LOCUS Exported 6429 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast Expression plasmid for SARS-CoV-2 3'-to-5' exonuclease. ACCESSION . VERSION . KEYWORDS pGBW-m4046579 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6429) TITLE SARS-CoV-2 protein Yeast expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6429) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6429 /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 2056..2073 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 2074..2139 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(2184..2202) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" CDS 2553..3356 /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 3439..4421 /label=2u ori /note="yeast 2u plasmid origin of replication" terminator 4499..4528 /label=T3Te terminator /note="phage T3 early transcription terminator" rep_origin 4550..5137 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 5149..5176 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(5200..5391) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5392..6252) /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 6015..6034 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6253..6355) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 6356..6387 /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 gaaaatgtaa caggactctt taaagattgt agtaaggtaa tcactgggtt acatcctaca 541 caggcaccta cacacctcag tgttgacact aaattcaaaa ctgaaggttt atgtgttgac 601 atacctggca tacctaagga catgacctat agaagactca tctctatgat gggttttaaa 661 atgaattatc aagttaatgg ttaccctaac atgtttatca cccgcgaaga agctataaga 721 catgtacgtg catggattgg cttcgatgtc gaggggtgtc atgctactag agaagctgtt 781 ggtaccaatt tacctttaca gctaggtttt tctacaggtg ttaacctagt tgctgtacct 841 acaggttatg ttgatacacc taataataca gatttttcca gagttagtgc taaaccaccg 901 cctggagatc aatttaaaca cctcatacca cttatgtaca aaggacttcc ttggaatgta 961 gtgcgtataa agattgtaca aatgttaagt gacacactta aaaatctctc tgacagagtc 1021 gtatttgtct tatgggcaca tggctttgag ttgacatcta tgaagtattt tgtgaaaata 1081 ggacctgagc gcacctgttg tctatgtgat agacgtgcca catgcttttc cactgcttca 1141 gacacttatg cctgttggca tcattctatt ggatttgatt acgtctataa tccgtttatg 1201 attgatgttc aacaatgggg ttttacaggt aacctacaaa gcaaccatga tctgtattgt 1261 caagtccatg gtaatgcaca tgtagctagt tgtgatgcaa tcatgactag gtgtctagct 1321 gtccacgagt gctttgttaa gcgtgttgac tggactattg aatatcctat aattggtgat 1381 gaactgaaga ttaatgcggc ttgtagaaag gttcaacaca tggttgttaa agctgcatta 1441 ttagcagaca aattcccagt tcttcacgac attggtaacc ctaaagctat taagtgtgta 1501 cctcaagctg atgtagaatg gaagttctat gatgcacagc cttgtagtga caaagcttat 1561 aaaatagaag aattattcta ttcttatgcc acacattctg acaaattcac agatggtgta 1621 tgcctatttt ggaattgcaa tgtcgataga tatcctgcta attccattgt ttgtagattt 1681 gacactagag tgctatctaa ccttaacttg cctggttgtg atggtggcag tttgtatgta 1741 aataaacatg cattccacac accagctttt gataaaagtg cttttgttaa tttaaaacaa 1801 ttaccatttt tctattactc tgacagtcca tgtgagtctc atggaaaaca agtagtgtca 1861 gatatagatt atgtaccact aaagtctgct acgtgtataa cacgttgcaa tttaggtggt 1921 gctgtctgta gacatcatgc taatgagtac agattgtatc tcgatgctta taacatgatg 1981 atctcagctg gctttagctt gtgggtttac aaacaatttg atacttataa cctctggaac 2041 acttttacaa gacttctcgt gccgagaggt tctgattata aagatcatga tggtgattat 2101 aaagatcatg atattgatta taaagatgat gatgataaat aaacaggccc cttttccttt 2161 gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctcctcc cacatccgct 2221 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta ttttttttaa 2281 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 2341 aagcggccgc cgaaccacta gtgcactgca gtacacggtt tccttgaaat ttttttgatt 2401 cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata 2461 tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca 2521 gaacaaaaac gtgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg 2581 ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa 2641 caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag 2701 cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca 2761 tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg 2821 aggacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat 2881 acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg 2941 ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt 3001 tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg 3061 acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg 3121 gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg 3181 gagatgcatt gggtcaacag tatagaaccg tggatgatgt ggtttctaca ggatctgaca 3241 ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac 3301 gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa 3361 ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat 3421 atcagttatt acccgagtaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac 3481 gcgagagcgc taattttwca aacaaagaat ctgagctgca tttttacaga acagaaatgc 3541 aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat 3601 gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga 3661 aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt ttgttctaca 3721 aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact ttttttctcc 3781 tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc cgttaaggtt 3841 agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga ctccacttcc 3901 cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa ggcatccccg 3961 attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg atagcgttga 4021 tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc tatatactac 4081 gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat agttcttact 4141 acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta gaggtcgagt 4201 ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga tatagcacag 4261 agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt cgcaatattt 4321 tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtca tcagagcgct 4381 tttggttttc aaaagcgctc tgaagttcct atactttcta gctagagaat aggaacttcc 4441 cgagcggccg cgtgttacaa gcttgactcc agcgtaactg gactgcaatc aactcactgg 4501 ctcaccttca cgggtgggcc tttcttcggt agaaaatcaa aggatcttct tgagatcctt 4561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 4621 gtttgccgga tcaagagcta ccaactcttt ttccgaggta actggcttca gcagagcgca 4681 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 4741 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 4801 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 4861 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 4921 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 4981 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggt 5041 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcatcgatt 5101 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cagaaaggcc 5161 cacccgaagg tgagccaggt gattacattt gggccctcat cagaggtttt caccgtcatc 5221 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 5281 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 5341 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca cttaccaatg 5401 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 5461 actccccgtc gtgtagataa ctacgatgcg ggagggctta ccatctggcc ccagtgctgc 5521 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 5581 cgggagggcc gagcgcagaa gtgatcctgc aactttatcc gcctccatcc agtctattaa 5641 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 5701 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 5761 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 5821 cttcggtcct ccgatcgttg ccagaagtaa gttggccgca gtgttatcac tcatggttat 5881 ggcagcactg cataattctc ttactgtcat gccatccgtg agatgctttt ctgtgactgg 5941 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 6001 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 6061 aaaacgttct tcggggcgta aactctcaag gatcttaccg ctgttgagat ccagttcgat 6121 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 6181 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 6241 ttgaatactc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc 6301 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaagtca 6361 aaagcctccg gtcggaggct tttgactttc tgctatggag gtcaggtatg atttaaatgg 6421 tcagttaga //