LOCUS       sequence-487601-        7409 bp    DNA     circular SYN 01-OCT-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 7409)
  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 7409)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 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..7409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        213..592
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        593..796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     747..767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     841..860
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        841..859
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      879..888
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             900..917
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             921..953
                     /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(957..965,966..980)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(987..989,990..992,993..1505)
                     /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(1032..1053)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1293..1312)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1640..1661
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2163..2873
                     /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(2940..2958)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2941..2958)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2978..2995)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2984..3208
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3254..3682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3341..3360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3551..3572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3696..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3938..3957
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4092..4886
                     /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(4146..4165)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4756..4775
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5060..5181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5097..5116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5151..5170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5230..5246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5230..5246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5243..5265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5254..5270
                     /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(5278..5284,5285..5302,5303..5308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5323..5344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5461..5478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5632..6220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5712..5731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6391..7182,7183..7251))
                     /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     7014..7033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7252..7356)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
       61 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      121 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      181 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      241 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      301 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      361 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      421 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      481 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      541 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      601 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      661 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      721 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      781 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
      841 taatacgact cactataggg agacccaagc ttgcggccgc caccatggtg cggggttctc
      901 atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc
      961 tgtacgacga tgacgataag gatcccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1021 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaggttc agcgtgtccg
     1081 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1141 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctggggc gtgcagtgct
     1201 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1261 gctacgtcca ggagcgtacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1321 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1381 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacatcagc cacaacgtct
     1441 atatcaccgc cgacaagcag aagaacggca tcaaggccca cttcaagatc cgccacaaca
     1501 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1561 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1621 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc cgcatgcaca
     1681 tggcggacga ggagaagctg ccgcccggct gggagaagcg catgagccgc agctcaggcc
     1741 gagtgtacta cttcaaccac atcactaacg ccagccagtg ggagcggccc agcggcaaca
     1801 gcagcagtgg tggcaaaaac gggcaggggg agcctgccag gggtaccagt gctggtggta
     1861 gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg
     1921 gtggtagtgc tggtggtagt accagtgctg gtggtagtgc tggtggtagt gctggtggta
     1981 gtgctggtgg tagtgctggt ggttccggca gtgctggtgg tagtgctggt ggtagtacca
     2041 gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggttcc ggaccagatg
     2101 tccctagaac tccagtggat aaagcaaagc tgtcattcca attcccccgc gcacgggagc
     2161 tctctaaagg tgaagaatta ttcactggtg ttgtcccaat tttggttgaa ttagatggtg
     2221 atgttaatgg tcacaaattt tctgtctccg gtgaaggtga aggtgatgct acgtacggta
     2281 aattgacctt aaaattactc tgtactactg gtaaattgcc agttccatgg ccaaccttag
     2341 tcactacttt aggttatggt gttcaatgtt ttgctagata cccagatcat atgaaacaac
     2401 atgacttttt caagtctgcc atgccagaag gttatgttca agaaagaact atttttttca
     2461 aagatgacgg taactacaag accagagctg aagtcaagtt tgaaggtgat accttagtta
     2521 atagaatcga attaaaaggt attgatttta aagaagatgg taacatttta ggtcacaaat
     2581 tggaatacaa ctataactct cacaatgttt acatcactgc tgacaaacaa aagaatggta
     2641 tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg tggtgttcaa ttagctgacc
     2701 attatcaaca aaatactcca attggtgatg gtccagtctt gttaccagac aaccattact
     2761 tatcctatca atctgcctta ttcaaagatc caaacgaaaa gagagaccac atggtcttgt
     2821 tagaattttt gactgctgct ggtattaccg agggtatgaa tgaattgtac aaatagtaag
     2881 aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga gggccctatt
     2941 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     3001 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3061 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3121 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3181 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3241 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3301 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3361 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3421 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3481 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3541 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3601 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3661 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4081 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4141 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4201 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4261 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4321 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4381 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4441 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4501 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4561 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4621 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4681 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4741 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4801 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4861 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4921 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4981 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5041 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5101 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5161 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5221 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5281 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5341 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5401 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5461 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5521 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5581 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5641 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5701 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5761 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5821 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5881 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5941 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6001 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6061 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6121 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6181 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6241 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6301 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6361 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6421 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6481 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6541 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6601 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6661 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6721 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6781 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6841 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6901 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6961 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7021 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7081 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7141 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7201 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7261 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7321 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7381 tgacgtcgac ggatcgggag atctcccga
//
