LOCUS       sequence-295537-        7220 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 7220)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7220)
  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-m4133808
FEATURES             Location/Qualifiers
     source          1..7220
                     /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             626..691
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    760..881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(771..790)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     825..844
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      996..1042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1072..1208
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1253..1324
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1550..2138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1630..1649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2638..3847
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3684..3703
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3851..4879
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    4906..4954
                     /note="synthetic polyadenylation signal"
     CDS             join(5220..5288,5289..6080)
                     /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(5438..5457)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6093..6229
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6374..6445
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6450..6496)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6514..6562
                     /note="synthetic polyadenylation signal"
     enhancer        6643..6946
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6948..7150
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7101..7121
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7219..7220,1..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 cgatggatta taaggaccac gatggagact acaaagatca
      661 tgacattgat tacaaggacg acgatgacaa ataaggatcc gcaggcctct gctagcttga
      721 ctgactgaga tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg
      781 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
      841 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
      901 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
      961 acaaatgtgg tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa
     1021 acgggtcttg aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg
     1081 cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag
     1141 caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt
     1201 atccttaagg tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt
     1261 taatggtctt ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc
     1321 cctaatctca ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact
     1381 tggcgagatc gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat
     1441 ctggtccttg ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa
     1501 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1561 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1621 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1681 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1741 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1801 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1861 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1921 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1981 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2041 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2101 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2161 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2221 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2281 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2341 cagcgatctg tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc
     2401 cctcgtcgga aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg
     2461 aactatcgca agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg
     2521 ccaggtatta ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct
     2581 acatcctcaa tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc
     2641 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca
     2701 cgtcagacga agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc
     2761 ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg
     2821 ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa
     2881 gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt
     2941 atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg
     3001 gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc
     3061 ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag
     3121 ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc
     3181 aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc
     3241 gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct
     3301 aatgcgggaa agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt
     3361 gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg
     3421 cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac
     3481 gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta
     3541 ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca
     3601 ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt
     3661 atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc
     3721 gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg
     3781 taattttcag tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt
     3841 gttagacaac atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     3901 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3961 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     4021 tttctacaaa gatcgttatg tttatcggca ctttgcatcc gccgcgctcc cgattccgga
     4081 agtgcttgac attggggagt tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     4141 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     4201 ggaggcaatg gatgccatcg ctgccgccga tcttagccag acgagcgggt tcggcccatt
     4261 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     4321 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     4381 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     4441 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     4501 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     4561 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     4621 tgcaggatcg ccccggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4681 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4741 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgccgccgt
     4801 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4861 tccgagggca aaggaataag ctagtatgta agcctagtct tagataataa aatcgctatc
     4921 catcgaagat ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac
     4981 cgagaacgat cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca
     5041 gtcggaatcc agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt
     5101 cacggcagcg taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata
     5161 atcgagtcct agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca
     5221 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5281 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc
     5341 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5401 aagaacgctt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5461 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5521 ttgagtattc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5581 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg
     5641 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5701 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5761 ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt
     5821 cccggcaaca gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5881 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5941 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6001 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6061 cactgattaa gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg
     6121 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     6181 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa
     6241 ctcgactctg gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct
     6301 catttgctcg tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga
     6361 tcgcggctcc cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc
     6421 ataacagcag cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc
     6481 ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc
     6541 ttcgatggat agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc
     6601 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     6661 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     6721 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     6781 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     6841 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     6901 tcctacttgg cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg
     6961 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     7021 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     7081 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     7141 aagcagagct ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg
     7201 acacacccgc cagcggccgc
//
