LOCUS       sequence-295720-        6745 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 6745)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6745)
  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-m4133945
FEATURES             Location/Qualifiers
     source          1..6745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             596..616
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             617..661
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    714..835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(725..744)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     779..798
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      950..996
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1026..1162
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1207..1278
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1504..2092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1584..1603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2592..3801
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3638..3657
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3805..4404
                     /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(3805..3824)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4301..4321
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4431..4479
                     /note="synthetic polyadenylation signal"
     CDS             join(4745..4813,4814..5605)
                     /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(4963..4982)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5618..5754
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5899..5970
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5975..6021)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6039..6087
                     /note="synthetic polyadenylation signal"
     enhancer        6168..6471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6473..6675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6626..6646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgata gcctcagagt ttagttccct tccatcatat gcagcttttg ctactgctca
       61 agaagcttat gagcaggctg ttgctaatgg tgattctgaa gttgttctta aaaagttgaa
      121 gaagtctttg aatgtggcta aatctgaatt tgaccgtgat gcagccatgc aacgtaagtt
      181 ggaaaagatg gctgatcaag ctatgaccca aatgtataaa caggctagat ctgaggacaa
      241 gagggcaaaa gttactagtg ctatgcagac aatgcttttc actatgctta gaaagttgga
      301 taatgatgca ctcaacaaca ttatcaacaa tgcaagagat ggttgtgttc ccttgaacat
      361 aatacctctt acaacagcag ccaaactaat ggttgtcata ccagactata acacatataa
      421 aaatacgtgt gatggtacaa catttactta tgcatcagca ttgtgggaaa tccaacaggt
      481 tgtagatgca gatagtaaaa ttgttcaact tagtgaaatt agtatggaca attcacctaa
      541 tttagcatgg cctcttattg taacagcttt aagggccaat tctgctgtca aattagaaaa
      601 cctttacttt cagtcgaaag aaaccgcggc agctaagttc gagaggcagc acatggacag
      661 ttaagctagc ttgactgact gagatacagc gtaccttcag ctcacagaca tgataagata
      721 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
      781 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
      841 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
      901 caagtaaaac ctctacaaat gtggtattgg cccatctcta tcggtatcgt agcataaccc
      961 cttggggcct ctaaacgggt cttgaggggt tttttgtgcc cctcgggccg gattgctatc
     1021 taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca
     1081 tatgccagat tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc
     1141 cttaccagaa atttatcctt aaggtcgtca gctatcctgc aggcgatctc tcgatttcga
     1201 tcaagacatt cctttaatgg tcttttctgg acaccactag gggtcagaag tagttcatca
     1261 aactttcttc cctccctaat ctcattggtt accttgggct atcgaaactt aattaaccag
     1321 tcaagtcagc tacttggcga gatcgacttg tctgggtttc gactacgctc agaattgcgt
     1381 cagtcaagtt cgatctggtc cttgctattg cacccgttct ccgattacga gtttcattta
     1441 aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1501 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1561 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1621 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1681 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1741 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1801 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1861 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1921 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     1981 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2041 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2101 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2161 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2221 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2281 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcat ttaaatttcc
     2341 gaactctcca aggccctcgt cggaaaatct tcaaaccttt cgtccgatcc atcttgcagg
     2401 ctacctctcg aacgaactat cgcaagtctc ttggccggcc ttgcgccttg gctattgctt
     2461 ggcagcgcct atcgccaggt attactccaa tcccgaatat ccgagatcgg gatcacccga
     2521 gagaagttca acctacatcc tcaatcccga tctatccgag atccgaggaa tatcgaaatc
     2581 ggggcgcgcc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcgtcac
     2641 ggcgagcgct gccacgtcag acgaagggcg caggagcgtc ctgatccttc cgcccggacg
     2701 ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag
     2761 gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga
     2821 acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt
     2881 gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg
     2941 ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg
     3001 ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg
     3061 gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt
     3121 ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg
     3181 cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc
     3241 ttgagccctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg ggcaccatct
     3301 ggggaccctg acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg
     3361 ggcggcagtt atggcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgccct
     3421 cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg cagggtgggg ccacctgccg
     3481 gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct agggtaggct
     3541 ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg gataagtgag gcgtcagttt
     3601 ctttggtcgg ttttatgtac ctatcttctt aagtagctga agctccggtt ttgaactatg
     3661 cgctcggggt tggcgagtgt gttttgtgaa gttttttagg caccttttga aatgtaatca
     3721 tttgggtcaa tatgtaattt tcagtgttag acttgtaaat tgtccgctaa attctggccg
     3781 tttttggctt ttttgttaga caacatgacc gagtacaagc ccacggtgcg cctcgccacc
     3841 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3901 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3961 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     4021 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     4081 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggagggcctc
     4141 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     4201 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggccgccgag
     4261 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     4321 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     4381 atgacccgca agcccggtgc ctaggctagt atgtaagcct agtcttagat aataaaatcg
     4441 ctatccatcg aagatggatg tgtgttggtt ttttgtgtgt gtaacgctag gcgcgcctgg
     4501 tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg cttggcagtc
     4561 agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt gtactcaagc
     4621 ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg atcggtcaac
     4681 gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag caggaggctt
     4741 tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4801 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4861 tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4921 ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4981 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5041 cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5101 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5161 gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5221 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5281 gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac tacttactct
     5341 agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag gaccacttct
     5401 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5461 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5521 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5581 tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc
     5641 tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct
     5701 tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaagatcgt
     5761 ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg
     5821 gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta cctttttggc
     5881 agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc ttccctctag
     5941 tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag acccgtttag
     6001 aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac caacacacat
     6061 ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag ccagatctag
     6121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     6181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     6241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     6301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     6361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     6421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg
     6481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     6541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     6601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     6661 tatataagca gagctggttt agtgaaccgt cagatcagat ctttgtcgat cctaccatcc
     6721 actcgacaca cccgccagcg gccgc
//
