LOCUS Exported 6264 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4046619 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6264) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6264) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6264 /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" primer_bind complement(2019..2037) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 2388..3191 /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 3274..4256 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 4334..4363 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 4385..4972 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4984..5011 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(5035..5226) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5227..6087) /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 5850..5869 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6088..6190) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 6191..6222 /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 attgttaata attggttgaa gcagttaatt aaagttacac ttgtgttcct ttttgttgct 541 gctattttct atttaataac acctgttcat gtcatgtcta aacatactga cttttcaagt 601 gaaatcatag gatacaaggc tattgatggt ggtgtcactc gtgacatagc atctacagat 661 acttgttttg ctaacaaaca tgctgatttt gacacatggt ttagccagcg tggtggtagt 721 tatactaatg acaaagcttg cccattgatt gctgcagtca taacaagaga agtgggtttt 781 gtcgtgcctg gtttgcctgg cacgatatta cgcacaacta atggtgactt tttgcatttc 841 ttacctagag tttttagtgc agttggtaac atctgttaca caccatcaaa acttatagag 901 tacactgact ttgcaacatc agcttgtgtt ttggctgctg aatgtacaat ttttaaagat 961 gcttctggta agccagtacc atattgttat gataccaatg tactagaagg ttctgttgct 1021 tatgaaagtt tacgccctga cacacgttat gtgctcatgg atggctctat tattcaattt 1081 cctaacacct accttgaagg ttctgttaga gtggtaacaa cttttgattc tgagtactgt 1141 aggcacggca cttgtgaaag atcagaagct ggtgtttgtg tatctactag tggtagatgg 1201 gtacttaaca atgattatta cagatcttta ccaggagttt tctgtggtgt agatgctgta 1261 aatttactta ctaatatgtt tacaccacta attcaaccta ttggtgcttt ggacatatca 1321 gcatctatag tagctggtgg tattgtagct atcgtagtaa catgccttgc ctactatttt 1381 atgaggttta gaagagcttt tggtgaatac agtcatgtag ttgcctttaa tactttacta 1441 ttccttatgt cattcactgt actctgttta acaccagttt actcattctt acctggtgtt 1501 tattctgtta tttacttgta cttgacattt tatcttacta atgatgtttc ttttttagca 1561 catattcagt ggatggttat gttcacacct ttagtacctt tctggataac aattgcttat 1621 atcatttgta tttccacaaa gcatttctat tggttcttta gtaattacct aaagagacgt 1681 gtagtcttta atggtgtttc ctttagtact tttgaagaag ctgcgctgtg cacctttttg 1741 ttaaataaag aaatgtatct aaagttgcgt agtgatgtgc tattacctct tacgcaatat 1801 aatagatact tagctcttta taataagtac aagtatttta gtggagcaat ggatacaact 1861 agctacagag aagctgcttg ttgtcatctc gcaaaggctc tcaatgactt cagtaactca 1921 ggttctgatg ttctttacca accaccacaa acctctatca cctcagctgt tttgtaaaca 1981 ggcccctttt cctttgtcga tatcatgtaa ttagttatgt cacgcttaca ttcacgccct 2041 cctcccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 2101 atttattttt tttaatagtt atgttagtat taagaacgtt atttatattt caaatttttc 2161 ttttttttct gtacaaagcg gccgccgaac cactagtgca ctgcagtaca cggtttcctt 2221 gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac 2281 ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct 2341 taacccaact gcacagaaca aaaacgtgca ggaaacgaag ataaatcatg tcgaaagcta 2401 catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca 2461 tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac 2521 tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatct 2581 tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca 2641 attttttact cttcgaggac agaaaatttg ctgacattgg taatacagtc aaattgcagt 2701 actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgg 2761 tgggcccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta 2821 gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata 2881 ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc 2941 aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg 3001 gtttagatga caagggagat gcattgggtc aacagtatag aaccgtggat gatgtggttt 3061 ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta 3121 aggtagaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc 3181 agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag 3241 cttcaattta attatatcag ttattacccg agtaacgaag catctgtgct tcattttgta 3301 gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 3361 acagaacaga aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg tgcttcattt 3421 ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca 3481 tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt 3541 cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga 3601 ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt 3661 aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag 3721 cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag 3781 ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga 3841 aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt 3901 gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta 3961 tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa 4021 atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat 4081 agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg 4141 gtattcgcaa tattttagta gctcgttaca gtccggtgcg tttttggttt tttgaaagtg 4201 cgtcatcaga gcgcttttgg ttttcaaaag cgctctgaag ttcctatact ttctagctag 4261 agaataggaa cttcccgagc ggccgcgtgt tacaagcttg actccagcgt aactggactg 4321 caatcaactc actggctcac cttcacgggt gggcctttct tcggtagaaa atcaaaggat 4381 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4441 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aggtaactgg 4501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 4561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4861 ggagcttcca ggggtaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4921 acttgagcat cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4981 caacgcagaa aggcccaccc gaaggtgagc caggtgatta catttgggcc ctcatcagag 5041 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 5101 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 5161 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 5221 ggtcacttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 5281 atccatagtt gcctgactcc ccgtcgtgta gataactacg atgcgggagg gcttaccatc 5341 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 5401 aataaaccag ccagccggga gggccgagcg cagaagtgat cctgcaactt tatccgcctc 5461 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 5521 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 5581 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 5641 aaaagcggtt agctccttcg gtcctccgat cgttgccaga agtaagttgg ccgcagtgtt 5701 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtgagatg 5761 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 5821 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 5881 agtgctcatc attggaaaac gttcttcggg gcgtaaactc tcaaggatct taccgctgtt 5941 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 6001 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 6061 ggcgacacgg aaatgttgaa tactcatttt agcttcctta gctcctgaaa atctcgataa 6121 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 6181 gtgccgatca agtcaaaagc ctccggtcgg aggcttttga ctttctgcta tggaggtcag 6241 gtatgattta aatggtcagt taga //