LOCUS Exported 6301 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4046300 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6301) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6301) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6301 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 10..417 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" CDS 1928..1945 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1946..2011 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(2056..2074) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 2425..3228 /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" terminator 4371..4400 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 4422..5009 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 5021..5048 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(5072..5263) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5264..6124) /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 5887..5906 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6125..6227) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 6228..6259 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" ORIGIN 1 gcggccgccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 61 tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 121 ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 181 aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 241 tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 301 tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 361 ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 421 taattacaaa atgattgtta ataattggtt gaagcagtta attaaagtta cacttgtgtt 481 cctttttgtt gctgctattt tctatttaat aacacctgtt catgtcatgt ctaaacatac 541 tgacttttca agtgaaatca taggatacaa ggctattgat ggtggtgtca ctcgtgacat 601 agcatctaca gatacttgtt ttgctaacaa acatgctgat tttgacacat ggtttagcca 661 gcgtggtggt agttatacta atgacaaagc ttgcccattg attgctgcag tcataacaag 721 agaagtgggt tttgtcgtgc ctggtttgcc tggcacgata ttacgcacaa ctaatggtga 781 ctttttgcat ttcttaccta gagtttttag tgcagttggt aacatctgtt acacaccatc 841 aaaacttata gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac 901 aatttttaaa gatgcttctg gtaagccagt accatattgt tatgatacca atgtactaga 961 aggttctgtt gcttatgaaa gtttacgccc tgacacacgt tatgtgctca tggatggctc 1021 tattattcaa tttcctaaca cctaccttga aggttctgtt agagtggtaa caacttttga 1081 ttctgagtac tgtaggcacg gcacttgtga aagatcagaa gctggtgttt gtgtatctac 1141 tagtggtaga tgggtactta acaatgatta ttacagatct ttaccaggag ttttctgtgg 1201 tgtagatgct gtaaatttac ttactaatat gtttacacca ctaattcaac ctattggtgc 1261 tttggacata tcagcatcta tagtagctgg tggtattgta gctatcgtag taacatgcct 1321 tgcctactat tttatgaggt ttagaagagc ttttggtgaa tacagtcatg tagttgcctt 1381 taatacttta ctattcctta tgtcattcac tgtactctgt ttaacaccag tttactcatt 1441 cttacctggt gtttattctg ttatttactt gtacttgaca ttttatctta ctaatgatgt 1501 ttctttttta gcacatattc agtggatggt tatgttcaca cctttagtac ctttctggat 1561 aacaattgct tatatcattt gtatttccac aaagcatttc tattggttct ttagtaatta 1621 cctaaagaga cgtgtagtct ttaatggtgt ttcctttagt acttttgaag aagctgcgct 1681 gtgcaccttt ttgttaaata aagaaatgta tctaaagttg cgtagtgatg tgctattacc 1741 tcttacgcaa tataatagat acttagctct ttataataag tacaagtatt ttagtggagc 1801 aatggataca actagctaca gagaagctgc ttgttgtcat ctcgcaaagg ctctcaatga 1861 cttcagtaac tcaggttctg atgttcttta ccaaccacca caaacctcta tcacctcagc 1921 tgttttgctc gtgccgagag gttctgatta taaagatcat gatggtgatt ataaagatca 1981 tgatattgat tataaagatg atgatgataa ataaacaggc cccttttcct ttgtcgatat 2041 catgtaatta gttatgtcac gcttacattc acgccctcct cccacatccg ctctaaccga 2101 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tatttttttt aatagttatg 2161 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta caaagcggcc 2221 gccgaaccac tagtgcactg cagtacacgg tttccttgaa atttttttga ttcggtaatc 2281 tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata tatacgcata 2341 tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca cagaacaaaa 2401 acgtgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg tgctgctact 2461 catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca aacaaacttg 2521 tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga agcattaggt 2581 cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc catggagggc 2641 acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt cgaggacaga 2701 aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt atacagaata 2761 gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt 2821 ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat gttagcagaa 2881 ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt tgacattgcg 2941 aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg tggaagagat 3001 gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa gggagatgca 3061 ttgggtcaac agtatagaac cgtggatgat gtggtttcta caggatctga cattattatt 3121 gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga 3181 aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta 3241 taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt atatcagtta 3301 ttacccgagt aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc 3361 gctaattttk caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa 3421 agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg 3481 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac 3541 gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca 3601 tcccgagann nnnntttttc taacaaagca tcttagatta ctttttttct cctttgtgcg 3661 ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag 3721 gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta 3781 ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt 3841 ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt 3901 cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga 3961 aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt 4021 tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc 4081 aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat 4141 agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct 4201 cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt catcagagcg cttttggttt 4261 tcaaaagcgc tctgaagttc ctatactttc tagctagaga ataggaactt cccgagcggc 4321 cgcgtgttac aagcttgact ccagcgtaac tggactgcaa tcaactcact ggctcacctt 4381 cacgggtggg cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg 4441 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 4501 gatcaagagc taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa 4561 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 4621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 4681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 4741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 4801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 4861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg gtaaacgcct 4921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat 4981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa 5041 ggtgagccag gtgattacat ttgggccctc atcagaggtt ttcaccgtca tcaccgaaac 5101 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg 5161 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga 5221 taaagcgggc catgttaagg gcggtttttt cctgtttggt cacttaccaa tgcttaatca 5281 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5341 tcgtgtagat aactacgatg cgggagggct taccatctgg ccccagtgct gcaatgatac 5401 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccgggaggg 5461 ccgagcgcag aagtgatcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 5521 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 5581 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 5641 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 5701 ctccgatcgt tgccagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 5761 tgcataattc tcttactgtc atgccatccg tgagatgctt ttctgtgact ggtgagtact 5821 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 5881 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 5941 cttcggggcg taaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6001 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6061 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6121 tcattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg 6181 atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaagt caaaagcctc 6241 cggtcggagg cttttgactt tctgctatgg aggtcaggta tgatttaaat ggtcagttag 6301 a //