LOCUS       sequence-325732-        1912 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Inducible expression of dominant negative HRas(N17) and P2A-coupled 
            reporter mKate2-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1912)
  AUTHORS   Ponsioen B, Post JB, Buissant des Amorie JR, Laskaris D, van Ineveld
            RL, Kersten S, Bertotti A, Sassi F, Sipieter F, Cappe B, Mertens S, 
            Verlaan-Klink I, Boj SF, Vries RGJ, Rehmann H, Vandenabeele P, 
            Riquet FB, Trusolino L, Bos JL, Snippert HJG
  TITLE     Quantifying single-cell ERK dynamics in colorectal cancer organoids 
            reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.
  JOURNAL   Nat Cell Biol. 2021 Apr;23(4):377-390. doi: 
            10.1038/s41556-021-00654-5. Epub 2021 Apr 1.
  PUBMED    33795873
REFERENCE   2  (bases 1 to 1912)
  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/s41556-021-00654-5"; journalName: "Nat Cell Biol"; date: 
            "2021-04"; volume: "23"; issue: "4"; pages: "377-390"
COMMENT     Sequence Label: pInducer(p20) x mKate2-NLS-P2A-HRas(N17)_ires-...
FEATURES             Location/Qualifiers
     source          1..1912
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    join(1..19,20..42,43..61,62..84,85..103,104..126,127..145,
                     146..168,169..187,188..210,211..229,230..252,253..271)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        304..342
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     339..363
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             461..1156
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             1157..1177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1187..1207
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1214..1234
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1280..1336
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1343..1912
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKNALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
ORIGIN
        1 tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag
       61 aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
      121 taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg
      181 atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt
      241 cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagctcg gtacccgggt
      301 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      361 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
      421 agcctccgcg gccccgaact agtccagacg cgtttccacc atggtgagcg agctgattaa
      481 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg
      541 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggcggt
      601 cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag
      661 caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccccga
      721 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
      781 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
      841 cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac
      901 cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga agctcgtggg
      961 cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac ccgctaagaa
     1021 cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca aggaggccga
     1081 caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag
     1141 caaactgggg cacagaccaa agaaaaagag aaaagtcgga ggtggaccaa agaagaagcg
     1201 aaaagttagc gctccaaaaa agaaaaggaa agtcagcatg ttaacggagc tgtacaaggc
     1261 tagagatatc ggatctggcg ccacaaactt ctccctgttg aagcaggcgg gagatgttga
     1321 agagaaccct ggacctgcta gcatgacgga atataagctt gtggtggtgg gcgccggcgg
     1381 tgtgggcaag aatgcgctga ccatccagct gatccagaac cattttgtgg acgaatacga
     1441 ccccactata gaggattcct accggaagca ggtggtcatt gatggggaga cgtgcctgtt
     1501 ggacatcctg gataccgccg gccaggagga gtacagcgcc atgcgggacc agtacatgcg
     1561 caccggggag ggcttcctgt gtgtgtttgc catcaacaac accaagtctt ttgaggacat
     1621 ccaccagtac agggagcaga tcaaacgggt gaaggactcg gatgacgtgc ccatggtgct
     1681 ggtggggaac aagtgtgacc tggctgcacg cactgtggaa tctcggcagg ctcaggacct
     1741 cgcccgaagc tacggcatcc cctacatcga gacctcggcc aagacccggc agggagtgga
     1801 ggatgccttc tacacgttgg tgcgtgagat ccggcagcac aagctgcgga agctgaaccc
     1861 tcctgatgag agtggccccg gctgcatgag ctgcaagtgt gtgctctcct ga
//
