LOCUS       sequence-306914-        7475 bp    DNA     circular SYN 15-APR-2025
DEFINITION  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 7475)
  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 7475)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Venus-FLARE-AKAR
FEATURES             Location/Qualifiers
     source          1..7475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(53..72)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        244..623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        624..827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     778..798
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     872..891
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        872..890
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      910..919
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             931..948
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             952..984
                     /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(988..996,997..1011)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1018..1020,1021..1023,1024..1734)
                     /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             join(2257..2259,2260..2262,2263..2976)
                     /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"
     promoter        complement(3037..3055)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3038..3055)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3075..3092)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3081..3305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3351..3779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3438..3457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3648..3669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3788..3808)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3793..4122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3973..4108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4035..4054
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4189..4983
                     /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(4243..4262)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4853..4872
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5157..5278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5194..5213)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5248..5267
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5327..5343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5327..5343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5340..5362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5351..5367
                     /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(5375..5381,5382..5399,5400..5405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5420..5441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5558..5575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5729..6317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5809..5828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6488..7279,7280..7348))
                     /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     7111..7130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7349..7453)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
       61 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      121 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
      181 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      241 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      301 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      361 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      421 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      481 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      541 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      601 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      661 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      721 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      781 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      841 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      901 cttgcggccg ccaccatggt gcggggttct catcatcatc atcatcatgg tatggctagc
      961 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcccatg
     1021 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1081 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1141 aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1201 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     1261 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1321 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1381 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1441 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     1501 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     1561 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1621 ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1681 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcgcatg
     1741 cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg
     1801 ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga
     1861 tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac
     1921 atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta
     1981 ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc
     2041 aatcagttac tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt
     2101 ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt
     2161 gatcgctctg caggtaagcc aggcagcggc gagggcagca ccaagggcct gcgtcgcgcc
     2221 accctggtgg acggcggcac cggcggcagc gagctcatgg tgagcaaggg cgaggagctg
     2281 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2341 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
     2401 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc
     2461 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2521 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2581 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2641 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2701 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     2761 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     2821 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaagctg
     2881 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2941 gggatcactc tcggcatgga cgagctgtac aagtaagaat tctgcagata tccatcacac
     3001 tggcggccgc tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag
     3061 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3121 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3181 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3241 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3301 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3601 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3661 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3721 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3781 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3841 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3901 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3961 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4021 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4081 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4141 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4201 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4261 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4321 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4381 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4441 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4501 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4561 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4621 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4681 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4741 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4801 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4861 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4921 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4981 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5041 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5101 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5161 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5221 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5281 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5341 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5401 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5461 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7261 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7441 taggggttcc gcgcacattt ccccgaaaag tgcca
//
