LOCUS       sequence-344828-        7221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Full-length P53 in E.coli with cleavable 
            expression/purification tag (ribose binding protein circular 
            permutant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  AUTHORS   Ha JH, Yu X, Carpizo DR, Loh SN
  TITLE     Urea Denaturation, Zinc Binding, and DNA Binding Assays of Mutant 
            p53 DNA-binding Domains and Full-length Proteins.
  JOURNAL   Bio Protoc. 2021 Oct 20;11(20):e4188. doi: 10.21769/BioProtoc.4188. 
            eCollection 2021 Oct 20.
  PUBMED    34786438
REFERENCE   2  (bases 1 to 7221)
  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: "Bio 
            Protoc."; date: "2021-10-20"; volume: "11(20):e4188. doi"; pages: " 
            10.21769/BioProtoc.4188. eCollection 2021 Oct 20"
COMMENT     Sequence Label: pKan cpRBP-FL P53
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     966..985
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1005..1082)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1288..1307
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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)."
     RBS             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1495..1512
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2113..2136
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2149..3327
                     /codon_start=1
                     /gene="Homo sapiens TP53"
                     /product="human tumor suppressor protein that is often 
                     mutated in cancers"
                     /label=p53
                     /note="responds to cellular stresses to regulate the 
                     expression of target genes"
                     /translation="MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLML
                     SPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQ
                     GSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIY
                     KQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEP
                     PEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRD
                     RRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEM
                     FRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD"
     CDS             3343..3366
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3697..3720
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3783..3801)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3797..3844
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3881..4336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3968..3987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4178..4199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4428..5243)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4428..5243)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5151..5170
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5235..5254
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5365..5953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5854..5873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6107..6124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6139..6278
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6204..6223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6364..6386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6380..6571)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7143..7221,1..1004))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
       61 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
      121 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
      181 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
      241 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
      301 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
      361 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
      421 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
      481 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      541 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      601 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      661 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      721 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      781 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      841 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
      901 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
      961 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     1021 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     1081 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1141 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     1201 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     1261 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     1321 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatgggtag ctcacaccat
     1501 caccatcacc attcgagctc gggcgacgta gtgtcgcata tcgcaagcga taacgtcaag
     1561 ggaggtgaga tggctgccga gttcattgcg aaagccttaa agggcaaagg gaacgtcgtg
     1621 gaattagagg gcatcccagg agcgtcggca gctcgtgacc gcggaaaagg gtttgacgag
     1681 gctattgcta agtacccaga catcaagatc gtggccaaac aagccgccga ctttgatcgc
     1741 agtaagggtt tgagcgttat ggaaaacatc ttacaggcgc aaccgaaaat tgatgcagtg
     1801 ttcgcgcaaa atgatgagat ggctttgggt gcaattaaag caattgaggc tgcgaaccgc
     1861 caaggtatta tcgtggtcgg cttcgacggt acagaggacg cgttgaaggc gattaaagaa
     1921 gggaagatgg cggcgaccat tgcacaacag ccggccttga tgggctcgct tggggtcgag
     1981 atggctgata aatatcttaa gggcgaaaaa atccctaatt ttatccccgc tgaattgaaa
     2041 ttgatcacta aagaaaatgt gcaagggggt tctgcctctg gagggacaag tggtggatcg
     2101 agtgcggccg gtttggaggt tttattccag ggtccagcgg ccgcaggcat ggaggagccg
     2161 cagtcagatc ctagcgtcga gccccctctg agtcaggaaa cattttcaga cctatggaaa
     2221 ctacttcctg aaaacaacgt tctgtccccc ttgccgtccc aagcaatgga tgatttgatg
     2281 ctgtccccgg acgatattga acaatggttc actgaagacc caggtccaga tgaagctccc
     2341 agaatgccag aggctgctcc ccccgtggcc cctgcaccag cagctcctac accggcggcc
     2401 cctgcaccag ccccctcctg gcccctgtca tcttctgtcc cttcccagaa aacctaccag
     2461 ggcagctacg gtttccgtct gggcttcttg cattctggga cagccaagtc tgtgacttgc
     2521 acgtactccc ctgccctcaa caagatgttt tgccaactgg ccaagacctg ccctgtgcag
     2581 ctgtgggttg attccacacc cccgcccggc acccgcgtcc gcgccatggc catctacaag
     2641 cagtcacagc acatgacgga ggttgtgagg cgctgccccc accatgagcg ctgctcagat
     2701 agcgatggtc tggcccctcc tcagcatctt atccgagtgg aaggaaattt gcgtgtggag
     2761 tatttggatg acagaaacac ttttcgacat agtgtggtgg tgccctatga gccgcctgag
     2821 gttggctctg actgtaccac catccactac aactacatgt gtaacagttc ctgcatgggc
     2881 ggcatgaacc ggaggcccat cctcaccatc atcacactgg aagactccag tggtaatcta
     2941 ctgggacgga acagctttga ggtgcgtgtt tgtgcctgtc ctgggagaga ccggcgcaca
     3001 gaggaagaga atctccgcaa gaaaggggag cctcaccacg agctgccccc agggagcact
     3061 aagcgagcac tgcccaacaa caccagctcc tctccccagc caaagaagaa accactggat
     3121 ggagaatatt tcacccttca gatccgtggg cgtgagcgct tcgagatgtt ccgagagctg
     3181 aatgaggcct tggaactcaa ggatgcccag gctgggaagg agccaggggg gagcagggct
     3241 cactccagcc acctgaagtc caaaaagggt cagtctacct cccgccataa aaaactcatg
     3301 ttcaagacag aagggcctga ctcagacggc ggatccggtg gcttagaagt attattccaa
     3361 ggtccaggtt cgtcaggagg gacggcatct ggagggaaag aaggcaaaac catcggtctg
     3421 gtcatcagta ccttgaataa ccccttcttt gtaaccttga aaaatggcgc tgaggagaaa
     3481 gcgaaggaac tgggatacaa gatcatcgta gaggattccc agaatgactc ctcgaaggag
     3541 ttatccaacg ttgaggattt aattcaacag aaggtcgacg tactgctgat taaccctgta
     3601 gattcggatg ctgttgtaac cgccattaag gaagcaaata gcaagaacat tccagttatt
     3661 actattgatc gctcggccaa cgggtaatga ctcgagcacc accaccacca ccaccaccac
     3721 taattgatta atacctaggc tgctaaacaa agcccgaaag gaagctgagt tggctgctgc
     3781 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3841 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     3901 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3961 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4021 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4081 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4141 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4201 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4261 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4321 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4381 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     4441 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     4501 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     4561 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     4621 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     4681 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     4741 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     4801 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     4861 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     4921 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     4981 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     5041 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     5101 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     5161 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     5221 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     5281 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6001 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6061 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6121 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6181 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     6241 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     6301 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6361 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     6421 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     6481 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     6541 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     6601 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     6661 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     6721 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     6781 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     6841 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     6901 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     6961 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     7021 cgatcatgct agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     7081 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     7141 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     7201 aacgcgcggg gagaggcggt t
//
