LOCUS       sequence-334000-        7052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculoviral transfer vector to express untagged nsp8 (SARS-CoV-2) in
            insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7052)
  AUTHORS   Bertolin AP, Weissmann F, Zeng J, Posse V, Milligan JC, Canal B, 
            Ulferts R, Wu M, Drury LS, Howell M, Beale R, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2425-2443. doi: 10.1042/BCJ20210200.
  PUBMED    34198323
REFERENCE   2  (bases 1 to 7052)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2021-07-16- 16"; volume: "478"; issue: "13"; 
            pages: "2425-2443"
COMMENT     Sequence Label: pBIG1c_nsp8 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..7052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(324..329,330..346,347..352))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(748..781)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(827..846)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    871..919
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        1014..1105
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     1030..1047
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      1149..1158
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1884..2018
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1921..1940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1975..1994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  2333..2498
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     CDS             3022..3813
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3854..4308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3941..3960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4150..4171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4334..4438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4439..4507,4508..5299)
                     /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(4657..4676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5470..6058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5959..5978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6212..6229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6363..6587
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(join(6654..7052,1..135))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
       61 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
      121 gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg
      181 cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga
      241 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
      301 agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttt
      361 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg
      421 aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg
      481 cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg cccttgcttc
      541 aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag
      601 tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct
      661 atagttctag tggttggcta cagctttgtt tgtactatca acaggttgaa ctgctgatca
      721 acagatcctc tacgcggccg cggtaccata acttcgtata gcatacatta tacgaagtta
      781 tctgggttta aactggatac tattgcacgt ttttatcgag gctaactgaa acacggaagg
      841 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
      901 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcaag
      961 tgaacgctct atggtctaaa gatttaaatc gacctactcc ggaatattaa tagatcatgg
     1021 agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt ttcgtaacag
     1081 ttttgtaata aaaaaaccta taaatattct ggattattca taccgtccca ccatcgggcg
     1141 cggatccagc caccatggct atcgcttccg agttctcctc tctgccctcc tacgctgctt
     1201 tcgctaccgc tcaagaggct tacgagcagg ctgtggctaa cggcgactct gaggtggtgc
     1261 tgaagaagct gaaaaagtcc ctgaacgtgg ccaagtccga gttcgaccgt gacgctgcta
     1321 tgcagcgcaa gctcgagaag atggctgacc aggctatgac ccagatgtac aagcaggctc
     1381 gttccgagga caagcgtgct aaagtgacct ccgctatgca gaccatgctg ttcaccatgc
     1441 tccgcaagct ggacaacgac gctctgaaca acatcatcaa caacgctcgt gacggttgcg
     1501 tgcccctgaa catcatccct ctgaccaccg ctgctaagct gatggtggtc atccccgact
     1561 acaacaccta caagaacact tgcgacggca ccaccttcac ctacgcttcc gctctgtggg
     1621 agatccagca agtggtggac gctgactcca agatcgtgca gctgtccgag atctccatgg
     1681 acaactctcc caacctggct tggcccctga tcgtgactgc tctgcgtgct aactccgctg
     1741 tgaagctgca gtaaaagctt gtcgagaagt actagaggat cataatcagc cataccacat
     1801 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1861 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1981 tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt gagcctagaa
     2041 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2101 taactatcat aaccatttaa ataaatctat atctcaatcg gggttacgtc gataccccac
     2161 cgagacccca ttgggaccaa tacgcccgcg tttcttcctt ttccccaccc caacccccaa
     2221 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaagccct gccatagcca
     2281 ctacgggtta attaaaaacc taatgatgcc tgatgtttaa acaggagatc cgaaccagat
     2341 aagtgaaatc tagttccaaa ctattttgtc atttttaatt ttcgtattag cttacgacgc
     2401 tacacccagt tcccatctat tttgtcactc ttccctaaat aatccttaaa aactccattt
     2461 ccacccctcc cagttcccaa ctattttgtc cgcccacagc ggggcatttt tcttcctgtt
     2521 atgtttttaa tcaaacatcc tgccaactcc atgtgacaaa ccgtcatctt cggctacttt
     2581 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
     2641 ctgaatatca acgcttattt gcagcctgaa ctgggaggca gaataaatga tcatatcgtc
     2701 aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac
     2761 ggtcacactg cttccggtag tcaataaacc ggtaagtagc gtatgcgctc acgcaactgg
     2821 tccagaacct tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt
     2881 tatgactgtt tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac
     2941 gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc
     3001 cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc gactcaacta
     3061 tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg
     3121 tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg
     3181 gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact
     3241 tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac
     3301 gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag
     3361 cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc
     3421 ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc
     3481 tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg
     3541 aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt
     3601 tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg
     3661 gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga
     3721 caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg
     3781 aaaggcgaga tcaccaaggt agtcggcaaa taatgtctaa caattcgttc aagccgacgg
     3841 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3901 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3961 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4021 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4081 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4141 ttcttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4201 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4261 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4321 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4381 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4441 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4501 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4561 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4621 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4681 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4741 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4801 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4861 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4921 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4981 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5041 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5101 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5161 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5221 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5281 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5341 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5401 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5461 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5521 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5581 aactggcttc agcagagcgc agataccaaa tactattctt ctagtgtagc cgtagttagg
     5641 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5701 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5761 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5821 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5881 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5941 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6001 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6061 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6121 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6181 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6241 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagacca
     6301 gccgcgtaac ctggcaaaat cggttacggt tgagtaataa atggatgccc tgcgtaagcg
     6361 ggtgtgggcg gacaataaag tcttaaactg aacaaaatag atctaaacta tgacaataaa
     6421 gtcttaaact agacagaata gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc
     6481 atactggact tttgttatgg ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg
     6541 tcgtattaaa gaggggcgtg gccaagggca tggtaaagac tatattcgcg gcgttgtgac
     6601 aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg
     6661 gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa ctttgtatag
     6721 agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata agcaccaagc
     6781 gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg cctccggtgc
     6841 tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc aaacttgggc
     6901 agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg
     6961 aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg ttgggagtag
     7021 gtggctacgt ctccgaactc acgaccgaaa ag
//
