LOCUS       sequence-334216-        7601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculoviral transfer vector for the expression of SARS-CoV-2 nsp15 
            in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7601)
  AUTHORS   Canal B, Fujisawa R, McClure AW, Deegan TD, Wu M, Ulferts R, 
            Weissmann F, Drury LS, Bertolin AP, Zeng J, Beale R, Howell M, Labib
            K, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of nsp15 endoribonuclease.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2465-2479. doi: 10.1042/BCJ20210199.
  PUBMED    34198324
REFERENCE   2  (bases 1 to 7601)
  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: "2465-2479"
COMMENT     Sequence Label: pBIG1a_3xFlag-6His-nsp15 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..7601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  659..824
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     polyA_signal    1139..1273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1163..1182)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1217..1236
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2459..2476)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2483..2548)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2548..2557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2601..2692)
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     complement(2659..2676)
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     polyA_signal    2787..2835
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2860..2879
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     protein_bind    2925..2958
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(3354..3359,3360..3376,3377..3382)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3571..4104
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  4171..4395
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     primer_bind     complement(4529..4546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4700..5288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4780..4799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5459..6250,6251..6319))
                     /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     6082..6101
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6320..6424)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6450..6904)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6587..6608)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6798..6817
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(6945..7601,1..135))
                     /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"
ORIGIN
        1 gtgtggcttc aggccgccat ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg
       61 ttcgagatgg cgctcgatga cgccaactac ctctgatagt tgagtcgata cttcggcgat
      121 caccgcttcc ctcatgatgt ttaactttgt tttagggcga ctgccctgct gcgtaacatc
      181 gttgctgctc cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc
      241 ccgaggcata gactgtaccc caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc
      301 gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta
      361 cttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc
      421 cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcat ttattctgcc
      481 tcccagttca ggctgcaaat aagcgttgat attcagtcaa ttacaaacat taataacgaa
      541 gagatgacag aaaaattttc attctgtgac agagaaaaag tagccgaaga tgacggtttg
      601 tcacatggag ttggcaggat gtttgattaa aaacataaca ggaagaaaaa tgccccgctg
      661 tgggcggaca aaatagttgg gaactgggag gggtggaaat ggagttttta aggattattt
      721 agggaagagt gacaaaatag atgggaactg ggtgtagcgt cgtaagctaa tacgaaaatt
      781 aaaaatgaca aaatagtttg gaactagatt tcacttatct ggttcggatc tcctgtttaa
      841 accccgattg agatatagtt tttaattaac ccgtagtggc tatggcaggg cttgccgccc
      901 cgacgttggc tgcgagccct gggccttcac ccgaacttgg gggttggggt ggggaaaagg
      961 aagaaacgcg ggcgtattgg tcccaatggg gtctcggtgg ggtatcgacg taaccccgat
     1021 tgagatatag atttatttaa atggttatga tagttattgc tcagcggtgg cagcagccaa
     1081 ctcagcttcc tttcgggctt tgttagcagc cggatcttct aggctcaagc agtgatcaga
     1141 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1201 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1261 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     1321 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc
     1381 tctagtactt ctcgacaagc ttttactgca gcttggggta gaaagtctcg acgtgaccgt
     1441 ccttgcacca cagcatgaag ctgatctcgg tgtagtcgat ggtcactttg accaccttgg
     1501 acaccacaga caggtcctgg gacttgatga tctcgacgaa gtcgtccagc agcaggtcga
     1561 tcacggagca cacgcactta gaggaaccgg tctgagcgtc ggtgatgaag tagttcttca
     1621 cagtggagtc catggggatg aagtcctcca actcgaatgg ggactctttg aagcgcttag
     1681 ccagtccgat caacaggtgc agtccaccga gctgagagtg ggagaagtcg ccgtacacga
     1741 tgtgctcgaa agcgtaaccc tcgagcttgt agcgctcgat gaactcgtcc atagccagct
     1801 cgaggaagtc gatttccatc tggctacggg gcttgaactc ttgcaggtta cgagactggg
     1861 tgaagtaagt ctcgggcagc tgctgcacga caccgtccac tttcttgtag tagttgaact
     1921 gggtcttgac agcctctccg atcagggtga caccgttcag ggaagcctgc ttaggaccca
     1981 cagaaggctg cagtcccttc acggaaccct cggtgatcag gacaccgtta cgagcgttac
     2041 ggaacaggtc cacctgtccg tccactctac cgtcgaagaa cacggtcagg ggagcgcaga
     2101 tagtctcggt aggcttctta gcaatgtcgg tcatggagca gacaccgatg gtggagatgt
     2161 gagcgggagc gtcacgcttg tagtcccaga tcacggtgtt agcagcgata tcgacaccca
     2221 ggttgttcag gatcttcact tcaggcacgg gcttgatgtt acgcttagcc cacaactcga
     2281 aggcgacgtt cacgggcaga gtggtcttgt tctcgaacag ttccacgtcc acgccgtcca
     2341 ccttggtgta cacggtgttg ttgatgatgg acacaggcac ttcgccttgc tggccgtcga
     2401 agtggccctt gttgaccacg ttgaaggcca cgttttccag ggactgcagc acagcagagt
     2461 gatggtggtg atggtgggaa cccttgtcat cgtcgtcttt gtagtcgatg tcatgatcct
     2521 tgtaatcgcc gtcgtggtcc ttgtagtcca tggtggctgg atccgcgccc gatggtggga
     2581 cggtatgaat aatccggaat atttataggt ttttttatta caaaactgtt acgaaaacag
     2641 taaaatactt atttatttgc gagatggtta tcattttaat tatctccatg atctattaat
     2701 attccggagt aggtcgattt aaatctttag accatagagc gttcacttga cagagtgcca
     2761 gccctgggac cgaaccccgc gtttatgaac aaacgaccca acacccgtgc gttttattct
     2821 gtctttttat tgccgtcata gcgcgggttc cttccggtat tgtctccttc cgtgtttcag
     2881 ttagcctcga taaaaacttt agaccataga gcgtttaaac ccagataact tcgtataatg
     2941 tatgctatac gaagttatgg taccgcggcc gcgtagagga tctgttgatc agcagttcaa
     3001 cctgttgata gtacaaacaa agctgtagcc aaccactaga actatagcta gagtcctggg
     3061 cgaacaaacg atgctcgcct tccagaaaac cgaggatgcg aaccacttca tccggggtca
     3121 gcaccaccgg caagcgccgc gacggccgag gtctaccgat ctcctgaagc aagggcagat
     3181 ccgtgcacag caccttgccg tagaagaaca gcaaggccgc caatgcctga cgatgcgtgg
     3241 agaccgaaac cttgcgctcg ttcgccagcc aggacagaaa tgcctcgact tcgctgctgc
     3301 ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacaaacc cagttgacat
     3361 aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca
     3421 gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg
     3481 actgtttttt tgtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg
     3541 gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagcaacga tgttacgcag
     3601 cagggcagtc gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt
     3661 aggctcggcc ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag
     3721 ttcggagacg tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg
     3781 ctccgtagta agacattcat cgcgcttgct gccttcgacc aagaagcggt tgttggcgct
     3841 ctcgcggctt acgttctgcc caagtttgag cagccgcgta gtgagatcta tatctatgat
     3901 ctcgcagtct ccggcgagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc
     3961 aagcatgagg ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac
     4021 gatcccgcag tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat
     4081 atcgacccaa gtaccgccac ctaacaattc gttcaagccg agatcggctt cccggccgcg
     4141 gagttgttcg gtaaattgtc acaacgccgc gaatatagtc tttaccatgc ccttggccac
     4201 gcccctcttt aatacgacgg gcaatttgca cttcagaaaa tgaagagttt gctttagcca
     4261 taacaaaagt ccagtatgct ttttcacagc ataactggac tgatttcagt ttacaactat
     4321 tctgtctagt ttaagacttt attgtcatag tttagatcta ttttgttcag tttaagactt
     4381 tattgtccgc ccacacccgc ttacgcaggg catccattta ttactcaacc gtaaccgatt
     4441 ttgccaggtt acgcggctgg tctatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4501 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4561 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4621 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4681 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4741 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4801 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4861 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4921 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4981 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5041 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5101 agttcttgaa gtggtggcct aactacggct acactagaag aatagtattt ggtatctgcg
     5161 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5221 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5281 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5341 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5401 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5461 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5521 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5581 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5641 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5701 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5761 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5821 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5881 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5941 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6001 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6061 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6121 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6181 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6241 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6301 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6361 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6421 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     6481 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     6541 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     6601 gagtccacta ttaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6661 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6721 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     6781 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     6841 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     6901 gcgtcccatt cgccaatccg tcggcttgaa cgaattgtta gacattattt gccgactacc
     6961 ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc
     7021 aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac
     7081 tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg
     7141 gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc
     7201 cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt
     7261 tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct
     7321 cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct
     7381 gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc
     7441 cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc
     7501 tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca
     7561 tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac t
//
