LOCUS       sequence-381344-        6604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 3CLpro with an active site mutation (C154A) for
            the expression of the mature protease in an inactive form.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6604)
  AUTHORS   Mazzei L, Greene-Cramer R, Bafna K, Jovanovic A, De Falco A, Acton 
            TB, Royer CA, Ciurli S, Montelione GT
  TITLE     Protocol for production and purification of SARS-CoV-2 3CL(pro).
  JOURNAL   STAR Protoc. 2023 May 5;4(2):102326. doi: 
            10.1016/j.xpro.2023.102326.
  PUBMED    37235475
REFERENCE   2  (bases 1 to 6604)
  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"; journalName: "STAR 
            Protoc."; date: "2023-05-5"; volume: "4(2):102326. doi"; pages: " 
            10.1016/j.xpro.2023.102326"
COMMENT     Sequence Label: pGTM_COV2_NSP5_C145A_001_SUMO
FEATURES             Location/Qualifiers
     source          1..6604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      307..895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     796..815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1049..1066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1081..1220
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1146..1165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1306..1328
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1322..1513)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2322..3404)
                     /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     3366..3385
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3405..3482)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3688..3707
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3791..3810
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3791..3809
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3810..3834
                     /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             3849..3871
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3884..3901
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3905..4195
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             5126..5143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5196..5214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5210..5257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5294..5749
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5381..5400)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5591..5612
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5775..5879
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5880..5948,5949..6604,1..136)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6098..6117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
       61 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
      121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
      181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
      241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
      301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
      361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
      421 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
      481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
      541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
      601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
      661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
      721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
      781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
      841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
      901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
      961 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1021 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     1081 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     1141 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     1201 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     1261 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     1321 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     1381 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     1441 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     1501 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     1561 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     1621 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     1681 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     1741 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     1801 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     1861 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     1921 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     1981 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     2041 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     2101 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     2161 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     2221 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     2281 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     2341 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2401 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     2461 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     2521 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     2581 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     2641 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     2701 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     2761 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     2821 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     2881 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     2941 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     3001 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     3061 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     3121 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     3181 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     3241 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     3301 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     3361 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     3421 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     3481 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     3541 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     3601 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     3661 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     3721 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     3781 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     3841 gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc atcatcatca
     3901 cgggtcggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc
     3961 tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa
     4021 aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat
     4081 ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcagg cccctgaaga
     4141 tttggacatg gaggataacg atattattga ggctcaccgc gaacagattg gaggtagcgg
     4201 tttccgtaag atggcgtttc cgagcggcaa agttgagggt tgcatggtgc aggttacctg
     4261 cggcaccacc accctgaacg gtctgtggct ggacgatgtg gtttactgcc cgcgtcacgt
     4321 tatctgcacc agcgaggaca tgctgaaccc gaactatgaa gatctgctga ttcgtaagag
     4381 caaccacaac ttcctggtgc aggcgggcaa cgtgcaactg cgtgttatcg gtcacagcat
     4441 gcagaactgc gtgctgaagc tgaaagttga caccgcgaac ccgaaaaccc cgaagtacaa
     4501 attcgtgcgt attcagccgg gccaaacctt tagcgttctg gcgtgctaca acggcagccc
     4561 gagcggtgtt tatcaatgcg cgatgcgtcc gaacttcacc atcaagggta gctttctgaa
     4621 cggtagcgcg ggcagcgtgg gtttcaacat tgactacgat tgcgttagct tttgctatat
     4681 gcaccatatg gagctgccga ccggcgttca tgcgggtacc gatctggaag gcaacttcta
     4741 cggtccgttt gttgaccgtc aaaccgcgca agcggcgggt accgatacca ccatcaccgt
     4801 gaacgttctg gcgtggctgt atgcggcggt gattaacggt gaccgttggt tcctgaaccg
     4861 ttttaccacc accctgaacg atttcaacct ggtggcgatg aagtacaact atgagccgct
     4921 gacccaggac cacgttgata tcctgggccc gctgagcgcg caaaccggta ttgcggtgct
     4981 ggacatgtgc gcgagcctga aagaactgct gcaaaacggc atgaacggtc gtaccattct
     5041 gggtagcgcg ctgctggagg acgaattcac cccgtttgat gtggttcgtc agtgcagcgg
     5101 tgtgaccttt caatagtaac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     5161 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     5221 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     5281 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5341 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5401 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5461 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5521 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5581 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5641 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5701 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5761 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5881 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5941 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     6001 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6061 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     6121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     6241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6421 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc
     6601 gcgg
//
