LOCUS       sequence-295364-        7630 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 7630)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7630)
  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-m4133647
FEATURES             Location/Qualifiers
     source          1..7630
                     /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..1546
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1599..1720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1610..1629)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1664..1683
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1835..1881
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1911..2047
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2092..2163
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2389..2977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2469..2488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3477..4686
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4523..4542
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4690..5289
                     /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(4690..4709)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5186..5206
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5316..5364
                     /note="synthetic polyadenylation signal"
     CDS             join(5630..5698,5699..6490)
                     /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(5848..5867)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6503..6639
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6784..6855
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6860..6906)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6924..6972
                     /note="synthetic polyadenylation signal"
     enhancer        7053..7356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7358..7560
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7511..7531
                     /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 ttcagtcgca ccatcaccat caccattaag ctagcttgac
     1561 tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg
     1621 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1681 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     1741 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     1801 caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa
     1861 cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc
     1921 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc
     1981 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta
     2041 tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt
     2101 aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc
     2161 ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt
     2221 ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc
     2281 tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa
     2341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2821 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3181 agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc
     3241 ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga
     3301 actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc
     3361 caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta
     3421 catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggcc
     3481 tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga gcgctgccac
     3541 gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc
     3601 cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg
     3661 gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag
     3721 tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta
     3781 tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg
     3841 ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg
     3901 gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg
     3961 gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca
     4021 aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg
     4081 ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag cccttcgcta
     4141 atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga ccctgacgtg
     4201 aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc
     4261 ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg
     4321 tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag
     4381 gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag
     4441 gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta
     4501 tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg
     4561 agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt
     4621 aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt ggcttttttg
     4681 ttagacaaca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     4741 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4801 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4861 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4921 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     4981 ttgagcggtt cccggctggc cgcgcagcaa cagatggagg gcctcctggc gccgcaccgg
     5041 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     5101 ggtctgggca gcgccgtcgt gctccccgga gtggaggccg ccgagcgcgc cggggtgccc
     5161 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     5221 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     5281 ggtgcctagg ctagtatgta agcctagtct tagataataa aatcgctatc catcgaagat
     5341 ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat
     5401 cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc
     5461 agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg
     5521 taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct
     5581 agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca tgagtattca
     5641 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5701 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta
     5761 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgctt
     5821 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5881 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc
     5941 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6001 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa
     6061 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6121 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6181 ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6241 gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6301 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6361 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6421 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6481 gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg
     6541 cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca
     6601 cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa ctcgactctg
     6661 gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct catttgctcg
     6721 tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga tcgcggctcc
     6781 cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc ataacagcag
     6841 cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt
     6901 atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc ttcgatggat
     6961 agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc aattacgggg
     7021 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     7081 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     7141 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     7201 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     7261 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     7321 cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg gcagtacatc
     7381 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     7441 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     7501 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     7561 ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg acacacccgc
     7621 cagcggccgc
//
