LOCUS       sequence-320764-        7682 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 7682)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7682)
  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-m4133850
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2080..2100
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2104..2169
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2222..2343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2233..2252)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2287..2306
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2458..2504
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2534..2670
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2715..2786
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3012..3600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3092..3111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4100..5309
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5146..5165
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5313..5912
                     /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(5313..5332)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5809..5829
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5939..5987
                     /note="synthetic polyadenylation signal"
     CDS             join(6253..6321,6322..7113)
                     /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(6471..6490)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7126..7262
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7414..7485
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7490..7536)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7554..7602
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc
      541 gatcctacca tccactcgac acacccgcca gcggccgcca ccatgattgt taataattgg
      601 ttgaagcagt taattaaagt tacacttgtg ttcctttttg ttgctgctat tttctattta
      661 ataacacctg ttcatgtcat gtctaaacat actgactttt caagtgaaat cataggatac
      721 aaggctattg atggtggtgt cactcgtgac atagcatcta cagatacttg ttttgctaac
      781 aaacatgctg attttgacac atggtttagc cagcgtggtg gtagttatac taatgacaaa
      841 gcttgcccat tgattgctgc agtcataaca agagaagtgg gttttgtcgt gcctggtttg
      901 cctggcacga tattacgcac aactaatggt gactttttgc atttcttacc tagagttttt
      961 agtgcagttg gtaacatctg ttacacacca tcaaaactta tagagtacac tgactttgca
     1021 acatcagctt gtgttttggc tgctgaatgt acaattttta aagatgcttc tggtaagcca
     1081 gtaccatatt gttatgatac caatgtacta gaaggttctg ttgcttatga aagtttacgc
     1141 cctgacacac gttatgtgct catggatggc tctattattc aatttcctaa cacctacctt
     1201 gaaggttctg ttagagtggt aacaactttt gattctgagt actgtaggca cggcacttgt
     1261 gaaagatcag aagctggtgt ttgtgtatct actagtggta gatgggtact taacaatgat
     1321 tattacagat ctttaccagg agttttctgt ggtgtagatg ctgtaaattt acttactaat
     1381 atgtttacac cactaattca acctattggt gctttggaca tatcagcatc tatagtagct
     1441 ggtggtattg tagctatcgt agtaacatgc cttgcctact attttatgag gtttagaaga
     1501 gcttttggtg aatacagtca tgtagttgcc tttaatactt tactattcct tatgtcattc
     1561 actgtactct gtttaacacc agtttactca ttcttacctg gtgtttattc tgttatttac
     1621 ttgtacttga cattttatct tactaatgat gtttcttttt tagcacatat tcagtggatg
     1681 gttatgttca cacctttagt acctttctgg ataacaattg cttatatcat ttgtatttcc
     1741 acaaagcatt tctattggtt ctttagtaat tacctaaaga gacgtgtagt ctttaatggt
     1801 gtttccttta gtacttttga agaagctgcg ctgtgcacct ttttgttaaa taaagaaatg
     1861 tatctaaagt tgcgtagtga tgtgctatta cctcttacgc aatataatag atacttagct
     1921 ctttataata agtacaagta ttttagtgga gcaatggata caactagcta cagagaagct
     1981 gcttgttgtc atctcgcaaa ggctctcaat gacttcagta actcaggttc tgatgttctt
     2041 taccaaccac cacaaacctc tatcacctca gctgttttgg aaaaccttta ctttcagtcg
     2101 atggattata aggaccacga tggagactac aaagatcatg acattgatta caaggacgac
     2161 gatgacaaat aagctagctt gactgactga gatacagcgt accttcagct cacagacatg
     2221 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     2281 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     2341 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     2401 ttttaaagca agtaaaacct ctacaaatgt ggtattggcc catctctatc ggtatcgtag
     2461 cataacccct tggggcctct aaacgggtct tgaggggttt tttgtgcccc tcgggccgga
     2521 ttgctatcta ccggcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata
     2581 ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc acttccatac
     2641 gtgtcctcct taccagaaat ttatccttaa ggtcgtcagc tatcctgcag gcgatctctc
     2701 gatttcgatc aagacattcc tttaatggtc ttttctggac accactaggg gtcagaagta
     2761 gttcatcaaa ctttcttccc tccctaatct cattggttac cttgggctat cgaaacttaa
     2821 ttaaccagtc aagtcagcta cttggcgaga tcgacttgtc tgggtttcga ctacgctcag
     2881 aattgcgtca gtcaagttcg atctggtcct tgctattgca cccgttctcc gattacgagt
     2941 ttcatttaaa tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3001 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3061 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3121 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3181 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3241 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3301 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3361 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3421 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3481 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3541 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3601 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3661 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3721 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3781 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt
     3841 aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat
     3901 cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc
     3961 tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga
     4021 tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata
     4081 tcgaaatcgg ggcgcgcctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc
     4141 ctcgtcacgg cgagcgctgc cacgtcagac gaagggcgca ggagcgtcct gatccttccg
     4201 cccggacgct caggacagcg gcccgctgct cataagactc ggccttagaa ccccagtatc
     4261 agcagaagga cattttagga cgggacttgg gtgactctag ggcactggtt ttctttccag
     4321 agagcggaac aggcgaggaa aagtagtccc ttctcggcga ttctgcggag ggatctccgt
     4381 ggggcggtga acgccgatga ttatataagg acgcgccggg tgtggcacag ctagttccgt
     4441 cgcagccggg atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga
     4501 gtagcgggct gctgggctgg ccggggcttt cgtggccgcc gggccgctcg gtgggacgga
     4561 agcgtgtgga gagaccgcca agggctgtag tctgggtccg cgagcaaggt tgccctgaac
     4621 tgggggttgg ggggagcgca gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc
     4681 ttgtgaggcg ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg cggcaagaac
     4741 ccaaggtctt gagcccttcg ctaatgcggg aaagctctta ttcgggtgag atgggctggg
     4801 caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct
     4861 gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg
     4921 cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc
     4981 acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
     5041 ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
     5101 gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt
     5161 gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa
     5221 tgtaatcatt tgggtcaata tgtaattttc agtgttagac ttgtaaattg tccgctaaat
     5281 tctggccgtt tttggctttt ttgttagaca acatgaccga gtacaagccc acggtgcgcc
     5341 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     5401 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     5461 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     5521 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     5581 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     5641 agggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     5701 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     5761 ccgccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     5821 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     5881 cctggtgcat gacccgcaag cccggtgcct aggctagtat gtaagcctag tcttagataa
     5941 taaaatcgct atccatcgaa gatggatgtg tgttggtttt ttgtgtgtgt aacgctaggc
     6001 gcgcctggtg taccgagaac gatcctctca gtgcgagtct cgacgatcca tatcgttgct
     6061 tggcagtcag ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt cagatattgt
     6121 actcaagcct ggtcacggca gcgtaccgat ctgtttaaac ctagatattg atagtctgat
     6181 cggtcaacgt ataatcgagt cctagctttt gcaaacatct atcaagagac aggatcagca
     6241 ggaggctttc gcatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6301 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6361 cagttgggtg cgcgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6421 agttttcgcc ccgaagaacg ctttccaatg atgagcactt ttaaagttct gctatgtggc
     6481 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6541 cagaatgact tggttgagta ttcaccagtc acagaaaagc atcttacgga tggcatgaca
     6601 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6661 ctgacaacga ttggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6721 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6781 gacaccacga tgcctgtagc aatggcaaca accttgcgta aactattaac tggcgaacta
     6841 cttactctag cttcccggca acagttgata gactggatgg aggcggataa agttgcagga
     6901 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6961 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     7021 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     7081 gagataggtg cctcactgat taagcattgg taaccgattc taggtgcatt ggcgcagaaa
     7141 aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg
     7201 atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt
     7261 aagatcccga atcgtttaaa ctcgactctg gctctatcga atctccgtcg tttcgagctt
     7321 acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca gctatcgtca
     7381 gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc tccacaggta
     7441 tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc aaaaaacccc
     7501 tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa
     7561 aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact gctgatcgag
     7621 tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     7681 cg
//
