LOCUS       sequence-295685-        7090 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 7090)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7090)
  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-m4133957
FEATURES             Location/Qualifiers
     source          1..7090
                     /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             965..982
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1059..1180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1070..1089)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1124..1143
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1295..1341
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1371..1507
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1552..1623
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1849..2437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1929..1948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2937..4146
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3983..4002
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4150..4749
                     /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(4150..4169)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4646..4666
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4776..4824
                     /note="synthetic polyadenylation signal"
     CDS             join(5090..5158,5159..5950)
                     /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(5308..5327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5963..6099
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6244..6315
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6320..6366)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6384..6432
                     /note="synthetic polyadenylation signal"
     enhancer        6513..6816
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6818..7020
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6971..6991
                     /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 cagtcgatga gtgacaaaat cattcatcta accgatgact cctttgatac
      661 agacgtgttg aaggccgatg gtgcgatact ggtagatttc tgggctgaat ggtgtggccc
      721 gtgcaagatg atcgcaccca ttctggacga gatcgccgac gagtatcaag gaaaattaac
      781 tgtcgctaag cttaatatag atcagaaccc agggacggca cctaaatacg gcatcagggg
      841 aatacctacc ctcctgctct ttaagaatgg ggaagttgcc gcgacaaaag tgggagccct
      901 gtctaagggc cagctaaaag agttcttgga cgctaacctg gcaggttcag gctcggggca
      961 catgcaccat caccatcacc atagcagcgg gctcgtgcca cgcggctaag ctagcttgac
     1021 tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg
     1081 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1141 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     1201 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     1261 caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa
     1321 cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc
     1381 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc
     1441 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta
     1501 tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt
     1561 aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc
     1621 ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt
     1681 ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc
     1741 tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa
     1801 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1861 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1921 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1981 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2041 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2101 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2161 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2221 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2281 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2341 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2401 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2461 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2521 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2581 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2641 agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc
     2701 ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga
     2761 actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc
     2821 caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta
     2881 catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggcc
     2941 tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga gcgctgccac
     3001 gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc
     3061 cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg
     3121 gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag
     3181 tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta
     3241 tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg
     3301 ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg
     3361 gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg
     3421 gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca
     3481 aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg
     3541 ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag cccttcgcta
     3601 atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga ccctgacgtg
     3661 aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc
     3721 ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg
     3781 tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag
     3841 gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag
     3901 gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta
     3961 tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg
     4021 agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt
     4081 aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt ggcttttttg
     4141 ttagacaaca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     4201 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4261 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4321 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4381 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     4441 ttgagcggtt cccggctggc cgcgcagcaa cagatggagg gcctcctggc gccgcaccgg
     4501 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     4561 ggtctgggca gcgccgtcgt gctccccgga gtggaggccg ccgagcgcgc cggggtgccc
     4621 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     4681 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     4741 ggtgcctagg ctagtatgta agcctagtct tagataataa aatcgctatc catcgaagat
     4801 ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat
     4861 cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc
     4921 agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg
     4981 taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct
     5041 agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca tgagtattca
     5101 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5161 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta
     5221 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgctt
     5281 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5341 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc
     5401 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5461 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa
     5521 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5581 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5641 ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5701 gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5761 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5821 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5881 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5941 gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg
     6001 cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca
     6061 cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa ctcgactctg
     6121 gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct catttgctcg
     6181 tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga tcgcggctcc
     6241 cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc ataacagcag
     6301 cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt
     6361 atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc ttcgatggat
     6421 agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc aattacgggg
     6481 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     6541 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     6601 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     6661 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     6721 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     6781 cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg gcagtacatc
     6841 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     6901 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     6961 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     7021 ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg acacacccgc
     7081 cagcggccgc
//
