LOCUS Exported 5353 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4046477 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5353) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5353) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5353 /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 1028..1045 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1046..1063 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1108..1126) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 1477..2280 /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 2363..3345 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 3423..3452 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 3474..4061 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 4073..4100 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(4124..4315) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4316..5176) /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 4939..4958 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5177..5279) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 5280..5311 /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 atggctatcg ccagtgaatt ttcctcatta cctagctatg cagctttcgc 481 tacggctcag gaggcctacg aacaagctgt agccaacggc gactctgaag ttgttcttaa 541 gaaattgaag aagtctttga atgtcgcgaa atctgaattt gatagagatg ctgctatgca 601 acgtaagcta gaaaagatgg ctgaccaagc aatgacccaa atgtacaaac aagccagaag 661 tgaggacaag cgagctaagg tgacttccgc tatgcaaaca atgctgttca ctatgttgag 721 aaagttggat aacgatgctc tgaacaatat tataaacaat gccagagatg gttgtgtccc 781 cctcaacatt atcccattaa ccactgccgc taaattgatg gttgttattc cggattataa 841 tacatacaaa aacacttgcg acggtaccac cttcacatac gcctctgctc tttgggaaat 901 ccaacaggta gtcgacgctg actccaagat agttcaattg agcgaaattt ctatggataa 961 ctcgccaaac ctagcttggc cattaatcgt cactgcgttg agagctaata gtgcggttaa 1021 gttgcaactc gtgccgagag gttctcacca tcatcaccat cattaaacag gccccttttc 1081 ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc ctcccacatc 1141 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt 1201 ttaatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 1261 tacaaagcgg ccgccgaacc actagtgcac tgcagtacac ggtttccttg aaattttttt 1321 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta 1381 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg 1441 cacagaacaa aaacgtgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa 1501 cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag 1561 caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt 1621 gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt 1681 tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc 1741 ttcgaggaca gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt 1801 gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt 1861 attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg 1921 atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact 1981 gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg 2041 ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac 2101 aagggagatg cattgggtca acagtataga accgtggatg atgtggtttc tacaggatct 2161 gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt 2221 gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa 2281 aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa 2341 ttatatcagt tattacccga gtaacgaagc atctgtgctt cattttgtag aacaaaaatg 2401 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa 2461 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa 2521 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa 2581 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc 2641 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt 2701 ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa 2761 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac 2821 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc 2881 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg 2941 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata 3001 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct 3061 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc 3121 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc 3181 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat 3241 attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcatcagag 3301 cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac 3361 ttcccgagcg gccgcgtgtt acaagcttga ctccagcgta actggactgc aatcaactca 3421 ctggctcacc ttcacgggtg ggcctttctt cggtagaaaa tcaaaggatc ttcttgagat 3481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 3541 gtttgtttgc cggatcaaga gctaccaact ctttttccga ggtaactggc ttcagcagag 3601 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 3661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3841 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3961 gggtaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcatc 4021 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcagaaa 4081 ggcccacccg aaggtgagcc aggtgattac atttgggccc tcatcagagg ttttcaccgt 4141 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt 4201 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg 4261 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcacttacc 4321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4381 cctgactccc cgtcgtgtag ataactacga tgcgggaggg cttaccatct ggccccagtg 4441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4501 cagccgggag ggccgagcgc agaagtgatc ctgcaacttt atccgcctcc atccagtcta 4561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4741 gctccttcgg tcctccgatc gttgccagaa gtaagttggc cgcagtgtta tcactcatgg 4801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtgagatgc ttttctgtga 4861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4921 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4981 ttggaaaacg ttcttcgggg cgtaaactct caaggatctt accgctgttg agatccagtt 5041 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 5101 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5161 aatgttgaat actcatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata 5221 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa 5281 gtcaaaagcc tccggtcgga ggcttttgac tttctgctat ggaggtcagg tatgatttaa 5341 atggtcagtt aga //