LOCUS       sequence-306276-        5772 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Yeast Expression plasmid for SARS-CoV-2 3C-like proteinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 5772)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4046203
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             1399..1416
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1417..1482
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1527..1545)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             1896..2699
                     /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      2782..3764
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      3842..3871
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      3893..4480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(4492..4519)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4543..4734)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(4735..5526,5527..5595))
                     /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     5358..5377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5596..5698)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(5699..5730)
                     /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 tcgggtttca gaaaaatggc ctttcctagt ggaaaggtcg agggctgtat ggtgcaagta
      541 acatgcggta cgaccacttt gaacggttta tggcttgacg atgttgttta ctgtccacgt
      601 catgtcatct gtacttcaga agatatgttg aatccaaact atgaagacct actgattcgc
      661 aagtctaacc acaatttctt agttcaggct ggtaacgttc aattgagagt tataggtcac
      721 agcatgcaaa attgtgtctt gaagttaaaa gttgataccg caaacccgaa gactccaaag
      781 tacaaatttg tcagaatcca acccggtcaa accttctccg tcttggcttg ttataacggg
      841 tccccatctg gtgtatacca gtgcgctatg aggccaaact tcactattaa gggttctttt
      901 ttgaatggtt cttgtggctc cgttggtttt aatatcgact acgattgcgt cagtttctgt
      961 tacatgcatc acatggaatt gcctacaggt gttcacgccg gaaccgacct agaagggaac
     1021 ttctatggtc cattcgttga tcggcaaact gctcaagctg cgggtacgga taccacaatt
     1081 actgtaaatg tgcttgcctg gttgtacgct gctgttataa acggtgaccg atggttttta
     1141 aacagattca ctaccacatt gaatgacttt aatctggtcg ctatgaaata taactacgaa
     1201 ccactcactc aagatcatgt cgatatttta ggtcctttgt ctgcacaaac tggcatcgct
     1261 gttttggaca tgtgtgcgtc ccttaaggaa ctactccaaa acggtatgaa cgggagaacg
     1321 attttgggtt cagccctatt ggaggacgaa tttaccccat tcgatgtggt tagacagtgc
     1381 tctggagtaa ccttccaact cgtgccgaga ggttctgatt ataaagatca tgatggtgat
     1441 tataaagatc atgatattga ttataaagat gatgatgata aataaacagg ccccttttcc
     1501 tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgccctcc tcccacatcc
     1561 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
     1621 taatagttat gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt
     1681 acaaagcggc cgccgaacca ctagtgcact gcagtacacg gtttccttga aatttttttg
     1741 attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt agattggtat
     1801 atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta acccaactgc
     1861 acagaacaaa aacgtgcagg aaacgaagat aaatcatgtc gaaagctaca tataaggaac
     1921 gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg cacgaaaagc
     1981 aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg gagttagttg
     2041 aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg actgattttt
     2101 ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat tttttactct
     2161 tcgaggacag aaaatttgct gacattggta atacagtcaa attgcagtac tctgcgggtg
     2221 tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg ggcccaggta
     2281 ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga ggccttttga
     2341 tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact aagggtactg
     2401 ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa agagacatgg
     2461 gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt ttagatgaca
     2521 agggagatgc attgggtcaa cagtatagaa ccgtggatga tgtggtttct acaggatctg
     2581 acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag gtagagggtg
     2641 aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag caaaactaaa
     2701 aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat
     2761 tatatcagtt attacccgag taacgaagca tctgtgcttc attttgtaga acaaaaatgc
     2821 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     2881 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa
     2941 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac
     3001 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct
     3061 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc
     3121 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag
     3181 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact
     3241 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc
     3301 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt
     3361 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac
     3421 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt
     3481 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg
     3541 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca
     3601 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata
     3661 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcatcagagc
     3721 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagctagag aataggaact
     3781 tcccgagcgg ccgcgtgtta caagcttgac tccagcgtaa ctggactgca atcaactcac
     3841 tggctcacct tcacgggtgg gcctttcttc ggtagaaaat caaaggatct tcttgagatc
     3901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3961 tttgtttgcc ggatcaagag ctaccaactc tttttccgag gtaactggct tcagcagagc
     4021 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4081 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4141 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4201 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4261 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4321 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4381 ggtaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcatcg
     4441 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcagaaag
     4501 gcccacccga aggtgagcca ggtgattaca tttgggccct catcagaggt tttcaccgtc
     4561 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4621 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4681 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcacttacca
     4741 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4801 ctgactcccc gtcgtgtaga taactacgat gcgggagggc ttaccatctg gccccagtgc
     4861 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4921 agccgggagg gccgagcgca gaagtgatcc tgcaacttta tccgcctcca tccagtctat
     4981 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5041 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5101 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5161 ctccttcggt cctccgatcg ttgccagaag taagttggcc gcagtgttat cactcatggt
     5221 tatggcagca ctgcataatt ctcttactgt catgccatcc gtgagatgct tttctgtgac
     5281 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5341 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5401 tggaaaacgt tcttcggggc gtaaactctc aaggatctta ccgctgttga gatccagttc
     5461 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5521 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5581 atgttgaata ctcattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     5641 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaag
     5701 tcaaaagcct ccggtcggag gcttttgact ttctgctatg gaggtcaggt atgatttaaa
     5761 tggtcagtta ga
//
