LOCUS       sequence-306911-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control mutant for Venus-cpVenus-FLARE-AKAR biosensor..
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(T/A)
FEATURES             Location/Qualifiers
     source          1..7493
                     /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             2257..2991
                     /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(3055..3073)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3056..3073)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3093..3110)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3099..3323
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3369..3797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3456..3475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3666..3687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3806..3826)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3811..4140
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3991..4126
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4053..4072
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4207..5001
                     /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(4261..4280)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4871..4890
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5175..5296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5212..5231)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5266..5285
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5345..5361)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5345..5361)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5358..5380)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5369..5385
                     /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(5393..5399,5400..5417,5418..5423))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5438..5459
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5576..5593)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5747..6335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5827..5846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6506..7297,7298..7366))
                     /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     7129..7148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7367..7471)
                     /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 gccctggtgg acggcggcac cggcggcagc gagctcatgg gcggcgtgca gctcgccgac
     2281 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2341 ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2401 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaggt
     2461 accggtggat ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2521 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2581 gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg
     2641 ccctggccca ccctcgtgac caccctgggc tacggccttc agtgcttcgc ccgctacccc
     2701 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     2761 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2821 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2881 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac
     2941 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga gtaagaattc
     3001 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata
     3061 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     3121 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3181 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3241 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3301 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3361 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3421 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3481 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3541 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3601 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3661 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3721 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3781 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3841 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3901 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3961 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4021 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4081 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4141 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4201 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4261 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4321 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4381 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4441 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4501 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4561 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4621 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4681 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4741 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4801 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4861 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4921 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4981 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5041 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5101 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5161 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5221 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5281 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5341 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5401 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5461 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5521 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5581 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5641 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5701 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5761 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5821 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5881 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5941 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6001 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6061 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6121 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6181 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6241 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6301 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6361 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6421 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6481 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6541 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6601 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6661 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6721 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6781 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6841 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6901 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6961 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7021 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7081 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7141 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7201 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7261 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7321 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7381 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7441 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg cca
//
