LOCUS       sequence-313436-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yellow-fluorescent homoFRET/anisotropy-based biosensor for 
            monitoring Protein Kinase A activity. Contains monomeric 
            Venus/cpVenus-E172 homoFRET pair..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  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-Venus-cpVenus-FLARE-AKAR
FEATURES             Location/Qualifiers
     source          1..7493
                     /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             793..810
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             814..846
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(850..858,859..873)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(880..882,883..885,886..1596)
                     /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             2119..2853
                     /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(2917..2935)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2918..2935)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2955..2972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2961..3185
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3231..3659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3318..3337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3528..3549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3668..3688)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3673..4002
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3853..3988
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3915..3934
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4069..4863
                     /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(4123..4142)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4733..4752
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5037..5158
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5074..5093)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5128..5147
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5207..5223)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5207..5223)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5220..5242)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5231..5247
                     /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(5255..5261,5262..5279,5280..5285))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5300..5321
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5438..5455)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5609..6197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5689..5708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6368..7159,7160..7228))
                     /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     6991..7010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7229..7333)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7408..7427)
                     /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 gtgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
      841 atgggtcggg atctgtacga cgatgacgat aaggatccca tggtgagcaa gggcgaggag
      901 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      961 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctg
     1021 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac
     1081 ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1141 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1201 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1261 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1321 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     1381 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
     1441 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaag
     1501 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1561 gccgggatca ctctcggcat ggacgagctg tacaagcgca tgcataagtt ttctcaagaa
     1621 cagatcggcg aaaacattgt gtgcagggtc atttgtacca cgggtcaaat tcccatccga
     1681 gatttgtcag ctgatatttc acaagtgctt aaggaaaaac gatccataaa gaaagtttgg
     1741 acatttggta gaaacccagc ctgtgactat catttaggaa acatttcaag actgtcaaat
     1801 aagcatttcc aaatactact aggagaagac ggtaaccttt tattgaatga catttccact
     1861 aatgggacct ggttaaatgg gcaaaaagtc gagaagaaca gcaatcagtt actgtctcaa
     1921 ggtgatgaaa taaccgttgg tgtaggcgtg gaatcagata ttttatctct ggtcattttc
     1981 ataaacgaca aatttaagca gtgcctcgag cagaacaaag ttgatcgctc tgcaggtaag
     2041 ccaggcagcg gcgagggcag caccaagggc ctgcgtcgcg ccaccctggt ggacggcggc
     2101 accggcggca gcgagctcat gggcggcgtg cagctcgccg accactacca gcagaacacc
     2161 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaag
     2221 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2281 gccgggatca ctctcggcat ggacgagctg tacaagggag gtaccggtgg atctatggtg
     2341 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2401 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2461 ctgaccctga agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2521 accaccctgg gctacggcct tcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     2581 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2641 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2701 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2761 gagtacaact acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     2821 aaggccaact tcaagatccg ccacaacatc gagtaagaat tctgcagata tccatcacac
     2881 tggcggccgc tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag
     2941 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3001 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3061 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3121 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3181 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3241 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3301 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3361 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3421 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3481 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3541 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3601 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3661 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3721 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3781 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3841 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3901 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     3961 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4021 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4081 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4141 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4201 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4261 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4321 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4381 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4441 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4501 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4561 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4621 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4681 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4741 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4801 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4861 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     4921 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4981 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5041 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5101 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5161 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5221 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5281 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5341 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5401 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5461 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5521 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5581 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5641 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5701 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5761 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5821 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5881 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5941 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6001 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6061 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6121 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6181 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6241 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6301 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6361 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6421 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6481 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6541 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6601 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6661 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6721 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6781 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6841 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6901 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6961 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7021 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7081 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7141 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7201 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7261 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7321 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7381 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7441 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaa
//
