LOCUS       sequence-295372-        8135 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 8135)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8135)
  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-m4133711
FEATURES             Location/Qualifiers
     source          1..8135
                     /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    1675..1796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1686..1705)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1740..1759
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1911..1957
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1987..2123
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2168..2239
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2465..3053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2545..2564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3553..4762
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4599..4618
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4766..5794
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5821..5869
                     /note="synthetic polyadenylation signal"
     CDS             join(6135..6203,6204..6995)
                     /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(6353..6372)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7008..7144
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7289..7360
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7365..7411)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7429..7477
                     /note="synthetic polyadenylation signal"
     enhancer        7558..7861
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7863..8065
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8016..8036
                     /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 gatccgcagg
     1621 cctctgctag cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat
     1681 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1741 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1801 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     1861 gcaagtaaaa cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc
     1921 ccttggggcc tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat
     1981 ctaccggcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac
     2041 atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct
     2101 ccttaccaga aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg
     2161 atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc
     2221 aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca
     2281 gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg
     2341 tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt
     2401 aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2461 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2521 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2581 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2641 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2701 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2761 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2821 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2881 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2941 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3001 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3061 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3121 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3181 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3241 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc
     3301 cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag
     3361 gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct
     3421 tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg
     3481 agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat
     3541 cggggcgcgc ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca
     3601 cggcgagcgc tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac
     3661 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa
     3721 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg
     3781 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg
     3841 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc
     3901 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg
     3961 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt
     4021 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt
     4081 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag
     4141 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt
     4201 cttgagccct tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc
     4261 tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg
     4321 gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc
     4381 tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc
     4441 ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc
     4501 tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt
     4561 tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat
     4621 gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc
     4681 atttgggtca atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc
     4741 gtttttggct tttttgttag acaacatggg taaaaagcct gaactcaccg cgacgtctgt
     4801 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4861 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4921 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catccgccgc
     4981 gctcccgatt ccggaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat
     5041 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     5101 tctgcagccg gtcgcggagg caatggatgc catcgctgcc gccgatctta gccagacgag
     5161 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     5221 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     5281 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     5341 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     5401 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     5461 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     5521 gaggcatccg gagcttgcag gatcgccccg gctccgggcg tatatgctcc gcattggtct
     5581 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     5641 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5701 cagaagcgcc gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5761 acgccccagc actcgtccga gggcaaagga ataagctagt atgtaagcct agtcttagat
     5821 aataaaatcg ctatccatcg aagatggatg tgtgttggtt ttttgtgtgt gtaacgctag
     5881 gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg
     5941 cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt
     6001 gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg
     6061 atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag
     6121 caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6181 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6241 atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6301 agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6361 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6421 ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga
     6481 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6541 ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6601 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6661 gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac
     6721 tacttactct agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag
     6781 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6841 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6901 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6961 ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga
     7021 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     7081 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     7141 ttaagatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg
     7201 aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta
     7261 cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc
     7321 ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag
     7381 acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac
     7441 caacacacat ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag
     7501 ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     7561 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7621 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     7681 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     7741 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     7801 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     7861 gctgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     7921 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     7981 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     8041 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcagat ctttgtcgat
     8101 cctaccatcc actcgacaca cccgccagcg gccgc
//
