LOCUS Exported 6270 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yeast expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4046697 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6270) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6270) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6270 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(316..1119) /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" primer_bind 1470..1488 /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(3047..3488) /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" primer_bind complement(3053..3076) /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" protein_bind 3371..3488 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" terminator 3555..3586 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" promoter 3587..3689 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 3690..4550 /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 complement(3908..3927) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" CDS 4551..4742 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" terminator 4766..4793 /label=T7Te terminator /note="phage T7 early transcription terminator" rep_origin complement(4805..5392) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 5414..5443 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 5521..233 /label=2u ori /note="yeast 2u plasmid origin of replication" ORIGIN 1 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc 61 tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc 121 gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 181 agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttactcggg 241 taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac 301 ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc 361 ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc 421 aacaataata atgtcagatc ctgtagaaac cacatcatcc acggttctat actgttgacc 481 caatgcatct cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc 541 ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct 601 cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga 661 caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt 721 caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc 781 tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa 841 ttttctgtcc tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac 901 tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt 961 gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc 1021 acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg 1081 atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca 1141 cgtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact 1201 acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc 1261 ggagattacc gaatcaaaaa aatttcaagg aaaccgtgta ctgcagtgca ctagtggttc 1321 ggcggccgct ttgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac 1381 taacataact attaaaaaaa ataaataggg acctagactt caggttgtct aactccttcc 1441 ttttcggtta gagcggatgt gggaggaggg cgtgaatgta agcgtgacat aactaattac 1501 atgatatcga caaaggaaaa ggggcctgtt tattgcaaaa cagcggaggt aatagacgtc 1561 tgaggtggtt gatacaagac gtcggaaccg gaattagaga aatcattcaa agccttagca 1621 aggtgacaac aagccgcttc acggtatgaa gtagtatcca tggcacccga gaagtatttg 1681 tacttgttgt ataaagctaa atatctgtta tattgggtta gcgggagtaa aacgtcagat 1741 ctcaacttca agtacatttc tttgttgagc aaaaaggtac acaaggcagc ttcttcgaag 1801 gtagaaaagc tgacaccatt aaagacaact cttctcttta gataatttga gaaaaaccaa 1861 tagaaatgtt tagtacttat gcaaatgatg taagcgatcg ttatccagaa gggaaccaat 1921 ggggtgaaca ttaccatcca ttgaatgtgt gccaaaaagc tgacatcgtt agtcaggtag 1981 aatgtcaggt ataaatagat aacggagtat acaccaggca agaaagagta tactggtgta 2041 agacacaaaa cagtgaacga catcaaaaat aatagagtgt tgaaagccac gacatgcgaa 2101 tactccccaa aggctcttct aaatctcatg aagtagtaag ccaaacaagt aacaacgata 2161 gcaacgattc caccggcgac aatagatgcg gaaatgtcca gcgcacctat tggttggatc 2221 aaaggggtaa acatgttagt caatagatta acagcatcga caccgcagaa gacacctgga 2281 agagaacggt aataatcgtt attcaacacc catctaccgc tggtagaaac acagacaccg 2341 gcttctgatc tttcacaagt accgtggcga cagtactcgg agtcgaaggt ggtgacaacc 2401 cgaacagaac cttcaaggta tgtgtttgga aattgaatga ttgatccatc cattaacaca 2461 tatctagtgt ccggtcgcaa ggattcgtaa gcaacagaac cttcgaggac gtttgtgtca 2521 taacagtatg gaactggctt accacttgca tccttgaaaa tagtgcattc agcagccaat 2581 acacaagcag aggtggcaaa atcagtgtat tcgatcaatt tgctaggggt ataacaaata 2641 ttgccaacag cagagaagac tcttggtaag aaatgcaaaa aatcaccatt ggtggtacgc 2701 agtatagtcc ctggtaaacc tggcacgacg aaaccaactt ctctagtgat aacagctgca 2761 atcaaggggc aggccttgtc gtttgtgtag gaaccacccc tttgagagaa ccaagtatca 2821 aagtccgcgt gcttgttagc gaaacaggtg tccgtcgaag caatatctct agtaactccg 2881 ccatcgatag ctttgtaacc aatgatctct gaggaaaagt cggtatgctt actcatcacg 2941 tggacaggtg taattagata gaagatggct gcaacgaaca aaaatactaa agtaaccttt 3001 atcaactgtt taagccaatt gttgacaatc ttcattatag ttttttctcc ttgacgttaa 3061 agtatagagg tatattaaca attttttgtt gatactttta tgacatttga ataagaagta 3121 atacaaactg aaaatgttga aagtattagt taaagtggtt atgcagcttt tccatttata 3181 tatctgttaa tagatcaaaa atcatcgctt cgctgattaa ttaccccaga aataaggcta 3241 aaaaactaat cgcattatca tcctatggtt gttaatttga ttcgttaatt tgaaggtttg 3301 tggggccagg ttactgccaa tttttcctct tcataaccat aaaagctagt attgtagaat 3361 ctttattgtt cggagcagtg cggcgcgagg cacatctgcg tttcaggaac gcgaccggtg 3421 aagacgagga cgcacggagg agagtcttcc gtcggagggc tgtcgcccgc tcggcggctt 3481 ctaatccgta cttcaatata gcaagcggcc gctctaactg accatttaaa tcatacctga 3541 cctccatagc agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa 3601 gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat 3661 cgagattttc aggagctaag gaagctaaaa tgagtattca acatttccgt gtcgccctta 3721 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 3781 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 3841 gcggtaagat ccttgagagt ttacgccccg aagaacgttt tccaatgatg agcactttta 3901 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 3961 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4021 tcacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4081 ctgcggccaa cttacttctg gcaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4141 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 4201 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 4261 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 4321 cggataaagt tgcaggatca cttctgcgct cggccctccc ggctggctgg tttattgctg 4381 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 4441 gtaagccctc ccgcatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 4501 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa gtgaccaaac 4561 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg 4621 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc 4681 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct 4741 gatgagggcc caaatgtaat cacctggctc accttcgggt gggcctttct gcgttgctgg 4801 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgatgc tcaagtcaga 4861 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tacccctgga agctccctcg 4921 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4981 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5041 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5101 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5161 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5221 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 5281 ttacctcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 5341 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5401 tttgattttc taccgaagaa aggcccaccc gtgaaggtga gccagtgagt tgattgcagt 5461 ccagttacgc tggagtcaag cttgtaacac gcggccgctc gggaagttcc tattctctag 5521 ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg atgacgcact 5581 ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct 5641 tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat 5701 aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt 5761 tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga 5821 gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa 5881 tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg 5941 ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg 6001 aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt 6061 ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag 6121 tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta 6181 agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt 6241 atagattctt tgttggtaaa atagcgctct //