LOCUS       sequence-66357-e        7586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOP Hupki-puro2A-G245S plasmid to introduce mutant p53 to PLF ESC or
            MEFs by IMCE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7586)
  AUTHORS   Wei QX, Odell AF, van der Hoeven F, Hollstein M
  TITLE     Rapid derivation of genetically related mutants from embryonic cells
            harboring a recombinase-specific Trp53 platform.
  JOURNAL   Cell Cycle. 2011 Apr 15;10(8):1261-70. Epub 2011 Apr 15.
  PUBMED    21445009
REFERENCE   2  (bases 1 to 7586)
  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: "Cell 
            Cycle."; date: "2011-04-15"; volume: "10(8)"; pages: "1261-70. Epub 
            2011 Apr 15"
COMMENT     Sequence Label: pX62
FEATURES             Location/Qualifiers
     source          1..7586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    672..705
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             1112..1711
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1611..1631
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1757..1810
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    5125..5194
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(5401..5419)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5440..5456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5440..5456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5453..5475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5464..5480
                     /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(5488..5494,5495..5512,5513..5518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5533..5554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5671..5688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5842..6430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5922..5941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6601..7392,7393..7461))
                     /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     7224..7243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7462..7566)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccta
      661 gccgcgggcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc
      721 atctgaagct tccgacttct tggatacttg taactttgcg ctttcccacc ctcgcataag
      781 tttcctgaaa taatgactct gaaactcaaa atatatttac aaacacctcg gctgtagctt
      841 tggtttgttc tctgacttgc ttataactta atatccctct tattctaacc taagttctgc
      901 cacgtggttg gttacctctg ctcagccccc ggcttctgtc ctccatgttc ctgggggaat
      961 ccctacactt tcagaattta atttccctac tggatgtccc accttctttt tattctaccc
     1021 tttcctataa gccatagggg tttgtttgtt tgtatgtttt ttaattgaca agttatgcat
     1081 ccatacagta cacaatctct tctctctaca gatgggcacc gagtacaagc ccacggtgcg
     1141 cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga
     1201 ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct
     1261 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     1321 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     1381 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     1441 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     1501 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     1561 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     1621 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     1681 cacctggtgc atgacccgca agcccggtgc cgggagcggc ggtggctctg gtggcggttc
     1741 aatggtgagg ctgctcgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
     1801 tcccggccct gcaccgggag ccaccatgac tgccatggag gagtcacagt cggatatcag
     1861 cctcgagctc cctctgagcc aggagacatt ttcaggctta tggaaactgt gagtggatct
     1921 ttttggggcc cttaagatac atcccgccat acctgtatcc tccccttgcc tgagagaaac
     1981 aaaaacagta gtgttcaaac atggtatggt gttgggtgtc tgtaaatcct gcggggcggg
     2041 gtggcggggg ggggggactg cagggtctca gaagtttgag gtcatcattg actacatagc
     2101 aagttggagg ccagcctggg ataagtgaga ttctgtcttc aaaaaatgga aggaaatcag
     2161 gaactaactc tctgctcttg ttttccagac ttcctccaga agatatcctg gtaaggccca
     2221 gaacagaaag ggacttgggc tttggtggat cctgaggacc tggtcctctg actgctcttt
     2281 tcacccatct acagtccccc ttgccgtccc aagcaatgga tgatttgatg ctgtccccgg
     2341 acgatattga acaatggttc actgaagacc caggtccaga tgaagctccc agaatgccag
     2401 aggctgctcc ccccgtggcc cctgcaccag cagctcctac accggcggcc cctgcaccag
     2461 ccccctcctg gcccctgtca tcttctgtcc cttcccagaa aacctaccag ggcagctacg
     2521 gtttccgtct gggcttcttg cattctggga cagccaagtc tgtgacttgc acggtcagtt
     2581 gccctgaggg gctggcttcc atgagacttc aatgcctggc cgtatccccc tgcatttctt
     2641 ttgtttggaa ctttgggatt cctcttcacc ctttggcttc ctgtcagtgt ttttttatag
     2701 tttacccact taatgtgtga tctctgactc ctgtcccaaa gttgaatatt ccccccttga
     2761 atttgggctt ttatccatcc catcacaccc tcagcatctc tcctggggat gcagaacttt
     2821 tctttttctt catccacgtg tattccttgg cttttgaaaa taagctcctg accaggcttg
     2881 gtggctcaca cctgcaatcc cagcactctc aaagaggcca aggcaggcag atcacctgag
     2941 cccaggagtt caagaccagc ctgggtaaca tgatgaaacc tcgtctctac aaaaaaatac
     3001 aaaaaattag ccaggcatgg tggtgcacac ctatagtccc agccacttag gaggctgagg
     3061 tgggaagatc acttgaggcc aggagatgga ggctgcagtg agctgtgatc acaccactgt
     3121 gctccagcct gagtgacaga gcaagaccct atctcaaaaa aaaaaaaaaa aaagaaaagc
     3181 tcctgaggtg tagacgccaa ctctctctag ctcgctagtg ggttgcagga ggtgcttacg
     3241 catgtttgtt tctttgctgc cgtcttccag ttgctttatc tgttcacttg tgccctgact
     3301 ttcaactctg tctccttcct cttcctacag tactcccctg ccctcaacaa gatgttttgc
     3361 caactggcca agacctgccc tgtgcagctg tgggttgatt ccacaccccc gcccggcacc
     3421 cgcgtccgcg ccatggccat ctacaagcag tcacagcaca tgacggaggt tgtgaggcgc
     3481 tgcccccacc atgagcgctg ctcagatagc gatggtgagc agctggggct ggagagacga
     3541 cagggctggt tgcccagggt ccccaggcct ctgattcctc actgattgct cttaggtctg
     3601 gcccctcctc agcatcttat ccgagtggaa ggaaatttgc gtgtggagta tttggatgac
     3661 agaaacactt ttcgacatag tgtggtggtg ccctatgagc cgcctgaggt ctggtttgca
     3721 actggggtct ctgggaggag gggttaaggg tggttgtcag tggccctcca ggtgagcagt
     3781 aggggggctt tctcctgctg cttatttgac ctccctataa ccccatgaga tgtgcaaagt
     3841 aaatgggttt aactattgca cagttgaaaa aactgaagct tacagaggct aagggcctcc
     3901 cctgcttggc tgggcgcagt ggctcatgcc tgtaatccca gcactttggg aggccaaggc
     3961 aggcggatca cgaggttggg agatcgagac catcctggct aacggtgaaa ccccgtctct
     4021 actgaaaaat acaaaaaaaa attagccggg cgtggtgctg ggcacctgta gtcccagcta
     4081 ctcgggaggc tgaggaagga gaatggcgtg aacctgggcg gtggagcttg cagtgagctg
     4141 agatcacgcc actgcactcc agcctgggcg acagagcgag attccatctc aaaaaaaaaa
     4201 aaaaggcctc ccctgcttgc cacaggtctc cccaaggcgc actggcctca tcttgggcct
     4261 gtgttatctc ctaggttggc tctgactgta ccaccatcca ctacaactac atgtgtaaca
     4321 gttcctgcat gggcagcatg aaccggaggc ccatcctcac catcatcaca ctggaagact
     4381 ccaggtcagg agccacttgc caccctgcac actggcctgc tgtgccccag cctctgcttg
     4441 cctctgaccc ctgggcccac ctcttaccga tttcttccat actactaccc atccacctct
     4501 catcacatcc ccggcgggga atctccttac tgctcccact cagttttctt ttctctggct
     4561 ttgggacctc ttaacctgtg gcttctcctc cacctacctg gagctggagc ttaggctcca
     4621 gaaaggacaa gggtggttgg gagtagatgg agtctggttt tttaaatggg acaggtagga
     4681 cctgatttcc ttactgcctc ttgcttctct tttcctatcc tgagtagtgg taatctactg
     4741 ggacggaaca gctttgaggt gcgtgtttgt gcctgtcctg ggagagaccg gcgcacagag
     4801 gaagagaatc tccgcaagaa aggggagcct caccacgagc tgcccccagg gagcactaag
     4861 cgaggtaagc aagcaggaca agaagcggtg gaggaggcca agggtgcagt tatgcctcag
     4921 attcactttt atcacctttc cttgcctctt tcctagcact gcccaacaac accagctcct
     4981 ctccccagcc aaagaagaaa ccactggatg gagaatattt cacccttcag gtactaagtc
     5041 ttgggacctc ttatcaagtg gaaagtttcc agtctaacac tcaaaatgcc gtgcggccgc
     5101 actagagacg atgtaggtca cggtctcgaa gccgcggtgc gggtgccagg gcgtgccctt
     5161 gggctccccg ggcgcgtact ccacctcacc catctggtcc atcatgatga acgggtcgag
     5221 gtggcggtag ttgatcccgg cgaacgcgcg gcgcaccggg aagccctcgc cctcgaaacc
     5281 gctgggcgcg gtggtcacgg tgagcacggg acgtgcgacg gcgtcggcgg gtgcggatac
     5341 gcggggcagc gtcagcgggt tctcgacggt cacggcgggc atgagctcca gcttttgttc
     5401 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5461 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5521 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5581 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5641 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5701 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5761 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5821 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5881 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5941 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6001 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6061 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6121 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6181 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6241 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6301 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6361 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6421 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6481 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6541 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6601 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6661 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6721 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6781 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6841 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6901 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6961 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7021 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7081 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7141 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7201 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7261 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7321 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7381 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7441 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7501 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7561 tccgcgcaca tttccccgaa aagtgc
//
