LOCUS Exported 7386 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4046160 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7386) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7386) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7386 /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 1981..1998 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" primer_bind complement(3141..3159) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 3510..4313 /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 4396..5378 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 5456..5485 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 5507..6094 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 6106..6133 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(6157..6348) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6349..7209) /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 6972..6991 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7210..7312) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 7313..7344 /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 aagattgtca acaattggct taaacagttg ataaaggtta ctttagtatt tttgttcgtt 541 gcagccatct tctatctaat tacacctgtc cacgtgatga gtaagcatac cgacttttcc 601 tcagagatca ttggttacaa agctatcgat ggcggagtta ctagagatat tgcttcgacg 661 gacacctgtt tcgctaacaa gcacgcggac tttgatactt ggttctctca aaggggtggt 721 tcctacacaa acgacaaggc ctgccccttg attgcagctg ttatcactag agaagttggt 781 ttcgtcgtgc caggtttacc agggactata ctgcgtacca ccaatggtga ttttttgcat 841 ttcttaccaa gagtcttctc tgctgttggc aatatttgtt atacccctag caaattgatc 901 gaatacactg attttgccac ctctgcttgt gtattggctg ctgaatgcac tattttcaag 961 gatgcaagtg gtaagccagt tccatactgt tatgacacaa acgtcctcga aggttctgtt 1021 gcttacgaat ccttgcgacc ggacactaga tatgtgttaa tggatggatc aatcattcaa 1081 tttccaaaca cataccttga aggttctgtt cgggttgtca ccaccttcga ctccgagtac 1141 tgtcgccacg gtacttgtga aagatcagaa gccggtgtct gtgtttctac cagcggtaga 1201 tgggtgttga ataacgatta ttaccgttct cttccaggtg tcttctgcgg tgtcgatgct 1261 gttaatctat tgactaacat gtttacccct ttgatccaac caataggtgc gctggacatt 1321 tccgcatcta ttgtcgccgg tggaatcgtt gctatcgttg ttacttgttt ggcttactac 1381 ttcatgagat ttagaagagc ctttggggag tattcgcatg tcgtggcttt caacactcta 1441 ttatttttga tgtcgttcac tgttttgtgt cttacaccag tatactcttt cttgcctggt 1501 gtatactccg ttatctattt atacctgaca ttctacctga ctaacgatgt cagctttttg 1561 gcacacattc aatggatggt aatgttcacc ccattggttc ccttctggat aacgatcgct 1621 tacatcattt gcataagtac taaacatttc tattggtttt tctcaaatta tctaaagaga 1681 agagttgtct ttaatggtgt cagcttttct accttcgaag aagctgcctt gtgtaccttt 1741 ttgctcaaca aagaaatgta cttgaagttg agatctgacg ttttactccc gctaacccaa 1801 tataacagat atttagcttt atacaacaag tacaaatact tctcgggtgc catggatact 1861 acttcatacc gtgaagcggc ttgttgtcac cttgctaagg ctttgaatga tttctctaat 1921 tccggttccg acgtcttgta tcaaccacct cagacgtcta ttacctccgc tgttttgcaa 1981 ctcgtgccga gaggttctaa aatcgaggaa ggtaagctag tcatttggat aaatggagat 2041 aagggttaca acggtttggc agaagtaggc aagaaatttg aaaaggacac aggtatcaag 2101 gtgacggttg aacatccaga caagttagaa gaaaaattcc ctcaagttgc tgctactggt 2161 gatggtcccg acattatttt ctgggcccac gatcgttttg gtggatatgc tcagtcaggt 2221 ttgctggctg agatcacccc agacaaggcc ttccaagata aattgtaccc attcacttgg 2281 gatgctgtca gatacaacgg taagcttata gcatatccaa ttgctgttga agccttatcg 2341 ttgatctaca ataaggactt gttacctaac ccaccaaaga catgggaaga aattccagct 2401 ttggataaag agctaaaggc caagggaaaa agcgctttga tgtttaactt acaagaacct 2461 tatttcacct ggccattaat cgctgctgac ggtggttacg catttaaata cgaaaacggt 2521 aagtatgaca ttaaggatgt cggcgttgat aatgctggtg caaaggctgg tttgaccttc 2581 ttggttgatt taataaagaa taaacacatg aacgctgaca ctgactactc tatcgccgaa 2641 gccgctttca acaagggtga aactgctatg accataaatg gtccatgggc ctggagtaac 2701 attgatactt ctaaagtaaa ctatggtgtc actgttttgc ctacattcaa aggccaacca 2761 tccaagccct ttgtcggtgt tctatccgct ggcatcaatg ctgcttctcc aaacaaggag 2821 ctagctaaag aatttttgga aaattacttg ttaactgacg aaggtttgga ggccgttaac 2881 aaggacaagc cattgggtgc tgtcgctttg aaatcttacg aagaagaatt ggctaaggat 2941 cccagaattg ccgctaccat ggagaacgcc caaaagggtg aaattatgcc aaacatccca 3001 caaatgtcag ccttctggta cgcagttagg actgccgtga ttaatgctgc ttccggtaga 3061 caaaccgtcg acgaagcatt aaaagatgct caaacgtaaa caggcccctt ttcctttgtc 3121 gatatcatgt aattagttat gtcacgctta cattcacgcc ctcctcccac atccgctcta 3181 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt tttttaatag 3241 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacaaag 3301 cggccgccga accactagtg cactgcagta cacggtttcc ttgaaatttt tttgattcgg 3361 taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 3421 gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 3481 caaaaacgtg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 3541 ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 3601 acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 3661 taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 3721 agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgagg 3781 acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 3841 gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 3901 gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 3961 cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 4021 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 4081 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 4141 atgcattggg tcaacagtat agaaccgtgg atgatgtggt ttctacagga tctgacatta 4201 ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 4261 acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 4321 tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 4381 agttattacc cgagtaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg 4441 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac 4501 gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca 4561 acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat 4621 gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa 4681 atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt 4741 gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga 4801 agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc 4861 gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt 4921 atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga 4981 ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta 5041 taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca 5101 atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta 5161 gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga 5221 tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag 5281 tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcatca gagcgctttt 5341 ggttttcaaa agcgctctga agttcctata ctttctagct agagaatagg aacttcccga 5401 gcggccgcgt gttacaagct tgactccagc gtaactggac tgcaatcaac tcactggctc 5461 accttcacgg gtgggccttt cttcggtaga aaatcaaagg atcttcttga gatccttttt 5521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 5581 tgccggatca agagctacca actctttttc cgaggtaact ggcttcagca gagcgcagat 5641 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 5701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 5761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc caggggtaaa 6001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc atcgattttt 6061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcag aaaggcccac 6121 ccgaaggtga gccaggtgat tacatttggg ccctcatcag aggttttcac cgtcatcacc 6181 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat 6241 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct 6301 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactt accaatgctt 6361 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 6421 ccccgtcgtg tagataacta cgatgcggga gggcttacca tctggcccca gtgctgcaat 6481 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6541 gagggccgag cgcagaagtg atcctgcaac tttatccgcc tccatccagt ctattaattg 6601 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6661 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6721 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6781 cggtcctccg atcgttgcca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6841 agcactgcat aattctctta ctgtcatgcc atccgtgaga tgcttttctg tgactggtga 6901 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6961 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 7021 acgttcttcg gggcgtaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 7081 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 7141 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 7201 aatactcatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg 7261 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caagtcaaaa 7321 gcctccggtc ggaggctttt gactttctgc tatggaggtc aggtatgatt taaatggtca 7381 gttaga //