LOCUS       sequence-451734-        4777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for 2A-Puro insertion into the N-terminus of the TP53
            locus. For selectable p53 knock-out. To be co-transfected with sgRNA
            plasmid px330-TP53 (Addgene #227318).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4777)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 4777)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: qTAG-KO-Puro-TP53
FEATURES             Location/Qualifiers
     source          1..4777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1034..1067)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1079..1135
                     /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             1142..1738
                     /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(1142..1161)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1638..1658
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(1745..1778)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1808..1929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1845..1864)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1899..1918
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2556..2572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2556..2572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2569..2591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2580..2596
                     /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(2604..2610,2611..2628,2629..2634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2649..2670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2787..2804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2958..3546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3038..3057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3717..4508,4509..4577))
                     /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     4340..4359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4578..4682)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4750..4768
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggctgaggc gggccgatca
      421 cctgaagtaa ggagttcgag accagcctgg ccaacatgca aagccctgtc tctactaaaa
      481 atacaaaaat tagctgggtg tggtggtact cgcctgtaat cccagctact cgggagactg
      541 aggcaggaga atggcttgaa cccggaaggt agaggttgca gtgagctgag atcatgccac
      601 tgtgctccag cctaggtgac agagagagac tccatctcaa aaaaaaaaaa aaatacagga
      661 agggagttgg gaatagggtg cacatttagg aagtcttggg gatttagtgg tgggaaggtt
      721 ggaagtccct ctctgattgt cttttcctca aagaagtgca tggctggtga ggggtggggc
      781 aggagtgctt gggttgtggt gaaacattgg aagagagaat gtgaagcagc cattcttttc
      841 ctgctccaca ggaagccgag ctgtctcaga cactggcatg gtgttggggg agggggttcc
      901 ttctctgcag gcccaggtga cccagggttg gaagtgtctc atgctggatc cccacttttc
      961 ctcttgcagc agccagactg ccttccgggt cactgccatg ggaggaagcg gaggatctgg
     1021 cggtaccacg cgttaccgtt cgtatagcat acattatacg aagttatctg gatccggagc
     1081 tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg gaccttctag
     1141 aatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     1201 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     1261 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     1321 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     1381 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     1441 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     1501 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     1561 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     1621 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     1681 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcccc
     1741 cgggataact tcgtatagca tacattatac gaacggtagc atgggtggcg gttcctgaca
     1801 gacatgaaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1861 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1921 tgtatcttac tcgagaccgg taagagcaag tgagatccta gcgtcgagcc ccctctgagt
     1981 caggaaacat tttcagacct atggaaactg tgagtggatc cattggaagg gcaggcccac
     2041 caccccgacc ccaaccccag ccccctagca gagacctgtg ggaagcgaaa attccatggg
     2101 actgactttc tgctcttgtc tttcagactt cctgaaaaca acgttctggt aaggacaagg
     2161 gttgggctgg ggacctggag ggctgggggg ctggggggct gaggacctgg tcctctgact
     2221 gctcttttca cccatctaca gtcccccttg ccgtcccaag caatggatga tttgatgctg
     2281 tccccggacg atattgaaca atggttcact gaagacccag gtccagatga agctcccaga
     2341 atgccagagg ctgctccccg cgtggcccct gcaccagcag ctcctacacc ggcggcccct
     2401 gcaccagccc cctcctggcc cctgtcatct tctgtccctt cccagaaaac ctaccagggc
     2461 agctacggtt tccgtctggg cttcttgcat tctgggacag ccaagtctgt gacttgcacg
     2521 gtcagttgcc ctgagggaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2581 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2641 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2701 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2761 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2821 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2881 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2941 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3001 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3061 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3121 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3181 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3241 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3301 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3361 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3421 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3481 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3541 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3601 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3661 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3721 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3781 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3841 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3901 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3961 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4021 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4081 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4141 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4201 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4261 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4321 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4381 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4441 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4501 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4561 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4621 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4681 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4741 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
