LOCUS Exported 5448 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4046231 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5448) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5448) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5448 /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 1075..1092 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1093..1158 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(1203..1221) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 1572..2375 /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 2458..3440 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 3518..3547 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 3569..4156 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4168..4195 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(4219..4410) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4411..5271) /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 5034..5053 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5272..5374) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 5375..5406 /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 gctatcgcca gtgaattttc ctcattacct agctatgcag ctttcgctac ggctcaggag 541 gcctacgaac aagctgtagc caacggcgac tctgaagttg ttcttaagaa attgaagaag 601 tctttgaatg tcgcgaaatc tgaatttgat agagatgctg ctatgcaacg taagctagaa 661 aagatggctg accaagcaat gacccaaatg tacaaacaag ccagaagtga ggacaagcga 721 gctaaggtga cttccgctat gcaaacaatg ctgttcacta tgttgagaaa gttggataac 781 gatgctctga acaatattat aaacaatgcc agagatggtt gtgtccccct caacattatc 841 ccattaacca ctgccgctaa attgatggtt gttattccgg attataatac atacaaaaac 901 acttgcgacg gtaccacctt cacatacgcc tctgctcttt gggaaatcca acaggtagtc 961 gacgctgact ccaagatagt tcaattgagc gaaatttcta tggataactc gccaaaccta 1021 gcttggccat taatcgtcac tgcgttgaga gctaatagtg cggttaagtt gcaactcgtg 1081 ccgagaggtt ctgattataa agatcatgat ggtgattata aagatcatga tattgattat 1141 aaagatgatg atgataaata aacaggcccc ttttcctttg tcgatatcat gtaattagtt 1201 atgtcacgct tacattcacg ccctcctccc acatccgctc taaccgaaaa ggaaggagtt 1261 agacaacctg aagtctaggt ccctatttat tttttttaat agttatgtta gtattaagaa 1321 cgttatttat atttcaaatt tttctttttt ttctgtacaa agcggccgcc gaaccactag 1381 tgcactgcag tacacggttt ccttgaaatt tttttgattc ggtaatctcc gaacagaagg 1441 aagaacgaag gaaggagcac agacttagat tggtatatat acgcatatgt agtgttgaag 1501 aaacatgaaa ttgcccagta ttcttaaccc aactgcacag aacaaaaacg tgcaggaaac 1561 gaagataaat catgtcgaaa gctacatata aggaacgtgc tgctactcat cctagtcctg 1621 ttgctgccaa gctatttaat atcatgcacg aaaagcaaac aaacttgtgt gcttcattgg 1681 atgttcgtac caccaaggaa ttactggagt tagttgaagc attaggtccc aaaatttgtt 1741 tactaaaaac acatgtggat atcttgactg atttttccat ggagggcaca gttaagccgc 1801 taaaggcatt atccgccaag tacaattttt tactcttcga ggacagaaaa tttgctgaca 1861 ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca gaatgggcag 1921 acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg aagcaggcgg 1981 cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg tcatgcaagg 2041 gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag agcgacaaag 2101 attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa ggttacgatt 2161 ggttgattat gacacccggt gtgggtttag atgacaaggg agatgcattg ggtcaacagt 2221 atagaaccgt ggatgatgtg gtttctacag gatctgacat tattattgtt ggaagaggac 2281 tatttgcaaa gggaagggat gctaaggtag agggtgaacg ttacagaaaa gcaggctggg 2341 aagcatattt gagaagatgc ggccagcaaa actaaaaaac tgtattataa gtaaatgcat 2401 gtatactaaa ctcacaaatt agagcttcaa tttaattata tcagttatta cccgagtaac 2461 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa 2521 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac 2581 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt 2641 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat 2701 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct 2761 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag 2821 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt 2881 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg 2941 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg 3001 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa 3061 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt 3121 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag 3181 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc 3241 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac 3301 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg 3361 tgcgtttttg gttttttgaa agtgcgtcat cagagcgctt ttggttttca aaagcgctct 3421 gaagttccta tactttctag ctagagaata ggaacttccc gagcggccgc gtgttacaag 3481 cttgactcca gcgtaactgg actgcaatca actcactggc tcaccttcac gggtgggcct 3541 ttcttcggta gaaaatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 3601 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 3661 caactctttt tccgaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 3721 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 3781 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 3841 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 3901 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 3961 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 4021 ggtcggaaca ggagagcgca cgagggagct tccaggggta aacgcctggt atctttatag 4081 tcctgtcggg tttcgccacc tctgacttga gcatcgattt ttgtgatgct cgtcaggggg 4141 gcggagccta tggaaaaacg ccagcaacgc agaaaggccc acccgaaggt gagccaggtg 4201 attacatttg ggccctcatc agaggttttc accgtcatca ccgaaacgcg cgaggcagct 4261 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc 4321 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat 4381 gttaagggcg gttttttcct gtttggtcac ttaccaatgc ttaatcagtg aggcacctat 4441 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 4501 tacgatgcgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 4561 ctcaccggct ccagatttat cagcaataaa ccagccagcc gggagggccg agcgcagaag 4621 tgatcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 4681 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 4741 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 4801 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgc 4861 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 4921 tactgtcatg ccatccgtga gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 4981 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 5041 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgtaa 5101 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 5161 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 5221 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca ttttagcttc 5281 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt 5341 atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg tcggaggctt 5401 ttgactttct gctatggagg tcaggtatga tttaaatggt cagttaga //