LOCUS       sequence-299927-        6307 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 6307)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6307)
  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-m4046182
FEATURES             Location/Qualifiers
     source          1..6307
                     /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..2017
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2062..2080)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2431..3234
                     /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      3317..4299
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4377..4406
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4428..5015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(5027..5054)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5078..5269)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5270..6061,6062..6130))
                     /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     5893..5912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6131..6233)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(6234..6265)
                     /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 tgattataaa gatcatgatg gtgattataa
     1981 agatcatgat attgattata aagatgatga tgataaataa acaggcccct tttcctttgt
     2041 cgatatcatg taattagtta tgtcacgctt acattcacgc cctcctccca catccgctct
     2101 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt ttttttaata
     2161 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaaa
     2221 gcggccgccg aaccactagt gcactgcagt acacggtttc cttgaaattt ttttgattcg
     2281 gtaatctccg aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata
     2341 cgcatatgta gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga
     2401 acaaaaacgt gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct
     2461 gctactcatc ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca
     2521 aacttgtgtg cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca
     2581 ttaggtccca aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg
     2641 gagggcacag ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgag
     2701 gacagaaaat ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac
     2761 agaatagcag aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt
     2821 agcggtttga agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta
     2881 gcagaattgt catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac
     2941 attgcgaaga gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga
     3001 agagatgaag gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga
     3061 gatgcattgg gtcaacagta tagaaccgtg gatgatgtgg tttctacagg atctgacatt
     3121 attattgttg gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt
     3181 tacagaaaag caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact
     3241 gtattataag taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat
     3301 cagttattac ccgagtaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc
     3361 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
     3421 cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc
     3481 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     3541 tgcaacgcga gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa
     3601 aatgcatccc gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt
     3661 tgtgcgctct ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag
     3721 aagaaggcta ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg
     3781 cgtttactga ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat
     3841 tatattctat accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg
     3901 attcttcatt ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt
     3961 ataggaaatg tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac
     4021 aatttttttg tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt
     4081 agatgcaagt tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag
     4141 atatatagca aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta
     4201 gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa gtgcgtcatc agagcgcttt
     4261 tggttttcaa aagcgctctg aagttcctat actttctagc tagagaatag gaacttcccg
     4321 agcggccgcg tgttacaagc ttgactccag cgtaactgga ctgcaatcaa ctcactggct
     4381 caccttcacg ggtgggcctt tcttcggtag aaaatcaaag gatcttcttg agatcctttt
     4441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4501 ttgccggatc aagagctacc aactcttttt ccgaggtaac tggcttcagc agagcgcaga
     4561 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4621 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4681 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4741 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4801 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4861 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccaggggtaa
     4921 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag catcgatttt
     4981 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgca gaaaggccca
     5041 cccgaaggtg agccaggtga ttacatttgg gccctcatca gaggttttca ccgtcatcac
     5101 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     5161 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     5221 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact taccaatgct
     5281 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5341 tccccgtcgt gtagataact acgatgcggg agggcttacc atctggcccc agtgctgcaa
     5401 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5461 ggagggccga gcgcagaagt gatcctgcaa ctttatccgc ctccatccag tctattaatt
     5521 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5581 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5641 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5701 tcggtcctcc gatcgttgcc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5761 cagcactgca taattctctt actgtcatgc catccgtgag atgcttttct gtgactggtg
     5821 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5881 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5941 aacgttcttc ggggcgtaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6001 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6061 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6121 gaatactcat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     6181 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaagtcaaa
     6241 agcctccggt cggaggcttt tgactttctg ctatggaggt caggtatgat ttaaatggtc
     6301 agttaga
//
