LOCUS       sequence-416330-        6251 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of Sars Cov 2 MPRO A191V 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 Apr 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 (A191V mutant - clini...
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1483..1500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1531..1551
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2505..2522
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2575..2593)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2589..2636
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2673..3128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2760..2779)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2970..2991
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3220..4035)
                     /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(3220..4035)
                     /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     3943..3962
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4027..4046
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4157..4745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4646..4665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4899..4916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4931..5070
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4996..5015)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5156..5178
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5172..5363)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6172..6251,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tataccatga aacatcacca tcaccatcac
     1501 cccatgagcg attacgacat ccccactact gagaatcttt attttcaggg cgccatgtca
     1561 gccgtgctgc agagtggttt tagaaaaatg gcattcccat ctggtaaagt tgagggttgt
     1621 atggtacaag taacttgtgg tacaactaca cttaacggtc tttggcttga tgacgtagtt
     1681 tactgtccaa gacatgtgat ctgcacctct gaagacatgc ttaaccctaa ttatgaagat
     1741 ttactcattc gtaagtctaa tcataatttc ttggtacagg ctggtaatgt tcaactcagg
     1801 gttattggac attctatgca aaattgtgta cttaagctta aggttgatac agccaatcct
     1861 aagacaccta agtataagtt tgttcgcatt caaccaggac agactttttc agtgttagct
     1921 tgttacaatg gttcaccatc tggtgtttac caatgtgcta tgaggcccaa tttcactatt
     1981 aagggttcat tccttaatgg ttcatgtggt agtgttggtt ttaacataga ttatgactgt
     2041 gtctcttttt gttacatgca ccatatggaa ttaccaactg gagttcatgc tggcacagac
     2101 ttagaaggta acttttatgg accttttgtt gacaggcaaa cagtacaagc agctggtacg
     2161 gacacaacta ttacagttaa tgttttagct tggttgtacg ctgctgttat aaatggagac
     2221 aggtggtttc tcaatcgatt taccacaact cttaatgact ttaaccttgt ggctatgaag
     2281 tacaattatg aacctctaac acaagaccat gttgacatac taggacctct ttctgctcaa
     2341 actggaattg ccgttttaga tatgtgtgct tcattaaaag aattactgca aaatggtatg
     2401 aatggacgta ccatattggg tagtgcttta ttagaagatg aatttacacc ttttgatgtt
     2461 gttagacaat gctcaggtgt tactttccaa taaccgcgtc gggtcaccac caccaccacc
     2521 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2581 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2641 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2701 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2761 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2821 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2881 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2941 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3001 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3061 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3121 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3181 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     3241 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     3301 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     3361 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     3421 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     3481 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     3541 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     3601 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     3661 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3721 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3781 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3841 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3901 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3961 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     4021 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     4081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4921 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4981 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     5041 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5101 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5161 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     5221 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     5281 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     5341 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5401 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5461 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5521 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5581 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5641 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5701 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5761 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5821 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5881 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5941 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     6001 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     6061 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6121 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6181 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6241 agaggcggtt t
//
