LOCUS       sequence-345238-        7508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear-targeted EKAR4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7508)
  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 7508)
  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: "2020-05-26"; volume: "9. pii: 57410. doi"; pages: " 
            10.7554/eLife.57410"
COMMENT     Sequence Label: pcDNA3-nuclear-EKAR4
FEATURES             Location/Qualifiers
     source          1..7508
                     /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     1662..1683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1870..2217
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGXGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             3013..3033
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3061..3079)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3062..3079)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3099..3116)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3105..3329
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3375..3803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3462..3481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3672..3693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3812..3832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3817..4146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3997..4132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4059..4078
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4213..5007
                     /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(4267..4286)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4877..4896
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5181..5302
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5218..5237)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5272..5291
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5351..5367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5351..5367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5364..5386)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5375..5391
                     /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(5399..5405,5406..5423,5424..5429))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5444..5465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5582..5599)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5753..6341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5833..5852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6512..7303,7304..7372))
                     /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     7135..7154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7373..7477)
                     /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 gtggttscgg 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 tcatgtctaa aggtgaagaa ttattcaccg gcgtggtgcc catcctggtg
     2341 gagctggacg gcgacgtgaa cggccacaag ttcagcgtga gcggcgaggg cgagggcgac
     2401 gccacctacg gcaagctgac cctgaagctg ctgtgcacca ccggcaagct gcccgtgccc
     2461 tggcccaccc tggtgaccac cctgggctac ggcgtgcagt gcttcgcccg gtaccccgac
     2521 cacatgaagc agcacgactt cttcaagagc gccatgcccg agggctacgt gcaggagcgg
     2581 accatcttct tcaaggacga cggcaactac aagacccggg ccgaggtgaa gttcgagggc
     2641 gacaccctgg tgaaccggat cgagctgaag ggcatcgact tcaaggagga cagcaacatc
     2701 ctgggccaca agctggagta caactacaac agccacaacg tgtacatcac cgccgacaag
     2761 cagaagaacg gcatcaaggc caacttcaag atccggcaca acatcgagga cggcggcgtg
     2821 cagctggccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2881 gataaccact acctgagcta ccagagcgcc ctgttcaagg accccaacga gaagcgggac
     2941 cacatggtgc tgctggagtt cctgaccgcc gccggcatca ccgagggtat gaatgaattg
     3001 tacaaagaat tccccaaaaa gaagagaaag gtggaagacg cataatctag agggccctat
     3061 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3121 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3181 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3241 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3301 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3361 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3421 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3481 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3541 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3601 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3661 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3721 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3781 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3841 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3901 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4081 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4141 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4201 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4261 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4321 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4381 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4441 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4501 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4561 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4621 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4681 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4741 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4801 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4861 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4921 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4981 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5041 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5101 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5161 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5221 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5281 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5341 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5401 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6181 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7501 ctgacgtc
//
