LOCUS       sequence-317517-        7646 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 7646)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7646)
  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-m4133478
FEATURES             Location/Qualifiers
     source          1..7646
                     /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             2086..2106
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2107..2133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2186..2307
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2197..2216)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2251..2270
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2422..2468
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2498..2634
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2679..2750
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2976..3564)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3056..3075)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4064..5273
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5110..5129
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5277..5876
                     /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(5277..5296)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5773..5793
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5903..5951
                     /note="synthetic polyadenylation signal"
     CDS             join(6217..6285,6286..7077)
                     /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(6435..6454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7090..7226
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7378..7449
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7454..7500)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7518..7566
                     /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 ccatgaagat tgtaaataac
      601 tggttgaaac aactaataaa ggttacttta gtgttcctgt ttgtcgccgc aatcttctat
      661 cttatcaccc cggtgcacgt gatgagtaaa catacagact tttcttcaga aattatcggg
      721 tacaaggcta tcgatggcgg tgtaacgcgc gacattgcct ccacagatac ctgcttcgcg
      781 aacaaacacg ctgactttga tacttggttc agccagcgag gaggctcgta caccaatgac
      841 aaagcctgtc cactgatcgc agccgtgata actagagagg ttggattcgt cgtccctggg
      901 ctccccggga caattctccg tacaaccaac ggcgactttc tgcattttct gccccgggtg
      961 ttctccgcag tcggtaatat ctgttatact ccatctaagc tcattgagta cacggatttt
     1021 gctaccagcg cgtgcgtgtt ggccgcagaa tgcaccatat ttaaggacgc ttctggaaaa
     1081 cctgtgccct attgttacga taccaatgtg cttgagggct cagttgccta tgaaagtctg
     1141 aggccagaca ctagatacgt tttaatggat ggaagcatca tccagttccc aaacacgtat
     1201 ctggaggggt ccgtgcgggt cgtgaccaca ttcgattcag aatactgccg ccacggtaca
     1261 tgcgagaggt ctgaggctgg cgtatgtgtg tcaactagcg gtcgatgggt tctaaataac
     1321 gattattatc gttctctgcc cggcgttttt tgtggagtgg acgccgttaa ccttttgacc
     1381 aacatgttta cacctctcat tcagcccatt ggtgcactgg atatctccgc gagtattgtg
     1441 gctgggggaa tagtcgccat tgtcgtaacc tgcttggcat actatttcat gcgtttcagg
     1501 cgagcttttg gcgaatactc acatgtcgtg gccttcaata ctcttctgtt tctgatgagc
     1561 ttcacggtac tctgtctgac cccagtatac agtttcctac ctggggtgta ttctgtgatc
     1621 tacttatacc tcacatttta cctgaccaat gacgtgtcct ttctggccca catccaatgg
     1681 atggttatgt tcacgccgct cgtgcctttc tggattacaa tcgcttatat aatctgtatt
     1741 agcacgaagc atttctattg gttcttttcc aactacttaa agcggcgcgt cgtatttaat
     1801 ggggtatcgt ttagcacttt tgaggaagcc gcgctctgca ctttcttgct taataaggag
     1861 atgtatttga aattaagatc tgacgttctg cttcctttaa cacagtacaa cagatacctg
     1921 gcgttgtata acaaatacaa atatttctcg ggagccatgg atacaacttc ctatagagag
     1981 gctgcttgtt gccaccttgc aaaggcactc aacgacttca gtaacagcgg cagtgatgtg
     2041 ctttatcagc cccctcagac ttcaataaca tccgccgtcc tccaagaaaa cctttacttt
     2101 cagtcgtacc cctatgacgt accagattat gcgtaagcta gcttgactga ctgagataca
     2161 gcgtaccttc agctcacaga catgataaga tacattgatg agtttggaca aaccacaact
     2221 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     2281 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     2341 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatt
     2401 ggcccatctc tatcggtatc gtagcataac cccttggggc ctctaaacgg gtcttgaggg
     2461 gttttttgtg cccctcgggc cggattgcta tctaccggca ttggcgcaga aaaaaatgcc
     2521 tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct
     2581 tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt
     2641 cagctatcct gcaggcgatc tctcgatttc gatcaagaca ttcctttaat ggtcttttct
     2701 ggacaccact aggggtcaga agtagttcat caaactttct tccctcccta atctcattgg
     2761 ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc gagatcgact
     2821 tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg tccttgctat
     2881 tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag
     2941 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3001 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3061 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3121 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3181 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3241 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3301 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3361 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3421 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3481 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3541 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3601 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3661 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3721 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3781 tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc gtcggaaaat
     3841 cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact atcgcaagtc
     3901 tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag gtattactcc
     3961 aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat cctcaatccc
     4021 gatctatccg agatccgagg aatatcgaaa tcggggcgcg cctggcctcc gcgccgggtt
     4081 ttggcgcctc ccgcgggcgc ccccctcgtc acggcgagcg ctgccacgtc agacgaaggg
     4141 cgcaggagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc tgctcataag
     4201 actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac ttgggtgact
     4261 ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag tcccttctcg
     4321 gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat aaggacgcgc
     4381 cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg
     4441 atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc
     4501 cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct gtagtctggg
     4561 tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa tggcggctgt
     4621 tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg
     4681 gggggcatgg tgggcggcaa gaacccaagg tcttgagccc ttcgctaatg cgggaaagct
     4741 cttattcggg tgagatgggc tgggcaccat ctggggaccc tgacgtgaag tttgtcactg
     4801 actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt gccgttgggc
     4861 agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt
     4921 tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct tttctccgtc
     4981 gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg ccggacctct
     5041 ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt acctatcttc
     5101 ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg
     5161 aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat tttcagtgtt
     5221 agacttgtaa attgtccgct aaattctggc cgtttttggc ttttttgtta gacaacatga
     5281 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     5341 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     5401 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     5461 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     5521 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     5581 ggctggccgc gcagcaacag atggagggcc tcctggcgcc gcaccggccc aaggagcccg
     5641 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     5701 ccgtcgtgct ccccggagtg gaggccgccg agcgcgccgg ggtgcccgcc ttcctggaga
     5761 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     5821 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctaggcta
     5881 gtatgtaagc ctagtcttag ataataaaat cgctatccat cgaagatgga tgtgtgttgg
     5941 ttttttgtgt gtgtaacgct aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga
     6001 gtctcgacga tccatatcgt tgcttggcag tcagccagtc ggaatccagc ttgggaccca
     6061 ggaagtccaa tcgtcagata ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt
     6121 aaacctagat attgatagtc tgatcggtca acgtataatc gagtcctagc ttttgcaaac
     6181 atctatcaag agacaggatc agcaggaggc tttcgcatga gtattcaaca tttccgtgtc
     6241 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6301 gtgaaagtaa aagatgctga agatcagttg ggtgcgcgag tgggttacat cgaactggat
     6361 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgctttcc aatgatgagc
     6421 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6481 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtattcacc agtcacagaa
     6541 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6601 gataacactg cggccaactt acttctgaca acgattggag gaccgaagga gctaaccgct
     6661 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6721 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaaccttg
     6781 cgtaaactat taactggcga actacttact ctagcttccc ggcaacagtt gatagactgg
     6841 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6901 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6961 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7021 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaaccg
     7081 attctaggtg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc
     7141 cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt
     7201 cctccttacc agaaatttat ccttaagatc ccgaatcgtt taaactcgac tctggctcta
     7261 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     7321 atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg ctcccgacat
     7381 cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca gcagcttcag
     7441 ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     7501 tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat ggatagcgat
     7561 tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac ggggtcatta
     7621 gttcatagcc catatatgga gttccg
//
