LOCUS       sequence-416321-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO R188K 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 (R188K mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1457..1474
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1505..1525
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2479..2496
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2549..2567)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2563..2610
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2647..3102
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2734..2753)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2944..2965
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3194..4009)
                     /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(3194..4009)
                     /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     3917..3936
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4001..4020
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4131..4719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4620..4639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4873..4890
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4905..5044
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4970..4989)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5130..5152
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5146..5337)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6146..6251,1..977))
                     /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 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatatacc atgaaacatc accatcacca tcaccccatg agcgattacg acatccccac
     1501 tactgagaat ctttattttc agggcgccat gtcagccgtg ctgcagagtg gttttagaaa
     1561 aatggcattc ccatctggta aagttgaggg ttgtatggta caagtaactt gtggtacaac
     1621 tacacttaac ggtctttggc ttgatgacgt agtttactgt ccaagacatg tgatctgcac
     1681 ctctgaagac atgcttaacc ctaattatga agatttactc attcgtaagt ctaatcataa
     1741 tttcttggta caggctggta atgttcaact cagggttatt ggacattcta tgcaaaattg
     1801 tgtacttaag cttaaggttg atacagccaa tcctaagaca cctaagtata agtttgttcg
     1861 cattcaacca ggacagactt tttcagtgtt agcttgttac aatggttcac catctggtgt
     1921 ttaccaatgt gctatgaggc ccaatttcac tattaagggt tcattcctta atggttcatg
     1981 tggtagtgtt ggttttaaca tagattatga ctgtgtctct ttttgttaca tgcaccatat
     2041 ggaattacca actggagttc atgctggcac agacttagaa ggtaactttt atggaccttt
     2101 tgttgacaag caaacagcac aagcagctgg tacggacaca actattacag ttaatgtttt
     2161 agcttggttg tacgctgctg ttataaatgg agacaggtgg tttctcaatc gatttaccac
     2221 aactcttaat gactttaacc ttgtggctat gaagtacaat tatgaacctc taacacaaga
     2281 ccatgttgac atactaggac ctctttctgc tcaaactgga attgccgttt tagatatgtg
     2341 tgcttcatta aaagaattac tgcaaaatgg tatgaatgga cgtaccatat tgggtagtgc
     2401 tttattagaa gatgaattta caccttttga tgttgttaga caatgctcag gtgttacttt
     2461 ccaataaccg cgtcgggtca ccaccaccac caccactgag atccggctgc taacaaagcc
     2521 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2581 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2641 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2701 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2761 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2821 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2881 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2941 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3001 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3061 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3121 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3181 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3241 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3301 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3361 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3421 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3481 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3541 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3601 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3661 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3721 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3781 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3841 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3901 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3961 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4021 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4081 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4141 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4201 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4261 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4321 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4381 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4441 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4501 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4561 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4621 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4681 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4741 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4801 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4861 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4921 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4981 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5041 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5101 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5161 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5221 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5281 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5341 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5401 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5461 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5521 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5581 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5641 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5701 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5761 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     5821 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5881 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5941 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6001 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     6061 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6121 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6181 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6241 gggtggtttt t
//
