LOCUS       sequence-295848-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7657)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7657)
  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-m4133496
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1508..1528
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1529..1573
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    1626..1747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1637..1656)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1691..1710
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1862..1908
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1938..2074
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2119..2190
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2416..3004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2496..2515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3504..4713
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4550..4569
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4717..5316
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4717..4736)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5213..5233
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5343..5391
                     /note="synthetic polyadenylation signal"
     CDS             join(5657..5725,5726..6517)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5875..5894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6530..6666
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6811..6882
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6887..6933)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6951..6999
                     /note="synthetic polyadenylation signal"
     enhancer        7080..7383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7385..7587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7538..7558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgaag attgtaaata actggttgaa acaactaata aaggttactt tagtgttcct
       61 gtttgtcgcc gcaatcttct atcttatcac cccggtgcac gtgatgagta aacatacaga
      121 cttttcttca gaaattatcg ggtacaaggc tatcgatggc ggtgtaacgc gcgacattgc
      181 ctccacagat acctgcttcg cgaacaaaca cgctgacttt gatacttggt tcagccagcg
      241 aggaggctcg tacaccaatg acaaagcctg tccactgatc gcagccgtga taactagaga
      301 ggttggattc gtcgtccctg ggctccccgg gacaattctc cgtacaacca acggcgactt
      361 tctgcatttt ctgccccggg tgttctccgc agtcggtaat atctgttata ctccatctaa
      421 gctcattgag tacacggatt ttgctaccag cgcgtgcgtg ttggccgcag aatgcaccat
      481 atttaaggac gcttctggaa aacctgtgcc ctattgttac gataccaatg tgcttgaggg
      541 ctcagttgcc tatgaaagtc tgaggccaga cactagatac gttttaatgg atggaagcat
      601 catccagttc ccaaacacgt atctggaggg gtccgtgcgg gtcgtgacca cattcgattc
      661 agaatactgc cgccacggta catgcgagag gtctgaggct ggcgtatgtg tgtcaactag
      721 cggtcgatgg gttctaaata acgattatta tcgttctctg cccggcgttt tttgtggagt
      781 ggacgccgtt aaccttttga ccaacatgtt tacacctctc attcagccca ttggtgcact
      841 ggatatctcc gcgagtattg tggctggggg aatagtcgcc attgtcgtaa cctgcttggc
      901 atactatttc atgcgtttca ggcgagcttt tggcgaatac tcacatgtcg tggccttcaa
      961 tactcttctg tttctgatga gcttcacggt actctgtctg accccagtat acagtttcct
     1021 acctggggtg tattctgtga tctacttata cctcacattt tacctgacca atgacgtgtc
     1081 ctttctggcc cacatccaat ggatggttat gttcacgccg ctcgtgcctt tctggattac
     1141 aatcgcttat ataatctgta ttagcacgaa gcatttctat tggttctttt ccaactactt
     1201 aaagcggcgc gtcgtattta atggggtatc gtttagcact tttgaggaag ccgcgctctg
     1261 cactttcttg cttaataagg agatgtattt gaaattaaga tctgacgttc tgcttccttt
     1321 aacacagtac aacagatacc tggcgttgta taacaaatac aaatatttct cgggagccat
     1381 ggatacaact tcctatagag aggctgcttg ttgccacctt gcaaaggcac tcaacgactt
     1441 cagtaacagc ggcagtgatg tgctttatca gccccctcag acttcaataa catccgccgt
     1501 cctccaagaa aacctttact ttcagtcgaa agaaaccgcg gcagctaagt tcgagaggca
     1561 gcacatggac agttaagcta gcttgactga ctgagataca gcgtaccttc agctcacaga
     1621 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     1681 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     1741 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     1801 ggttttttaa agcaagtaaa acctctacaa atgtggtatt ggcccatctc tatcggtatc
     1861 gtagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgtg cccctcgggc
     1921 cggattgcta tctaccggca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct
     1981 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc
     2041 atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt cagctatcct gcaggcgatc
     2101 tctcgatttc gatcaagaca ttcctttaat ggtcttttct ggacaccact aggggtcaga
     2161 agtagttcat caaactttct tccctcccta atctcattgg ttaccttggg ctatcgaaac
     2221 ttaattaacc agtcaagtca gctacttggc gagatcgact tgtctgggtt tcgactacgc
     2281 tcagaattgc gtcagtcaag ttcgatctgg tccttgctat tgcacccgtt ctccgattac
     2341 gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2401 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2461 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2521 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2581 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2641 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2701 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2761 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2821 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2881 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2941 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3001 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3061 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3121 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3181 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3241 atttaaattt ccgaactctc caaggccctc gtcggaaaat cttcaaacct ttcgtccgat
     3301 ccatcttgca ggctacctct cgaacgaact atcgcaagtc tcttggccgg ccttgcgcct
     3361 tggctattgc ttggcagcgc ctatcgccag gtattactcc aatcccgaat atccgagatc
     3421 gggatcaccc gagagaagtt caacctacat cctcaatccc gatctatccg agatccgagg
     3481 aatatcgaaa tcggggcgcg cctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc
     3541 ccccctcgtc acggcgagcg ctgccacgtc agacgaaggg cgcaggagcg tcctgatcct
     3601 tccgcccgga cgctcaggac agcggcccgc tgctcataag actcggcctt agaaccccag
     3661 tatcagcaga aggacatttt aggacgggac ttgggtgact ctagggcact ggttttcttt
     3721 ccagagagcg gaacaggcga ggaaaagtag tcccttctcg gcgattctgc ggagggatct
     3781 ccgtggggcg gtgaacgccg atgattatat aaggacgcgc cgggtgtggc acagctagtt
     3841 ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg
     3901 gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc cgccgggccg ctcggtggga
     3961 cggaagcgtg tggagagacc gccaagggct gtagtctggg tccgcgagca aggttgccct
     4021 gaactggggg ttggggggag cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag
     4081 acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa
     4141 gaacccaagg tcttgagccc ttcgctaatg cgggaaagct cttattcggg tgagatgggc
     4201 tgggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc
     4261 gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg
     4321 agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg
     4381 ggccacctgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc
     4441 ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg
     4501 aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg
     4561 ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt
     4621 gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agacttgtaa attgtccgct
     4681 aaattctggc cgtttttggc ttttttgtta gacaacatga ccgagtacaa gcccacggtg
     4741 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     4801 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     4861 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     4921 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     4981 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     5041 atggagggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     5101 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     5161 gaggccgccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     5221 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     5281 cgcacctggt gcatgacccg caagcccggt gcctaggcta gtatgtaagc ctagtcttag
     5341 ataataaaat cgctatccat cgaagatgga tgtgtgttgg ttttttgtgt gtgtaacgct
     5401 aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga gtctcgacga tccatatcgt
     5461 tgcttggcag tcagccagtc ggaatccagc ttgggaccca ggaagtccaa tcgtcagata
     5521 ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt aaacctagat attgatagtc
     5581 tgatcggtca acgtataatc gagtcctagc ttttgcaaac atctatcaag agacaggatc
     5641 agcaggaggc tttcgcatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5701 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5761 agatcagttg ggtgcgcgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5821 tgagagtttt cgccccgaag aacgctttcc aatgatgagc acttttaaag ttctgctatg
     5881 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5941 ttctcagaat gacttggttg agtattcacc agtcacagaa aagcatctta cggatggcat
     6001 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6061 acttctgaca acgattggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6121 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6181 gcgtgacacc acgatgcctg tagcaatggc aacaaccttg cgtaaactat taactggcga
     6241 actacttact ctagcttccc ggcaacagtt gatagactgg atggaggcgg ataaagttgc
     6301 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6361 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6421 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6481 cgctgagata ggtgcctcac tgattaagca ttggtaaccg attctaggtg cattggcgca
     6541 gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca
     6601 acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat
     6661 ccttaagatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt cgagcttacg
     6721 cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct atcgtcagct
     6781 tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc acaggtatct
     6841 tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa aaacccctca
     6901 agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca cacacaaaaa
     6961 accaacacac atccatcttc gatggatagc gattttatta tctaactgct gatcgagtgt
     7021 agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     7081 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     7141 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     7201 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     7261 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     7321 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     7381 atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     7441 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     7501 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     7561 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag atctttgtcg
     7621 atcctaccat ccactcgaca cacccgccag cggccgc
//
