LOCUS       sequence-416356-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO G143S 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 (G143S mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     916..935
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(955..1032)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1238..1257
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1341..1360
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1341..1359
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1360..1384
                     /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             1399..1421
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1434..1451
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1482..1502
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2456..2473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2526..2544)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2540..2587
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2624..3079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2711..2730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2921..2942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3171..3986)
                     /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(3171..3986)
                     /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     3894..3913
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3978..3997
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4108..4696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4597..4616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4850..4867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4882..5021
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4947..4966)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5107..5129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5123..5314)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6123..6251,1..954))
                     /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 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
       61 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
      121 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
      181 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
      241 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
      301 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
      361 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
      421 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
      481 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
      541 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
      601 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
      661 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
      721 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
      781 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
      841 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
      901 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
      961 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     1021 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     1081 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     1141 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     1201 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     1261 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     1321 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1381 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg aaacatcacc
     1441 atcaccatca ccccatgagc gattacgaca tccccactac tgagaatctt tattttcagg
     1501 gcgccatgtc agccgtgctg cagagtggtt ttagaaaaat ggcattccca tctggtaaag
     1561 ttgagggttg tatggtacaa gtaacttgtg gtacaactac acttaacggt ctttggcttg
     1621 atgacgtagt ttactgtcca agacatgtga tctgcacctc tgaagacatg cttaacccta
     1681 attatgaaga tttactcatt cgtaagtcta atcataattt cttggtacag gctggtaatg
     1741 ttcaactcag ggttattgga cattctatgc aaaattgtgt acttaagctt aaggttgata
     1801 cagccaatcc taagacacct aagtataagt ttgttcgcat tcaaccagga cagacttttt
     1861 cagtgttagc ttgttacaat ggttcaccat ctggtgttta ccaatgtgct atgaggccca
     1921 atttcactat taagggttca ttccttaata gttcatgtgg tagtgttggt tttaacatag
     1981 attatgactg tgtctctttt tgttacatgc accatatgga attaccaact ggagttcatg
     2041 ctggcacaga cttagaaggt aacttttatg gaccttttgt tgacaggcaa acagcacaag
     2101 cagctggtac ggacacaact attacagtta atgttttagc ttggttgtac gctgctgtta
     2161 taaatggaga caggtggttt ctcaatcgat ttaccacaac tcttaatgac tttaaccttg
     2221 tggctatgaa gtacaattat gaacctctaa cacaagacca tgttgacata ctaggacctc
     2281 tttctgctca aactggaatt gccgttttag atatgtgtgc ttcattaaaa gaattactgc
     2341 aaaatggtat gaatggacgt accatattgg gtagtgcttt attagaagat gaatttacac
     2401 cttttgatgt tgttagacaa tgctcaggtg ttactttcca ataaccgcgt cgggtcacca
     2461 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2521 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2581 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     2641 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2701 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2761 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2821 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2881 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2941 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3001 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3061 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     3181 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     3241 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     3301 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     3361 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     3421 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     3481 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     3541 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     3601 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     3661 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     3721 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     3781 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     3841 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     3901 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     3961 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     4021 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4081 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4141 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4201 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4261 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4321 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4381 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4441 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4501 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4561 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4621 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4681 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4741 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4801 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4861 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4921 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4981 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     5041 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5101 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5161 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     5221 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     5281 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     5341 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     5401 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     5461 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     5521 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     5581 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     5641 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     5701 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     5761 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     5821 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5881 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5941 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     6001 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     6061 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     6121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6181 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     6241 gagacgggca a
//
