LOCUS       sequence-295584-        6267 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 6267)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6267)
  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-m4134254
FEATURES             Location/Qualifiers
     source          1..6267
                     /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             1562..1639
                     /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"
     CDS             1667..1687
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    2136..2257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2147..2166)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2201..2220
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2372..2418
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2448..2584
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2629..2700
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2926..3514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3006..3025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(4260..4328,4329..5120)
                     /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(4478..4497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5133..5269
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5421..5492
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5497..5543)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    5561..5609
                     /note="synthetic polyadenylation signal"
     enhancer        5690..5993
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5995..6197
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6148..6168
                     /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 ttcagtcggg acggcgaatc ccggggctga taaatccctg
     1561 gaaaaggaga tggaagaaga actttattgc agtgtctgcg gctaaccggt tcaaaaagat
     1621 cagttcaagc ggcgccctag attacgacat tcctacgacc gcctccgaga atctctattt
     1681 ccagggtgaa tttggcttgg cacaacatga cgaggccgtc gataacaagt ttaataaaga
     1741 acagcagaac gctttctatg agatccttca cttaccaaat ctgaacgaag agcaacgcaa
     1801 cgctttcatt cagagcctga aagacgatcc ctcgcagtct gcaaatctcc tggccgaagc
     1861 caagaagctg aatgacgcac aggctccaaa agttgataac aagtttaaca aggagcagca
     1921 gaatgctttc tacgagatat tgcaccttcc taatctgaat gaggaacaaa gaaatgcatt
     1981 tatccagtcc ctaaaagacg atccatcaca gagtgccaac ctgcttgcgg aagccaaaaa
     2041 gctcaatgat gcccaggccc cgaaagtaga cgcgaaccat caataagcta gcttgactga
     2101 ctgagataca gcgtaccttc agctcacaga catgataaga tacattgatg agtttggaca
     2161 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2221 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     2281 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     2341 atgtggtatt ggcccatctc tatcggtatc gtagcataac cccttggggc ctctaaacgg
     2401 gtcttgaggg gttttttgtg cccctcgggc cggattgcta tctaccggca ttggcgcaga
     2461 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     2521 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     2581 ttaaggtcgt cagctatcct gcaggcgatc tctcgatttc gatcaagaca ttcctttaat
     2641 ggtcttttct ggacaccact aggggtcaga agtagttcat caaactttct tccctcccta
     2701 atctcattgg ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc
     2761 gagatcgact tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg
     2821 tccttgctat tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg
     2881 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2941 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3001 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3061 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3121 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3181 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3241 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3301 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3361 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3421 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3481 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3541 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3601 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3661 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3721 gatctgtcta tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc
     3781 gtcggaaaat cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact
     3841 atcgcaagtc tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag
     3901 gtattactcc aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat
     3961 cctcaatccc gatctatccg agatccgagg aatatcgaaa tcggggcgcg cctggtgtac
     4021 cgagaacgat cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca
     4081 gtcggaatcc agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt
     4141 cacggcagcg taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata
     4201 atcgagtcct agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca
     4261 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4321 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc
     4381 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4441 aagaacgctt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4501 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4561 ttgagtattc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4621 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg
     4681 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4741 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4801 ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt
     4861 cccggcaaca gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4921 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4981 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5041 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5101 cactgattaa gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg
     5161 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     5221 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag atcccgaatc
     5281 gtttaaactc gactctggct ctatcgaatc tccgtcgttt cgagcttacg cgaacagccg
     5341 tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct atcgtcagct tacctttttg
     5401 gcagcgatcg cggctcccga catcttggac cattagctcc acaggtatct tcttccctct
     5461 agtggtcata acagcagctt cagctacctc tcaattcaaa aaacccctca agacccgttt
     5521 agaggcccca aggggttatg ctatcaatcg ttgcgttaca cacacaaaaa accaacacac
     5581 atccatcttc gatggatagc gattttatta tctaactgct gatcgagtgt agccagatct
     5641 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     5701 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     5761 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     5821 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     5881 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     5941 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atgctgatgc
     6001 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     6061 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     6121 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     6181 tctatataag cagagctggt ttagtgaacc gtcagatcag atctttgtcg atcctaccat
     6241 ccactcgaca cacccgccag cggccgc
//
