LOCUS       sequence-416944-        6248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO M49T mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6248)
  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 (M49T mutant - clinic...
FEATURES             Location/Qualifiers
     source          1..6248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     309..328
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        309..327
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    328..352
                     /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             367..389
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             402..419
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             450..470
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1491..1509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1505..1552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1589..2044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1676..1695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1886..1907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2136..2951)
                     /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(2136..2951)
                     /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     2859..2878
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2943..2962
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3073..3661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3562..3581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3815..3832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3847..3986
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3912..3931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4072..4094
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4088..4279)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5088..6170)
                     /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     6132..6151
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6171..6248)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctacattag gaagcagccc agtagtaggt
       61 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      121 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      181 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      241 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      301 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      361 aataattttg tttaacttta agaaggagat ataccatgaa acatcaccat caccatcacc
      421 ccatgagcga ttacgacatc cccactactg agaatcttta ttttcagggc gccatgtcag
      481 ccgtgctgca gagtggtttt agaaaaatgg cattcccatc tggtaaagtt gagggttgta
      541 tggtacaagt aacttgtggt acaactacac ttaacggtct ttggcttgat gacgtagttt
      601 actgtccaag acatgtgatc tgcacctctg aagacacgct taaccctaat tatgaagatt
      661 tactcattcg taagtctaat cataatttct tggtacaggc tggtaatgtt caactcaggg
      721 ttattggaca ttctatgcaa aattgtgtac ttaagcttaa ggttgataca gccaatccta
      781 agacacctaa gtataagttt gttcgcattc aaccaggaca gactttttca gtgttagctt
      841 gttacaatgg ttcaccatct ggtgtttacc aatgtgctat gaggcccaat ttcactatta
      901 agggttcatt ccttaatggt tcatgtggta gtgttggttt taacatagat tatgactgtg
      961 tctctttttg ttacatgcac catatggaat taccaactgg agttcatgct ggcacagact
     1021 tagaaggtaa cttttatgga ccttttgttg acaggcaaac agcacaagca gctggtacgg
     1081 acacaactat tacagttaat gttttagctt ggttgtacgc tgctgttata aatggagaca
     1141 ggtggtttct caatcgattt accacaactc ttaatgactt taaccttgtg gctatgaagt
     1201 acaattatga acctctaaca caagaccatg ttgacatact aggacctctt tctgctcaaa
     1261 ctggaattgc cgttttagat atgtgtgctt cattaaaaga attactgcaa aatggtatga
     1321 atggacgtac catattgggt agtgctttat tagaagatga atttacacct tttgatgttg
     1381 ttagacaatg ctcaggtgtt actttccaat aaccgcgtcg ggtcaccacc accaccactg
     1441 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1501 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1561 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1621 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1681 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1741 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1801 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1861 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1921 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1981 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2041 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2101 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2161 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2221 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2281 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2341 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2401 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2461 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2521 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2581 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2641 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2701 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2761 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     2821 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     2881 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     2941 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3001 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3061 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3121 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3181 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3241 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3301 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3361 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3421 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3481 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3541 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3601 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3661 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3721 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3781 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3841 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     3901 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     3961 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4021 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4081 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4141 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4201 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4261 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4321 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4381 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4441 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4501 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4561 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4621 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4681 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4741 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4801 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4861 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     4921 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     4981 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5041 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5101 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5161 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5221 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5281 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5341 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5401 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5461 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5521 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5581 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5641 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5701 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5761 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5821 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     5881 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     5941 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6001 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6061 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6121 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6181 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6241 atggtgtc
//
