LOCUS       sequence-66356-e        7590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOP Hupki-puro2A-Hupki-wt plasmid .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7590)
  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 7590)
  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: pX61
FEATURES             Location/Qualifiers
     source          1..7590
                     /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    5129..5198
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(5405..5423)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5444..5460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5444..5460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5457..5479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5468..5484
                     /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(5492..5498,5499..5516,5517..5522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5537..5558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5675..5692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5846..6434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5926..5945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6605..7396,7397..7465))
                     /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     7228..7247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7466..7570)
                     /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 gagcagaaag 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 agccactcag gaggctgagg
     3061 tgggaagatc acttgaggcc aggagatgga ggctgcagtg agctgtgatc acaccactgt
     3121 gctccagcct gagtgacaga gcaagaccct atctcaaaaa aaaaaaaaaa aagaaaagct
     3181 cctgaggtgt agacgccaac tctctctagc tcgctagtgg gttgcaggag gtgcttacac
     3241 atgtttgttt ctttgctgcc gtcttccagt tgctttatct gttcacttgt gccctgactt
     3301 tcaactctgt ctccttcctc ttcctacagt actcccctgc cctcaacaag atgttttgcc
     3361 aactggccaa gacctgccct gtgcagctgt gggttgattc cacacccccg cccggcaccc
     3421 gcgtccgcgc catggccatc tacaagcagt cacagcacat gacggaggtt gtgaggcgct
     3481 gcccccacca tgagcgctgc tcagatagcg atggtgagca gctggggctg gagagacgac
     3541 agggctggtt gcccagggtc cccaggcctc tgattcctca ctgattgctc ttaggtctgg
     3601 cccctcctca gcatcttatc cgagtggaag gaaatttgcg tgtggagtat ttggatgaca
     3661 gaaacacttt tcgacatagt gtggtggtgc cctatgagcc gcctgaggtc tggtttgcaa
     3721 ctggggtctc tgggaggagg ggttaagggt ggttgtcagt ggccctccag gtgagcagta
     3781 ggggggcttt ctcctgctgc ttatttgacc tcccctccct ataaccccat gagatgtgca
     3841 aagtaaatgg gtttaactat tgcacagttg aaaaaactga agcttacaga ggctaagggc
     3901 ctcccctgct tggctgggcg cagtggctca tgcctgtaat cccagcactt tgggaggcca
     3961 aggcaggcgg atcacgaggt tgggagatcg agaccatcct ggctaacggt gaaaccccgt
     4021 ctctactgaa aaatacaaaa aaaaattagc cgggcgtggt gctgggcacc tgtagtccca
     4081 gctactcggg aggctgagga aggagaatgg cgtgaacctg ggcggtggag cttgcagtga
     4141 gctgagatca cgccactgca ctccagcctg ggcgacagag cgagattcca tctcaaaaaa
     4201 aaaaaaaagg cctcccctgc ttgccacagg tctccccaag gcgcactggc ctcatcttgg
     4261 gcctgtgtta tctcctaggt tggctctgac tgtaccacca tccactacaa ctacatgtgt
     4321 aacagttcct gcatgggcgg catgaaccgg aggcccatcc tcaccatcat cacactggaa
     4381 gactccaggt caggagccac ttgccaccct gcacactggc ctgctgtgcc ccagcctctg
     4441 cttgcctctg acccctgggc ccacctctta ccgatttctt ccatactact acccatccac
     4501 ctctcatcac atccccggcg gggaatctcc ttactgctcc cactcagttt tcttttctct
     4561 ggctttggga cctcttaacc tgtggcttct cctccaccta cctggagctg gagcttaggc
     4621 tccagaaagg acaagggtgg ttgggagtag atggagtctg gttttttaaa tgggacaggt
     4681 aggacctgat ttccttactg cctcttgctt ctcttttcct atcctgagta gtggtaatct
     4741 actgggacgg aacagctttg aggtgcgtgt ttgtgcctgt cctgggagag accggcgcac
     4801 agaggaagag aatctccgca agaaagggga gcctcaccac gagctgcccc cagggagcac
     4861 taagcgaggt aagcaagcag gacaagaagc ggtggaggag gccaagggtg cagttatgcc
     4921 tcagattcac ttttatcacc tttccttgcc tctttcctag cactgcccaa caacaccagc
     4981 tcctctcccc agccaaagaa gaaaccactg gatggagaat atttcaccct tcaggtacta
     5041 agtcttggga cctcttatca agtggaaagt ttccagtcta acactcaaaa tgccgtgcgg
     5101 ccgcactaga gacgatgtag gtcacggtct cgaagccgcg gtgcgggtgc cagggcgtgc
     5161 ccttgggctc cccgggcgcg tactccacct cacccatctg gtccatcatg atgaacgggt
     5221 cgaggtggcg gtagttgatc ccggcgaacg cgcggcgcac cgggaagccc tcgccctcga
     5281 aaccgctggg cgcggtggtc acggtgagca cgggacgtgc gacggcgtcg gcgggtgcgg
     5341 atacgcgggg cagcgtcagc gggttctcga cggtcacggc gggcatgagc tccagctttt
     5401 gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg
     5461 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5521 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5581 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5641 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5701 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5761 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5821 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5881 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5941 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6001 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6061 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6121 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6181 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6241 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     6301 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6361 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6421 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6481 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6541 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6601 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6661 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6721 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6781 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6841 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6901 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6961 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7021 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7081 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7141 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7201 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7261 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7321 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7381 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7441 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7501 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7561 gggttccgcg cacatttccc cgaaaagtgc
//
