LOCUS       sequence-295883-        7996 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 7996)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7996)
  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-m4133665
FEATURES             Location/Qualifiers
     source          1..7996
                     /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             1871..1888
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1965..2086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1976..1995)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2030..2049
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2201..2247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2277..2413
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2458..2529
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2755..3343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2835..2854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3843..5052
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4889..4908
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5056..5655
                     /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(5056..5075)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5552..5572
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5682..5730
                     /note="synthetic polyadenylation signal"
     CDS             join(5996..6064,6065..6856)
                     /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(6214..6233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6869..7005
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7150..7221
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7226..7272)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7290..7338
                     /note="synthetic polyadenylation signal"
     enhancer        7419..7722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7724..7926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7877..7897
                     /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 cgatgagtga caaaatcatt catctaaccg atgactcctt
     1561 tgatacagac gtgttgaagg ccgatggtgc gatactggta gatttctggg ctgaatggtg
     1621 tggcccgtgc aagatgatcg cacccattct ggacgagatc gccgacgagt atcaaggaaa
     1681 attaactgtc gctaagctta atatagatca gaacccaggg acggcaccta aatacggcat
     1741 caggggaata cctaccctcc tgctctttaa gaatggggaa gttgccgcga caaaagtggg
     1801 agccctgtct aagggccagc taaaagagtt cttggacgct aacctggcag gttcaggctc
     1861 ggggcacatg caccatcacc atcaccatag cagcgggctc gtgccacgcg gctaagctag
     1921 cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat acattgatga
     1981 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     2041 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2101 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     2161 cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc
     2221 tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat
     2281 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga
     2341 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga
     2401 aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat
     2461 tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt
     2521 ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag
     2581 ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt
     2641 tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg
     2701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3541 tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc
     3601 aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc
     3661 gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc
     3721 tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc
     3781 aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc
     3841 ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc
     3901 tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca
     3961 gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta
     4021 ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag
     4081 gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga
     4141 tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg
     4201 tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc
     4261 tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg
     4321 ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc
     4381 gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg
     4441 aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct
     4501 tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct
     4561 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt
     4621 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc
     4681 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg
     4741 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat
     4801 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg
     4861 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg
     4921 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca
     4981 atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct
     5041 tttttgttag acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     5101 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     5161 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     5221 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     5281 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     5341 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg
     5401 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     5461 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg
     5521 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     5581 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     5641 aagcccggtg cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc
     5701 gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag
     5761 aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg
     5821 gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg
     5881 gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg
     5941 agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag
     6001 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6061 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt
     6121 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6181 acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6241 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6301 gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6361 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg
     6421 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6481 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6541 agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg
     6601 gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6661 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6721 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6781 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6841 gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac
     6901 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact
     6961 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg tttaaactcg
     7021 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt
     7081 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc
     7141 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa
     7201 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa
     7261 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg
     7321 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt
     7381 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     7441 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     7501 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     7561 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     7621 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     7681 acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag
     7741 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     7801 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     7861 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     7921 agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc cactcgacac
     7981 acccgccagc ggccgc
//
