LOCUS       sequence-295834-        8126 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 8126)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8126)
  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-m4134419
FEATURES             Location/Qualifiers
     source          1..8126
                     /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             1532..1597
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1666..1787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1677..1696)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1731..1750
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1902..1948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1978..2114
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2159..2230
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2456..3044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2536..2555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3544..4753
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4590..4609
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4757..5785
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5812..5860
                     /note="synthetic polyadenylation signal"
     CDS             join(6126..6194,6195..6986)
                     /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(6344..6363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6999..7135
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7280..7351
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7356..7402)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7420..7468
                     /note="synthetic polyadenylation signal"
     enhancer        7549..7852
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7854..8056
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8007..8027
                     /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 ttcagtcgat ggattataag gaccacgatg gagactacaa
     1561 agatcatgac attgattaca aggacgacga tgacaaataa ggatccgcag gcctctgcta
     1621 gcttgactga ctgagataca gcgtaccttc agctcacaga catgataaga tacattgatg
     1681 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1741 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1801 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     1861 acctctacaa atgtggtatt ggcccatctc tatcggtatc gtagcataac cccttggggc
     1921 ctctaaacgg gtcttgaggg gttttttgtg cccctcgggc cggattgcta tctaccggca
     1981 ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag
     2041 attcagcaac ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag
     2101 aaatttatcc ttaaggtcgt cagctatcct gcaggcgatc tctcgatttc gatcaagaca
     2161 ttcctttaat ggtcttttct ggacaccact aggggtcaga agtagttcat caaactttct
     2221 tccctcccta atctcattgg ttaccttggg ctatcgaaac ttaattaacc agtcaagtca
     2281 gctacttggc gagatcgact tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag
     2341 ttcgatctgg tccttgctat tgcacccgtt ctccgattac gagtttcatt taaatcatgt
     2401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc atttaaattt ccgaactctc
     3301 caaggccctc gtcggaaaat cttcaaacct ttcgtccgat ccatcttgca ggctacctct
     3361 cgaacgaact atcgcaagtc tcttggccgg ccttgcgcct tggctattgc ttggcagcgc
     3421 ctatcgccag gtattactcc aatcccgaat atccgagatc gggatcaccc gagagaagtt
     3481 caacctacat cctcaatccc gatctatccg agatccgagg aatatcgaaa tcggggcgcg
     3541 cctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcgtc acggcgagcg
     3601 ctgccacgtc agacgaaggg cgcaggagcg tcctgatcct tccgcccgga cgctcaggac
     3661 agcggcccgc tgctcataag actcggcctt agaaccccag tatcagcaga aggacatttt
     3721 aggacgggac ttgggtgact ctagggcact ggttttcttt ccagagagcg gaacaggcga
     3781 ggaaaagtag tcccttctcg gcgattctgc ggagggatct ccgtggggcg gtgaacgccg
     3841 atgattatat aaggacgcgc cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg
     3901 gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg
     3961 ctggccgggg ctttcgtggc cgccgggccg ctcggtggga cggaagcgtg tggagagacc
     4021 gccaagggct gtagtctggg tccgcgagca aggttgccct gaactggggg ttggggggag
     4081 cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag acgcttgtga ggcgggctgt
     4141 gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgagccc
     4201 ttcgctaatg cgggaaagct cttattcggg tgagatgggc tgggcaccat ctggggaccc
     4261 tgacgtgaag tttgtcactg actggagaac tcggtttgtc gtctgttgcg ggggcggcag
     4321 ttatggcggt gccgttgggc agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt
     4381 cgtgacgtca cccgttctgt tggcttataa tgcagggtgg ggccacctgc cggtaggtgt
     4441 gcggtaggct tttctccgtc gcaggacgca gggttcgggc ctagggtagg ctctcctgaa
     4501 tcgacaggcg ccggacctct ggtgagggga gggataagtg aggcgtcagt ttctttggtc
     4561 ggttttatgt acctatcttc ttaagtagct gaagctccgg ttttgaacta tgcgctcggg
     4621 gttggcgagt gtgttttgtg aagtttttta ggcacctttt gaaatgtaat catttgggtc
     4681 aatatgtaat tttcagtgtt agacttgtaa attgtccgct aaattctggc cgtttttggc
     4741 ttttttgtta gacaacatgg gtaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     4801 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     4861 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     4921 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatccgccg cgctcccgat
     4981 tccggaagtg cttgacattg gggagttcag cgagagcctg acctattgca tctcccgccg
     5041 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     5101 ggtcgcggag gcaatggatg ccatcgctgc cgccgatctt agccagacga gcgggttcgg
     5161 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     5221 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     5281 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     5341 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     5401 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     5461 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     5521 ggagcttgca ggatcgcccc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     5581 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     5641 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     5701 cgccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     5761 cactcgtccg agggcaaagg aataagctag tatgtaagcc tagtcttaga taataaaatc
     5821 gctatccatc gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg
     5881 gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt
     5941 cagccagtcg gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag
     6001 cctggtcacg gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa
     6061 cgtataatcg agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct
     6121 ttcgcatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6241 gtgcgcgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6301 gccccgaaga acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6421 acttggttga gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6541 cgattggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6661 cgatgcctgt agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc
     6721 tagcttcccg gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc
     6781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6961 gtgcctcact gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc
     7021 ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc
     7081 ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg
     7141 tttaaactcg actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt
     7201 ggcgctcatt tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg
     7261 cagcgatcgc ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta
     7321 gtggtcataa cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta
     7381 gaggccccaa ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca
     7441 tccatcttcg atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta
     7501 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     7561 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     7621 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     7681 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     7741 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     7801 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg
     7861 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     7921 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     7981 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     8041 ctatataagc agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc
     8101 cactcgacac acccgccagc ggccgc
//
