LOCUS Exported 6474 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4046363 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6474) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6474) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6474 /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 1069..1086 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" primer_bind complement(2229..2247) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 2598..3401 /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 3484..4466 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 4544..4573 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 4595..5182 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 5194..5221 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(5245..5436) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5437..6297) /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 6060..6079 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6298..6400) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 6401..6432 /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 atagcctcag agtttagttc ccttccatca tatgcagctt ttgctactgc tcaagaagct 541 tatgagcagg ctgttgctaa tggtgattct gaagttgttc ttaaaaagtt gaagaagtct 601 ttgaatgtgg ctaaatctga atttgaccgt gatgcagcca tgcaacgtaa gttggaaaag 661 atggctgatc aagctatgac ccaaatgtat aaacaggcta gatctgagga caagagggca 721 aaagttacta gtgctatgca gacaatgctt ttcactatgc ttagaaagtt ggataatgat 781 gcactcaaca acattatcaa caatgcaaga gatggttgtg ttcccttgaa cataatacct 841 cttacaacag cagccaaact aatggttgtc ataccagact ataacacata taaaaatacg 901 tgtgatggta caacatttac ttatgcatca gcattgtggg aaatccaaca ggttgtagat 961 gcagatagta aaattgttca acttagtgaa attagtatgg acaattcacc taatttagca 1021 tggcctctta ttgtaacagc tttaagggcc aattctgctg tcaaattact cgtgccgaga 1081 ggttctaaaa tcgaggaagg taagctagtc atttggataa atggagataa gggttacaac 1141 ggtttggcag aagtaggcaa gaaatttgaa aaggacacag gtatcaaggt gacggttgaa 1201 catccagaca agttagaaga aaaattccct caagttgctg ctactggtga tggtcccgac 1261 attattttct gggcccacga tcgttttggt ggatatgctc agtcaggttt gctggctgag 1321 atcaccccag acaaggcctt ccaagataaa ttgtacccat tcacttggga tgctgtcaga 1381 tacaacggta agcttatagc atatccaatt gctgttgaag ccttatcgtt gatctacaat 1441 aaggacttgt tacctaaccc accaaagaca tgggaagaaa ttccagcttt ggataaagag 1501 ctaaaggcca agggaaaaag cgctttgatg tttaacttac aagaacctta tttcacctgg 1561 ccattaatcg ctgctgacgg tggttacgca tttaaatacg aaaacggtaa gtatgacatt 1621 aaggatgtcg gcgttgataa tgctggtgca aaggctggtt tgaccttctt ggttgattta 1681 ataaagaata aacacatgaa cgctgacact gactactcta tcgccgaagc cgctttcaac 1741 aagggtgaaa ctgctatgac cataaatggt ccatgggcct ggagtaacat tgatacttct 1801 aaagtaaact atggtgtcac tgttttgcct acattcaaag gccaaccatc caagcccttt 1861 gtcggtgttc tatccgctgg catcaatgct gcttctccaa acaaggagct agctaaagaa 1921 tttttggaaa attacttgtt aactgacgaa ggtttggagg ccgttaacaa ggacaagcca 1981 ttgggtgctg tcgctttgaa atcttacgaa gaagaattgg ctaaggatcc cagaattgcc 2041 gctaccatgg agaacgccca aaagggtgaa attatgccaa acatcccaca aatgtcagcc 2101 ttctggtacg cagttaggac tgccgtgatt aatgctgctt ccggtagaca aaccgtcgac 2161 gaagcattaa aagatgctca aacgtaaaca ggcccctttt cctttgtcga tatcatgtaa 2221 ttagttatgt cacgcttaca ttcacgccct cctcccacat ccgctctaac cgaaaaggaa 2281 ggagttagac aacctgaagt ctaggtccct atttattttt tttaatagtt atgttagtat 2341 taagaacgtt atttatattt caaatttttc ttttttttct gtacaaagcg gccgccgaac 2401 cactagtgca ctgcagtaca cggtttcctt gaaatttttt tgattcggta atctccgaac 2461 agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc atatgtagtg 2521 ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca aaaacgtgca 2581 ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct actcatccta 2641 gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac ttgtgtgctt 2701 cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta ggtcccaaaa 2761 tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag ggcacagtta 2821 agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaggac agaaaatttg 2881 ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga atagcagaat 2941 gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc ggtttgaagc 3001 aggcggcaga agaagtaaca aaggaaccta gaggcctttt gatgttagca gaattgtcat 3061 gcaagggctc cctatctact ggagaatata ctaagggtac tgttgacatt gcgaagagcg 3121 acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga gatgaaggtt 3181 acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagat gcattgggtc 3241 aacagtatag aaccgtggat gatgtggttt ctacaggatc tgacattatt attgttggaa 3301 gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac agaaaagcag 3361 gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta ttataagtaa 3421 atgcatgtat actaaactca caaattagag cttcaattta attatatcag ttattacccg 3481 agtaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt 3541 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta 3601 ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc 3661 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag 3721 cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag 3781 agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata 3841 atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt 3901 tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta 3961 ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc 4021 gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt 4081 cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt 4141 acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct 4201 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 4261 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 4321 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 4381 gtccggtgcg tttttggttt tttgaaagtg cgtcatcaga gcgcttttgg ttttcaaaag 4441 cgctctgaag ttcctatact ttctagctag agaataggaa cttcccgagc ggccgcgtgt 4501 tacaagcttg actccagcgt aactggactg caatcaactc actggctcac cttcacgggt 4561 gggcctttct tcggtagaaa atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4621 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4681 agctaccaac tctttttccg aggtaactgg cttcagcaga gcgcagatac caaatactgt 4741 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 4801 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 4861 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 4921 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 4981 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 5041 cggcagggtc ggaacaggag agcgcacgag ggagcttcca ggggtaaacg cctggtatct 5101 ttatagtcct gtcgggtttc gccacctctg acttgagcat cgatttttgt gatgctcgtc 5161 aggggggcgg agcctatgga aaaacgccag caacgcagaa aggcccaccc gaaggtgagc 5221 caggtgatta catttgggcc ctcatcagag gttttcaccg tcatcaccga aacgcgcgag 5281 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc 5341 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg 5401 ggccatgtta agggcggttt tttcctgttt ggtcacttac caatgcttaa tcagtgaggc 5461 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5521 gataactacg atgcgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5581 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggga gggccgagcg 5641 cagaagtgat cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 5701 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 5761 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5821 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 5881 cgttgccaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 5941 ttctcttact gtcatgccat ccgtgagatg cttttctgtg actggtgagt actcaaccaa 6001 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6061 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6121 gcgtaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6181 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6241 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatttt 6301 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat 6361 ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc ctccggtcgg 6421 aggcttttga ctttctgcta tggaggtcag gtatgattta aatggtcagt taga //