LOCUS       sequence-333229-        6631 bp    DNA     circular SYN 15-DEC-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 6631)
  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 6631)
  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: "2517-2531"
COMMENT     Sequence Label: pET11a_His-TEV-PLpro (nsp3) (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..6631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             34..51
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             52..72
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1074..1092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1088..1135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1310..1315,1316..1332,1333..1338))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1365..1383)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1451..1555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1556..1624,1625..2416)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1774..1793)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2587..3175
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3076..3095
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3329..3346
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3361..3500
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3426..3445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3586..3608
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3602..3793)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5105..6187)
                     /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     6149..6168
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6188..6265)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6471..6490
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6574..6593
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6574..6592
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6593..6617
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacat atgcatcacc atcaccatca cgaaaacttg
       61 tacttccaag gtgaagaagt gcgtaccatt aaagttttta ccaccgtgga taacatcaac
      121 ctgcataccc aggttgttga tatgagcatg acctatggtc agcagtttgg tccgacctat
      181 ctggatggtg cagatgttac caaaatcaaa ccgcataata gccatgaagg caaaaccttt
      241 tatgtgctgc cgaatgatga taccctgcgt gttgaagcat ttgagtatta tcataccacc
      301 gatccgagct ttctgggtcg ttatatgagc gcactgaatc ataccaaaaa atggaaatat
      361 ccgcaggtta atggtctgac cagcattaaa tgggcagata ataactgtta tctggcaacc
      421 gcactgctga cactgcagca gattgaactg aaatttaacc ctccggcact gcaggatgcc
      481 tattatcgtg cacgtgccgg tgaagcagca aatttttgtg cactgattct ggcctattgc
      541 aacaaaaccg ttggtgaact gggtgatgtt cgtgaaacca tgagctacct gtttcagcat
      601 gcaaatctgg atagctgtaa acgtgttctg aatgttgttt gtaaaacctg cggtcagcag
      661 cagaccacac tgaaaggtgt ggaagcagtt atgtatatgg gcaccctgag ctatgaacag
      721 ttcaaaaaag gtgttcagat tccgtgtacc tgtggtaaac aggcaaccaa atatctggtt
      781 cagcaagaaa gcccgtttgt gatgatgagt gcaccgcctg cacagtatga gctgaaacat
      841 ggcaccttta cctgtgcaag cgaatacacc ggtaattatc agtgtggcca ttataaacac
      901 atcaccagca aagaaaccct gtattgtatt gatggtgccc tgctgacaaa aagcagcgaa
      961 tataaaggtc cgattaccga cgtgttctat aaagaaaata gctacaccac caccattaaa
     1021 taaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1081 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1141 aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct
     1201 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc
     1261 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta
     1321 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
     1381 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1441 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1501 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1561 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1621 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1681 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1741 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     1801 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1861 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1921 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1981 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2041 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2101 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2161 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2221 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2281 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2341 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2401 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2461 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2521 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2581 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2641 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2701 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2761 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2821 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2881 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2941 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3001 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3061 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3121 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3181 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3241 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3301 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3361 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3421 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3481 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3541 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3601 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3661 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     3721 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3781 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     3841 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     3901 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     3961 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4021 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4081 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4141 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4201 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4261 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     4321 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     4381 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     4441 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     4501 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     4561 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     4621 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     4681 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     4741 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     4801 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4861 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4921 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4981 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5041 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5101 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5161 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5221 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5281 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5341 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5401 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5461 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5521 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5581 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5641 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5701 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5761 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5821 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5881 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5941 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6001 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6061 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6121 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6181 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6241 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6301 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6361 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6421 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6481 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6541 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     6601 gagcggataa caattcccct ctagaaataa t
//
