LOCUS       sequence-291496-        6301 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 6301)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6301)
  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-m4046300
FEATURES             Location/Qualifiers
     source          1..6301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..417
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             1928..1945
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1946..2011
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2056..2074)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2425..3228
                     /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      3311..4293
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4371..4400
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4422..5009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(5021..5048)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5072..5263)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5264..6055,6056..6124))
                     /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     5887..5906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6125..6227)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6228..6259)
                     /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 atgattgtta ataattggtt gaagcagtta attaaagtta cacttgtgtt
      481 cctttttgtt gctgctattt tctatttaat aacacctgtt catgtcatgt ctaaacatac
      541 tgacttttca agtgaaatca taggatacaa ggctattgat ggtggtgtca ctcgtgacat
      601 agcatctaca gatacttgtt ttgctaacaa acatgctgat tttgacacat ggtttagcca
      661 gcgtggtggt agttatacta atgacaaagc ttgcccattg attgctgcag tcataacaag
      721 agaagtgggt tttgtcgtgc ctggtttgcc tggcacgata ttacgcacaa ctaatggtga
      781 ctttttgcat ttcttaccta gagtttttag tgcagttggt aacatctgtt acacaccatc
      841 aaaacttata gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac
      901 aatttttaaa gatgcttctg gtaagccagt accatattgt tatgatacca atgtactaga
      961 aggttctgtt gcttatgaaa gtttacgccc tgacacacgt tatgtgctca tggatggctc
     1021 tattattcaa tttcctaaca cctaccttga aggttctgtt agagtggtaa caacttttga
     1081 ttctgagtac tgtaggcacg gcacttgtga aagatcagaa gctggtgttt gtgtatctac
     1141 tagtggtaga tgggtactta acaatgatta ttacagatct ttaccaggag ttttctgtgg
     1201 tgtagatgct gtaaatttac ttactaatat gtttacacca ctaattcaac ctattggtgc
     1261 tttggacata tcagcatcta tagtagctgg tggtattgta gctatcgtag taacatgcct
     1321 tgcctactat tttatgaggt ttagaagagc ttttggtgaa tacagtcatg tagttgcctt
     1381 taatacttta ctattcctta tgtcattcac tgtactctgt ttaacaccag tttactcatt
     1441 cttacctggt gtttattctg ttatttactt gtacttgaca ttttatctta ctaatgatgt
     1501 ttctttttta gcacatattc agtggatggt tatgttcaca cctttagtac ctttctggat
     1561 aacaattgct tatatcattt gtatttccac aaagcatttc tattggttct ttagtaatta
     1621 cctaaagaga cgtgtagtct ttaatggtgt ttcctttagt acttttgaag aagctgcgct
     1681 gtgcaccttt ttgttaaata aagaaatgta tctaaagttg cgtagtgatg tgctattacc
     1741 tcttacgcaa tataatagat acttagctct ttataataag tacaagtatt ttagtggagc
     1801 aatggataca actagctaca gagaagctgc ttgttgtcat ctcgcaaagg ctctcaatga
     1861 cttcagtaac tcaggttctg atgttcttta ccaaccacca caaacctcta tcacctcagc
     1921 tgttttgctc gtgccgagag gttctgatta taaagatcat gatggtgatt ataaagatca
     1981 tgatattgat tataaagatg atgatgataa ataaacaggc cccttttcct ttgtcgatat
     2041 catgtaatta gttatgtcac gcttacattc acgccctcct cccacatccg ctctaaccga
     2101 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tatttttttt aatagttatg
     2161 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta caaagcggcc
     2221 gccgaaccac tagtgcactg cagtacacgg tttccttgaa atttttttga ttcggtaatc
     2281 tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata tatacgcata
     2341 tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca cagaacaaaa
     2401 acgtgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg tgctgctact
     2461 catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca aacaaacttg
     2521 tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga agcattaggt
     2581 cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc catggagggc
     2641 acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt cgaggacaga
     2701 aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt atacagaata
     2761 gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt
     2821 ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat gttagcagaa
     2881 ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt tgacattgcg
     2941 aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg tggaagagat
     3001 gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa gggagatgca
     3061 ttgggtcaac agtatagaac cgtggatgat gtggtttcta caggatctga cattattatt
     3121 gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga
     3181 aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta
     3241 taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt atatcagtta
     3301 ttacccgagt aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc
     3361 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa
     3421 agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg
     3481 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac
     3541 gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca
     3601 tcccgagagc gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg
     3661 ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag
     3721 gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta
     3781 ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt
     3841 ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt
     3901 cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga
     3961 aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt
     4021 tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc
     4081 aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat
     4141 agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct
     4201 cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt catcagagcg cttttggttt
     4261 tcaaaagcgc tctgaagttc ctatactttc tagctagaga ataggaactt cccgagcggc
     4321 cgcgtgttac aagcttgact ccagcgtaac tggactgcaa tcaactcact ggctcacctt
     4381 cacgggtggg cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg
     4441 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4501 gatcaagagc taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa
     4561 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg gtaaacgcct
     4921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat
     4981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa
     5041 ggtgagccag gtgattacat ttgggccctc atcagaggtt ttcaccgtca tcaccgaaac
     5101 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     5161 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     5221 taaagcgggc catgttaagg gcggtttttt cctgtttggt cacttaccaa tgcttaatca
     5281 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5341 tcgtgtagat aactacgatg cgggagggct taccatctgg ccccagtgct gcaatgatac
     5401 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccgggaggg
     5461 ccgagcgcag aagtgatcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5521 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5581 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5641 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5701 ctccgatcgt tgccagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5761 tgcataattc tcttactgtc atgccatccg tgagatgctt ttctgtgact ggtgagtact
     5821 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5881 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5941 cttcggggcg taaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6001 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6061 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6121 tcattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg
     6181 atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaagt caaaagcctc
     6241 cggtcggagg cttttgactt tctgctatgg aggtcaggta tgatttaaat ggtcagttag
     6301 a
//
