LOCUS       sequence-297918-        8127 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 8127)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8127)
  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-m4134072
FEATURES             Location/Qualifiers
     source          1..8127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1776..1796
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1800..1865
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1934..2055
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1945..1964)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1999..2018
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2170..2216
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2246..2382
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2427..2498
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2724..3312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2804..2823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3812..5021
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4858..4877
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5025..6053
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    6080..6128
                     /note="synthetic polyadenylation signal"
     CDS             join(6394..6462,6463..7254)
                     /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(6612..6631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7267..7403
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7555..7626
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7631..7677)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7695..7743
                     /note="synthetic polyadenylation signal"
     enhancer        7824..8127
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatcagatc tttgtcgatc
      241 ctaccatcca ctcgacacac ccgccagcgg ccgccaccat gattgttaat aattggttga
      301 agcagttaat taaagttaca cttgtgttcc tttttgttgc tgctattttc tatttaataa
      361 cacctgttca tgtcatgtct aaacatactg acttttcaag tgaaatcata ggatacaagg
      421 ctattgatgg tggtgtcact cgtgacatag catctacaga tacttgtttt gctaacaaac
      481 atgctgattt tgacacatgg tttagccagc gtggtggtag ttatactaat gacaaagctt
      541 gcccattgat tgctgcagtc ataacaagag aagtgggttt tgtcgtgcct ggtttgcctg
      601 gcacgatatt acgcacaact aatggtgact ttttgcattt cttacctaga gtttttagtg
      661 cagttggtaa catctgttac acaccatcaa aacttataga gtacactgac tttgcaacat
      721 cagcttgtgt tttggctgct gaatgtacaa tttttaaaga tgcttctggt aagccagtac
      781 catattgtta tgataccaat gtactagaag gttctgttgc ttatgaaagt ttacgccctg
      841 acacacgtta tgtgctcatg gatggctcta ttattcaatt tcctaacacc taccttgaag
      901 gttctgttag agtggtaaca acttttgatt ctgagtactg taggcacggc acttgtgaaa
      961 gatcagaagc tggtgtttgt gtatctacta gtggtagatg ggtacttaac aatgattatt
     1021 acagatcttt accaggagtt ttctgtggtg tagatgctgt aaatttactt actaatatgt
     1081 ttacaccact aattcaacct attggtgctt tggacatatc agcatctata gtagctggtg
     1141 gtattgtagc tatcgtagta acatgccttg cctactattt tatgaggttt agaagagctt
     1201 ttggtgaata cagtcatgta gttgccttta atactttact attccttatg tcattcactg
     1261 tactctgttt aacaccagtt tactcattct tacctggtgt ttattctgtt atttacttgt
     1321 acttgacatt ttatcttact aatgatgttt cttttttagc acatattcag tggatggtta
     1381 tgttcacacc tttagtacct ttctggataa caattgctta tatcatttgt atttccacaa
     1441 agcatttcta ttggttcttt agtaattacc taaagagacg tgtagtcttt aatggtgttt
     1501 cctttagtac ttttgaagaa gctgcgctgt gcaccttttt gttaaataaa gaaatgtatc
     1561 taaagttgcg tagtgatgtg ctattacctc ttacgcaata taatagatac ttagctcttt
     1621 ataataagta caagtatttt agtggagcaa tggatacaac tagctacaga gaagctgctt
     1681 gttgtcatct cgcaaaggct ctcaatgact tcagtaactc aggttctgat gttctttacc
     1741 aaccaccaca aacctctatc acctcagctg ttttggaaaa cctttacttt cagtcgatgg
     1801 attataagga ccacgatgga gactacaaag atcatgacat tgattacaag gacgacgatg
     1861 acaaataagg atccgcaggc ctctgctagc ttgactgact gagatacagc gtaccttcag
     1921 ctcacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     1981 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2041 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     2101 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtattgg cccatctcta
     2161 tcggtatcgt agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgtgcc
     2221 cctcgggccg gattgctatc taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct
     2281 gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc cccaacttgc
     2341 ccacttccat acgtgtcctc cttaccagaa atttatcctt aaggtcgtca gctatcctgc
     2401 aggcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg acaccactag
     2461 gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt accttgggct
     2521 atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg tctgggtttc
     2581 gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg cacccgttct
     2641 ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2701 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2761 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2821 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2881 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2941 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3001 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3061 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3121 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3181 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3241 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3301 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3361 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3421 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3481 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3541 atagttgcat ttaaatttcc gaactctcca aggccctcgt cggaaaatct tcaaaccttt
     3601 cgtccgatcc atcttgcagg ctacctctcg aacgaactat cgcaagtctc ttggccggcc
     3661 ttgcgccttg gctattgctt ggcagcgcct atcgccaggt attactccaa tcccgaatat
     3721 ccgagatcgg gatcacccga gagaagttca acctacatcc tcaatcccga tctatccgag
     3781 atccgaggaa tatcgaaatc ggggcgcgcc tggcctccgc gccgggtttt ggcgcctccc
     3841 gcgggcgccc ccctcgtcac ggcgagcgct gccacgtcag acgaagggcg caggagcgtc
     3901 ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag
     3961 aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg
     4021 ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg
     4081 agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac
     4141 agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc
     4201 gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct
     4261 cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag
     4321 gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg
     4381 aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg
     4441 ggcggcaaga acccaaggtc ttgagccctt cgctaatgcg ggaaagctct tattcgggtg
     4501 agatgggctg ggcaccatct ggggaccctg acgtgaagtt tgtcactgac tggagaactc
     4561 ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag tgcacccgta
     4621 cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg
     4681 cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg
     4741 gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg
     4801 gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt aagtagctga
     4861 agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa gttttttagg
     4921 caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttag acttgtaaat
     4981 tgtccgctaa attctggccg tttttggctt ttttgttaga caacatgggt aaaaagcctg
     5041 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     5101 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     5161 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     5221 ggcactttgc atccgccgcg ctcccgattc cggaagtgct tgacattggg gagttcagcg
     5281 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     5341 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc aatggatgcc atcgctgccg
     5401 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     5461 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     5521 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     5581 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     5641 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     5701 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     5761 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccccgg ctccgggcgt
     5821 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     5881 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     5941 ggcgtacaca aatcgcccgc agaagcgccg ccgtctggac cgatggctgt gtagaagtac
     6001 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taagctagta
     6061 tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt gtgttggttt
     6121 tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc
     6181 tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg ggacccagga
     6241 agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga tctgtttaaa
     6301 cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt tgcaaacatc
     6361 tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc
     6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6481 aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga actggatctc
     6541 aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat gatgagcact
     6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt cacagaaaag
     6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6781 aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct aaccgctttt
     6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aaccttgcgt
     6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat agactggatg
     7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7081 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaaccgatt
     7261 ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac
     7321 atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct
     7381 ccttaccaga aatttatcct taagatcccg aatcgtttaa actcgactct ggctctatcg
     7441 aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc
     7501 gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt
     7561 ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta
     7621 cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca
     7681 atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt
     7741 attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt
     7801 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     7861 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     7921 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     7981 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     8041 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     8101 tacgtattag tcatcgctat taccatg
//
