LOCUS       sequence-325733-        3233 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 3233)
  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 3233)
  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..3233
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        6..1217
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1054..1073
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(1276..1893,1894..1899,1900..1935,1936..1938,1939..1974,
                     1975..1977,1978..2013,2014..2022)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    2055..2607
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2199..2216)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2426..2445
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2634..3233
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2634..2653)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3130..3150
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 gatctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag
       61 cgctgccacg tcagacgaag ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag
      121 gacagcggcc cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat
      181 tttaggacgg gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg
      241 cgaggaaaag tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg
      301 ccgatgatta tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt
      361 tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct
      421 gggctggccg gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag
      481 accgccaagg gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg
      541 gagcgcagca aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc
      601 tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag
      661 gccttcgcta atgcgggaaa gctcttattc gggtgagatg ggctggggca ccatctgggg
      721 accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg
      781 gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc
      841 gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag
      901 gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc
      961 tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt
     1021 ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct
     1081 cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg
     1141 ggtcaatatg taattttcag tgttagacta gtaaattgtc cgctaaattc tggccgtttt
     1201 tggctttttt gttagacgaa gcttggtacc gagctcggat ctccaccccg taccggtcct
     1261 gcagtcgaat tcaccatgtc tagactggac aagagcaaag tcataaacgg agctctggaa
     1321 ttactcaatg gtgtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga
     1381 gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg
     1441 ccaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg
     1501 caagactttc tgcggaacaa cgccaagtca taccgctgtg ctctcctctc acatcgcgac
     1561 ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat
     1621 cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc
     1681 gccgtgggcc actttacact gggctgcgta ttggaggaac aggagcatca agtagcaaaa
     1741 gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca agcaattgag
     1801 ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt
     1861 ggcctggaga aacagctaaa gtgcgaaagc ggcgggccga ccgacgccct tgacgatttt
     1921 gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct
     1981 gacgctcttg acgattttga ccttgacatg ctccccgggt aactaagtaa ggatccgcgg
     2041 ccgcatcgat actaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     2101 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     2161 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     2221 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     2281 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     2341 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     2401 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     2461 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     2521 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     2581 tttcctttga aaaacacgat gataatatgg atatggccac aacggtcgcc accatgaccg
     2641 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2701 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2761 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2821 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     2881 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2941 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3001 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3061 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3121 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3181 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tga
//
