LOCUS       sequence-175151-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes negative-control C-terminal (substrate) fragment of 
            bimolecular ERK activity reporter (bimEKAR); cytosol targeted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6341)
  AUTHORS   Depry C, Mehta S, Li R, Zhang J
  TITLE     Visualization of Compartmentalized Kinase Activity Dynamics Using 
            Adaptable BimKARs.
  JOURNAL   Chem Biol. 2015 Nov 19;22(11):1470-9. doi: 
            10.1016/j.chembiol.2015.10.004. Epub 2015 Nov 5.
  PUBMED    26548610
REFERENCE   2  (bases 1 to 6341)
  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"; doi: 
            "10.1016/j.chembiol.2015.10.004"; journalName: "Chem Biol"; date: 
            "2015-11-19- 19"; volume: "22"; issue: "11"; pages: "1470-9"
COMMENT     Sequence Label: pcDNA3-ERKsub(TA)DD-Venus-NES
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             16..33
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             37..69
                     /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(73..81,82..96)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(220..222,223..225,226..936)
                     /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"
     primer_bind     complement(265..286)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(526..545)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     873..894
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             943..969
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(988..1006)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(989..1006)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1026..1043)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1032..1256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1302..1730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1389..1408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1599..1620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1739..1759)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1744..2073
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1924..2059
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1986..2005
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2140..2934
                     /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(2194..2213)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2804..2823
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3108..3229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3145..3164)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3199..3218
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3278..3294)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3278..3294)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3291..3313)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3302..3318
                     /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(3326..3332,3333..3350,3351..3356))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3371..3392
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3509..3526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3680..4268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3760..3779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4439..5230,5231..5299))
                     /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     5062..5081
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5300..5404)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5479..5498)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5670..6049
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6050..6253
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6204..6224
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6298..6317
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6298..6316
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(6336..6341,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgcggg gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag
       61 caaatgggtc gggatctgta cgacgatgac gataaggatc ccatggctga ccaactgact
      121 gaagagtggt cgacggtacc gcggaccggt cctgacgtac cacgtgcacc agtaggaaag
      181 gcaaagctgt cattccaatt cccccgcgca cgggagctca tggtgagcaa gggcgaggag
      241 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      301 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctg
      361 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac
      421 ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      481 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
      541 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      601 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
      661 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
      721 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
      781 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaag
      841 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      901 gccgggatca ctctcggcat ggacgagctg tacaaggaat tcctgccccc cctggagcgc
      961 ctgaccctgt aatctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     1021 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1081 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1141 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1201 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     1261 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     1321 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1381 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1441 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1501 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1561 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1621 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1681 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     1741 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1801 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     1861 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     1921 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     1981 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2041 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     2101 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2161 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2221 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2281 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     2341 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2401 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2461 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2521 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2581 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2641 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     2701 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2761 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2821 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2881 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     2941 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3001 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3061 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3121 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3181 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3241 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3301 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3361 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3421 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3481 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3541 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3601 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3661 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3721 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3781 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3841 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3901 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3961 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4021 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4081 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4141 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4201 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4261 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4321 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4381 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4441 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4501 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4561 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4621 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4681 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4741 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4801 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4861 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4921 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4981 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5041 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5101 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5161 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5221 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5281 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5341 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5401 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5461 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5521 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5581 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     5641 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     5701 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     5761 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     5821 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     5881 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     5941 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     6001 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     6061 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     6121 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     6181 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     6241 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     6301 tacgactcac tatagggaga cccaagcttg cggccgccac c
//
