LOCUS Exported 5724 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4046332 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5724) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5724) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5724 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 25..466 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 25..142 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" primer_bind 437..460 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" CDS 1399..1416 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1417..1434 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1479..1497) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 1848..2651 /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" rep_origin 2734..3716 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 3794..3823 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 3845..4432 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4444..4471 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(4495..4686) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4687..5547) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5310..5329 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5548..5650) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 5651..5682 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" ORIGIN 1 gcggccgctt gctatattga agtacggatt agaagccgcc gagcgggcga cagccctccg 61 acggaagact ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg 121 tgcctcgcgc cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat 181 gaagaggaaa aattggcagt aacctggccc cacaaacctt caaattaacg aatcaaatta 241 acaaccatag gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag 301 cgaagcgatg atttttgatc tattaacaga tatataaatg gaaaagctgc ataaccactt 361 taactaatac tttcaacatt ttcagtttgt attacttctt attcaaatgt cataaaagta 421 tcaacaaaaa attgttaata tacctctata ctttaacgtc aaggagaaaa aactataatg 481 tcgggtttca gaaaaatggc ctttcctagt ggaaaggtcg agggctgtat ggtgcaagta 541 acatgcggta cgaccacttt gaacggttta tggcttgacg atgttgttta ctgtccacgt 601 catgtcatct gtacttcaga agatatgttg aatccaaact atgaagacct actgattcgc 661 aagtctaacc acaatttctt agttcaggct ggtaacgttc aattgagagt tataggtcac 721 agcatgcaaa attgtgtctt gaagttaaaa gttgataccg caaacccgaa gactccaaag 781 tacaaatttg tcagaatcca acccggtcaa accttctccg tcttggcttg ttataacggg 841 tccccatctg gtgtatacca gtgcgctatg aggccaaact tcactattaa gggttctttt 901 ttgaatggtt cttgtggctc cgttggtttt aatatcgact acgattgcgt cagtttctgt 961 tacatgcatc acatggaatt gcctacaggt gttcacgccg gaaccgacct agaagggaac 1021 ttctatggtc cattcgttga tcggcaaact gctcaagctg cgggtacgga taccacaatt 1081 actgtaaatg tgcttgcctg gttgtacgct gctgttataa acggtgaccg atggttttta 1141 aacagattca ctaccacatt gaatgacttt aatctggtcg ctatgaaata taactacgaa 1201 ccactcactc aagatcatgt cgatatttta ggtcctttgt ctgcacaaac tggcatcgct 1261 gttttggaca tgtgtgcgtc ccttaaggaa ctactccaaa acggtatgaa cgggagaacg 1321 attttgggtt cagccctatt ggaggacgaa tttaccccat tcgatgtggt tagacagtgc 1381 tctggagtaa ccttccaact cgtgccgaga ggttctcacc atcatcacca tcattaaaca 1441 ggcccctttt cctttgtcga tatcatgtaa ttagttatgt cacgcttaca ttcacgccct 1501 cctcccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 1561 atttattttt tttaatagtt atgttagtat taagaacgtt atttatattt caaatttttc 1621 ttttttttct gtacaaagcg gccgccgaac cactagtgca ctgcagtaca cggtttcctt 1681 gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac 1741 ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct 1801 taacccaact gcacagaaca aaaacgtgca ggaaacgaag ataaatcatg tcgaaagcta 1861 catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca 1921 tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac 1981 tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatct 2041 tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca 2101 attttttact cttcgaggac agaaaatttg ctgacattgg taatacagtc aaattgcagt 2161 actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgg 2221 tgggcccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta 2281 gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata 2341 ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc 2401 aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg 2461 gtttagatga caagggagat gcattgggtc aacagtatag aaccgtggat gatgtggttt 2521 ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta 2581 aggtagaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc 2641 agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag 2701 cttcaattta attatatcag ttattacccg agtaacgaag catctgtgct tcattttgta 2761 gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 2821 acagaacaga aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg tgcttcattt 2881 ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca 2941 tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt 3001 cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga 3061 ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt 3121 aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag 3181 cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag 3241 ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga 3301 aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt 3361 gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta 3421 tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa 3481 atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat 3541 agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg 3601 gtattcgcaa tattttagta gctcgttaca gtccggtgcg tttttggttt tttgaaagtg 3661 cgtcatcaga gcgcttttgg ttttcaaaag cgctctgaag ttcctatact ttctagctag 3721 agaataggaa cttcccgagc ggccgcgtgt tacaagcttg actccagcgt aactggactg 3781 caatcaactc actggctcac cttcacgggt gggcctttct tcggtagaaa atcaaaggat 3841 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 3901 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aggtaactgg 3961 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 4021 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4081 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4141 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4201 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4261 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4321 ggagcttcca ggggtaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4381 acttgagcat cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4441 caacgcagaa aggcccaccc gaaggtgagc caggtgatta catttgggcc ctcatcagag 4501 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 4561 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 4621 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 4681 ggtcacttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 4741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atgcgggagg gcttaccatc 4801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 4861 aataaaccag ccagccggga gggccgagcg cagaagtgat cctgcaactt tatccgcctc 4921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 4981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 5041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 5101 aaaagcggtt agctccttcg gtcctccgat cgttgccaga agtaagttgg ccgcagtgtt 5161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtgagatg 5221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 5281 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 5341 agtgctcatc attggaaaac gttcttcggg gcgtaaactc tcaaggatct taccgctgtt 5401 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 5461 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 5521 ggcgacacgg aaatgttgaa tactcatttt agcttcctta gctcctgaaa atctcgataa 5581 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 5641 gtgccgatca agtcaaaagc ctccggtcgg aggcttttga ctttctgcta tggaggtcag 5701 gtatgattta aatggtcagt taga //