LOCUS       sequence-295529-        8102 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 8102)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8102)
  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-m4133849
FEATURES             Location/Qualifiers
     source          1..8102
                     /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    1642..1763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1653..1672)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1707..1726
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1878..1924
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1954..2090
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2135..2206
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2432..3020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2512..2531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3520..4729
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4566..4585
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4733..5761
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5788..5836
                     /note="synthetic polyadenylation signal"
     CDS             join(6102..6170,6171..6962)
                     /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(6320..6339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6975..7111
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7256..7327
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7332..7378)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7396..7444
                     /note="synthetic polyadenylation signal"
     enhancer        7525..7828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7830..8032
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7983..8003
                     /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 agttaaggat ccgcaggcct ctgctagctt gactgactga gatacagcgt
     1621 accttcagct cacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     1681 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     1741 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     1801 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtattggcc
     1861 catctctatc ggtatcgtag cataacccct tggggcctct aaacgggtct tgaggggttt
     1921 tttgtgcccc tcgggccgga ttgctatcta ccggcattgg cgcagaaaaa aatgcctgat
     1981 gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc
     2041 caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa ggtcgtcagc
     2101 tatcctgcag gcgatctctc gatttcgatc aagacattcc tttaatggtc ttttctggac
     2161 accactaggg gtcagaagta gttcatcaaa ctttcttccc tccctaatct cattggttac
     2221 cttgggctat cgaaacttaa ttaaccagtc aagtcagcta cttggcgaga tcgacttgtc
     2281 tgggtttcga ctacgctcag aattgcgtca gtcaagttcg atctggtcct tgctattgca
     2341 cccgttctcc gattacgagt ttcatttaaa tcatgtgagc aaaaggccag caaaaggcca
     2401 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2461 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2521 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2581 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2641 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2701 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2761 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2821 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2881 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2941 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3001 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3061 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3121 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3181 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3241 gttcatccat agttgcattt aaatttccga actctccaag gccctcgtcg gaaaatcttc
     3301 aaacctttcg tccgatccat cttgcaggct acctctcgaa cgaactatcg caagtctctt
     3361 ggccggcctt gcgccttggc tattgcttgg cagcgcctat cgccaggtat tactccaatc
     3421 ccgaatatcc gagatcggga tcacccgaga gaagttcaac ctacatcctc aatcccgatc
     3481 tatccgagat ccgaggaata tcgaaatcgg ggcgcgcctg gcctccgcgc cgggttttgg
     3541 cgcctcccgc gggcgccccc ctcgtcacgg cgagcgctgc cacgtcagac gaagggcgca
     3601 ggagcgtcct gatccttccg cccggacgct caggacagcg gcccgctgct cataagactc
     3661 ggccttagaa ccccagtatc agcagaagga cattttagga cgggacttgg gtgactctag
     3721 ggcactggtt ttctttccag agagcggaac aggcgaggaa aagtagtccc ttctcggcga
     3781 ttctgcggag ggatctccgt ggggcggtga acgccgatga ttatataagg acgcgccggg
     3841 tgtggcacag ctagttccgt cgcagccggg atttgggtcg cggttcttgt ttgtggatcg
     3901 ctgtgatcgt cacttggtga gtagcgggct gctgggctgg ccggggcttt cgtggccgcc
     3961 gggccgctcg gtgggacgga agcgtgtgga gagaccgcca agggctgtag tctgggtccg
     4021 cgagcaaggt tgccctgaac tgggggttgg ggggagcgca gcaaaatggc ggctgttccc
     4081 gagtcttgaa tggaagacgc ttgtgaggcg ggctgtgagg tcgttgaaac aaggtggggg
     4141 gcatggtggg cggcaagaac ccaaggtctt gagcccttcg ctaatgcggg aaagctctta
     4201 ttcgggtgag atgggctggg caccatctgg ggaccctgac gtgaagtttg tcactgactg
     4261 gagaactcgg tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg
     4321 cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc
     4381 ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag
     4441 gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg
     4501 aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa
     4561 gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt
     4621 tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac
     4681 ttgtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagaca acatgggtaa
     4741 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     4801 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     4861 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     4921 tgtttatcgg cactttgcat ccgccgcgct cccgattccg gaagtgcttg acattgggga
     4981 gttcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     5041 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcaa tggatgccat
     5101 cgctgccgcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     5161 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     5221 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     5281 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     5341 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     5401 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     5461 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccccggct
     5521 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     5581 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     5641 gactgtcggg cgtacacaaa tcgcccgcag aagcgccgcc gtctggaccg atggctgtgt
     5701 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     5761 agctagtatg taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt
     5821 gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag
     5881 tgcgagtctc gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg
     5941 acccaggaag tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc
     6001 tgtttaaacc tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg
     6061 caaacatcta tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc
     6121 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6181 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac
     6241 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga
     6301 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     6361 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca
     6421 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6481 tgagtgataa cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa
     6541 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6601 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6661 ccttgcgtaa actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag
     6721 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6781 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6841 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6901 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6961 aaccgattct aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat
     7021 actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata
     7081 cgtgtcctcc ttaccagaaa tttatcctta agatcgttta aactcgactc tggctctatc
     7141 gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat
     7201 cgaatctcgt cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct
     7261 tggaccatta gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct
     7321 acctctcaat tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc
     7381 aatcgttgcg ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt
     7441 tattatctaa ctgctgatcg agtgtagcca gatctagtaa tcaattacgg ggtcattagt
     7501 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     7561 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     7621 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     7681 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     7741 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     7801 ctacgtatta gtcatcgcta ttaccatgct gatgcggttt tggcagtaca tcaatgggcg
     7861 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     7921 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     7981 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     8041 gaaccgtcag atcagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc
     8101 gc
//
