LOCUS       sequence-295929-        8096 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 8096)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8096)
  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-m4133592
FEATURES             Location/Qualifiers
     source          1..8096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1502..1522
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1523..1567
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    1636..1757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1647..1666)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1701..1720
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1872..1918
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1948..2084
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2129..2200
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2426..3014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2506..2525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3514..4723
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4560..4579
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4727..5755
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5782..5830
                     /note="synthetic polyadenylation signal"
     CDS             join(6096..6164,6165..6956)
                     /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(6314..6333)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6969..7105
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7250..7321
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7326..7372)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7390..7438
                     /note="synthetic polyadenylation signal"
     enhancer        7519..7822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7824..8026
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7977..7997
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgatt gttaataatt ggttgaagca gttaattaaa gttacacttg tgttcctttt
       61 tgttgctgct attttctatt taataacacc tgttcatgtc atgtctaaac atactgactt
      121 ttcaagtgaa atcataggat acaaggctat tgatggtggt gtcactcgtg acatagcatc
      181 tacagatact tgttttgcta acaaacatgc tgattttgac acatggttta gccagcgtgg
      241 tggtagttat actaatgaca aagcttgccc attgattgct gcagtcataa caagagaagt
      301 gggttttgtc gtgcctggtt tgcctggcac gatattacgc acaactaatg gtgacttttt
      361 gcatttctta cctagagttt ttagtgcagt tggtaacatc tgttacacac catcaaaact
      421 tatagagtac actgactttg caacatcagc ttgtgttttg gctgctgaat gtacaatttt
      481 taaagatgct tctggtaagc cagtaccata ttgttatgat accaatgtac tagaaggttc
      541 tgttgcttat gaaagtttac gccctgacac acgttatgtg ctcatggatg gctctattat
      601 tcaatttcct aacacctacc ttgaaggttc tgttagagtg gtaacaactt ttgattctga
      661 gtactgtagg cacggcactt gtgaaagatc agaagctggt gtttgtgtat ctactagtgg
      721 tagatgggta cttaacaatg attattacag atctttacca ggagttttct gtggtgtaga
      781 tgctgtaaat ttacttacta atatgtttac accactaatt caacctattg gtgctttgga
      841 catatcagca tctatagtag ctggtggtat tgtagctatc gtagtaacat gccttgccta
      901 ctattttatg aggtttagaa gagcttttgg tgaatacagt catgtagttg cctttaatac
      961 tttactattc cttatgtcat tcactgtact ctgtttaaca ccagtttact cattcttacc
     1021 tggtgtttat tctgttattt acttgtactt gacattttat cttactaatg atgtttcttt
     1081 tttagcacat attcagtgga tggttatgtt cacaccttta gtacctttct ggataacaat
     1141 tgcttatatc atttgtattt ccacaaagca tttctattgg ttctttagta attacctaaa
     1201 gagacgtgta gtctttaatg gtgtttcctt tagtactttt gaagaagctg cgctgtgcac
     1261 ctttttgtta aataaagaaa tgtatctaaa gttgcgtagt gatgtgctat tacctcttac
     1321 gcaatataat agatacttag ctctttataa taagtacaag tattttagtg gagcaatgga
     1381 tacaactagc tacagagaag ctgcttgttg tcatctcgca aaggctctca atgacttcag
     1441 taactcaggt tctgatgttc tttaccaacc accacaaacc tctatcacct cagctgtttt
     1501 ggaaaacctt tactttcagt cgaaagaaac cgcggcagct aagttcgaga ggcagcacat
     1561 ggacagttaa ggatccgcag gcctctgcta gcttgactga ctgagataca gcgtaccttc
     1621 agctcacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     1681 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     1741 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     1801 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatt ggcccatctc
     1861 tatcggtatc gtagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgtg
     1921 cccctcgggc cggattgcta tctaccggca ttggcgcaga aaaaaatgcc tgatgcgacg
     1981 ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct tccccaactt
     2041 gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt cagctatcct
     2101 gcaggcgatc tctcgatttc gatcaagaca ttcctttaat ggtcttttct ggacaccact
     2161 aggggtcaga agtagttcat caaactttct tccctcccta atctcattgg ttaccttggg
     2221 ctatcgaaac ttaattaacc agtcaagtca gctacttggc gagatcgact tgtctgggtt
     2281 tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg tccttgctat tgcacccgtt
     2341 ctccgattac gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag gccaggaacc
     2401 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2461 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2521 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2581 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2641 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2701 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2761 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2821 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2881 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2941 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3001 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3061 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3121 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3181 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3241 ccatagttgc atttaaattt ccgaactctc caaggccctc gtcggaaaat cttcaaacct
     3301 ttcgtccgat ccatcttgca ggctacctct cgaacgaact atcgcaagtc tcttggccgg
     3361 ccttgcgcct tggctattgc ttggcagcgc ctatcgccag gtattactcc aatcccgaat
     3421 atccgagatc gggatcaccc gagagaagtt caacctacat cctcaatccc gatctatccg
     3481 agatccgagg aatatcgaaa tcggggcgcg cctggcctcc gcgccgggtt ttggcgcctc
     3541 ccgcgggcgc ccccctcgtc acggcgagcg ctgccacgtc agacgaaggg cgcaggagcg
     3601 tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag actcggcctt
     3661 agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact ctagggcact
     3721 ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg gcgattctgc
     3781 ggagggatct ccgtggggcg gtgaacgccg atgattatat aaggacgcgc cgggtgtggc
     3841 acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg atcgctgtga
     3901 tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc cgccgggccg
     3961 ctcggtggga cggaagcgtg tggagagacc gccaagggct gtagtctggg tccgcgagca
     4021 aggttgccct gaactggggg ttggggggag cgcagcaaaa tggcggctgt tcccgagtct
     4081 tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg
     4141 tgggcggcaa gaacccaagg tcttgagccc ttcgctaatg cgggaaagct cttattcggg
     4201 tgagatgggc tgggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac
     4261 tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg
     4321 tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa
     4381 tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca
     4441 gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga
     4501 gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct
     4561 gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta
     4621 ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agacttgtaa
     4681 attgtccgct aaattctggc cgtttttggc ttttttgtta gacaacatgg gtaaaaagcc
     4741 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     4801 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4861 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4921 tcggcacttt gcatccgccg cgctcccgat tccggaagtg cttgacattg gggagttcag
     4981 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     5041 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gcaatggatg ccatcgctgc
     5101 cgccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     5161 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     5221 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     5281 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     5341 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     5401 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     5461 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgcccc ggctccgggc
     5521 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     5581 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     5641 cgggcgtaca caaatcgccc gcagaagcgc cgccgtctgg accgatggct gtgtagaagt
     5701 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aataagctag
     5761 tatgtaagcc tagtcttaga taataaaatc gctatccatc gaagatggat gtgtgttggt
     5821 tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag
     5881 tctcgacgat ccatatcgtt gcttggcagt cagccagtcg gaatccagct tgggacccag
     5941 gaagtccaat cgtcagatat tgtactcaag cctggtcacg gcagcgtacc gatctgttta
     6001 aacctagata ttgatagtct gatcggtcaa cgtataatcg agtcctagct tttgcaaaca
     6061 tctatcaaga gacaggatca gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg
     6121 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6181 tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt gggttacatc gaactggatc
     6241 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgctttcca atgatgagca
     6301 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6361 tcggtcgccg catacactat tctcagaatg acttggttga gtattcacca gtcacagaaa
     6421 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6481 ataacactgc ggccaactta cttctgacaa cgattggagg accgaaggag ctaaccgctt
     6541 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6601 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaaccttgc
     6661 gtaaactatt aactggcgaa ctacttactc tagcttcccg gcaacagttg atagactgga
     6721 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6781 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6841 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6901 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaaccga
     6961 ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc
     7021 acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc
     7081 ctccttacca gaaatttatc cttaagatcg tttaaactcg actctggctc tatcgaatct
     7141 ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt tgctcgtcgg gcatcgaatc
     7201 tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc ggctcccgac atcttggacc
     7261 attagctcca caggtatctt cttccctcta gtggtcataa cagcagcttc agctacctct
     7321 caattcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tatcaatcgt
     7381 tgcgttacac acacaaaaaa ccaacacaca tccatcttcg atggatagcg attttattat
     7441 ctaactgctg atcgagtgta gccagatcta gtaatcaatt acggggtcat tagttcatag
     7501 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     7561 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     7621 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     7681 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     7741 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     7801 attagtcatc gctattacca tgctgatgcg gttttggcag tacatcaatg ggcgtggata
     7861 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     7921 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     7981 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     8041 tcagatcaga tctttgtcga tcctaccatc cactcgacac acccgccagc ggccgc
//
