LOCUS       sequence-344972-        6575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted ERK monobody binder for local inhibition of
            ERK activity in live cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6575)
  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 6575)
  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-pmEMBer
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        255..634
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        635..838
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     789..809
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     883..902
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        883..901
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      921..930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             942..959
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             963..995
                     /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(999..1007,1008..1022)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1332..1361
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1377..2084
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1522..1540)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1790..1809
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(2148..2166)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2149..2166)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2186..2203)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2192..2416
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2462..2890
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2549..2568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2759..2780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2899..2919)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2904..3233
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3084..3219
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3146..3165
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3300..4094
                     /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(3354..3373)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3964..3983
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4268..4389
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4305..4324)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4359..4378
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4438..4454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4438..4454)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4451..4473)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4462..4478
                     /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(4486..4492,4493..4510,4511..4516))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4531..4552
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4669..4686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4840..5428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4920..4939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5599..6390,6391..6459))
                     /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     6222..6241
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6460..6564)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
       61 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
      121 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
      181 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      241 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      301 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      361 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      421 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      481 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      541 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      601 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      661 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      721 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      781 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      841 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
      901 ggagacccaa gcttgcggcc gccaccatgg tgcggggttc tcatcatcat catcatcatg
      961 gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac gatgacgata
     1021 aggatcccat ggctagcgtt tctgatgttc cgagggacct ggaagttgtt gctgcgaccc
     1081 ccaccagcct actgatcagc tggtacgatc agtcgccttc gtattcttac aggatcactt
     1141 acggagaaac aggaggaaat agccctgtcc aggagttcac tgtgcctagt tctgtttaca
     1201 ccgctaccat cagcggcctt aaacctggag ttgattatac catcactgtg tatgctgtca
     1261 cttgtaggcc gcgctctctc ccaatttcca ttaattaccg aacagaaatt gacaaaccat
     1321 cccagggatc agaacaaaaa cttatttctg aagaggactt gggtaccttt gtcgacatgg
     1381 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1441 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1501 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     1561 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1621 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1681 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1741 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1801 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1861 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     1921 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     1981 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     2041 ccgagggccg ccactccacc ggcggcatgg acgagctgta caaggaattc aagaagaaga
     2101 agaagagcaa gaccaagtgc gtgatcatgt aatctagagg gccctattct atagtgtcac
     2161 ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2221 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2281 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2341 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2401 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2461 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2521 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2581 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2641 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2701 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2761 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2821 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2881 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2941 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3001 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3061 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3121 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3181 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3241 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3301 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3361 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3421 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3481 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3541 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3601 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3661 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3721 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3781 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     3841 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3901 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3961 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4021 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4081 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4141 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4201 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4261 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4321 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4381 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4441 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4501 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4561 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4621 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4681 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4741 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4801 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4861 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4921 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4981 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5041 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5101 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5161 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5221 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5281 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5341 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5401 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5461 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5521 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5581 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5641 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5701 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5761 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5821 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5881 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5941 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6001 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6061 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6121 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6181 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6241 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6301 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6361 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6421 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6481 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6541 aaataaacaa ataggggttc cgcgcacatt tcccc
//
