LOCUS       sequence-307135-        7433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control mutant for Venus-cpVenus-FLARE-EKAR-EV biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7433)
  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 7433)
  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-EKAR-EV(T/A)
FEATURES             Location/Qualifiers
     source          1..7433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1009..1011,1012..1014,1015..1725)
                     /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             2188..2922
                     /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(2986..3004)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2987..3004)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3024..3041)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3030..3254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3300..3728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3387..3406)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3597..3618
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3737..3757)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3742..4071
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3922..4057
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3984..4003
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4138..4932
                     /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(4192..4211)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4802..4821
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5106..5227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5143..5162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5197..5216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5276..5292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5289..5311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5300..5316
                     /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(5324..5330,5331..5348,5349..5354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5507..5524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5678..6266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5758..5777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6437..7228,7229..7297))
                     /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     7060..7079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7298..7402)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     1621 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcacatggcg
     1741 gacgaggaga agctgccgcc cggctgggag aagcgcatga gccgcagctc aggccgagtg
     1801 tactacttca accacatcac taacgccagc cagtgggagc ggcccagcgg caacagcagc
     1861 agtggtggca aaaacgggca gggggagcct gccaggggta ccagtgctgg tggtagtgct
     1921 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt
     1981 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     2041 tccggcagtg ctggtggtag tgctggtggt agtaccagtg ctggtggtag tgctggtggt
     2101 agtgctggtg gttccggacc agatgtccct agagccccag tggataaagc aaagctgtca
     2161 ttccaattcc cccgcgcacg ggagctcatg ggcggcgtgc agctcgccga ccactaccag
     2221 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2281 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2341 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg taccggtgga
     2401 tctatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2461 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2521 tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc
     2581 accctcgtga ccaccctggg ctacggcctt cagtgcttcg cccgctaccc cgaccacatg
     2641 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2701 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2761 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2821 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag
     2881 aacggcatca aggccaactt caagatccgc cacaacatcg agtaagaatt ctgcagatat
     2941 ccatcacact ggcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct
     3001 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3061 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3121 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3181 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3241 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3301 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3361 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3421 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3481 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3541 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3601 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3661 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3721 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3781 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3841 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3901 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3961 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4021 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     4081 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     4141 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4201 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4261 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4321 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4381 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4441 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4501 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4561 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4621 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4681 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4741 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4801 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4861 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4921 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4981 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5041 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5101 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5161 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5221 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5341 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6541 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
