LOCUS       sequence-416326-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO A191T 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 (A191T mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1284..1475
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1469..1491)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1577..1716
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1632..1651
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1731..1748)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1902..2490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1982..2001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2601..2620)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2612..3427
                     /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             2612..3427
                     /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(2685..2704)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3519..3974)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3656..3677)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3868..3887
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4011..4058)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4054..4072
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4125..4142)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5096..5116)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5147..5164)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5177..5199
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5214..5238
                     /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(5238..5257)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5239..5257)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5341..5360)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5566..5643
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5644..6251,1..475)
                     /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(5663..5682)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
       61 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
      121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
      181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
      241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
      301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
      361 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
      541 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
      601 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
      661 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
      721 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
      781 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
      841 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
      901 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
      961 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     1021 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     1081 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     1141 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     1201 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     1261 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     1321 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     1381 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     1441 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     1501 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     1561 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     1621 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     1681 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     1741 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1801 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1861 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1921 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1981 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2041 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2101 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2161 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2221 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2281 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2341 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2401 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2461 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2521 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc
     2581 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
     2641 ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     2701 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
     2761 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     2821 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     2881 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     2941 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     3001 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     3061 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     3121 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     3181 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     3241 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     3301 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     3361 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     3421 tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3481 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     3541 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3601 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3661 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3721 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3781 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3841 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3901 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3961 gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag caaaaaaccc
     4021 ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca
     4081 gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg gtggtggtgg
     4141 tgacccgacg cggttattgg aaagtaacac ctgagcattg tctaacaaca tcaaaaggtg
     4201 taaattcatc ttctaataaa gcactaccca atatggtacg tccattcata ccattttgca
     4261 gtaattcttt taatgaagca cacatatcta aaacggcaat tccagtttga gcagaaagag
     4321 gtcctagtat gtcaacatgg tcttgtgtta gaggttcata attgtacttc atagccacaa
     4381 ggttaaagtc attaagagtt gtggtaaatc gattgagaaa ccacctgtct ccatttataa
     4441 cagcagcgta caaccaagct aaaacattaa ctgtaatagt tgtgtccgta ccagctgctt
     4501 gtgttgtttg cctgtcaaca aaaggtccat aaaagttacc ttctaagtct gtgccagcat
     4561 gaactccagt tggtaattcc atatggtgca tgtaacaaaa agagacacag tcataatcta
     4621 tgttaaaacc aacactacca catgaaccat taaggaatga acccttaata gtgaaattgg
     4681 gcctcatagc acattggtaa acaccagatg gtgaaccatt gtaacaagct aacactgaaa
     4741 aagtctgtcc tggttgaatg cgaacaaact tatacttagg tgtcttagga ttggctgtat
     4801 caaccttaag cttaagtaca caattttgca tagaatgtcc aataaccctg agttgaacat
     4861 taccagcctg taccaagaaa ttatgattag acttacgaat gagtaaatct tcataattag
     4921 ggttaagcat gtcttcagag gtgcagatca catgtcttgg acagtaaact acgtcatcaa
     4981 gccaaagacc gttaagtgta gttgtaccac aagttacttg taccatacaa ccctcaactt
     5041 taccagatgg gaatgccatt tttctaaaac cactctgcag cacggctgac atggcgccct
     5101 gaaaataaag attctcagta gtggggatgt cgtaatcgct catggggtga tggtgatggt
     5161 gatgtttcat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     5221 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
     5281 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     5341 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     5401 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     5461 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     5521 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     5581 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     5641 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     5701 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     5761 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     5821 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     5881 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     5941 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     6001 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     6061 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     6121 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     6181 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     6241 ggctggcata a
//
