LOCUS Exported 6253 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4046322 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6253) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6253) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6253 /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..1963 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2008..2026) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 2377..3180 /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 3263..4245 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 4323..4352 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 4374..4961 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4973..5000 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(5024..5215) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5216..6076) /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 5839..5858 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6077..6179) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 6180..6211 /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 gttctcacca tcatcaccat cattaaacag gccccttttc 1981 ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc ctcccacatc 2041 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt 2101 ttaatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 2161 tacaaagcgg ccgccgaacc actagtgcac tgcagtacac ggtttccttg aaattttttt 2221 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta 2281 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg 2341 cacagaacaa aaacgtgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa 2401 cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag 2461 caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt 2521 gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt 2581 tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc 2641 ttcgaggaca gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt 2701 gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt 2761 attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg 2821 atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact 2881 gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg 2941 ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac 3001 aagggagatg cattgggtca acagtataga accgtggatg atgtggtttc tacaggatct 3061 gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt 3121 gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa 3181 aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa 3241 ttatatcagt tattacccga gtaacgaagc atctgtgctt cattttgtag aacaaaaatg 3301 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa 3361 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa 3421 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa 3481 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc 3541 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt 3601 ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa 3661 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac 3721 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc 3781 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg 3841 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata 3901 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct 3961 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc 4021 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc 4081 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat 4141 attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcatcagag 4201 cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac 4261 ttcccgagcg gccgcgtgtt acaagcttga ctccagcgta actggactgc aatcaactca 4321 ctggctcacc ttcacgggtg ggcctttctt cggtagaaaa tcaaaggatc ttcttgagat 4381 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 4441 gtttgtttgc cggatcaaga gctaccaact ctttttccga ggtaactggc ttcagcagag 4501 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 4561 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 4621 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 4681 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 4741 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 4801 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 4861 gggtaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcatc 4921 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcagaaa 4981 ggcccacccg aaggtgagcc aggtgattac atttgggccc tcatcagagg ttttcaccgt 5041 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt 5101 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg 5161 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcacttacc 5221 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 5281 cctgactccc cgtcgtgtag ataactacga tgcgggaggg cttaccatct ggccccagtg 5341 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 5401 cagccgggag ggccgagcgc agaagtgatc ctgcaacttt atccgcctcc atccagtcta 5461 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 5521 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 5581 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 5641 gctccttcgg tcctccgatc gttgccagaa gtaagttggc cgcagtgtta tcactcatgg 5701 ttatggcagc actgcataat tctcttactg tcatgccatc cgtgagatgc ttttctgtga 5761 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 5821 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 5881 ttggaaaacg ttcttcgggg cgtaaactct caaggatctt accgctgttg agatccagtt 5941 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 6001 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 6061 aatgttgaat actcatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata 6121 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa 6181 gtcaaaagcc tccggtcgga ggcttttgac tttctgctat ggaggtcagg tatgatttaa 6241 atggtcagtt aga //