LOCUS       sequence-299971-        6635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible expression of SARS PLpro protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  AUTHORS   Lee H, Cao S, Hevener KE, Truong L, Gatuz JL, Patel K, Ghosh AK, 
            Johnson ME
  TITLE     Synergistic inhibitor binding to the papain-like protease of human 
            SARS coronavirus: mechanistic and inhibitor design implications.
  JOURNAL   ChemMedChem. 2013 Aug;8(8):1361-72. doi: 10.1002/cmdc.201300134. 
            Epub 2013 Jun 20.
  PUBMED    23788528
REFERENCE   2  (bases 1 to 6635)
  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: 
            "10.1002/cmdc.201300134"; journalName: "ChemMedChem"; date: 
            "2013-08"; volume: "8"; issue: "8"; pages: "1361-72"
COMMENT     Sequence Label: pET15b SARS-CoV PLpro
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1036..1059
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1060..1077
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1136..1154)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1150..1197
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1372..1377,1378..1394,1395..1400))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1427..1445)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1513..1617
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1618..1686,1687..2478)
                     /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(1836..1855)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2649..3237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3138..3157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3391..3408
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3423..3562
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3488..3507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3648..3670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3664..3855)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5167..6249)
                     /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     6211..6230
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6250..6327)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6533..6552
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gaagtaaaaa ccattaaagt gtttaccact
      121 gtggataaca cgaatctgca cacccagctg gtggacatga gcatgacgta tggtcagcag
      181 ttcggcccga cttacctgga cggtgcggac gtaaccaaaa ttaaaccgca cgtcaaccac
      241 gaaggcaaaa cctttttcgt tctgccgagc gacgataccc tgcgctctga agcgttcgaa
      301 tactaccaca ccctggacga atctttcctg ggtcgttaca tgtctgcgct gaaccacact
      361 aaaaaatgga aattcccgca ggttggcggt ctgaccagca ttaaatgggc agacaacaac
      421 tgctacctgt ccagcgttct gctggccctg caacagctgg aggttaagtt caacgcgccg
      481 gctctgcagg aagcgtatta ccgtgcacgc gcgggtgatg cggcaaactt ctgtgctctg
      541 atcctggctt attccaacaa gaccgtgggc gaactgggcg atgttcgtga aaccatgacc
      601 cacctgctgc aacatgctaa tctggaatcc gctaaacgcg tactgaacgt ggtctgtaaa
      661 cattgcggcc agaaaactac caccctgacc ggcgttgaag cagtgatgta tatgggtact
      721 ctgtcctatg acaacctgaa aacgggcgtt tctatcccgt gcgtttgtgg tcgtgatgca
      781 actcagtacc tggtacagca ggaatcctcc ttcgttatga tgtctgctcc acctgccgaa
      841 tataaactgc agcaaggcac ctttctgtgc gcaaacgagt atactggtaa ctaccagtgc
      901 ggtcactaca ctcacatcac ggccaaggag accctgtacc gtatcgatgg tgcccacctg
      961 accaaaatgt ctgaatacaa aggcccggta acggacgtct tctacaaaga gaccagctac
     1021 accactacta tcaagctgga agttctgttt cagggtccac accatcacca ccatcattaa
     1081 ctcgaggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1141 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1201 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     1261 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     1321 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     1381 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     1441 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1501 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1561 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1621 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1681 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1741 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1801 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1861 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1921 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1981 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2041 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2101 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2161 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2221 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2281 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2341 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2401 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2461 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2521 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2581 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2641 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2701 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2761 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2821 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2881 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2941 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3001 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3061 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3121 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3181 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3241 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3301 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3361 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3481 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3541 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3601 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3661 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3721 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3781 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3841 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3901 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3961 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4021 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4081 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4141 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4201 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4261 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4321 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     4381 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     4441 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     4501 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     4561 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     4621 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     4681 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     4741 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     4801 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     4861 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4921 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4981 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5041 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5101 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5161 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5221 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5281 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5341 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5401 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5461 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     5521 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5581 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     5641 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     5701 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     5761 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5821 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5881 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5941 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6001 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6061 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6121 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6181 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6241 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6301 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     6361 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6421 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6481 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6541 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6601 cggcgtagag gatcgagatc tcgatcccgc gaaat
//
