LOCUS       sequence-416377-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO R188S 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 R188S mutantSARS-CoV-2 Mpro pr...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1270..1461
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1455..1477)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1563..1702
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1618..1637
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1717..1734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1888..2476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1968..1987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2587..2606)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2598..3413
                     /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             2598..3413
                     /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(2671..2690)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3505..3960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3642..3663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3854..3873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3997..4044)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4040..4058
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4111..4128)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5082..5102)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5133..5150)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5163..5185
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5200..5224
                     /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)."
     primer_bind     complement(5224..5243)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5225..5243)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5327..5346)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5552..5629
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5630..6251,1..461)
                     /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     complement(5649..5668)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
       61 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
      121 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
      181 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
      241 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
      301 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
      361 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      421 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
      481 agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca
      541 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
      601 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
      661 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
      721 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
      781 attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
      841 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
      901 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
      961 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     1021 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     1081 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     1141 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     1201 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     1261 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     1321 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     1381 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     1441 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     1501 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     1561 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     1621 catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt
     1681 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     1741 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     1801 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     1861 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     1921 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1981 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2041 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2101 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2161 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2221 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2281 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     2341 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2401 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2461 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2521 cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca
     2581 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
     2641 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     2701 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     2761 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     2821 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     2881 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     2941 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     3001 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     3061 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     3121 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     3181 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     3241 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     3301 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     3361 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     3421 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     3481 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     3541 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3721 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3781 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3841 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3901 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3961 cccattcgcc aatccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt
     4021 agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc
     4081 ctttcgggct ttgttagcag ccggatctca gtggtggtgg tggtggtgac ccgacgcggt
     4141 tattggaaag taacacctga gcattgtcta acaacatcaa aaggtgtaaa ttcatcttct
     4201 aataaagcac tacccaatat ggtacgtcca ttcataccat tttgcagtaa ttcttttaat
     4261 gaagcacaca tatctaaaac ggcaattcca gtttgagcag aaagaggtcc tagtatgtca
     4321 acatggtctt gtgttagagg ttcataattg tacttcatag ccacaaggtt aaagtcatta
     4381 agagttgtgg taaatcgatt gagaaaccac ctgtctccat ttataacagc agcgtacaac
     4441 caagctaaaa cattaactgt aatagttgtg tccgtaccag ctgcttgtgc tgtttggctg
     4501 tcaacaaaag gtccataaaa gttaccttct aagtctgtgc cagcatgaac tccagttggt
     4561 aattccatat ggtgcatgta acaaaaagag acacagtcat aatctatgtt aaaaccaaca
     4621 ctaccacatg aaccattaag gaatgaaccc ttaatagtga aattgggcct catagcacat
     4681 tggtaaacac cagatggtga accattgtaa caagctaaca ctgaaaaagt ctgtcctggt
     4741 tgaatgcgaa caaacttata cttaggtgtc ttaggattgg ctgtatcaac cttaagctta
     4801 agtacacaat tttgcataga atgtccaata accctgagtt gaacattacc agcctgtacc
     4861 aagaaattat gattagactt acgaatgagt aaatcttcat aattagggtt aagcatgtct
     4921 tcagaggtgc agatcacatg tcttggacag taaactacgt catcaagcca aagaccgtta
     4981 agtgtagttg taccacaagt tacttgtacc atacaaccct caactttacc agatgggaat
     5041 gccatttttc taaaaccact ctgcagcacg gctgacatgg cgccctgaaa ataaagattc
     5101 tcagtagtgg ggatgtcgta atcgctcatg gggtgatggt gatggtgatg tttcatggta
     5161 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
     5221 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
     5281 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     5341 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     5401 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     5461 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     5521 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     5581 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     5641 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     5701 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     5761 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     5821 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     5881 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     5941 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     6001 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     6061 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     6121 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     6181 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     6241 tctcactcgc a
//
