LOCUS       sequence-333988-        5236 bp    DNA     circular SYN 14-APR-2025
DEFINITION  To express SARS-CoV-2 Papain-like protease in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5236)
  AUTHORS   Lim CT, Tan KW, Wu M, Ulferts R, Armstrong LA, Ozono E, Drury LS, 
            Milligan JC, Zeisner TU, Zeng J, Weissmann F, Canal B, Bineva-Todd 
            G, Howell M, O'Reilly N, Beale R, Kulathu Y, Labib K, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of Nsp3 papain-like protease.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2517-2531. doi: 10.1042/BCJ20210244.
  PUBMED    34198325
REFERENCE   2  (bases 1 to 5236)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: "2517-2531"
COMMENT     Sequence Label: pK27Sumo_His-SUMO-PLpro (nsp3) (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..5236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     680..698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(722..736,737..742,743..759,760..766)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    742..758
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    774..790
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             809..820
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             905..1195
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     terminator      2193..2287
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2335..2354)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2346..3161
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2346..3161
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2419..2438)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3380..3466
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3559..4641)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4603..4622
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4642..4719)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4803..4825)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4911..5051
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4966..4985
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5066..5083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc acctcgagaa
      721 atcataaaaa atttatttgc tttgtgagcg gataacaatt ataatagatt caattgtgag
      781 cggataacaa tttcacacag aattcattaa agaggagaaa ttaaccatga gcaagcatca
      841 ccatcattca ggccatcacc ataccggaca ccaccatcat tcaggcagtc atcaccatac
      901 cggatcggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc
      961 tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa
     1021 aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat
     1081 ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga
     1141 tttggacatg gaggataacg atattattga ggctcacaga gaacagattg gtggcgaagt
     1201 gcgtaccatt aaagttttta ccaccgtgga taacatcaac ctgcataccc aggttgttga
     1261 tatgagcatg acctatggtc agcagtttgg tccgacctat ctggatggtg cagatgttac
     1321 caaaatcaaa ccgcataata gccatgaagg caaaaccttt tatgtgctgc cgaatgatga
     1381 taccctgcgt gttgaagcat ttgagtatta tcataccacc gatccgagct ttctgggtcg
     1441 ttatatgagc gcactgaatc ataccaaaaa atggaaatat ccgcaggtta atggtctgac
     1501 cagcattaaa tgggcagata ataactgtta tctggcaacc gcactgctga cactgcagca
     1561 gattgaactg aaatttaacc ctccggcact gcaggatgcc tattatcgtg cacgtgccgg
     1621 tgaagcagca aatttttgtg cactgattct ggcctattgc aacaaaaccg ttggtgaact
     1681 gggtgatgtt cgtgaaacca tgagctacct gtttcagcat gcaaatctgg atagctgtaa
     1741 acgtgttctg aatgttgttt gtaaaacctg cggtcagcag cagaccacac tgaaaggtgt
     1801 ggaagcagtt atgtatatgg gcaccctgag ctatgaacag ttcaaaaaag gtgttcagat
     1861 tccgtgtacc tgtggtaaac aggcaaccaa atatctggtt cagcaagaaa gcccgtttgt
     1921 gatgatgagt gcaccgcctg cacagtatga gctgaaacat ggcaccttta cctgtgcaag
     1981 cgaatacacc ggtaattatc agtgtggcca ttataaacac atcaccagca aagaaaccct
     2041 gtattgtatt gatggtgccc tgctgacaaa aagcagcgaa tataaaggtc cgattaccga
     2101 cgtgttctat aaagaaaata gctacaccac caccattaaa taataataag cggccgcaca
     2161 tcaccatcac taagcttaat tagctgagct tggactcctg ttgatagatc cagtaatgac
     2221 ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg
     2281 tgagaatcca agctagccat gaaaataaac tgtctgctta cataaacagt aatacaaggg
     2341 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     2401 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     2461 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     2521 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     2581 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcttgg ttactcacga
     2641 ctgcgatccc cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2701 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     2761 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2821 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     2881 ggaaagaaat gcacaaactt ttgccattct caccggattc agtcgtcact catggtgatt
     2941 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     3001 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     3061 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     3121 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgggcaaa
     3181 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     3241 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     3301 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg
     3361 actctctagc ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     3421 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cctctagatt
     3481 acgtgcagtc gatgataagc tgtcaaacat gagaattgtg cctaatgagt gagctaactt
     3541 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3601 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattggggg ccagggtggt
     3661 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     3721 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     3781 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     3841 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     3901 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     3961 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4021 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4081 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4141 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4201 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4261 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4321 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4381 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4441 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4501 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4561 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4621 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     4681 tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gaatttcggg cagcgttggg
     4741 tcctggccac gggtgcgcat gatctagagc tgcctcgcgc gtttcggtga tgacggtgaa
     4801 aacctctgac acatgcagct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4861 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4921 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4981 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     5041 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5101 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5161 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5221 ccgcgttgct ggcgtt
//
