LOCUS       sequence-297741-        6253 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 6253)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6253)
  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-m4046322
FEATURES             Location/Qualifiers
     source          1..6253
                     /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..1963
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2008..2026)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2377..3180
                     /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      3263..4245
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4323..4352
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4374..4961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(4973..5000)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5024..5215)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5216..6007,6008..6076))
                     /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     5839..5858
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6077..6179)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6180..6211)
                     /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 gttctcacca tcatcaccat cattaaacag gccccttttc
     1981 ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc ctcccacatc
     2041 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     2101 ttaatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     2161 tacaaagcgg ccgccgaacc actagtgcac tgcagtacac ggtttccttg aaattttttt
     2221 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta
     2281 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg
     2341 cacagaacaa aaacgtgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa
     2401 cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag
     2461 caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt
     2521 gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt
     2581 tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc
     2641 ttcgaggaca gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt
     2701 gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt
     2761 attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg
     2821 atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact
     2881 gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg
     2941 ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac
     3001 aagggagatg cattgggtca acagtataga accgtggatg atgtggtttc tacaggatct
     3061 gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt
     3121 gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa
     3181 aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa
     3241 ttatatcagt tattacccga gtaacgaagc atctgtgctt cattttgtag aacaaaaatg
     3301 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa
     3361 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa
     3421 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     3481 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc
     3541 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt
     3601 ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa
     3661 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac
     3721 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc
     3781 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg
     3841 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata
     3901 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct
     3961 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc
     4021 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc
     4081 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat
     4141 attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcatcagag
     4201 cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagctaga gaataggaac
     4261 ttcccgagcg gccgcgtgtt acaagcttga ctccagcgta actggactgc aatcaactca
     4321 ctggctcacc ttcacgggtg ggcctttctt cggtagaaaa tcaaaggatc ttcttgagat
     4381 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4441 gtttgtttgc cggatcaaga gctaccaact ctttttccga ggtaactggc ttcagcagag
     4501 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4561 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4621 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4681 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4741 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4801 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4861 gggtaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcatc
     4921 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcagaaa
     4981 ggcccacccg aaggtgagcc aggtgattac atttgggccc tcatcagagg ttttcaccgt
     5041 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     5101 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     5161 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcacttacc
     5221 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5281 cctgactccc cgtcgtgtag ataactacga tgcgggaggg cttaccatct ggccccagtg
     5341 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5401 cagccgggag ggccgagcgc agaagtgatc ctgcaacttt atccgcctcc atccagtcta
     5461 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5521 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5581 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5641 gctccttcgg tcctccgatc gttgccagaa gtaagttggc cgcagtgtta tcactcatgg
     5701 ttatggcagc actgcataat tctcttactg tcatgccatc cgtgagatgc ttttctgtga
     5761 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5821 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5881 ttggaaaacg ttcttcgggg cgtaaactct caaggatctt accgctgttg agatccagtt
     5941 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6001 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6061 aatgttgaat actcatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     6121 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     6181 gtcaaaagcc tccggtcgga ggcttttgac tttctgctat ggaggtcagg tatgatttaa
     6241 atggtcagtt aga
//
