LOCUS       sequence-204835-        7918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A PKA biosensor based on fluorescence resonance energy transfer 
            (FRET) and those based on bioluminescence energy transfer (BRET).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  AUTHORS   Komatsu N, Terai K, Imanishi A, Kamioka Y, Sumiyama K, Jin T, Okada 
            Y, Nagai T, Matsuda M
  TITLE     A platform of BRET-FRET hybrid biosensors for optogenetics, chemical
            screening, and in vivo imaging.
  JOURNAL   Sci Rep. 2018 Jun 12;8(1):8984. doi: 10.1038/s41598-018-27174-x.
  PUBMED    29895862
REFERENCE   2  (bases 1 to 7918)
  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: "Sci Rep."; 
            date: "2018-06-12"; volume: "8(1):8984. doi"; pages: " 
            10.1038/s41598-018-27174-x"
COMMENT     Sequence Label: pCAGGS-6017nes
FEATURES             Location/Qualifiers
     source          1..7918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             join(1735..1737,1738..1740,1741..2448)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     CDS             2884..3231
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             4900..4926
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(4942..4961)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5007..5062
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5061..5080)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5421..5437)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5421..5437)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5434..5456)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5445..5461
                     /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(5469..5475,5476..5493,5494..5499))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5514..5535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5594..5789
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5640..5775
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5795..5929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5832..5851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5886..5905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5997..6014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6168..6756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6248..6267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6927..7718,7719..7787))
                     /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     7550..7569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7788..7892)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgatcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cgggggcgag
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1501 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt ggcaagaatt cggcatggtg
     1741 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1801 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1861 ctgaccctga agcttctatg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1921 accaccctgg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1981 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2041 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2101 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2161 gagtacaact acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     2221 aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac
     2281 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2341 agctaccagt ccgccctgtt caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2401 gagttcctga ccgccgccgg gatcactgag ggcatgaacg agctgtacct cgagaagttt
     2461 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt
     2521 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag
     2581 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga
     2641 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac
     2701 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta
     2761 ctgtcccaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg
     2821 gtcattttca taaacgacaa atttaagcag tgcctggagc agaacaaagt tgatcgcggt
     2881 accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     2941 ggttccggca gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg tagtgctggt
     3001 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc tggtggtagt
     3061 gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     3121 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt
     3181 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggattg
     3241 aggcgcgcga cgctggttga cggcggccgc atggtgagca agggcgagga gctgttcacc
     3301 ggggtggtgc ccatcctggt cgagctggac ggcgacgtca acggccacaa gttcagcgtg
     3361 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3421 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgtcctg gggcgtgcag
     3481 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3541 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3601 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3661 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactactt cagcgggaac
     3721 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccgccac
     3781 aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3841 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cttaagcaaa
     3901 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcttgaccgc cgccgggggt
     3961 accaaggtgt acgaccccga gcagaggaag aggatgatca ccggccccca gtggtgggcc
     4021 aggtgcaagc agatgaacgt gctggacagc ttcatcaact actacgacag cgagaagcac
     4081 gccgagaacg ccgtgatctt cctgcacggc aacgccacta gcagctacct gtggaggcac
     4141 gtggtgcccc acatcgagcc cgtggccagg tgcatcatcc ccgatctgat cggcatgggc
     4201 aagagcggca agagcggcaa cggcagctac aggctgctgg accactacaa gtacctgacc
     4261 gcctggttcg agcttctgaa cctgcccaag aagatcatct tcgtgggcca cgactggggc
     4321 gccgccctgg ccttccacta cgcctacgag caccaggaca ggatcaaggc catcgtgcac
     4381 atggagagcg tggtggacgt gatcgagagc tgggacgagt ggccagacat cgaggaggac
     4441 atcgccctga tcaagagcga ggagggcgag aagatggtgc tggagaacaa cttcttcgtg
     4501 gagaccgtgc tgcccagcaa gatcatgaga aagctggagc ccgaggagtt cgccgcctac
     4561 ctggagccct tcaaggagaa gggcgaggtg agaagaccca ccctgagctg gcccagagag
     4621 atccccctgg tgaagggcgg caagcccgac gtggtgcaga tcgtgagaaa ctacaacgcc
     4681 tacctgagag ccagcgacga cctgcccaag ctgttcatcg agggcgaccc cggcttcttc
     4741 agcaacgcca tcgtggaggg cgccaagaag ttccccaaca ccgagttcgt gaaggtgaag
     4801 ggcctgcact tcctccagga ggacgccccc gacgagatgg gcaagtacat caagagcttc
     4861 gtggagagag tgctgaagaa cgagcagtct agactgcagc tgcctcctct ggaacgcctg
     4921 actctggatt aagtcgacgg atctttttcc ctctgccaaa aattatgggg acatcatgaa
     4981 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     5041 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     5101 tcagaatgag tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac
     5161 aaaggtggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     5221 tccatagaaa agccttgact tgaggttaga ttttttttta tattttgttt gtgttatttt
     5281 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     5341 cctgactact cccagtcata gctgtccctc ttctcttatg aagatccctc gacctgcagc
     5401 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5461 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5521 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5581 tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     5641 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     5701 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     5761 gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg
     5821 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5881 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa
     5941 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6001 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6061 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6361 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6601 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
