LOCUS       sequence-333223-        5340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculoviral transfer vector to express SARS-CoV-2 nsp9 with 
            nsp5-cleavable 3xFlag-His6 tag in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5340)
  AUTHORS   Milligan JC, Zeisner TU, Papageorgiou G, Joshi D, Soudy C, Ulferts 
            R, Wu M, Lim CT, Tan KW, Weissmann F, Canal B, Fujisawa R, Deegan T,
            Nagaraj H, Bineva-Todd G, Basier C, Curran JF, Howell M, Beale R, 
            Labib K, O'Reilly N, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of Nsp5 main protease.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2499-2515. doi: 10.1042/BCJ20210197.
  PUBMED    34198327
REFERENCE   2  (bases 1 to 5340)
  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: "2499-2515"
COMMENT     Sequence Label: pLIB_3xFlag-His-nsp5CS-nsp9 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..5340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(446..451,452..468,469..474))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(870..903)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        951..1042
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     967..984
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      1086..1095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1095..1160
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1167..1184
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1671..1805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1708..1727)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1762..1781
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  1915..2080
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2264..2718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2351..2370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2560..2581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2744..2848
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2849..2917,2918..3709)
                     /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(3067..3086)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3880..4468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4369..4388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4622..4639
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  4773..4997
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(join(5064..5340,1..257))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 gcttcttggt cgaaggcagc aagcgcgatg aatgtcttac tacggagcaa gttcccgagg
       61 taatcggagt ccggctgatg ttgggagtag gtggctacgt ctccgaactc acgaccgaaa
      121 agatcaagag cagcccgcat ggatttgact tggtcagggc cgagcctaca tgtgcgaatg
      181 atgcccatac ttgagccacc taactttgtt ttagggcgac tgccctgctg cgtaacatcg
      241 ttgctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca cggcgtaacg
      301 cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa aaacagtcat aacaagccat
      361 gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg
      421 tgagcgcata cgctacttgc attacagttt acgaaccgaa caggcttatg tcaactgggt
      481 ttgtgccttc atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt
      541 cgaggcattt ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca
      601 ggcattggcg gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgcccttgct
      661 tcaggagatc ggtagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga
      721 agtggttcgc atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc aggactctag
      781 ctatagttct agtggttggc tacagctttg tttgtactat caacaggttg aactgctgat
      841 caacagatcc tctacgcggc cgcggtacca taacttcgta tagcatacat tatacgaagt
      901 tatctggttt tactagtatc gattcgcgac ctactccgga atattaatag atcatggaga
      961 taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt
     1021 tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg
     1081 atccagccac catggactac aaggaccacg acggcgatta caaggatcat gacatcgact
     1141 acaaagacga cgatgacaag ggttcccacc atcaccacca tcactctgct gtgctgcaga
     1201 acaacgagct gtcccctgtg gctctgcgtc agatgtcttg cgctgctggt actacccaga
     1261 ccgcttgcac tgacgacaac gctctggctt actacaacac caccaaaggt ggtcgtttcg
     1321 tgctggctct gctgagcgac ctgcaagact tgaagtgggc tcgtttcccc aagtccgacg
     1381 gcaccggtac tatctacacc gagctggaac ctccatgccg tttcgtgacc gacactccca
     1441 agggtcccaa agtgaagtac ctctacttca tcaagggcct gaacaacctg aaccgtggca
     1501 tggtgctggg ttccctggct gctactgtgc gtctgcagta aaagcttgtc gagaagtact
     1561 agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     1621 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     1681 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1741 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     1801 ggatctgatc actgcttgag cctagaagat ccggctgcta acaaagcccg aaaggaagct
     1861 gagttggctg ctgccaccgc tgagcaataa ctatcataac ccctaggaga tccgaaccag
     1921 ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt agcttacgac
     1981 gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta aaaactccat
     2041 ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt tttcttcctg
     2101 ttatgttttt aatcaaacat cctgccaact ccatgtgaca aaccgtcatc ttcggctact
     2161 ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat gtttgtaatt
     2221 gactgaatat caacgcttat ttgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     2281 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2341 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2401 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2461 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2521 gtttttcgcc ctttgacgtt ggagtccacg ttcttaatag tggactcttg ttccaaactg
     2581 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2641 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2701 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2761 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2821 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2881 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2941 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3001 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3061 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3121 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3181 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3241 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3301 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3361 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3421 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3481 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3541 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3601 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3661 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3721 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3781 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3841 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3901 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3961 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4021 tactattctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4081 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4141 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4201 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4261 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4321 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4381 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4441 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4501 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4561 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4621 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4681 tctgtgcggt atttcacacc gcatagacca gccgcgtaac ctggcaaaat cggttacggt
     4741 tgagtaataa atggatgccc tgcgtaagcg ggtgtgggcg gacaataaag tcttaaactg
     4801 aacaaaatag atctaaacta tgacaataaa gtcttaaact agacagaata gttgtaaact
     4861 gaaatcagtc cagttatgct gtgaaaaagc atactggact tttgttatgg ctaaagcaaa
     4921 ctcttcattt tctgaagtgc aaattgcccg tcgtattaaa gaggggcgtg gccaagggca
     4981 tggtaaagac tatattcgcg gcgttgtgac aatttaccga acaactccgc ggccgggaag
     5041 ccgatctcgg cttgaacgaa ttgttaggtg gcggtacttg ggtcgatatc aaagtgcatc
     5101 acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc accgtaatct
     5161 gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag gagattgatg
     5221 agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc atagatatag
     5281 atctcactac gcggctgctc aaacttgggc agaacgtaag ccgcgagagc gccaacaacc
//
