LOCUS       sequence-416361-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO M165I 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 (M165I mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     907..926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(946..1023)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1229..1248
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1332..1351
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1332..1350
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1351..1375
                     /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             1390..1412
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1425..1442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1473..1493
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2447..2464
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2517..2535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2531..2578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2615..3070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2702..2721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2912..2933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3162..3977)
                     /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(3162..3977)
                     /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     3885..3904
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3969..3988
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4099..4687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4588..4607
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4841..4858
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4873..5012
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4938..4957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5098..5120
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5114..5305)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6114..6251,1..945))
                     /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 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
       61 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
      121 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
      181 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
      241 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
      301 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
      361 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
      421 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
      481 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
      541 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
      601 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
      661 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
      721 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
      781 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
      841 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
      901 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
      961 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     1021 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     1081 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     1141 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     1201 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     1261 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     1321 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1381 agaaataatt ttgtttaact ttaagaagga gatataccat gaaacatcac catcaccatc
     1441 accccatgag cgattacgac atccccacta ctgagaatct ttattttcag ggcgccatgt
     1501 cagccgtgct gcagagtggt tttagaaaaa tggcattccc atctggtaaa gttgagggtt
     1561 gtatggtaca agtaacttgt ggtacaacta cacttaacgg tctttggctt gatgacgtag
     1621 tttactgtcc aagacatgtg atctgcacct ctgaagacat gcttaaccct aattatgaag
     1681 atttactcat tcgtaagtct aatcataatt tcttggtaca ggctggtaat gttcaactca
     1741 gggttattgg acattctatg caaaattgtg tacttaagct taaggttgat acagccaatc
     1801 ctaagacacc taagtataag tttgttcgca ttcaaccagg acagactttt tcagtgttag
     1861 cttgttacaa tggttcacca tctggtgttt accaatgtgc tatgaggccc aatttcacta
     1921 ttaagggttc attccttaat ggttcatgtg gtagtgttgg ttttaacata gattatgact
     1981 gtgtctcttt ttgttacatg caccatattg aattaccaac tggagttcat gctggcacag
     2041 acttagaagg taacttttat ggaccttttg ttgacaggca aacagcacaa gcagctggta
     2101 cggacacaac tattacagtt aatgttttag cttggttgta cgctgctgtt ataaatggag
     2161 acaggtggtt tctcaatcga tttaccacaa ctcttaatga ctttaacctt gtggctatga
     2221 agtacaatta tgaacctcta acacaagacc atgttgacat actaggacct ctttctgctc
     2281 aaactggaat tgccgtttta gatatgtgtg cttcattaaa agaattactg caaaatggta
     2341 tgaatggacg taccatattg ggtagtgctt tattagaaga tgaatttaca ccttttgatg
     2401 ttgttagaca atgctcaggt gttactttcc aataaccgcg tcgggtcacc accaccacca
     2461 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2521 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2581 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2641 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2701 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2761 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2821 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2881 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2941 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3001 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3061 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3121 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     3181 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     3241 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     3301 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     3361 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     3421 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     3481 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     3541 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     3601 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     3661 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     3721 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3781 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3841 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3901 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     3961 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     4021 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4081 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4141 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4201 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4261 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4321 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4381 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4441 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4501 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4561 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4621 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4681 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4741 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4801 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4861 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4921 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4981 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     5041 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5101 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     5161 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     5221 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     5281 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     5341 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     5401 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     5461 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     5521 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5581 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5641 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5701 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5761 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5821 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5881 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5941 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     6001 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     6061 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     6121 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6181 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     6241 aacagctgat t
//
