LOCUS       sequence-300257-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast Expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6354)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4046479
FEATURES             Location/Qualifiers
     source          1..6354
                     /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             1981..1998
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1999..2064
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2109..2127)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2478..3281
                     /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      3364..4346
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4424..4453
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4475..5062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(5074..5101)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5125..5316)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5317..6108,6109..6177))
                     /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     5940..5959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6178..6280)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6281..6312)
                     /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 aagattgtca acaattggct taaacagttg ataaaggtta ctttagtatt tttgttcgtt
      541 gcagccatct tctatctaat tacacctgtc cacgtgatga gtaagcatac cgacttttcc
      601 tcagagatca ttggttacaa agctatcgat ggcggagtta ctagagatat tgcttcgacg
      661 gacacctgtt tcgctaacaa gcacgcggac tttgatactt ggttctctca aaggggtggt
      721 tcctacacaa acgacaaggc ctgccccttg attgcagctg ttatcactag agaagttggt
      781 ttcgtcgtgc caggtttacc agggactata ctgcgtacca ccaatggtga ttttttgcat
      841 ttcttaccaa gagtcttctc tgctgttggc aatatttgtt atacccctag caaattgatc
      901 gaatacactg attttgccac ctctgcttgt gtattggctg ctgaatgcac tattttcaag
      961 gatgcaagtg gtaagccagt tccatactgt tatgacacaa acgtcctcga aggttctgtt
     1021 gcttacgaat ccttgcgacc ggacactaga tatgtgttaa tggatggatc aatcattcaa
     1081 tttccaaaca cataccttga aggttctgtt cgggttgtca ccaccttcga ctccgagtac
     1141 tgtcgccacg gtacttgtga aagatcagaa gccggtgtct gtgtttctac cagcggtaga
     1201 tgggtgttga ataacgatta ttaccgttct cttccaggtg tcttctgcgg tgtcgatgct
     1261 gttaatctat tgactaacat gtttacccct ttgatccaac caataggtgc gctggacatt
     1321 tccgcatcta ttgtcgccgg tggaatcgtt gctatcgttg ttacttgttt ggcttactac
     1381 ttcatgagat ttagaagagc ctttggggag tattcgcatg tcgtggcttt caacactcta
     1441 ttatttttga tgtcgttcac tgttttgtgt cttacaccag tatactcttt cttgcctggt
     1501 gtatactccg ttatctattt atacctgaca ttctacctga ctaacgatgt cagctttttg
     1561 gcacacattc aatggatggt aatgttcacc ccattggttc ccttctggat aacgatcgct
     1621 tacatcattt gcataagtac taaacatttc tattggtttt tctcaaatta tctaaagaga
     1681 agagttgtct ttaatggtgt cagcttttct accttcgaag aagctgcctt gtgtaccttt
     1741 ttgctcaaca aagaaatgta cttgaagttg agatctgacg ttttactccc gctaacccaa
     1801 tataacagat atttagcttt atacaacaag tacaaatact tctcgggtgc catggatact
     1861 acttcatacc gtgaagcggc ttgttgtcac cttgctaagg ctttgaatga tttctctaat
     1921 tccggttccg acgtcttgta tcaaccacct cagacgtcta ttacctccgc tgttttgcaa
     1981 ctcgtgccga gaggttctga ttataaagat catgatggtg attataaaga tcatgatatt
     2041 gattataaag atgatgatga taaataaaca ggcccctttt cctttgtcga tatcatgtaa
     2101 ttagttatgt cacgcttaca ttcacgccct cctcccacat ccgctctaac cgaaaaggaa
     2161 ggagttagac aacctgaagt ctaggtccct atttattttt tttaatagtt atgttagtat
     2221 taagaacgtt atttatattt caaatttttc ttttttttct gtacaaagcg gccgccgaac
     2281 cactagtgca ctgcagtaca cggtttcctt gaaatttttt tgattcggta atctccgaac
     2341 agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc atatgtagtg
     2401 ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca aaaacgtgca
     2461 ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct actcatccta
     2521 gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac ttgtgtgctt
     2581 cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta ggtcccaaaa
     2641 tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag ggcacagtta
     2701 agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaggac agaaaatttg
     2761 ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga atagcagaat
     2821 gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc ggtttgaagc
     2881 aggcggcaga agaagtaaca aaggaaccta gaggcctttt gatgttagca gaattgtcat
     2941 gcaagggctc cctatctact ggagaatata ctaagggtac tgttgacatt gcgaagagcg
     3001 acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga gatgaaggtt
     3061 acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagat gcattgggtc
     3121 aacagtatag aaccgtggat gatgtggttt ctacaggatc tgacattatt attgttggaa
     3181 gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac agaaaagcag
     3241 gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta ttataagtaa
     3301 atgcatgtat actaaactca caaattagag cttcaattta attatatcag ttattacccg
     3361 agtaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt
     3421 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta
     3481 ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc
     3541 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag
     3601 cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag
     3661 agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata
     3721 atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt
     3781 tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta
     3841 ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc
     3901 gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt
     3961 cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt
     4021 acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct
     4081 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca
     4141 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag
     4201 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca
     4261 gtccggtgcg tttttggttt tttgaaagtg cgtcatcaga gcgcttttgg ttttcaaaag
     4321 cgctctgaag ttcctatact ttctagctag agaataggaa cttcccgagc ggccgcgtgt
     4381 tacaagcttg actccagcgt aactggactg caatcaactc actggctcac cttcacgggt
     4441 gggcctttct tcggtagaaa atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4501 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4561 agctaccaac tctttttccg aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4621 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4681 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4741 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4801 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4861 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4921 cggcagggtc ggaacaggag agcgcacgag ggagcttcca ggggtaaacg cctggtatct
     4981 ttatagtcct gtcgggtttc gccacctctg acttgagcat cgatttttgt gatgctcgtc
     5041 aggggggcgg agcctatgga aaaacgccag caacgcagaa aggcccaccc gaaggtgagc
     5101 caggtgatta catttgggcc ctcatcagag gttttcaccg tcatcaccga aacgcgcgag
     5161 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     5221 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     5281 ggccatgtta agggcggttt tttcctgttt ggtcacttac caatgcttaa tcagtgaggc
     5341 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5401 gataactacg atgcgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5461 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggga gggccgagcg
     5521 cagaagtgat cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5581 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5641 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5701 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5761 cgttgccaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5821 ttctcttact gtcatgccat ccgtgagatg cttttctgtg actggtgagt actcaaccaa
     5881 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5941 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6001 gcgtaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6061 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6121 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatttt
     6181 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     6241 ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc ctccggtcgg
     6301 aggcttttga ctttctgcta tggaggtcag gtatgattta aatggtcagt taga
//
