LOCUS       Exported                6643 bp DNA     circular SYN 10-OCT-2025
DEFINITION  Expresses biosensor for cAMP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    Flamindo2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6643)
  AUTHORS   Odaka H, Arai S, Inoue T, Kitaguchi T
  TITLE     Genetically-encoded yellow fluorescent cAMP indicator with an 
            expanded dynamic range for dual-color imaging.
  JOURNAL   PLoS One. 2014 Jun 24;9(6):e100252. doi: 
            10.1371/journal.pone.0100252. eCollection 2014.
  PUBMED    24959857
REFERENCE   2  (bases 1 to 6643)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 10, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2014-06-24"; volume: "9(6):e100252. doi"; pages: " 
            10.1371/journal.pone.0100252. eCollection 2014"
FEATURES             Location/Qualifiers
     source          1..6643
                     /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             750..1973
                     /codon_start=1
                     /label=Flamindo2
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLRGALKKEELVEAMALLS
                     QRGPDALLTVALRKPPGQRTDEELDLIFEELLHIKAVAHLSNSVKRELAAVLLFEPHSK
                     AGTVLFSQGDKGTSWYIIWKGSVNVVTHGKGLVTTLHEGDDFGQLALVNDAPRAATIIL
                     RENNCHFLRVDKQDFNRIIKDVEAKTMRLEEEFNSHNVYIMADKQKNGIKVNFKIRHNI
                     EDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGIT
                     LGMDELYK"
     primer_bind     complement(795..816)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1056..1075)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1719..1970
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     1907..1928
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2000..2017)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2006..2230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2276..2704
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2363..2382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2573..2594
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2713..2733)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2718..3047
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2898..3033
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2960..2979
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3114..3908
                     /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(3168..3187)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3778..3797
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4082..4203
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4119..4138)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4173..4192
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4252..4268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4252..4268)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4265..4287)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4276..4292
                     /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(4300..4330)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4345..4366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4483..4500)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4654..5242)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4734..4753)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5413..6273)
                     /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     6036..6055
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6274..6378)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6453..6472)
                     /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 acccaagctg
      661 gctagcgttt aaacgggccc tctagactcg agcggccgcc actgtgctgg atatctgcag
      721 aattccacca cactggacta gtggatccca tggtgagcaa gggcgaggag ctgttcaccg
      781 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      841 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      901 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgatgt
      961 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1021 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1081 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1141 tcaaggagga cggcaacatc ctggggcaca agctggagta caacctccgc ggggcactga
     1201 agaaagagga gcttgttgag gctatggccc tcctgtccca gcgagggcct gatgccctac
     1261 tcaccgtggc gctccggaag cccccaggtc agcgtacgga tgaagaactg gacctgatct
     1321 ttgaagagct gctgcatatc aaggcggtgg cacacctttc taactcggtg aagcgggaac
     1381 tagctgctgt tctgctcttt gaaccacaca gcaaggcagg aactgtgttg ttcagccagg
     1441 gggacaaggg tacctcgtgg tacattatct ggaagggatc tgtcaatgtg gtgacccatg
     1501 gcaaggggct ggtgaccacg ttgcacgagg gagatgactt tggacagctg gctctggtga
     1561 acgacgcacc tcgggcagcc accatcatcc ttcgagaaaa taactgtcac tttctgcgtg
     1621 tggacaagca ggacttcaac cgcatcatca aggatgtgga agcaaaaacc atgagactgg
     1681 aagaagaatt caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1741 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1801 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1861 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1921 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaaagctta
     1981 agtttaaacc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2041 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2101 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2161 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2221 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2281 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2341 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2401 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2461 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2521 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2581 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2641 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2701 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2761 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2821 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2881 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2941 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3001 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3061 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3121 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3181 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3241 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3301 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3361 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3421 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3481 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3541 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3601 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3661 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3721 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3781 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3841 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3901 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3961 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4021 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4081 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4141 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4201 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4261 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4321 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4381 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4441 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4501 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4561 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4621 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4681 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4741 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4801 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4861 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4921 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4981 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5041 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5101 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5161 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5221 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5281 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5341 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5401 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5461 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5521 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5581 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5641 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5701 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5761 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5821 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5881 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5941 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6001 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6061 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6121 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6181 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6241 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6301 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6361 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     6421 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6481 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     6541 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     6601 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
