LOCUS       sequence-295887-        7651 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 7651)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7651)
  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-m4133795
FEATURES             Location/Qualifiers
     source          1..7651
                     /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..1567
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    1620..1741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1631..1650)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1685..1704
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1856..1902
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1932..2068
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2113..2184
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2410..2998)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2490..2509)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3498..4707
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4544..4563
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4711..5310
                     /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(4711..4730)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5207..5227
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5337..5385
                     /note="synthetic polyadenylation signal"
     CDS             join(5651..5719,5720..6511)
                     /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(5869..5888)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6524..6660
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6805..6876
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6881..6927)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6945..6993
                     /note="synthetic polyadenylation signal"
     enhancer        7074..7377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7379..7581
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7532..7552
                     /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 cgaaagaaac cgcggcagct aagttcgaga ggcagcacat
     1561 ggacagttaa gctagcttga ctgactgaga tacagcgtac cttcagctca cagacatgat
     1621 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1681 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1741 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     1801 ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg tatcgtagca
     1861 taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc gggccggatt
     1921 gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact
     1981 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt
     2041 gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc gatctctcga
     2101 tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt cagaagtagt
     2161 tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg aaacttaatt
     2221 aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact acgctcagaa
     2281 ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga ttacgagttt
     2341 catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2401 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2461 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2521 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2581 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2641 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2701 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2761 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2821 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2881 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2941 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3001 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3061 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3121 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3181 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa
     3241 atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc cgatccatct
     3301 tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc gccttggcta
     3361 ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga gatcgggatc
     3421 acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc gaggaatatc
     3481 gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct
     3541 cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga tccttccgcc
     3601 cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag
     3661 cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag
     3721 agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg
     3781 ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg
     3841 cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt
     3901 agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag
     3961 cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg
     4021 ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt
     4081 gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc
     4141 aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat gggctgggca
     4201 ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt
     4261 tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg
     4321 cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac
     4381 ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg
     4441 taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt
     4501 cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga
     4561 actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg
     4621 taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc cgctaaattc
     4681 tggccgtttt tggctttttt gttagacaac atgaccgagt acaagcccac ggtgcgcctc
     4741 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4801 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     4861 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4921 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4981 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggag
     5041 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     5101 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcc
     5161 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5221 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5281 tggtgcatga cccgcaagcc cggtgcctag gctagtatgt aagcctagtc ttagataata
     5341 aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc
     5401 gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata tcgttgcttg
     5461 gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca gatattgtac
     5521 tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat agtctgatcg
     5581 gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag gatcagcagg
     5641 aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5701 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5761 gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5821 ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5881 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5941 gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt
     6001 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6061 gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6121 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6181 caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg gcgaactact
     6241 tactctagct tcccggcaac agttgataga ctggatggag gcggataaag ttgcaggacc
     6301 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6361 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6421 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6481 gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg cgcagaaaaa
     6541 aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat
     6601 acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa
     6661 gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct tacgcgaaca
     6721 gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc agcttacctt
     6781 tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt atcttcttcc
     6841 ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc
     6901 gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca aaaaaccaac
     6961 acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga gtgtagccag
     7021 atctagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     7081 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     7141 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     7201 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     7261 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     7321 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg
     7381 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     7441 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     7501 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     7561 gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt gtcgatccta
     7621 ccatccactc gacacacccg ccagcggccg c
//
