LOCUS       sequence-295231-        8129 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 8129)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8129)
  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-m4133959
FEATURES             Location/Qualifiers
     source          1..8129
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1502..1522
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1523..1600
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     polyA_signal    1669..1790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1680..1699)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1734..1753
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1905..1951
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1981..2117
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2162..2233
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2459..3047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2539..2558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3547..4756
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4593..4612
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4760..5788
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5815..5863
                     /note="synthetic polyadenylation signal"
     CDS             join(6129..6197,6198..6989)
                     /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(6347..6366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7002..7138
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7283..7354
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7359..7405)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7423..7471
                     /note="synthetic polyadenylation signal"
     enhancer        7552..7855
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7857..8059
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8010..8030
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgatt gttaataatt ggttgaagca gttaattaaa gttacacttg tgttcctttt
       61 tgttgctgct attttctatt taataacacc tgttcatgtc atgtctaaac atactgactt
      121 ttcaagtgaa atcataggat acaaggctat tgatggtggt gtcactcgtg acatagcatc
      181 tacagatact tgttttgcta acaaacatgc tgattttgac acatggttta gccagcgtgg
      241 tggtagttat actaatgaca aagcttgccc attgattgct gcagtcataa caagagaagt
      301 gggttttgtc gtgcctggtt tgcctggcac gatattacgc acaactaatg gtgacttttt
      361 gcatttctta cctagagttt ttagtgcagt tggtaacatc tgttacacac catcaaaact
      421 tatagagtac actgactttg caacatcagc ttgtgttttg gctgctgaat gtacaatttt
      481 taaagatgct tctggtaagc cagtaccata ttgttatgat accaatgtac tagaaggttc
      541 tgttgcttat gaaagtttac gccctgacac acgttatgtg ctcatggatg gctctattat
      601 tcaatttcct aacacctacc ttgaaggttc tgttagagtg gtaacaactt ttgattctga
      661 gtactgtagg cacggcactt gtgaaagatc agaagctggt gtttgtgtat ctactagtgg
      721 tagatgggta cttaacaatg attattacag atctttacca ggagttttct gtggtgtaga
      781 tgctgtaaat ttacttacta atatgtttac accactaatt caacctattg gtgctttgga
      841 catatcagca tctatagtag ctggtggtat tgtagctatc gtagtaacat gccttgccta
      901 ctattttatg aggtttagaa gagcttttgg tgaatacagt catgtagttg cctttaatac
      961 tttactattc cttatgtcat tcactgtact ctgtttaaca ccagtttact cattcttacc
     1021 tggtgtttat tctgttattt acttgtactt gacattttat cttactaatg atgtttcttt
     1081 tttagcacat attcagtgga tggttatgtt cacaccttta gtacctttct ggataacaat
     1141 tgcttatatc atttgtattt ccacaaagca tttctattgg ttctttagta attacctaaa
     1201 gagacgtgta gtctttaatg gtgtttcctt tagtactttt gaagaagctg cgctgtgcac
     1261 ctttttgtta aataaagaaa tgtatctaaa gttgcgtagt gatgtgctat tacctcttac
     1321 gcaatataat agatacttag ctctttataa taagtacaag tattttagtg gagcaatgga
     1381 tacaactagc tacagagaag ctgcttgttg tcatctcgca aaggctctca atgacttcag
     1441 taactcaggt tctgatgttc tttaccaacc accacaaacc tctatcacct cagctgtttt
     1501 ggaaaacctt tactttcagt cgaagaggcg atggaaaaag aattttatcg cggtgagtgc
     1561 agccaacaga ttcaaaaaga tatcctcgtc tggtgccctc taaggatccg caggcctctg
     1621 ctagcttgac tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg
     1681 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1741 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1801 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     1861 aaaacctcta caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg
     1921 ggcctctaaa cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg
     1981 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc
     2041 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac
     2101 cagaaattta tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag
     2161 acattccttt aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt
     2221 tcttccctcc ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag
     2281 tcagctactt ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc
     2341 aagttcgatc tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca
     2401 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2461 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2521 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2581 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2641 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2701 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2761 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2821 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2881 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2941 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3001 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3061 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3121 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3181 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3241 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact
     3301 ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc
     3361 tctcgaacga actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag
     3421 cgcctatcgc caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa
     3481 gttcaaccta catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc
     3541 gcgcctggcc tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga
     3601 gcgctgccac gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag
     3661 gacagcggcc cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat
     3721 tttaggacgg gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg
     3781 cgaggaaaag tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg
     3841 ccgatgatta tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt
     3901 tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct
     3961 gggctggccg gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag
     4021 accgccaagg gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg
     4081 gagcgcagca aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc
     4141 tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag
     4201 cccttcgcta atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga
     4261 ccctgacgtg aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg
     4321 cagttatggc ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg
     4381 tgtcgtgacg tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg
     4441 tgtgcggtag gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct
     4501 gaatcgacag gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg
     4561 gtcggtttta tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc
     4621 ggggttggcg agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg
     4681 gtcaatatgt aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt
     4741 ggcttttttg ttagacaaca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     4801 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     4861 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     4921 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatccg ccgcgctccc
     4981 gattccggaa gtgcttgaca ttggggagtt cagcgagagc ctgacctatt gcatctcccg
     5041 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     5101 gccggtcgcg gaggcaatgg atgccatcgc tgccgccgat cttagccaga cgagcgggtt
     5161 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     5221 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     5281 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     5341 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     5401 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     5461 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     5521 tccggagctt gcaggatcgc cccggctccg ggcgtatatg ctccgcattg gtcttgacca
     5581 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     5641 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     5701 cgccgccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     5761 cagcactcgt ccgagggcaa aggaataagc tagtatgtaa gcctagtctt agataataaa
     5821 atcgctatcc atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc
     5881 ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac gatccatatc gttgcttggc
     5941 agtcagccag tcggaatcca gcttgggacc caggaagtcc aatcgtcaga tattgtactc
     6001 aagcctggtc acggcagcgt accgatctgt ttaaacctag atattgatag tctgatcggt
     6061 caacgtataa tcgagtccta gcttttgcaa acatctatca agagacagga tcagcaggag
     6121 gctttcgcat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6241 tgggtgcgcg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6301 ttcgccccga agaacgcttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6421 atgacttggt tgagtattca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6541 caacgattgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6661 ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact attaactggc gaactactta
     6721 ctctagcttc ccggcaacag ttgatagact ggatggaggc ggataaagtt gcaggaccac
     6781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6961 taggtgcctc actgattaag cattggtaac cgattctagg tgcattggcg cagaaaaaaa
     7021 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac
     7081 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaaga
     7141 tcgtttaaac tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc
     7201 cgtggcgctc atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt
     7261 tggcagcgat cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct
     7321 ctagtggtca taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt
     7381 ttagaggccc caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac
     7441 acatccatct tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccagat
     7501 ctagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     7561 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     7621 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     7681 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     7741 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     7801 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctgat
     7861 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     7921 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     7981 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     8041 ggtctatata agcagagctg gtttagtgaa ccgtcagatc agatctttgt cgatcctacc
     8101 atccactcga cacacccgcc agcggccgc
//
