LOCUS       sequence-317522-        7688 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 7688)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7688)
  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-m4134439
FEATURES             Location/Qualifiers
     source          1..7688
                     /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             2110..2175
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2228..2349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2239..2258)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2293..2312
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2464..2510
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2540..2676
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2721..2792
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3018..3606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3098..3117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4106..5315
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5152..5171
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5319..5918
                     /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(5319..5338)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5815..5835
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5945..5993
                     /note="synthetic polyadenylation signal"
     CDS             join(6259..6327,6328..7119)
                     /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(6477..6496)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7132..7268
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7420..7491
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7496..7542)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7560..7608
                     /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 cagtcgatgg attataagga ccacgatgga gactacaaag atcatgacat tgattacaag
     2161 gacgacgatg acaaataagc tagcttgact gactgagata cagcgtacct tcagctcaca
     2221 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     2281 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     2341 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     2401 gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta
     2461 tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg tgcccctcgg
     2521 gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt
     2581 cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt
     2641 ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc ctgcaggcga
     2701 tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca
     2761 gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg ggctatcgaa
     2821 acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg tttcgactac
     2881 gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg ttctccgatt
     2941 acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3601 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3661 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3721 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3841 gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg
     3901 atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc
     3961 cttggctatt gcttggcagc gcctatcgcc aggtattact ccaatcccga atatccgaga
     4021 tcgggatcac ccgagagaag ttcaacctac atcctcaatc ccgatctatc cgagatccga
     4081 ggaatatcga aatcggggcg cgcctggcct ccgcgccggg ttttggcgcc tcccgcgggc
     4141 gcccccctcg tcacggcgag cgctgccacg tcagacgaag ggcgcaggag cgtcctgatc
     4201 cttccgcccg gacgctcagg acagcggccc gctgctcata agactcggcc ttagaacccc
     4261 agtatcagca gaaggacatt ttaggacggg acttgggtga ctctagggca ctggttttct
     4321 ttccagagag cggaacaggc gaggaaaagt agtcccttct cggcgattct gcggagggat
     4381 ctccgtgggg cggtgaacgc cgatgattat ataaggacgc gccgggtgtg gcacagctag
     4441 ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact
     4501 tggtgagtag cgggctgctg ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg
     4561 gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc
     4621 ctgaactggg ggttgggggg agcgcagcaa aatggcggct gttcccgagt cttgaatgga
     4681 agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc
     4741 aagaacccaa ggtcttgagc ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg
     4801 gctgggcacc atctggggac cctgacgtga agtttgtcac tgactggaga actcggtttg
     4861 tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc cgtacctttg
     4921 ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat aatgcagggt
     4981 ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg cagggttcgg
     5041 gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg gagggataag
     5101 tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag ctgaagctcc
     5161 ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt taggcacctt
     5221 ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagacttgt aaattgtccg
     5281 ctaaattctg gccgtttttg gcttttttgt tagacaacat gaccgagtac aagcccacgg
     5341 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     5401 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     5461 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     5521 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     5581 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     5641 agatggaggg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     5701 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     5761 tggaggccgc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     5821 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     5881 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaggc tagtatgtaa gcctagtctt
     5941 agataataaa atcgctatcc atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg
     6001 ctaggcgcgc ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac gatccatatc
     6061 gttgcttggc agtcagccag tcggaatcca gcttgggacc caggaagtcc aatcgtcaga
     6121 tattgtactc aagcctggtc acggcagcgt accgatctgt ttaaacctag atattgatag
     6181 tctgatcggt caacgtataa tcgagtccta gcttttgcaa acatctatca agagacagga
     6241 tcagcaggag gctttcgcat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6301 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6361 gaagatcagt tgggtgcgcg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6421 cttgagagtt ttcgccccga agaacgcttt ccaatgatga gcacttttaa agttctgcta
     6481 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6541 tattctcaga atgacttggt tgagtattca ccagtcacag aaaagcatct tacggatggc
     6601 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6661 ttacttctga caacgattgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6721 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6781 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact attaactggc
     6841 gaactactta ctctagcttc ccggcaacag ttgatagact ggatggaggc ggataaagtt
     6901 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6961 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7021 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7081 atcgctgaga taggtgcctc actgattaag cattggtaac cgattctagg tgcattggcg
     7141 cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag
     7201 caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt
     7261 atccttaaga tcccgaatcg tttaaactcg actctggctc tatcgaatct ccgtcgtttc
     7321 gagcttacgc gaacagccgt ggcgctcatt tgctcgtcgg gcatcgaatc tcgtcagcta
     7381 tcgtcagctt acctttttgg cagcgatcgc ggctcccgac atcttggacc attagctcca
     7441 caggtatctt cttccctcta gtggtcataa cagcagcttc agctacctct caattcaaaa
     7501 aacccctcaa gacccgttta gaggccccaa ggggttatgc tatcaatcgt tgcgttacac
     7561 acacaaaaaa ccaacacaca tccatcttcg atggatagcg attttattat ctaactgctg
     7621 atcgagtgta gccagatcta gtaatcaatt acggggtcat tagttcatag cccatatatg
     7681 gagttccg
//
