LOCUS       sequence-295229-        7684 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 7684)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7684)
  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-m4133771
FEATURES             Location/Qualifiers
     source          1..7684
                     /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..1600
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     polyA_signal    1653..1774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1664..1683)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1718..1737
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1889..1935
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1965..2101
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2146..2217
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2443..3031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2523..2542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3531..4740
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4577..4596
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4744..5343
                     /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(4744..4763)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5240..5260
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5370..5418
                     /note="synthetic polyadenylation signal"
     CDS             join(5684..5752,5753..6544)
                     /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(5902..5921)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6557..6693
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6838..6909
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6914..6960)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6978..7026
                     /note="synthetic polyadenylation signal"
     enhancer        7107..7410
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7412..7614
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7565..7585
                     /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 cgaagaggcg atggaaaaag aattttatcg cggtgagtgc
     1561 agccaacaga ttcaaaaaga tatcctcgtc tggtgccctc taagctagct tgactgactg
     1621 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
     1681 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1741 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1801 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1861 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
     1921 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     1981 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     2041 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     2101 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     2161 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     2221 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     2281 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     2341 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     2401 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2461 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2521 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2581 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2641 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2701 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2761 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2821 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2881 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2941 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3001 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3061 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3121 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3181 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3241 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc
     3301 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc
     3361 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta
     3421 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct
     3481 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg
     3541 ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga
     3601 cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc
     3661 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg
     3721 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc
     3781 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag
     3841 gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg
     3901 tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt
     3961 tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta
     4021 gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg
     4081 cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa
     4141 caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg
     4201 gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt
     4261 gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc
     4321 gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc
     4381 gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg gtaggctttt
     4441 ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg
     4501 gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc
     4561 tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg
     4621 ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt
     4681 cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac
     4741 aacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     4801 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4861 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4921 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4981 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     5041 ggttcccggc tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag
     5101 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     5161 ggcagcgccg tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc
     5221 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     5281 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     5341 taggctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt
     5401 gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc
     5461 agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg
     5521 ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga
     5581 tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt
     5641 tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt
     5701 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5761 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga
     5821 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat
     5881 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5941 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt
     6001 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6061 catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct
     6121 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6181 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6241 aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat
     6301 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6361 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6421 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6481 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6541 gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt
     6601 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca
     6661 tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac tctggctcta
     6721 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     6781 atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg ctcccgacat
     6841 cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca gcagcttcag
     6901 ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     6961 tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat ggatagcgat
     7021 tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac ggggtcatta
     7081 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     7141 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     7201 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     7261 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     7321 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     7381 atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta catcaatggg
     7441 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     7501 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     7561 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
     7621 gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac ccgccagcgg
     7681 ccgc
//
