LOCUS       sequence-333228-        6121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a PLpro substrate (GST-PLproCS-MBP) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6121)
  AUTHORS   Lim CT, Tan KW, Wu M, Ulferts R, Armstrong LA, Ozono E, Drury LS, 
            Milligan JC, Zeisner TU, Zeng J, Weissmann F, Canal B, Bineva-Todd 
            G, Howell M, O'Reilly N, Beale R, Kulathu Y, Labib K, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of Nsp3 papain-like protease.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2517-2531. doi: 10.1042/BCJ20210244.
  PUBMED    34198325
REFERENCE   2  (bases 1 to 6121)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2021-07-16- 16"; volume: "478"; issue: "13"; 
            pages: "2517-2531"
COMMENT     Sequence Label: pGEX4T1_GST-PLproCS-MBP
FEATURES             Location/Qualifiers
     source          1..6121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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     219..235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     219..235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             231..758
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(251..268)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(251..267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(260..282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(860..865,866..881,882..888)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    896..912
                     /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)."
     CDS             935..1588
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1546..1568
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1595..1612
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1652..1669
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1679..2779
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2753..2776
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(2848..2870)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3015..3033)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3101..3205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3206..3274,3275..4066)
                     /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     complement(3424..3443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4237..4825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4726..4745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4979..4996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5069..5146
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5147..6121,1..108)
                     /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(5166..5185)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      241 ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
      301 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
      361 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt
      421 ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata
      481 ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg
      541 tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt
      601 actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt
      661 ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc
      721 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
      781 tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca
      841 aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
      901 agcggataac aatttcacac aggaaacagt attcatgtcc cctatactag gttattggaa
      961 aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga
     1021 agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg
     1081 tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat
     1141 ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg
     1201 tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat
     1261 tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat
     1321 gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac
     1381 ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg
     1441 cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat
     1501 tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac
     1561 gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt ccgcgtggat ccactctgaa
     1621 aggcggcgct cccaccaaag tgaaatcttc tcaccatcac catcaccatg gttcttctat
     1681 gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct
     1741 cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc
     1801 ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat
     1861 cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac
     1921 cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa
     1981 cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga
     2041 tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa
     2101 agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct
     2161 gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga
     2221 cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa
     2281 aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg
     2341 cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt
     2401 gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg
     2461 cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct
     2521 cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg
     2581 tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac
     2641 tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg
     2701 gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc
     2761 cctgaaagac gcgcagactt aactcgagcg gccgcatcgt gactgactga cgatctgcct
     2821 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2881 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2941 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     3001 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     3061 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3121 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3181 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3241 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3301 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3361 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3421 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3481 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3541 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3601 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3661 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3721 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3781 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3841 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3901 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3961 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4021 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4081 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4141 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4201 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4261 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4321 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4381 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4441 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4501 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4561 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4621 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4681 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4741 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4801 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4861 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4921 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4981 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5041 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     5101 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     5161 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     5221 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     5281 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     5341 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     5401 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     5461 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     5521 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     5581 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     5641 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     5701 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     5761 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     5821 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     5881 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     5941 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     6001 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     6061 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     6121 a
//
