LOCUS Exported 7590 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION TOP Hupki-puro2A-Hupki-wt plasmid . ACCESSION . VERSION . KEYWORDS pX61 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7590 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(6..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(5492..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(6605..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 //