LOCUS       sequence-234664-        7386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Yellow Cameleon-Nano50, an ultrasensitive 
            Ca2+ indicator with Kd = 50 nM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7386)
  AUTHORS   Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-ura
            T, Miyawaki A, Michikawa T, Mikoshiba K, Nagai T
  TITLE     Spontaneous network activity visualized by ultrasensitive Ca(2+) 
            indicators, yellow Cameleon-Nano.
  JOURNAL   Nat Methods. 2010 Sep;7(9):729-32. doi: 10.1038/nmeth.1488. Epub 
            2010 Aug 8.
  PUBMED    20693999
REFERENCE   2  (bases 1 to 7386)
  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"; doi: 
            "10.1038/nmeth.1488"; journalName: "Nat Methods"; date: "2010-09"; 
            volume: "7"; issue: "9"; pages: "729-32"
COMMENT     Sequence Label: Yellow Cameleon-Nano50/pcDNA3
FEATURES             Location/Qualifiers
     source          1..7386
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(916..918,919..921,922..1434)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             2059..2136
                     /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"
     CDS             2143..2877
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(2941..2959)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2942..2959)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2979..2996)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2985..3209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3255..3683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3342..3361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3552..3573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3692..3712)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3697..4026
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3877..4012
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3939..3958
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4093..4887
                     /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(4147..4166)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4757..4776
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5061..5182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5098..5117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5152..5171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5231..5247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5231..5247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5244..5266)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5255..5271
                     /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(5279..5285,5286..5303,5304..5309))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5324..5345
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5462..5479)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5633..6221)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5713..5732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6392..7183,7184..7252))
                     /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     7015..7034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7253..7357)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta
      901 ccgagctcgg atcccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      961 ctggtcgagc tggacggcga cgtaaacggc cacaggttca gcgtgtccgg cgagggcgag
     1021 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1081 gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt cagccgctac
     1141 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1201 gagcgtacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1261 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1321 aacatcctgg ggcacaagct ggagtacaac tacatcagcc acaacgtcta tatcaccgcc
     1381 gacaagcaga agaacggcat caaggcccac ttcaagatcc gccacaacat cgaggacggc
     1441 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1501 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1561 cgcgatcaca tggtcctgct ggagttcgtg accgccgccc gcatgcatga ccaactgaca
     1621 gaagagcaga ttgcagagtt caaagaagcc ttctcattat tcgacaagga tggggacggc
     1681 accatcacca caaaggaact tggcaccgtt atgaggtcgc ttggacaaaa cccaacggaa
     1741 gcagaattgc aggatatgat caatgaagtc gatgctgatg gcaatggaac gatttacttt
     1801 cctgaatttc ttactatgat ggctagaaaa atgaaggaca cagacagcga agaggaaatc
     1861 cgagaagcat tccgtgtttt tgacaaggat gggaacggct acatcagcgc tgctgaatta
     1921 cgtcacgtca tgacaaacct cggggagaag ttaacagatg aagaagttga tgaaatgata
     1981 agggaagcag atatcgatgg tgatggccaa gtaaactatg aagagtttgt acaaatgatg
     2041 acagcaaagg ggggctccaa gaggcgctgg aagaaaaact tcattgccgt cagcgctgcc
     2101 aaccggttca agaagatctc cagctccggg gcactggagc tcatggacgg cggcgtgcag
     2161 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2221 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2281 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2341 aagggtggca gcggtggcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2401 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2461 gagggcgatg ccacctacgg caagctgacc ctgaagctga tctgcaccac cggcaagctg
     2521 cccgtgccct ggcccaccct cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc
     2581 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2641 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2701 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2761 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc
     2821 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgagtaa
     2881 gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag agggccctat
     2941 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3001 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3061 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3121 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3181 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3241 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3301 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3361 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3421 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3481 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3541 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3601 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3661 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3781 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3841 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3901 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3961 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4021 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4081 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4141 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4201 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4261 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4321 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4381 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4441 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4501 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4561 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4621 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4681 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4741 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4801 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4861 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4921 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4981 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5041 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5101 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5161 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5221 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5281 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5341 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5401 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5461 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5521 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5581 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5641 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5701 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5761 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5821 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5881 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5941 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6001 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6061 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6121 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6181 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6241 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6301 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6361 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6421 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6481 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6541 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6601 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6661 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6721 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6781 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6841 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6901 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6961 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7021 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7081 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7141 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7201 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7261 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7321 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7381 ctgacg
//
