LOCUS       sequence-308749-        7655 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 7655)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7655)
  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-m4134104
FEATURES             Location/Qualifiers
     source          1..7655
                     /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             2101..2142
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    2195..2316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2206..2225)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2260..2279
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2431..2477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2507..2643
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2688..2759
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2985..3573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3065..3084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4073..5282
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5119..5138
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5286..5885
                     /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(5286..5305)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5782..5802
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5912..5960
                     /note="synthetic polyadenylation signal"
     CDS             join(6226..6294,6295..7086)
                     /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(6444..6463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7099..7235
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7387..7458
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7463..7509)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7527..7575
                     /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 gggaaaccta tacccaatcc acttctcggc ttagattcta cctaagctag cttgactgac
     2161 tgagatacag cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa
     2221 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2281 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     2341 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     2401 tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg
     2461 tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa
     2521 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg
     2581 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct
     2641 taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg
     2701 gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa
     2761 tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg
     2821 agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt
     2881 ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc
     2941 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3001 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3061 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3121 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3181 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3241 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3301 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3361 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3421 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3481 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3541 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3601 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3661 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3721 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3781 atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc aaggccctcg
     3841 tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc gaacgaacta
     3901 tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc tatcgccagg
     3961 tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc aacctacatc
     4021 ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc ctggcctccg
     4081 cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc tgccacgtca
     4141 gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct
     4201 gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact
     4261 tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt
     4321 cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata
     4381 aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct
     4441 tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc
     4501 tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg
     4561 tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat
     4621 ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga
     4681 aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct tcgctaatgc
     4741 gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct gacgtgaagt
     4801 ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg
     4861 ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac
     4921 ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt
     4981 ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc
     5041 cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta
     5101 cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg
     5161 tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt
     5221 ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag
     5281 acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     5341 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     5401 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     5461 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     5521 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     5581 gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg caccggccca
     5641 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     5701 tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg gtgcccgcct
     5761 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     5821 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     5881 cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc gaagatggat
     5941 gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag aacgatcctc
     6001 tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg gaatccagct
     6061 tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg gcagcgtacc
     6121 gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg agtcctagct
     6181 tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag tattcaacat
     6241 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6301 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt gggttacatc
     6361 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgctttcca
     6421 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6481 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtattcacca
     6541 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6601 accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg accgaaggag
     6661 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6721 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6781 acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg gcaacagttg
     6841 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6901 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6961 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     7021 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7081 tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc
     7141 ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc
     7201 catacgtgtc ctccttacca gaaatttatc cttaagatcc cgaatcgttt aaactcgact
     7261 ctggctctat cgaatctccg tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc
     7321 tcgtcgggca tcgaatctcg tcagctatcg tcagcttacc tttttggcag cgatcgcggc
     7381 tcccgacatc ttggaccatt agctccacag gtatcttctt ccctctagtg gtcataacag
     7441 cagcttcagc tacctctcaa ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     7501 gttatgctat caatcgttgc gttacacaca caaaaaacca acacacatcc atcttcgatg
     7561 gatagcgatt ttattatcta actgctgatc gagtgtagcc agatctagta atcaattacg
     7621 gggtcattag ttcatagccc atatatggag ttccg
//
