LOCUS       sequence-370049-        5977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SARS-CoV-2 Mpro C145A inactive mutant fused 
            to SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5977)
  AUTHORS   MacDonald EA, Frey G, Namchuk MN, Harrison SC, Hinshaw SM, Windsor 
            IW
  TITLE     Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main 
            Protease.
  JOURNAL   ACS Infect Dis. 2021 Sep 10;7(9):2591-2595. doi: 
            10.1021/acsinfecdis.1c00237. Epub 2021 Aug 26.
  PUBMED    34437808
REFERENCE   2  (bases 1 to 5977)
  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"; doi: 
            "10.1021/acsinfecdis.1c00237"; journalName: "ACS Infect Dis"; date: 
            "2021-09-10- 10"; volume: "7"; issue: "9"; pages: "2591-2595"
COMMENT     Sequence Label: pET-His6-SUMO-MproC145A
FEATURES             Location/Qualifiers
     source          1..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             186..479
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(1507..1525)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1521..1568
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1934..1939,1940..1956,1957..1962))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1989..2007)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2075..2179
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2180..2248,2249..3040)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2398..2417)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3211..3799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3700..3719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3953..3970
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3985..4124
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4050..4069)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4210..4232
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4226..4417)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5875..5894
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
       61 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatggt tcttctatgg
      181 ctagcatgtc ggactcagaa gtcaatcaag aagctaagcc agaggtcaag ccagaagtca
      241 agcctgagac tcacatcaat ttaaaggtgt ccgatggatc ttcagagatc ttcttcaaga
      301 tcaaaaagac cactccttta agaaggctga tggaagcgtt cgctaaaaga cagggtaagg
      361 aaatggactc cttaagattc ttgtacgacg gtattagaat tcaagctgat cagacccctg
      421 aagatttgga catggaggat aacgatatta ttgaggctca cagagaacag attggtgggt
      481 cggggtttcg caaaatggcg tttccgtccg gcaaagtgga aggttgtatg gtacaggtga
      541 catgcggcac cacaaccctg aatgggttgt ggttagacga cgtagtctat tgccctcgtc
      601 atgtcatctg cacctctgag gacatgctca atccgaatta cgaagatctc ctgatccgca
      661 aatccaacca caacttcctc gttcaagctg gcaatgttca actgcgcgtt attggtcata
      721 gcatgcagaa ttgcgtcctt aaactgaaag ttgataccgc caacccgaaa accccgaagt
      781 acaagtttgt gcgtattcag cctggtcaga ccttttcagt gttagcgtgc tataacggca
      841 gtccctctgg tgtgtatcag tgtgctatgc gtccaaactt cacgatcaaa ggcagcttcc
      901 ttaatggcag cgcgggttcg gtgggcttta acatcgacta cgattgcgtt agcttctgct
      961 atatgcacca catggaattg ccgactggtg tacatgccgg gacggactta gaaggcaact
     1021 tttatggtcc gtttgtcgat cgccaaacag cccaagccgc aggaaccgat acgaccatta
     1081 ctgtgaatgt tctggcatgg ctttatgcgg cggtaatcaa cggagatcgg tggtttctga
     1141 accgctttac caccaccttg aacgatttca acctggtcgc gatgaagtac aactacgagc
     1201 cactgacgca ggatcatgtt gacattctgg gaccgttgtc agcacagacg ggcattgctg
     1261 tgctggatat gtgtgccagt ctgaaagaac tgctgcagaa tgggatgaat ggccgcacta
     1321 ttctgggtag tgcgttactg gaagatgagt tcactccctt cgatgtggtg cgtcagtgta
     1381 gcggtgtcac gtttcagtaa tagcggatcc gaattcgagc gccacctggt ggccggccgg
     1441 taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1501 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1561 gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg
     1621 atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag
     1681 cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct
     1741 agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc
     1801 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg
     1861 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     1921 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag
     1981 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2041 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2101 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2161 acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2221 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2281 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2341 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2401 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     2461 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2521 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2581 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2641 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     2701 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2761 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2821 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2881 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2941 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3001 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3061 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3121 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3181 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3241 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3301 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3361 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3421 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3481 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3541 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3601 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3661 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3721 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3781 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3841 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3901 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3961 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4021 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4081 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4141 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4201 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4261 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4321 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4381 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4441 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4501 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4561 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4621 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4681 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4741 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4801 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4861 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     4921 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     4981 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     5041 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     5101 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     5161 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     5221 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     5281 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     5341 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     5401 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5461 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5521 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5581 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5641 ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc
     5701 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5761 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5821 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5881 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5941 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
