LOCUS       sequence-295730-        7261 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 7261)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7261)
  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-m4134258
FEATURES             Location/Qualifiers
     source          1..7261
                     /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             656..733
                     /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"
     CDS             761..781
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    1230..1351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1241..1260)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1295..1314
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1466..1512
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1542..1678
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1723..1794
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2020..2608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2100..2119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3108..4317
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4154..4173
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4321..4920
                     /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(4321..4340)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4817..4837
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4947..4995
                     /note="synthetic polyadenylation signal"
     CDS             join(5261..5329,5330..6121)
                     /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(5479..5498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6134..6270
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6415..6486
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6491..6537)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6555..6603
                     /note="synthetic polyadenylation signal"
     enhancer        6684..6987
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6989..7191
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7142..7162
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7260..7261,1..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 cgggacggcg aatcccgggg ctgataaatc cctggaaaag
      661 gagatggaag aagaacttta ttgcagtgtc tgcggctaac cggttcaaaa agatcagttc
      721 aagcggcgcc ctagattacg acattcctac gaccgcctcc gagaatctct atttccaggg
      781 tgaatttggc ttggcacaac atgacgaggc cgtcgataac aagtttaata aagaacagca
      841 gaacgctttc tatgagatcc ttcacttacc aaatctgaac gaagagcaac gcaacgcttt
      901 cattcagagc ctgaaagacg atccctcgca gtctgcaaat ctcctggccg aagccaagaa
      961 gctgaatgac gcacaggctc caaaagttga taacaagttt aacaaggagc agcagaatgc
     1021 tttctacgag atattgcacc ttcctaatct gaatgaggaa caaagaaatg catttatcca
     1081 gtccctaaaa gacgatccat cacagagtgc caacctgctt gcggaagcca aaaagctcaa
     1141 tgatgcccag gccccgaaag tagacgcgaa ccatcaataa gctagcttga ctgactgaga
     1201 tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac
     1261 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1321 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     1381 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     1441 tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg
     1501 aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa
     1561 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac
     1621 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg
     1681 tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt
     1741 ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca
     1801 ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc
     1861 gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg
     1921 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca
     1981 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2041 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2101 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2161 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2221 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2281 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2341 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2401 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2461 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2521 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2581 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2641 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2701 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2761 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2821 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga
     2881 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca
     2941 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta
     3001 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa
     3061 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg
     3121 ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca cgtcagacga
     3181 agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc ccgctgctca
     3241 taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg ggacttgggt
     3301 gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa gtagtccctt
     3361 ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt atataaggac
     3421 gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg gttcttgttt
     3481 gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc ggggctttcg
     3541 tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag ggctgtagtc
     3601 tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg
     3661 ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa
     3721 ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa
     3781 agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt gaagtttgtc
     3841 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt
     3901 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt
     3961 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc
     4021 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac
     4081 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat
     4141 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt
     4201 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag
     4261 tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacaac
     4321 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     4381 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     4441 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     4501 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     4561 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     4621 tcccggctgg ccgcgcagca acagatggag ggcctcctgg cgccgcaccg gcccaaggag
     4681 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     4741 agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc cgccttcctg
     4801 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4861 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag
     4921 gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga tggatgtgtg
     4981 ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga tcctctcagt
     5041 gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc cagcttggga
     5101 cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc gtaccgatct
     5161 gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc
     5221 aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc aacatttccg
     5281 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5341 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt acatcgaact
     5401 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct ttccaatgat
     5461 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5521 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt caccagtcac
     5581 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5641 gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga aggagctaac
     5701 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5761 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5821 cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac agttgataga
     5881 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5941 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6001 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6061 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6121 accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata
     6181 ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc acttccatac
     6241 gtgtcctcct taccagaaat ttatccttaa gatcgtttaa actcgactct ggctctatcg
     6301 aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc
     6361 gaatctcgtc agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt
     6421 ggaccattag ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta
     6481 cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca
     6541 atcgttgcgt tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt
     6601 attatctaac tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt
     6661 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     6721 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     6781 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     6841 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     6901 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     6961 tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacat caatgggcgt
     7021 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     7081 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     7141 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
     7201 aaccgtcaga tcagatcttt gtcgatccta ccatccactc gacacacccg ccagcggccg
     7261 c
//
