LOCUS       sequence-313437-        7430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted yellow-fluorescent 
            homoFRET/anisotropy-based biosensor for monitoring Protein Kinase A 
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7430)
  AUTHORS   Ross BL, Tenner B, Markwardt ML, Zviman A, Shi G, Kerr JP, Snell NE,
            McFarland JJ, Mauban JR, Ward CW, Rizzo MA, Zhang J
  TITLE     Single-color, ratiometric biosensors for detecting signaling 
            activities in live cells.
  JOURNAL   eLife. 2018 Jul 3;7. pii: 35458. doi: 10.7554/eLife.35458
  PUBMED    29968564
REFERENCE   2  (bases 1 to 7430)
  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: "eLife."; 
            date: "2018-07-3"; volume: "7. pii: 35458. doi"; pages: " 
            10.7554/eLife.3545"
COMMENT     Sequence Label: pcDNA3-Lyn-Venus-cpVenus-FLARE-AKAR
FEATURES             Location/Qualifiers
     source          1..7430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        106..485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        486..689
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     640..660
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     734..753
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        734..752
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      772..781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(817..819,820..822,823..1533)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2056..2790
                     /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="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(2854..2872)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2855..2872)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2892..2909)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2898..3122
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3168..3596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3255..3274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3465..3486
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3605..3625)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3610..3939
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3790..3925
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3852..3871
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4006..4800
                     /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(4060..4079)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4670..4689
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4974..5095
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5011..5030)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5065..5084
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5144..5160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5144..5160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5157..5179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5168..5184
                     /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(5192..5198,5199..5216,5217..5222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5237..5258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5375..5392)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5546..6134)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5626..5645)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6305..7096,7097..7165))
                     /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     6928..6947
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7166..7270)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7345..7364)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
       61 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
      121 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      181 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      241 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      301 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      361 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      421 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      481 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      541 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      601 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      661 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      721 ggcttatcga aattaatacg actcactata gggagaccca agcttgcggc cgccaccatg
      781 ggctgcatca agagcaagcg caaggacaag gatcccatgg tgagcaaggg cgaggagctg
      841 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      901 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
      961 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc
     1021 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1081 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1141 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1201 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1261 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     1321 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     1381 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaagctg
     1441 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1501 gggatcactc tcggcatgga cgagctgtac aagcgcatgc ataagttttc tcaagaacag
     1561 atcggcgaaa acattgtgtg cagggtcatt tgtaccacgg gtcaaattcc catccgagat
     1621 ttgtcagctg atatttcaca agtgcttaag gaaaaacgat ccataaagaa agtttggaca
     1681 tttggtagaa acccagcctg tgactatcat ttaggaaaca tttcaagact gtcaaataag
     1741 catttccaaa tactactagg agaagacggt aaccttttat tgaatgacat ttccactaat
     1801 gggacctggt taaatgggca aaaagtcgag aagaacagca atcagttact gtctcaaggt
     1861 gatgaaataa ccgttggtgt aggcgtggaa tcagatattt tatctctggt cattttcata
     1921 aacgacaaat ttaagcagtg cctcgagcag aacaaagttg atcgctctgc aggtaagcca
     1981 ggcagcggcg agggcagcac caagggcctg cgtcgcgcca ccctggtgga cggcggcacc
     2041 ggcggcagcg agctcatggg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     2101 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaagctg
     2161 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2221 gggatcactc tcggcatgga cgagctgtac aagggaggta ccggtggatc tatggtgagc
     2281 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2341 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2401 accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2461 accctgggct acggccttca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     2521 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2581 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2641 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2701 tacaactaca acagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag
     2761 gccaacttca agatccgcca caacatcgag taagaattct gcagatatcc atcacactgg
     2821 cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc
     2881 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2941 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3001 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3061 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3121 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3181 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3241 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3301 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3361 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3421 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3481 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3541 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3601 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3661 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3721 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3781 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3841 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3901 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3961 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4021 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4081 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4141 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4201 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4261 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4321 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4381 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4441 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4501 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4561 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4621 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4681 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4741 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4801 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4861 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4921 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4981 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5041 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5101 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5161 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5221 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5281 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5341 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5401 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5461 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5521 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5581 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5641 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5701 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5761 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5821 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5881 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5941 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6001 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6061 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6121 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6181 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6241 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6301 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6361 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6421 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6481 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6541 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6601 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6661 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6721 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6781 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6841 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6901 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6961 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7021 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7081 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7141 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7201 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7261 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7321 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     7381 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
//
