LOCUS       sequence-215111-        7407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of PCMT1_PCMT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  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 7407)
  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_PCMT1_PCMT
FEATURES             Location/Qualifiers
     source          1..7407
                     /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      1602..1649
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3242..3697)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3379..3400)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3591..3610
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3739..3761)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3847..3987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3902..3921
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4002..4019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4173..4760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4931..5722,5723..5791))
                     /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     5554..5573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5792..5896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5964..5982
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6005..6082
                     /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             6083..7165
                     /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(6102..6121)
                     /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 cggccatggc atggaaaagc ggtggtgcaa gccatagcga actgattcat aatctgcgta
      961 aaaacggcat catcaaaacc gataaagtgt ttgaagttat gctggcaacc gatcgtagcc
     1021 attatgcaaa atgtaatccg tatatggata gtccgcagag cattggtttt caggcaacca
     1081 ttagcgcacc gcatatgcac gcctatgcac tggaactgct gtttgatcag ctgcatgaag
     1141 gtgcaaaagc actggatgtt ggtagcggta gtggtattct gaccgcatgt tttgcacgta
     1201 tggttggttg taccggtaaa gtgattggca tcgatcatat caaagaactg gttgatgata
     1261 gcgtgaacaa cgtgcgtaaa gatgatccga ccctgctgag tagcggtcgt gttcagctgg
     1321 ttgttggtga tggtcgtatg ggttatgccg aagaggcacc gtatgatgca attcatgttg
     1381 gtgcagcagc accggttgtt ccgcaggcac tgattgatca actgaaaccg ggtggtcgtc
     1441 tgattctgcc ggtgggtccg gcaggcggta atcagatgct ggaacagtat gataaactgc
     1501 aggatggctc cattaaaatg aaaccgctga tgggtgttat ttatgttccg ctgacagaca
     1561 aagaaaaaca gtggtcacgt tggaaagcgg ccgcataata aaccgataca attaaaggct
     1621 ccttttggag cctttttttt ggagattttc aacgtgaaaa aattattatt cgcaattcct
     1681 ttagttgttc ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc
     1741 catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct
     1801 aactatgagg gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact
     1861 cagtgtctag ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg
     1921 caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg
     1981 ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg
     2041 gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt
     2101 cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat
     2161 atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac
     2221 catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc tttgcgtttc
     2281 ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata
     2341 aggagtctta atcatgccag ttcttttggc tagcgccgcc ctataccttg tctgcctccc
     2401 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac
     2461 ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg
     2521 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc
     2581 acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg
     2641 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2701 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     2761 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     2821 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     2881 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     2941 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3001 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3061 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3121 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3181 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg caggatccgc
     3241 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3301 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3361 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3421 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3481 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3541 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3601 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3661 acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc tcgcgcgttt
     3721 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3781 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3841 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3901 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     3961 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     4021 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4081 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4141 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4201 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4261 cagggtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4321 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4381 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4441 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4501 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4561 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4621 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4681 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4741 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4801 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4861 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4921 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4981 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5041 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5101 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5161 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5221 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     5281 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5341 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5401 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5461 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5521 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     5581 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5641 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5701 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5761 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5821 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5881 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5941 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     6001 tcccgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     6061 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     6121 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     6181 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     6241 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     6301 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     6361 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     6421 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     6481 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     6541 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     6601 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     6661 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     6721 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     6781 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     6841 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     6901 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     6961 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     7021 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     7081 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     7141 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     7201 aggcacaatt ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg
     7261 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     7321 cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg
     7381 ttctggcaaa tattctgaaa tgagctg
//
