LOCUS       sequence-213791-        7209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of PPWD1_Pro-isomerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7209)
  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 7209)
  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_PPWD1_Pro-isomerase
FEATURES             Location/Qualifiers
     source          1..7209
                     /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      1404..1451
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3044..3499)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3181..3202)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3393..3412
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3541..3563)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3649..3789
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3704..3723
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3804..3821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3975..4562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4733..5524,5525..5593))
                     /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     5356..5375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5594..5698)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5766..5784
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5807..5884
                     /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             5885..6967
                     /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(5904..5923)
                     /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 cggccggtcc gaaacgtgtt tctgatagcg caattattca tacttccatg ggtgatatcc
      961 acaccaaact gttcccggtg gaatgtccaa aaactgtgga gaacttttgc gttcacagcc
     1021 gtaacggtta ttacaatggt cacaccttcc accgtatcat taaaggtttt atgatccaga
     1081 ccggtgaccc gaccggcact ggcatgggcg gcgaatccat ctggggtggc gagttcgaag
     1141 acgaattcca ctctaccctg cgtcacgacc gcccgtacac tctgtctatg gcgaacgctg
     1201 gctctaacac taacggctcc caattcttta tcaccgtagt gcctaccccg tggctggata
     1261 ataaacatac cgttttcggc cgtgtcacga agggtatgga agtagttcag cgcatcagca
     1321 acgttaaagt gaacccgaaa accgacaagc cgtacgaaga tgtatccatc attaacatca
     1381 cggttaaagc ggccgcataa taaaccgata caattaaagg ctccttttgg agcctttttt
     1441 ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt tcctttctat
     1501 tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga aaattcattt
     1561 actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg
     1621 tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct agctagagtg
     1681 gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg
     1741 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     1801 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     1861 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     1921 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tccctccctc
     1981 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg
     2041 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     2101 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc
     2161 agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     2221 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     2281 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     2341 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     2401 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2461 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2521 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     2581 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     2641 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     2701 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     2761 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     2821 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     2881 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     2941 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     3001 tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt aagcgttaat
     3061 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3121 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3181 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3241 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3301 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3361 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3421 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3481 gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3541 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3601 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3661 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3721 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3781 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3841 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3901 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3961 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4021 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt ccccctggaa
     4081 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4141 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4201 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4261 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4321 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4381 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4441 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4501 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4561 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4621 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4681 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4741 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4801 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4861 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4921 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4981 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5041 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5101 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5161 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5221 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5281 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5341 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5401 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5461 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5521 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5581 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5641 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5701 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5761 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca ccatcgaatg
     5821 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     5881 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     5941 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     6001 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     6061 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     6121 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     6181 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     6241 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     6301 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     6361 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     6421 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     6481 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     6541 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     6601 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     6661 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     6721 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     6781 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     6841 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     6901 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     6961 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt
     7021 tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     7081 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     7141 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     7201 aatgagctg
//
