LOCUS       sequence-339792-        7509 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 7509)
  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 7509)
  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..7509
                     /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
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     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..4147
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3998..4133
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4060..4079
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4214..5008
                     /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(4268..4287)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4878..4897
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5182..5303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5219..5238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5273..5292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5352..5368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5352..5368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5365..5387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5376..5392
                     /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(5400..5406,5407..5424,5425..5430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5445..5466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5583..5600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5754..6342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5834..5853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6513..7304,7305..7373))
                     /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     7136..7155
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7374..7478)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 tcatgtctaa aggtgaagaa ttattcaccg gcgtggtgcc catcctggtg
     2341 gagctggacg gcgacgtgaa cggccacaag ttcagcgtga gcggcgaggg cgagggcgac
     2401 gccacntacg 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 gcatcccttt
     3541 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3601 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3661 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3721 ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat
     3781 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3841 tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3901 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3961 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4021 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4081 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4141 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     4201 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4261 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4321 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4381 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4441 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4501 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4561 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4621 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4681 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4741 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4801 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4861 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4921 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4981 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5041 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5101 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5161 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5221 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5281 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5341 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5401 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5461 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5521 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5941 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6181 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6421 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6481 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6541 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6601 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6661 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6721 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6781 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6841 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6901 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6961 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7021 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7081 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7141 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7201 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7261 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7321 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7381 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7441 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7501 cctgacgtc
//
