LOCUS       sequence-295200-        7096 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 7096)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7096)
  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-m4133869
FEATURES             Location/Qualifiers
     source          1..7096
                     /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             971..988
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1065..1186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1076..1095)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1130..1149
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1301..1347
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1377..1513
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1558..1629
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1855..2443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1935..1954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2943..4152
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3989..4008
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4156..4755
                     /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(4156..4175)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4652..4672
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4782..4830
                     /note="synthetic polyadenylation signal"
     CDS             join(5096..5164,5165..5956)
                     /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(5314..5333)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5969..6105
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6250..6321
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6326..6372)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6390..6438
                     /note="synthetic polyadenylation signal"
     enhancer        6519..6822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6824..7026
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6977..6997
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7095..7096,1..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 cgatgagtga caaaatcatt catctaaccg atgactcctt
      661 tgatacagac gtgttgaagg ccgatggtgc gatactggta gatttctggg ctgaatggtg
      721 tggcccgtgc aagatgatcg cacccattct ggacgagatc gccgacgagt atcaaggaaa
      781 attaactgtc gctaagctta atatagatca gaacccaggg acggcaccta aatacggcat
      841 caggggaata cctaccctcc tgctctttaa gaatggggaa gttgccgcga caaaagtggg
      901 agccctgtct aagggccagc taaaagagtt cttggacgct aacctggcag gttcaggctc
      961 ggggcacatg caccatcacc atcaccatag cagcgggctc gtgccacgcg gctaagctag
     1021 cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat acattgatga
     1081 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1141 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1201 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     1261 cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc
     1321 tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat
     1381 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga
     1441 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga
     1501 aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat
     1561 tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt
     1621 ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag
     1681 ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt
     1741 tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg
     1801 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1861 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1921 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1981 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2041 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2101 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2161 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2221 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2281 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2341 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2401 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2461 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2521 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2581 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2641 tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc cgaactctcc
     2701 aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag gctacctctc
     2761 gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct tggcagcgcc
     2821 tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg agagaagttc
     2881 aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat cggggcgcgc
     2941 ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc
     3001 tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca
     3061 gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta
     3121 ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag
     3181 gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga
     3241 tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg
     3301 tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc
     3361 tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg
     3421 ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc
     3481 gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg
     3541 aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgagccct
     3601 tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc tggggaccct
     3661 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt
     3721 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc
     3781 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg
     3841 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat
     3901 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg
     3961 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg
     4021 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca
     4081 atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc gtttttggct
     4141 tttttgttag acaacatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4201 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4261 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4321 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4381 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4441 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggagggcct cctggcgccg
     4501 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     4561 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggccgccga gcgcgccggg
     4621 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     4681 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4741 aagcccggtg cctaggctag tatgtaagcc tagtcttaga taataaaatc gctatccatc
     4801 gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta ggcgcgcctg gtgtaccgag
     4861 aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt gcttggcagt cagccagtcg
     4921 gaatccagct tgggacccag gaagtccaat cgtcagatat tgtactcaag cctggtcacg
     4981 gcagcgtacc gatctgttta aacctagata ttgatagtct gatcggtcaa cgtataatcg
     5041 agtcctagct tttgcaaaca tctatcaaga gacaggatca gcaggaggct ttcgcatgag
     5101 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5161 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcgcgagt
     5221 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5281 acgctttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5341 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5401 gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5461 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgattggagg
     5521 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5581 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5641 agcaatggca acaaccttgc gtaaactatt aactggcgaa ctacttactc tagcttcccg
     5701 gcaacagttg atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5761 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5821 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5881 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5941 gattaagcat tggtaaccga ttctaggtgc attggcgcag aaaaaaatgc ctgatgcgac
     6001 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact
     6061 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaagatcg tttaaactcg
     6121 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt
     6181 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc
     6241 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa
     6301 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa
     6361 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg
     6421 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt
     6481 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     6541 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     6601 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     6661 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     6721 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     6781 acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag
     6841 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     6901 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     6961 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     7021 agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc cactcgacac
     7081 acccgccagc ggccgc
//
