LOCUS       sequence-295071-        8161 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 8161)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8161)
  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-m4134032
FEATURES             Location/Qualifiers
     source          1..8161
                     /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             1556..1633
                     /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             1661..1681
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    2130..2251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2141..2160)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2195..2214
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2366..2412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2442..2578
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2623..2694
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2920..3508)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3000..3019)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4008..5217
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5054..5073
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5221..5820
                     /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(5221..5240)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5717..5737
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5847..5895
                     /note="synthetic polyadenylation signal"
     CDS             join(6161..6229,6230..7021)
                     /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(6379..6398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7034..7170
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7315..7386
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7391..7437)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7455..7503
                     /note="synthetic polyadenylation signal"
     enhancer        7584..7887
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7889..8091
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8042..8062
                     /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 cgggacggcg aatcccgggg ctgataaatc cctggaaaag
     1561 gagatggaag aagaacttta ttgcagtgtc tgcggctaac cggttcaaaa agatcagttc
     1621 aagcggcgcc ctagattacg acattcctac gaccgcctcc gagaatctct atttccaggg
     1681 tgaatttggc ttggcacaac atgacgaggc cgtcgataac aagtttaata aagaacagca
     1741 gaacgctttc tatgagatcc ttcacttacc aaatctgaac gaagagcaac gcaacgcttt
     1801 cattcagagc ctgaaagacg atccctcgca gtctgcaaat ctcctggccg aagccaagaa
     1861 gctgaatgac gcacaggctc caaaagttga taacaagttt aacaaggagc agcagaatgc
     1921 tttctacgag atattgcacc ttcctaatct gaatgaggaa caaagaaatg catttatcca
     1981 gtccctaaaa gacgatccat cacagagtgc caacctgctt gcggaagcca aaaagctcaa
     2041 tgatgcccag gccccgaaag tagacgcgaa ccatcaataa gctagcttga ctgactgaga
     2101 tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac
     2161 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2221 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     2281 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     2341 tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg
     2401 aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa
     2461 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac
     2521 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg
     2581 tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt
     2641 ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca
     2701 ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc
     2761 gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg
     2821 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca
     2881 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2941 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3001 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3061 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3121 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3181 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3241 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3301 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3361 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3421 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3481 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3541 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3601 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3661 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3721 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga
     3781 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca
     3841 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta
     3901 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa
     3961 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg
     4021 ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca cgtcagacga
     4081 agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc ccgctgctca
     4141 taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg ggacttgggt
     4201 gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa gtagtccctt
     4261 ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt atataaggac
     4321 gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg gttcttgttt
     4381 gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc ggggctttcg
     4441 tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag ggctgtagtc
     4501 tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg
     4561 ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa
     4621 ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa
     4681 agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt gaagtttgtc
     4741 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt
     4801 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt
     4861 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc
     4921 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac
     4981 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat
     5041 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt
     5101 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag
     5161 tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacaac
     5221 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     5281 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     5341 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     5401 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     5461 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     5521 tcccggctgg ccgcgcagca acagatggag ggcctcctgg cgccgcaccg gcccaaggag
     5581 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     5641 agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc cgccttcctg
     5701 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     5761 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag
     5821 gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga tggatgtgtg
     5881 ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga tcctctcagt
     5941 gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc cagcttggga
     6001 cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc gtaccgatct
     6061 gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc
     6121 aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc aacatttccg
     6181 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6241 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt acatcgaact
     6301 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct ttccaatgat
     6361 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6421 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt caccagtcac
     6481 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6541 gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga aggagctaac
     6601 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6661 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6721 cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac agttgataga
     6781 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6841 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6901 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6961 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     7021 accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata
     7081 ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc acttccatac
     7141 gtgtcctcct taccagaaat ttatccttaa gatcgtttaa actcgactct ggctctatcg
     7201 aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc
     7261 gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt
     7321 ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta
     7381 cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca
     7441 atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt
     7501 attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt
     7561 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     7621 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     7681 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     7741 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     7801 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     7861 tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacat caatgggcgt
     7921 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     7981 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     8041 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
     8101 aaccgtcaga tcagatcttt gtcgatccta ccatccactc gacacacccg ccagcggccg
     8161 c
//
