LOCUS       sequence-451895-        6986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to transiently express the pErk reporter in LABEL-seq MAPK 
            activity assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  AUTHORS   Simon JJ, Fowler DM, Maly DJ
  TITLE     Multiplexed profiling of intracellular protein abundance, activity, 
            interactions and druggability with LABEL-seq.
  JOURNAL   Nat Methods. 2024 Nov;21(11):2094-2106. doi: 
            10.1038/s41592-024-02456-7. Epub 2024 Oct 21.
  PUBMED    39433876
REFERENCE   2  (bases 1 to 6986)
  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"; doi: 
            "10.1038/s41592-024-02456-7"; journalName: "Nat Methods"; date: 
            "2024-11"; volume: "21"; issue: "11"; pages: "2094-2106"
COMMENT     Sequence Label: 3xFlag-tdMCP-Erk2 (pErk reporter)
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(404..1195,1196..1264))
                     /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     1027..1046
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1265..1369)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1444..1463)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1635..2014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2015..2218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2169..2189
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2220..2238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2241..2259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2269..2293
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2387..2396
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2396..2461
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4262..4297
                     /codon_start=1
                     /product="epitope tag derived from MAP kinase 2"
                     /label=Tag-100
                     /translation="EETARFQPGYRS"
     primer_bind     complement(4338..4355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4344..4568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4701..4720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4852..4899
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4907..5929
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4907..5929
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    6059..6180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6096..6115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6150..6169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6229..6245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6229..6245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6242..6264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6253..6269
                     /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(6277..6283,6284..6301,6302..6307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6322..6343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6460..6477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6631..6986,1..233))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6711..6730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
       61 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      121 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      181 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      241 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      301 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      361 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      421 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
      481 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
      541 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
      601 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
      661 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      721 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      781 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      841 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
      901 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
      961 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1021 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1081 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1141 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1201 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1261 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1321 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1381 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     1441 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     1501 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     1561 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     1621 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     1681 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     1741 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     1801 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     1861 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     1921 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     1981 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     2041 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     2101 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     2161 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     2221 ccctatcagt gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt
     2281 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
     2341 ggaccgatcc agcctccgga ctctagcgtt taaacttaag cttgccgcca ccatggatta
     2401 taaagatcat gacggggact ataaggatca cgatattgac tacaaggatg acgacgacaa
     2461 aggttctggc agtggcagcg gagcttctaa ctttactcag ttcgttctcg tcgacaatgg
     2521 cggaactggc gacgtgactg tcgccccaag caacttcgct aacgggatcg ctgaatggat
     2581 cagctctaac tcgcgttcac aggcttacaa agtaacctgt agcgttcgtc agagctctgc
     2641 gcagaatcgc aaatacacca tcaaagtcga ggtgcctaaa ggcgcctggc gttcgtactt
     2701 aaatatggaa ctaaccattc caattttcgc cacgaattcc gactgcgagc ttattgttaa
     2761 ggcaatgcaa ggtctcctaa aagatggaaa cccgattccc tcagcaatcg cagcaaactc
     2821 cggcatctac gcaatggctt ctaactttac tcagttcgtt ctcgtcgaca atggcggaac
     2881 tggcgacgtg actgtcgccc caagcaactt cgctaacggg atcgctgaat ggatcagctc
     2941 taactcgcgt tcacaggctt acaaagtaac ctgtagcgtt cgtcagagct ctgcgcagaa
     3001 tcgcaaatac accatcaaag tcgaggtgcc taaaggcgcc tggcgttcgt acttaaatat
     3061 ggaactaacc attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat
     3121 gcaaggtctc ctaaaagatg gaaacccgat tccctcagca atcgcagcaa actccggcat
     3181 ctacggctcc ggaggctctg gtagcggatc cggtggcatg gcggcagccg cagcggcagg
     3241 ggctgggcca gagatggtcc gcgggcaggt gttcgacgtg gggccgcgct acaccaacct
     3301 ctcgtacatc ggcgagggcg cctacggcat ggtgtgctct gcttatgata atgtcaacaa
     3361 agttcgagta gctatcaaga aaatcagccc ctttgagcac cagacctact gccagagaac
     3421 cctgagggag ataaaaatct tactgcgctt cagacatgag aacatcattg gaatcaatga
     3481 cattattcga gcaccaacca tcgagcaaat gaaagatgta tatatagtac aggacctcat
     3541 ggaaacagat ctttacaagc tcttgaagac acaacacctc agcaatgacc atatctgcta
     3601 ttttctctac cagatcctca gagggttaaa atatatccat tcagctaacg ttctgcaccg
     3661 tgacctcaag ccttccaacc tgctgctcaa caccacctgt gatctcaaga tctgtgactt
     3721 tggcctggcc cgtgttgcag atccagacca tgatcacaca gggttcctga cagaatatgt
     3781 ggccacacgt tggtacaggg ctccagaaat tatgttgaat tccaagggct acaccaagtc
     3841 cattgatatt tggtctgtag gctgcattct ggcagaaatg ctttctaaca ggcccatctt
     3901 tccagggaag cattatcttg accagctgaa ccacattttg ggtattcttg gatccccatc
     3961 acaagaagac ctgaattgta taataaattt aaaagctagg aactatttgc tttctcttcc
     4021 acacaaaaat aaggtgccat ggaacaggct gttcccaaat gctgactcca aagctctgga
     4081 cttattggac aaaatgttga cattcaaccc acacaagagg attgaagtag aacaggctct
     4141 ggcccaccca tatctggagc agtattacga cccgagtgac gagcccatcg ccgaagcacc
     4201 attcaagttc gacatggaat tggatgactt gcctaaggaa aagctcaaag aactaatttt
     4261 tgaagagact gctagattcc agccaggata cagatcttaa ctcgagtcta gagggcccgt
     4321 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4381 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4441 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4501 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4561 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     4621 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4681 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4741 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4801 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     4861 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     4921 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     4981 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     5041 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     5101 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     5161 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     5221 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     5281 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     5341 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     5401 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     5461 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     5521 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     5581 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     5641 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     5701 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     5761 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     5821 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     5881 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     5941 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     6001 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6061 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6121 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6181 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     6241 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6301 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6361 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6421 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6481 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6541 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6601 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6661 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6721 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6781 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6841 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6901 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6961 cgacttatcg ccactggcag cagcca
//
