LOCUS       sequence-314083-        7389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Yellow Cameleon-Nano140, an ultrasensitive 
            Ca2+ indicator with Kd = 140 nM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7389)
  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 7389)
  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-Nano140/pcDNA3
FEATURES             Location/Qualifiers
     source          1..7389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(680..682,683..685,686..1198)
                     /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             1826..1903
                     /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             1910..2644
                     /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(2708..2726)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2709..2726)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2746..2763)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2752..2976
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3022..3450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3109..3128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3319..3340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3459..3479)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3464..3793
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3644..3779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3706..3725
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3860..4654
                     /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(3914..3933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4524..4543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4828..4949
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4865..4884)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4919..4938
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4998..5014)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4998..5014)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5011..5033)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5022..5038
                     /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(5046..5052,5053..5070,5071..5076))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5091..5112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5229..5246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5400..5988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5480..5499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6159..6950,6951..7019))
                     /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     6782..6801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7020..7124)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7199..7218)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      721 catcctggtc gagctggacg gcgacgtaaa cggccacagg ttcagcgtgt ccggcgaggg
      781 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      841 gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt gcttcagccg
      901 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      961 ccaggagcgt accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1021 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1081 cggcaacatc ctggggcaca agctggagta caactacatc agccacaacg tctatatcac
     1141 cgccgacaag cagaagaacg gcatcaaggc ccacttcaag atccgccaca acatcgagga
     1201 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1261 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1321 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gcccgcatgc atgaccaact
     1381 gacagaagag cagattgcag agttcaaaga agccttctca ttattcgaca aggatgggga
     1441 cggcaccatc accacaaagg aacttggcac cgttatgagg tcgcttggac aaaacccaac
     1501 ggaagcagaa ttgcaggata tgatcaatga agtcgatgct gatggcaatg gaacgattta
     1561 ctttcctgaa tttcttacta tgatggctag aaaaatgaag gacacagaca gcgaagagga
     1621 aatccgagaa gcattccgtg tttttgacaa ggatgggaac ggctacatca gcgctgctca
     1681 attacgtcac gtcatgacaa acctcgggga gaagttaaca gatgaagaag ttgatgaaat
     1741 gataagggaa gcagatatcg atggtgatgg ccaagtaaac tatgaagagt ttgtacaaat
     1801 gatgacagca aaggggggcg ggtccaagag gcgctggaag aaaaacttca ttgccgtcag
     1861 cgctgccaac cggttcaaga agatctccag ctccggggca ctggagctca tggacggcgg
     1921 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1981 gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg
     2041 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2101 gctgtacaag ggtggcagcg gtggcatggt gagcaagggc gaggagctgt tcaccggggt
     2161 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2221 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct gcaccaccgg
     2281 caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcc tgcagtgctt
     2341 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2401 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2461 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2521 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2581 tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat
     2641 cgagtaagaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
     2701 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     2761 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2821 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2881 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2941 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3001 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3061 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3121 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3181 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3241 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3301 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3361 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3421 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3661 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3721 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3781 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3841 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3901 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3961 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4021 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4081 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4141 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4201 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4261 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4321 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4381 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4441 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4501 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4561 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4621 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4681 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4741 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4801 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4861 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4921 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4981 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5041 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5101 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5161 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5461 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5581 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5821 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7021 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7081 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7141 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     7201 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7261 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     7321 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     7381 atacgcgtt
//
