LOCUS       sequence-97644-e        2600 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for HR-mediated recombination to insert puromycin 
            resistance and append a destabilization domain to the N-terminus of 
            TCOF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2600)
  AUTHORS   Park A, Won ST, Pentecost M, Bartkowski W, Lee B
  TITLE     CRISPR/Cas9 allows efficient and complete knock-in of a 
            destabilization domain-tagged essential protein in a human cell 
            line, allowing rapid knockdown of protein function.
  JOURNAL   PLoS One. 2014 Apr 17;9(4):e95101. doi: 
            10.1371/journal.pone.0095101. eCollection 2014.
  PUBMED    24743236
REFERENCE   2  (bases 1 to 2600)
  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: "PLoS One.";
            date: "2014-04-17"; volume: "9(4):e95101. doi"; pages: " 
            10.1371/journal.pone.0095101. eCollection 2014"
COMMENT     Sequence Label: PuroR-P2A-DD-TCOF1-donor
FEATURES             Location/Qualifiers
     source          1..2600
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             759..1355
                     /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(759..778)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1255..1275
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1365..1421
                     /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             join(1422..1424,1425..1745)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
ORIGIN
        1 ggatcccagc aatttcgggt gtctttatgc ctcagcttag atatctctct ggggttcggt
       61 gctcctcctt ggggcgccca taggctgcat ctgtccctga caaggcacac aagatattga
      121 actatagttg cttggttacg gacagtcgtc tctgtgagct ttgagggcaa ctgccatgta
      181 ttatgcatct tttgaatccc cctagcctag ccgggtgcta gcccaagtta ggtgcttatt
      241 acagatttgt tcagttagcg aatgaatcaa tgaataaact ggcaattcgt gtctggttca
      301 tgggcaccaa aaaccgtgcg aaatggataa atgagcaggt aatggcagta caaagttcgg
      361 atctagagaa agatccagat tagggagttc agagatgagt aaaacgcaga cccagccatc
      421 agagaattaa caaggaagat aaatctcttc aagaagccag ccggaaggat taaaagagag
      481 tcgtccaagt cccggccggc cccgggggcg ggtccctgtc tagccccgca tgctccgcgc
      541 caatgggcgg tattgttgat cacgagtctc cgcccccttc gccttgaggg gcggggccgg
      601 ccactcccgg agaggggact acgtttcccg gcgcgccgcg cggccggagc gaaagaggag
      661 ccggaagtgg ggcgcgcgag gtctaagggc gcgagggaag tggcgggcgg ggactaaggc
      721 ggggcgtgca ggtagccggc cggccggggg tcgcgggtat gaccgagtac aagcccacgg
      781 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
      841 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
      901 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
      961 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     1021 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     1081 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     1141 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     1201 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     1261 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     1321 cgcgcacctg gtgcatgacc cgcaagcccg gtgccggatc cggagccacg aacttctctc
     1381 tgttaaagca agcaggagac gtggaagaaa accccggtcc catgggagtg caggtggaaa
     1441 ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg ccagacctgt gtggtgcact
     1501 acaccgggat gcttgaagat ggaaagaaag tcgattcctc ccgggacaga aacaagccct
     1561 ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga
     1621 tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg
     1681 ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac
     1741 cggaaggtgg aggtggatct ggtggaggtg gatctggtgg cggcggttca atggccgagg
     1801 ccaggaagcg gcgggagcta cttcccctga tctaccacca tctgctgcgg gctggctatg
     1861 tgcgtgcggc gcgggaagtg aaggagcaga gcggccaggt aagcgttcgt gggccgtgtg
     1921 cgagggccgc gtgcaagatg tggagatcag cggcccgcgg cccgcgcccc gtccccaggc
     1981 gacccggcag gcgcccggag ccgggtcccg cagtgctcga cggcgcggcc aggggtaccg
     2041 gaggagccgc aatctctgcc ttcccactgc gacttcagtt cccctgggcc tcactttcct
     2101 catccgggca gtgggttggg ccagaaagtg tgctcgcagg ggccgaactt tgtaattccc
     2161 gcttccctta cctccacgcc cttcctggta gcgggtattt taagtttcct aagtctcccg
     2221 ccacgtggct aggctctgcg cggccccccc tggggcaagg aggttgctgc gagtctcggg
     2281 ggtggattgc gaccagcccc tcgggggagc gcctggaaaa gggaccctac agcctcttag
     2341 ttgagctgga actcaggcct ttagcgattg tctgtactga gacggtcagc tttggtactc
     2401 gaactcgtgg tacagtcatt tcttacttga ttaattcaac tgttaatccg gtactcaggg
     2461 tgcttcctgg tgttcgaatg gtgagcacca tctctcagcc ctcctggagc ttacactcta
     2521 gtaaggtaga caaatcaaag aatcacaaaa gtgactatat gcttagcacc gcaataaggg
     2581 ccggccaaaa gggcgaattc
//
