LOCUS       sequence-492002-        7517 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  Improved cyan/yellow FRET-based ERK kinase activity reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7517)
  AUTHORS   Keyes J, Ganesan A, Molinar-Inglis O, Hamidzadeh A, Zhang J, Ling M,
            Trejo J, Levchenko A, Zhang J
  TITLE     Signaling diversity enabled by Rap1-regulated plasma membrane ERK 
            with distinct temporal dynamics.
  JOURNAL   eLife. 2020 May 26;9. pii: 57410. doi: 10.7554/eLife.57410.
  PUBMED    32452765
REFERENCE   2  (bases 1 to 7517)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife."; 
            date: "2020-05-26"; volume: "9. pii: 57410. doi"; pages: " 
            10.7554/eLife.57410"
COMMENT     Sequence Label: pcDNA3-EKAR4
FEATURES             Location/Qualifiers
     source          1..7517
                     /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..1527)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     primer_bind     complement(1054..1075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1315..1334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1662..1683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2293..3003
                     /codon_start=1
                     /product="yellow fluorescent protein optimized for FRET 
                     with CyPet (Nguyen and Daugherty, 2005)"
                     /label=YPet
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
                     LTAAGITEGMNELYK"
     promoter        complement(3070..3088)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3071..3088)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3108..3125)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3114..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3384..3812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3471..3490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3681..3702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3821..3841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3826..4155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4006..4141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4068..4087
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4222..5016
                     /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(4276..4295)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4886..4905
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5190..5311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5227..5246)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5281..5300
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5360..5376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5360..5376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5373..5395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5384..5400
                     /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(5408..5414,5415..5432,5433..5438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5453..5474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5591..5608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5762..6350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5842..5861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6521..7312,7313..7381))
                     /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     7144..7163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7382..7486)
                     /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 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgacctggg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aactacatca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1621 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg cccgcatgca catggcggac gaggagaagc tgccgcccgg ctgggagaag
     1741 cgcatgagcc gcagctcagg ccgagtgtac tacttcaacc acatcactaa cgccagccag
     1801 tgggagcggc ccagcggcaa cagcagcagt ggtggcaaaa acgggcaggg ggagcctgcc
     1861 aggggtacca gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggtagt
     1921 gctggtggtt ccggcagtgc tggtggtagt gctggtggta gtaccagtgc tggtggtagt
     1981 gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggttccgg cagtgctggt
     2041 ggtagtgctg gtggtagtac cagtgctggt ggtagtgctg gtggtagtgc tggtggtagt
     2101 gctggtggta gtgctggtgg ttccggcagt gctggtggta gtgctggtgg tagtaccagt
     2161 gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggttcc
     2221 ggaccagatg tccctagaac tccagtggat aaagcaaagc tgtcattcca attcccccgc
     2281 gcacgggagc tctctaaagg tgaagaatta ttcactggtg ttgtcccaat tttggttgaa
     2341 ttagatggtg atgttaatgg tcacaaattt tctgtctccg gtgaaggtga aggtgatgct
     2401 acgtacggta aattgacctt aaaattactc tgtactactg gtaaattgcc agttccatgg
     2461 ccaaccttag tcactacttt aggttatggt gttcaatgtt ttgctagata cccagatcat
     2521 atgaaacaac atgacttttt caagtctgcc atgccagaag gttatgttca agaaagaact
     2581 atttttttca aagatgacgg taactacaag accagagctg aagtcaagtt tgaaggtgat
     2641 accttagtta atagaatcga attaaaaggt attgatttta aagaagatgg taacatttta
     2701 ggtcacaaat tggaatacaa ctataactct cacaatgttt acatcactgc tgacaaacaa
     2761 aagaatggta tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg tggtgttcaa
     2821 ttagctgacc attatcaaca aaatactcca attggtgatg gtccagtctt gttaccagac
     2881 aaccattact tatcctatca atctgcctta ttcaaagatc caaacgaaaa gagagaccac
     2941 atggtcttgt tagaattttt gactgctgct ggtattaccg agggtatgaa tgaattgtac
     3001 aaatagtaag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     3061 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     3121 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3181 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3241 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3301 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3361 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3421 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3481 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3541 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3601 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3661 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3721 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3781 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3901 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3961 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4021 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4081 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4141 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4201 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4261 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4321 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4381 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4441 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4501 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4561 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4621 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4681 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4741 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4801 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4861 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4921 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4981 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5041 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5101 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5161 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5281 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5341 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5401 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6181 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7381 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7441 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7501 aagtgccacc tgacgtc
//
