LOCUS       sequence-215934-        7509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of PCNA_PCNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7509)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7509)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_PCNA_PCNA
FEATURES             Location/Qualifiers
     source          1..7509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     primer_bind     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1704..1751
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3344..3799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3481..3502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3693..3712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3841..3863)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3949..4089
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4004..4023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4104..4121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4275..4862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5033..5824,5825..5893))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5656..5675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5894..5998)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6066..6084
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6107..6184
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6185..7267
                     /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"
     primer_bind     complement(6204..6223)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccatgtt tgaagcgcgt ctggttcagg gctctattct gaaaaaagtc ctggaagccc
      961 tgaaagatct gattaacgaa gcatgctggg acatcagctc tagtggtgtt aatctgcagt
     1021 ccatggattc ctcacatgtg tcactggttc aactgaccct gcgtagcgaa ggctttgata
     1081 cgtatcgttg cgaccgcaac ctggctatgg gtgtcaatct gacctctatg agtaaaatcc
     1141 tgaaatgtgc gggcaacgaa gatattatca ccctgcgcgc ggaagataat gccgacacgc
     1201 tggcactggt cttcgaagct ccgaatcagg aaaaagtgtc cgattatgaa atgaaactga
     1261 tggatctgga cgtggaacag ctgggtattc cggaacaaga atactcgtgc gtggttaaaa
     1321 tgccgagcgg cgaatttgcc cgtatctgtc gcgatctgtc tcatattggt gacgctgtcg
     1381 tgatcagttg tgcgaaagat ggcgtgaaat tctccgcctc aggtgaactg ggcaacggta
     1441 atattaaact gtctcagacc agtaacgtcg acaaagaaga agaagcggtg accatcgaaa
     1501 tgaatgaacc ggttcaactg acgttcgctc tgcgctatct gaactttttc accaaagcaa
     1561 cgccgctgtc gagcaccgtg acgctgtcga tgagcgcgga tgttccgctg gttgtcgaat
     1621 acaaaattgc cgacatgggc cacctgaaat attacctggc accgaaaatc gaagatgaag
     1681 aaggtagcgc ggccgcataa taaaccgata caattaaagg ctccttttgg agcctttttt
     1741 ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt tcctttctat
     1801 tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga aaattcattt
     1861 actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg
     1921 tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct agctagagtg
     1981 gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg
     2041 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     2101 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     2161 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     2221 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tccctccctc
     2281 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg
     2341 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     2401 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc
     2461 agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     2521 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     2581 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     2641 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     2701 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2761 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2821 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     2881 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     2941 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     3001 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     3061 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     3121 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     3181 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     3241 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     3301 tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt aagcgttaat
     3361 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3421 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3481 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3541 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3601 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3661 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3721 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3781 gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3841 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3901 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3961 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     4021 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4081 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt ccccctggaa
     4381 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4441 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4501 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4561 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4621 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4681 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4741 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4801 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4861 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4921 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4981 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5041 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5101 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5161 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5221 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5281 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5341 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5401 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5461 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5521 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5581 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5641 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5701 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5761 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5821 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5881 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6001 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6061 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca ccatcgaatg
     6121 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     6181 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     6241 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     6301 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     6361 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     6421 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     6481 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     6541 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     6601 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     6661 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     6721 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     6781 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     6841 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     6901 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     6961 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     7021 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     7081 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     7141 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     7201 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7261 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt
     7321 tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     7381 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     7441 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     7501 aatgagctg
//
