LOCUS       sequence-295436-        6724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6724)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6724)
  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-m4133990
FEATURES             Location/Qualifiers
     source          1..6724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             602..622
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             623..640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    693..814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(704..723)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     758..777
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      929..975
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1005..1141
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1186..1257
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1483..2071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1563..1582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2571..3780
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3617..3636
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3784..4383
                     /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(3784..3803)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4280..4300
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4410..4458
                     /note="synthetic polyadenylation signal"
     CDS             join(4724..4792,4793..5584)
                     /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(4942..4961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5597..5733
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5878..5949
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5954..6000)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6018..6066
                     /note="synthetic polyadenylation signal"
     enhancer        6147..6450
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6452..6654
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6605..6625
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6723..6724,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggca attgcctcag aatttagttc cctcccctcg tacgctgcgt tcgctaccgc
       61 ccaagaggca tatgagcagg ccgtagccaa cggggacagc gaagtggttc ttaagaaact
      121 gaagaaatct ttgaatgtgg caaagagcga gtttgatcgg gacgcggcaa tgcagaggaa
      181 gctggaaaaa atggctgatc aggctatgac acagatgtac aaacaagccc gatccgagga
      241 caagagagct aaagtcactt ctgccatgca aacgatgcta ttcaccatgt tacgtaagct
      301 ggataacgac gcactcaata acataatcaa taacgcccgc gatggttgtg tgccattgaa
      361 tatcattcct cttactacag cggccaagct gatggtcgtt atcccggact ataacacata
      421 caaaaatacc tgcgacggca ccactttcac gtatgctagc gccctgtggg aaatacagca
      481 ggtagtggat gccgattcaa agattgtgca gcttagtgag atctccatgg acaacagtcc
      541 caacttggct tggcctttaa tcgtcacagc tctcagagcc aatagcgcag ttaaactgca
      601 agaaaacctt tactttcagt cgcaccatca ccatcaccat taagctagct tgactgactg
      661 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
      721 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
      781 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
      841 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
      901 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
      961 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     1021 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     1081 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     1141 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     1201 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     1261 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     1321 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     1381 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     1441 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1501 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1561 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1621 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1681 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1741 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1801 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1861 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1921 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1981 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2041 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2101 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2161 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2221 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2281 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc
     2341 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc
     2401 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta
     2461 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct
     2521 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg
     2581 ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga
     2641 cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc
     2701 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg
     2761 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc
     2821 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag
     2881 gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg
     2941 tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt
     3001 tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta
     3061 gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg
     3121 cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa
     3181 caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg
     3241 gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt
     3301 gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc
     3361 gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc
     3421 gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg gtaggctttt
     3481 ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg
     3541 gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc
     3601 tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg
     3661 ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt
     3721 cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac
     3781 aacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3841 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     3901 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3961 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4021 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4081 ggttcccggc tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag
     4141 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4201 ggcagcgccg tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc
     4261 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4321 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4381 taggctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt
     4441 gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc
     4501 agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg
     4561 ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga
     4621 tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt
     4681 tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt
     4741 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4801 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga
     4861 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat
     4921 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4981 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt
     5041 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5101 catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct
     5161 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5221 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5281 aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat
     5341 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5401 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5461 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5521 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5581 gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt
     5641 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca
     5701 tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac tctggctcta
     5761 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     5821 atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg ctcccgacat
     5881 cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca gcagcttcag
     5941 ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     6001 tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat ggatagcgat
     6061 tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac ggggtcatta
     6121 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     6181 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     6241 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     6301 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     6361 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     6421 atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta catcaatggg
     6481 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     6541 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     6601 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
     6661 gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac ccgccagcgg
     6721 ccgc
//
