatgggcaa gtacatcaag agcttcgtgg agagagtgct gaagaacgag cagtaagaat
     3121 tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg ccctattcta
     3181 tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     3241 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3301 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3361 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3421 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3481 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3541 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3601 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3661 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3721 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3781 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3841 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3901 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4021 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4081 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4201 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4261 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4321 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4381 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4441 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4501 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4561 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4621 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4681 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4741 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4801 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4861 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4921 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4981 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5041 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5101 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5161 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5221 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5281 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5341 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5401 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5461 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5521 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5581 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5641 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5701 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5761 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5821 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5881 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5941 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6001 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6061 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6121 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6181 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6241 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6301 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6361 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6421 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6481 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6541 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6601 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6661 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6721 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6781 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6841 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6901 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6961 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7021 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7081 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7141 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7201 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7261 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7321 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7381 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7441 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7501 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7561 tatttagaaa aataaacaaa taggggttcc
//
