LOCUS       sequence-295558-        6784 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 6784)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6784)
  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-m4133776
FEATURES             Location/Qualifiers
     source          1..6784
                     /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..700
                     /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    753..874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(764..783)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     818..837
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      989..1035
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1065..1201
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1246..1317
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1543..2131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1623..1642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2631..3840
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3677..3696
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3844..4443
                     /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(3844..3863)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4340..4360
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4470..4518
                     /note="synthetic polyadenylation signal"
     CDS             join(4784..4852,4853..5644)
                     /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(5002..5021)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5657..5793
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5938..6009
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6014..6060)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6078..6126
                     /note="synthetic polyadenylation signal"
     enhancer        6207..6510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6512..6714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6665..6685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6783..6784,1..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 cgaagaggcg atggaaaaag aattttatcg cggtgagtgc
      661 agccaacaga ttcaaaaaga tatcctcgtc tggtgccctc taagctagct tgactgactg
      721 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
      781 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
      841 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
      901 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
      961 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
     1021 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     1081 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     1141 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     1201 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     1261 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     1321 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     1381 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     1441 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     1501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1981 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2341 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc
     2401 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc
     2461 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta
     2521 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct
     2581 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg
     2641 ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga
     2701 cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc
     2761 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg
     2821 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc
     2881 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag
     2941 gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg
     3001 tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt
     3061 tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta
     3121 gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg
     3181 cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa
     3241 caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg
     3301 gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt
     3361 gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc
     3421 gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc
     3481 gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg gtaggctttt
     3541 ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg
     3601 gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc
     3661 tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg
     3721 ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt
     3781 cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac
     3841 aacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3901 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     3961 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4021 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4081 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4141 ggttcccggc tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag
     4201 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4261 ggcagcgccg tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc
     4321 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4381 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4441 taggctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt
     4501 gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc
     4561 agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg
     4621 ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga
     4681 tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt
     4741 tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt
     4801 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4861 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga
     4921 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat
     4981 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5041 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt
     5101 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5161 catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct
     5221 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5281 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5341 aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat
     5401 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5461 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5521 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5581 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5641 gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt
     5701 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca
     5761 tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac tctggctcta
     5821 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     5881 atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg ctcccgacat
     5941 cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca gcagcttcag
     6001 ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     6061 tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat ggatagcgat
     6121 tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac ggggtcatta
     6181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     6241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     6301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     6361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     6421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     6481 atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta catcaatggg
     6541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     6601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     6661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
     6721 gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac ccgccagcgg
     6781 ccgc
//
