LOCUS       sequence-295978-        7229 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 7229)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7229)
  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-m4133736
FEATURES             Location/Qualifiers
     source          1..7229
                     /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    769..890
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(780..799)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     834..853
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1005..1051
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1081..1217
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1262..1333
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1559..2147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1639..1658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2647..3856
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3693..3712
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3860..4888
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    4915..4963
                     /note="synthetic polyadenylation signal"
     CDS             join(5229..5297,5298..6089)
                     /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(5447..5466)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6102..6238
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6383..6454
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6459..6505)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6523..6571
                     /note="synthetic polyadenylation signal"
     enhancer        6652..6955
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6957..7159
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7110..7130
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7228..7229,1..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 taaggatccg caggcctctg
      721 ctagcttgac tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg
      781 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
      841 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
      901 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
      961 aaaacctcta caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg
     1021 ggcctctaaa cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg
     1081 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc
     1141 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac
     1201 cagaaattta tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag
     1261 acattccttt aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt
     1321 tcttccctcc ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag
     1381 tcagctactt ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc
     1441 aagttcgatc tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca
     1501 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1561 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1621 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1681 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1741 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1801 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1861 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1921 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1981 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2041 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2101 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2161 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2221 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2281 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2341 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact
     2401 ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc
     2461 tctcgaacga actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag
     2521 cgcctatcgc caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa
     2581 gttcaaccta catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc
     2641 gcgcctggcc tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga
     2701 gcgctgccac gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag
     2761 gacagcggcc cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat
     2821 tttaggacgg gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg
     2881 cgaggaaaag tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg
     2941 ccgatgatta tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt
     3001 tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct
     3061 gggctggccg gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag
     3121 accgccaagg gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg
     3181 gagcgcagca aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc
     3241 tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag
     3301 cccttcgcta atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga
     3361 ccctgacgtg aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg
     3421 cagttatggc ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg
     3481 tgtcgtgacg tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg
     3541 tgtgcggtag gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct
     3601 gaatcgacag gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg
     3661 gtcggtttta tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc
     3721 ggggttggcg agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg
     3781 gtcaatatgt aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt
     3841 ggcttttttg ttagacaaca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     3901 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     3961 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     4021 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatccg ccgcgctccc
     4081 gattccggaa gtgcttgaca ttggggagtt cagcgagagc ctgacctatt gcatctcccg
     4141 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     4201 gccggtcgcg gaggcaatgg atgccatcgc tgccgccgat cttagccaga cgagcgggtt
     4261 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     4321 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     4381 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     4441 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     4501 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     4561 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     4621 tccggagctt gcaggatcgc cccggctccg ggcgtatatg ctccgcattg gtcttgacca
     4681 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     4741 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     4801 cgccgccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     4861 cagcactcgt ccgagggcaa aggaataagc tagtatgtaa gcctagtctt agataataaa
     4921 atcgctatcc atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc
     4981 ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac gatccatatc gttgcttggc
     5041 agtcagccag tcggaatcca gcttgggacc caggaagtcc aatcgtcaga tattgtactc
     5101 aagcctggtc acggcagcgt accgatctgt ttaaacctag atattgatag tctgatcggt
     5161 caacgtataa tcgagtccta gcttttgcaa acatctatca agagacagga tcagcaggag
     5221 gctttcgcat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5281 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5341 tgggtgcgcg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5401 ttcgccccga agaacgcttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5461 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5521 atgacttggt tgagtattca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5581 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5641 caacgattgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5701 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5761 ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact attaactggc gaactactta
     5821 ctctagcttc ccggcaacag ttgatagact ggatggaggc ggataaagtt gcaggaccac
     5881 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5941 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6001 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6061 taggtgcctc actgattaag cattggtaac cgattctagg tgcattggcg cagaaaaaaa
     6121 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac
     6181 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaaga
     6241 tcgtttaaac tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc
     6301 cgtggcgctc atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt
     6361 tggcagcgat cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct
     6421 ctagtggtca taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt
     6481 ttagaggccc caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac
     6541 acatccatct tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccagat
     6601 ctagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     6661 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     6721 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     6781 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     6841 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     6901 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctgat
     6961 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     7021 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     7081 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     7141 ggtctatata agcagagctg gtttagtgaa ccgtcagatc agatctttgt cgatcctacc
     7201 atccactcga cacacccgcc agcggccgc
//
