LOCUS       sequence-297627-        6259 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 6259)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6259)
  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-m4046439
FEATURES             Location/Qualifiers
     source          1..6259
                     /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             1934..1951
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1952..1969
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2014..2032)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2383..3186
                     /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      3269..4251
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4329..4358
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4380..4967
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(4979..5006)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5030..5221)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5222..6013,6014..6082))
                     /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     5845..5864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6083..6185)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6186..6217)
                     /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 atgaagattg tcaacaattg gcttaaacag ttgataaagg ttactttagt
      481 atttttgttc gttgcagcca tcttctatct aattacacct gtccacgtga tgagtaagca
      541 taccgacttt tcctcagaga tcattggtta caaagctatc gatggcggag ttactagaga
      601 tattgcttcg acggacacct gtttcgctaa caagcacgcg gactttgata cttggttctc
      661 tcaaaggggt ggttcctaca caaacgacaa ggcctgcccc ttgattgcag ctgttatcac
      721 tagagaagtt ggtttcgtcg tgccaggttt accagggact atactgcgta ccaccaatgg
      781 tgattttttg catttcttac caagagtctt ctctgctgtt ggcaatattt gttatacccc
      841 tagcaaattg atcgaataca ctgattttgc cacctctgct tgtgtattgg ctgctgaatg
      901 cactattttc aaggatgcaa gtggtaagcc agttccatac tgttatgaca caaacgtcct
      961 cgaaggttct gttgcttacg aatccttgcg accggacact agatatgtgt taatggatgg
     1021 atcaatcatt caatttccaa acacatacct tgaaggttct gttcgggttg tcaccacctt
     1081 cgactccgag tactgtcgcc acggtacttg tgaaagatca gaagccggtg tctgtgtttc
     1141 taccagcggt agatgggtgt tgaataacga ttattaccgt tctcttccag gtgtcttctg
     1201 cggtgtcgat gctgttaatc tattgactaa catgtttacc cctttgatcc aaccaatagg
     1261 tgcgctggac atttccgcat ctattgtcgc cggtggaatc gttgctatcg ttgttacttg
     1321 tttggcttac tacttcatga gatttagaag agcctttggg gagtattcgc atgtcgtggc
     1381 tttcaacact ctattatttt tgatgtcgtt cactgttttg tgtcttacac cagtatactc
     1441 tttcttgcct ggtgtatact ccgttatcta tttatacctg acattctacc tgactaacga
     1501 tgtcagcttt ttggcacaca ttcaatggat ggtaatgttc accccattgg ttcccttctg
     1561 gataacgatc gcttacatca tttgcataag tactaaacat ttctattggt ttttctcaaa
     1621 ttatctaaag agaagagttg tctttaatgg tgtcagcttt tctaccttcg aagaagctgc
     1681 cttgtgtacc tttttgctca acaaagaaat gtacttgaag ttgagatctg acgttttact
     1741 cccgctaacc caatataaca gatatttagc tttatacaac aagtacaaat acttctcggg
     1801 tgccatggat actacttcat accgtgaagc ggcttgttgt caccttgcta aggctttgaa
     1861 tgatttctct aattccggtt ccgacgtctt gtatcaacca cctcagacgt ctattacctc
     1921 cgctgttttg caactcgtgc cgagaggttc tcaccatcat caccatcatt aaacaggccc
     1981 cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctcctcc
     2041 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     2101 ttttttttaa tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     2161 tttctgtaca aagcggccgc cgaaccacta gtgcactgca gtacacggtt tccttgaaat
     2221 ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga
     2281 ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc
     2341 caactgcaca gaacaaaaac gtgcaggaaa cgaagataaa tcatgtcgaa agctacatat
     2401 aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac
     2461 gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag
     2521 ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact
     2581 gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt
     2641 ttactcttcg aggacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct
     2701 gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc
     2761 ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc
     2821 cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag
     2881 ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga
     2941 gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta
     3001 gatgacaagg gagatgcatt gggtcaacag tatagaaccg tggatgatgt ggtttctaca
     3061 ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta
     3121 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa
     3181 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca
     3241 atttaattat atcagttatt acccgagtaa cgaagcatct gtgcttcatt ttgtagaaca
     3301 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     3361 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta
     3421 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt
     3481 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt
     3541 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact
     3601 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc
     3661 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga
     3721 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa
     3781 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg
     3841 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc
     3901 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat
     3961 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta
     4021 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga
     4081 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt
     4141 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtca
     4201 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gctagagaat
     4261 aggaacttcc cgagcggccg cgtgttacaa gcttgactcc agcgtaactg gactgcaatc
     4321 aactcactgg ctcaccttca cgggtgggcc tttcttcggt agaaaatcaa aggatcttct
     4381 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4441 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaggta actggcttca
     4501 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4561 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4621 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4681 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4741 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4801 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4861 ttccaggggt aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4921 agcatcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4981 cagaaaggcc cacccgaagg tgagccaggt gattacattt gggccctcat cagaggtttt
     5041 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     5101 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     5161 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     5221 cttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5281 tagttgcctg actccccgtc gtgtagataa ctacgatgcg ggagggctta ccatctggcc
     5341 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5401 accagccagc cgggagggcc gagcgcagaa gtgatcctgc aactttatcc gcctccatcc
     5461 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5521 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5581 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5641 cggttagctc cttcggtcct ccgatcgttg ccagaagtaa gttggccgca gtgttatcac
     5701 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgtg agatgctttt
     5761 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5821 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5881 tcatcattgg aaaacgttct tcggggcgta aactctcaag gatcttaccg ctgttgagat
     5941 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6001 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6061 cacggaaatg ttgaatactc attttagctt ccttagctcc tgaaaatctc gataactcaa
     6121 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     6181 gatcaagtca aaagcctccg gtcggaggct tttgactttc tgctatggag gtcaggtatg
     6241 atttaaatgg tcagttaga
//
