LOCUS       sequence-417052-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO Q189K mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6251)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6251)
  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"
COMMENT     Sequence Label: SARS-CoV-2 Mpro protease (Q189K mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             1415..1437
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1450..1467
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1498..1518
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2472..2489
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2542..2560)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2556..2603
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2640..3095
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2727..2746)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2937..2958
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3187..4002)
                     /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             complement(3187..4002)
                     /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     3910..3929
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3994..4013
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4124..4712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4613..4632
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4866..4883
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4898..5037
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4963..4982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5123..5145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5139..5330)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6139..6251,1..970))
                     /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"
ORIGIN
        1 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     1441 accatgaaac atcaccatca ccatcacccc atgagcgatt acgacatccc cactactgag
     1501 aatctttatt ttcagggcgc catgtcagcc gtgctgcaga gtggttttag aaaaatggca
     1561 ttcccatctg gtaaagttga gggttgtatg gtacaagtaa cttgtggtac aactacactt
     1621 aacggtcttt ggcttgatga cgtagtttac tgtccaagac atgtgatctg cacctctgaa
     1681 gacatgctta accctaatta tgaagattta ctcattcgta agtctaatca taatttcttg
     1741 gtacaggctg gtaatgttca actcagggtt attggacatt ctatgcaaaa ttgtgtactt
     1801 aagcttaagg ttgatacagc caatcctaag acacctaagt ataagtttgt tcgcattcaa
     1861 ccaggacaga ctttttcagt gttagcttgt tacaatggtt caccatctgg tgtttaccaa
     1921 tgtgctatga ggcccaattt cactattaag ggttcattcc ttaatggttc atgtggtagt
     1981 gttggtttta acatagatta tgactgtgtc tctttttgtt acatgcacca tatggaatta
     2041 ccaactggag ttcatgctgg cacagactta gaaggtaact tttatggacc ttttgttgac
     2101 aggaaaacag cacaagcagc tggtacggac acaactatta cagttaatgt tttagcttgg
     2161 ttgtacgctg ctgttataaa tggagacagg tggtttctca atcgatttac cacaactctt
     2221 aatgacttta accttgtggc tatgaagtac aattatgaac ctctaacaca agaccatgtt
     2281 gacatactag gacctctttc tgctcaaact ggaattgccg ttttagatat gtgtgcttca
     2341 ttaaaagaat tactgcaaaa tggtatgaat ggacgtacca tattgggtag tgctttatta
     2401 gaagatgaat ttacaccttt tgatgttgtt agacaatgct caggtgttac tttccaataa
     2461 ccgcgtcggg tcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     2521 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2581 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2641 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2701 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2761 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2821 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2881 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2941 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3001 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3061 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3121 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     3181 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3241 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3301 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3361 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3421 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3481 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3541 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     3601 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     3661 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     3721 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     3781 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     3841 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     3901 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3961 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     4021 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     4081 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4141 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4201 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4261 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4321 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4381 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4441 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4501 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4561 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4621 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4681 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4741 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4801 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4861 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4921 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4981 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     5041 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5101 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5161 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     5221 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5281 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5341 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5401 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5461 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5521 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5581 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5641 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5701 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5761 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     5821 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5881 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5941 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     6001 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     6061 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     6121 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6181 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6241 ttttcttttc a
//
