LOCUS       sequence-208324-        7919 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 7919)
  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 7919)
  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..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          161..1173
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1093..1115
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             join(1230..1232,1233..1235,1236..1943)
                     /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             2379..2726
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             4395..4421
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(4437..4456)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4502..4557
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4556..4575)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4916..4932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4916..4932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4929..4951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4940..4956
                     /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(4964..4970,4971..4988,4989..4994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5009..5030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5089..5284
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5135..5270
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5290..5424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5327..5346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5381..5400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5492..5509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5663..6251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5743..5762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6422..7213,7214..7282))
                     /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     7045..7064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7283..7387)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7422..7801
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7804..7919,1..160)
                     /label=chicken beta-actin promoter
ORIGIN
        1 ggggcgcgcg ccaggcgggg cggggcgggg gcgagggcgg ggcggggcga ggcggagagg
       61 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg
      121 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc
      181 gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      241 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
      301 ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg
      361 ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
      421 ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg
      481 tgcgctccgc gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
      541 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
      601 tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac
      661 ggcccggctt cgggtgcggg gctccgtgcg gggcgtggcg cggggctcgc cgtgccgggc
      721 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
      781 tcgggggagg ggcgcggcgg ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
      841 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
      901 ggcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg ggcgaagcgg
      961 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1021 ccccttctcc atctccagcc tcggggctgc cgcaggggga cggctgcctt cgggggggac
     1081 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagctag agcctctgct
     1141 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttgttgtg
     1201 ctgtctcatc attttggcaa gaattcggca tggtgagcaa gggcgaggag ctgttcaccg
     1261 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1321 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctt ctatgcacca
     1381 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac ggcctgcagt
     1441 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1501 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1561 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1621 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1681 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     1741 acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1801 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgttcaaag
     1861 accccaacga gaagcgcgat cacatggtcc tgctggagtt cctgaccgcc gccgggatca
     1921 ctgagggcat gaacgagctg tacctcgaga agttttctca agaacagatc ggcgaaaaca
     1981 ttgtgtgcag ggtcatttgt accacgggtc aaattcccat ccgagatttg tcagctgata
     2041 tttcacaagt gcttaaggaa aaacgatcca taaagaaagt ttggacattt ggtagaaacc
     2101 cagcctgtga ctatcattta ggaaacattt caagactgtc aaataagcat ttccaaatac
     2161 tactaggaga agacggtaac cttttattga atgacatttc cactaatggg acctggttaa
     2221 atgggcaaaa agtcgagaag aacagcaatc agttactgtc ccaaggtgat gaaataaccg
     2281 ttggtgtagg cgtggaatca gatattttat ctctggtcat tttcataaac gacaaattta
     2341 agcagtgcct ggagcagaac aaagttgatc gcggtaccag tgctggtggt agtgctggtg
     2401 gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc cggcagtgct ggtggtagtg
     2461 ctggtggtag taccagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     2521 gtagtgctgg tggttccggc agtgctggtg gtagtgctgg tggtagtacc agtgctggtg
     2581 gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt tccggcagtg
     2641 ctggtggtag tgctggtggt agtaccagtg ctggtggtag tgctggtggt agtgctggtg
     2701 gtagtgctgg tggtagtgct ggtggttccg gattgaggcg cgcgacgctg gttgacggcg
     2761 gccgcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2821 tggacggcga cgtcaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2881 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2941 ccaccctcgt gaccaccctg tcctggggcg tgcagtgctt cgcccgctac cccgaccaca
     3001 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3061 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     3121 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3181 ggcacaagct ggagtacaac tacttcagcg ggaacgtcta tatcaccgcc gacaagcaga
     3241 agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc
     3301 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3361 accactacct gagcacccag tccgccttaa gcaaagaccc caacgagaag cgcgatcaca
     3421 tggtcctgct ggagttcttg accgccgccg ggggtaccaa ggtgtacgac cccgagcaga
     3481 ggaagaggat gatcaccggc ccccagtggt gggccaggtg caagcagatg aacgtgctgg
     3541 acagcttcat caactactac gacagcgaga agcacgccga gaacgccgtg atcttcctgc
     3601 acggcaacgc cactagcagc tacctgtgga ggcacgtggt gccccacatc gagcccgtgg
     3661 ccaggtgcat catccccgat ctgatcggca tgggcaagag cggcaagagc ggcaacggca
     3721 gctacaggct gctggaccac tacaagtacc tgaccgcctg gttcgagctt ctgaacctgc
     3781 ccaagaagat catcttcgtg ggccacgact ggggcgccgc cctggccttc cactacgcct
     3841 acgagcacca ggacaggatc aaggccatcg tgcacatgga gagcgtggtg gacgtgatcg
     3901 agagctggga cgagtggcca gacatcgagg aggacatcgc cctgatcaag agcgaggagg
     3961 gcgagaagat ggtgctggag aacaacttct tcgtggagac cgtgctgccc agcaagatca
     4021 tgagaaagct ggagcccgag gagttcgccg cctacctgga gcccttcaag gagaagggcg
     4081 aggtgagaag acccaccctg agctggccca gagagatccc cctggtgaag ggcggcaagc
     4141 ccgacgtggt gcagatcgtg agaaactaca acgcctacct gagagccagc gacgacctgc
     4201 ccaagctgtt catcgagggc gaccccggct tcttcagcaa cgccatcgtg gagggcgcca
     4261 agaagttccc caacaccgag ttcgtgaagg tgaagggcct gcacttcctc caggaggacg
     4321 cccccgacga gatgggcaag tacatcaaga gcttcgtgga gagagtgctg aagaacgagc
     4381 agtctagact gcagctgcct cctctggaac gcctgactct ggattaagtc gacggatctt
     4441 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     4501 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     4561 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     4621 ttggcaacat atgccatatg ctggctgcca tgaacaaagg tggctataaa gaggtcatca
     4681 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
     4741 ttagattttt ttttatattt tgtttgtgtt atttttttct ttaacatccc taaaattttc
     4801 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
     4861 ccctcttctc ttatgaagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat
     4921 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4981 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5041 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     5101 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     5161 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     5221 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     5281 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5341 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5401 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag
     5461 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5521 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5581 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5641 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5701 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5761 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5821 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca
     5881 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5941 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6001 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6061 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6121 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6181 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6241 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6301 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6361 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6421 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6481 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6541 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6601 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6661 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6721 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6781 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6841 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6901 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6961 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7021 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7081 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7141 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7201 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7261 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7321 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7381 ttccgcgcac atttccccga aaagtgccac ctgggtcgat cgacattgat tattgactag
     7441 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     7501 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7561 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     7621 ggtggactat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     7681 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     7741 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     7801 gggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     7861 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg ggggggggg
//
