LOCUS Exported 6121 bp ds-DNA circular SYN 19-MAY-2021 DEFINITION To express a PLpro substrate (GST-PLproCS-MBP) in E. coli. ACCESSION . VERSION . KEYWORDS pGEX4T1_GST-PLproCS-MBP 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, Binerva-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 bioRxiv https://www.biorxiv.org/content/10.1101/2021.04.07.438804v1 REFERENCE 2 (bases 1 to 6121) AUTHORS . TITLE Direct Submission JOURNAL Exported May 19, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6121 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 157..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 860..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 3206..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 5147..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 //