LOCUS       sequence-295766-        7690 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 7690)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7690)
  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-m4133740
FEATURES             Location/Qualifiers
     source          1..7690
                     /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..1606
                     /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    1659..1780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1670..1689)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1724..1743
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1895..1941
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1971..2107
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2152..2223
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2449..3037)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2529..2548)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3537..4746
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4583..4602
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4750..5349
                     /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(4750..4769)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5246..5266
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5376..5424
                     /note="synthetic polyadenylation signal"
     CDS             join(5690..5758,5759..6550)
                     /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(5908..5927)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6563..6699
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6844..6915
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6920..6966)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6984..7032
                     /note="synthetic polyadenylation signal"
     enhancer        7113..7416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7418..7620
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7571..7591
                     /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 gaggcgatgg aaaaagaatt ttatcgcggt
     1561 gagtgcagcc aacagattca aaaagatatc ctcgtctggt gccctctaag ctagcttgac
     1621 tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg
     1681 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1741 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     1801 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     1861 caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa
     1921 cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc
     1981 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc
     2041 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta
     2101 tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt
     2161 aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc
     2221 ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt
     2281 ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc
     2341 tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa
     2401 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2461 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2521 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2581 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2641 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2701 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2761 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2821 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2881 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2941 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3001 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3061 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3121 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3181 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3241 agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc
     3301 ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga
     3361 actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc
     3421 caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta
     3481 catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggcc
     3541 tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga gcgctgccac
     3601 gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc
     3661 cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg
     3721 gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag
     3781 tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta
     3841 tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg
     3901 ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg
     3961 gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg
     4021 gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca
     4081 aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg
     4141 ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag cccttcgcta
     4201 atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga ccctgacgtg
     4261 aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc
     4321 ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg
     4381 tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag
     4441 gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag
     4501 gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta
     4561 tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg
     4621 agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt
     4681 aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt ggcttttttg
     4741 ttagacaaca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     4801 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4861 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4921 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4981 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     5041 ttgagcggtt cccggctggc cgcgcagcaa cagatggagg gcctcctggc gccgcaccgg
     5101 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     5161 ggtctgggca gcgccgtcgt gctccccgga gtggaggccg ccgagcgcgc cggggtgccc
     5221 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     5281 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     5341 ggtgcctagg ctagtatgta agcctagtct tagataataa aatcgctatc catcgaagat
     5401 ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat
     5461 cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc
     5521 agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg
     5581 taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct
     5641 agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca tgagtattca
     5701 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5761 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta
     5821 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgctt
     5881 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5941 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc
     6001 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6061 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa
     6121 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6181 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6241 ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6301 gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6361 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6421 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6481 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6541 gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg
     6601 cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca
     6661 cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa ctcgactctg
     6721 gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct catttgctcg
     6781 tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga tcgcggctcc
     6841 cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc ataacagcag
     6901 cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt
     6961 atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc ttcgatggat
     7021 agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc aattacgggg
     7081 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     7141 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     7201 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     7261 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     7321 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     7381 cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg gcagtacatc
     7441 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     7501 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     7561 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     7621 ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg acacacccgc
     7681 cagcggccgc
//
