LOCUS Exported 5730 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 3C-like proteinase. ACCESSION . VERSION . KEYWORDS pGBW-m4046400 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5730) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5730) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5730 /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..1440 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" primer_bind complement(1485..1503) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 1854..2657 /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 2740..3722 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 3800..3829 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 3851..4438 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4450..4477 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(4501..4692) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4693..5553) /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 5316..5335 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5554..5656) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 5657..5688 /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 ggttcttgga gccatccgca gtttgaaaaa 1441 taaacaggcc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca 1501 cgccctcctc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 1561 gtccctattt atttttttta atagttatgt tagtattaag aacgttattt atatttcaaa 1621 tttttctttt ttttctgtac aaagcggccg ccgaaccact agtgcactgc agtacacggt 1681 ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga aggaaggagc 1741 acagacttag attggtatat atacgcatat gtagtgttga agaaacatga aattgcccag 1801 tattcttaac ccaactgcac agaacaaaaa cgtgcaggaa acgaagataa atcatgtcga 1861 aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc aagctattta 1921 atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt accaccaagg 1981 aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa acacatgtgg 2041 atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca ttatccgcca 2101 agtacaattt tttactcttc gaggacagaa aatttgctga cattggtaat acagtcaaat 2161 tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg aatgcacacg 2221 gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa gtaacaaagg 2281 aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta tctactggag 2341 aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt atcggcttta 2401 ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt atgacacccg 2461 gtgtgggttt agatgacaag ggagatgcat tgggtcaaca gtatagaacc gtggatgatg 2521 tggtttctac aggatctgac attattattg ttggaagagg actatttgca aagggaaggg 2581 atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat ttgagaagat 2641 gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta aactcacaaa 2701 ttagagcttc aatttaatta tatcagttat tacccgagta acgaagcatc tgtgcttcat 2761 tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 2821 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct 2881 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga 2941 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct 3001 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat 3061 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg 3121 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa 3181 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt 3241 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg 3301 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc 3361 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc 3421 actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta gagataaaca 3481 taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg gatgggtagg 3541 ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc aatgtttgtg 3601 gaagcggtat tcgcaatatt ttagtagctc gttacagtcc ggtgcgtttt tggttttttg 3661 aaagtgcgtc atcagagcgc ttttggtttt caaaagcgct ctgaagttcc tatactttct 3721 agctagagaa taggaacttc ccgagcggcc gcgtgttaca agcttgactc cagcgtaact 3781 ggactgcaat caactcactg gctcaccttc acgggtgggc ctttcttcgg tagaaaatca 3841 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3901 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaggt 3961 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 4021 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 4081 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 4141 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 4201 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 4261 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4321 cacgagggag cttccagggg taaacgcctg gtatctttat agtcctgtcg ggtttcgcca 4381 cctctgactt gagcatcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 4441 cgccagcaac gcagaaaggc ccacccgaag gtgagccagg tgattacatt tgggccctca 4501 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 4561 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 4621 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 4681 ctgtttggtc acttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 4741 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatgc gggagggctt 4801 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 4861 atcagcaata aaccagccag ccgggagggc cgagcgcaga agtgatcctg caactttatc 4921 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 4981 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 5041 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 5101 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gccagaagta agttggccgc 5161 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 5221 gagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 5281 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 5341 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcgt aaactctcaa ggatcttacc 5401 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 5461 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 5521 aataagggcg acacggaaat gttgaatact cattttagct tccttagctc ctgaaaatct 5581 cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc 5641 tcttacgtgc cgatcaagtc aaaagcctcc ggtcggaggc ttttgacttt ctgctatgga 5701 ggtcaggtat gatttaaatg gtcagttaga //