LOCUS       sequence-348602-        6480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of active SARS-CoV-2 3C-like proteinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6480)
  AUTHORS   Pablos I, Machado Y, de Jesus HCR, Mohamud Y, Kappelhoff R, Lindskog
            C, Vlok M, Bell PA, Butler GS, Grin PM, Cao QT, Nguyen JP, Solis N, 
            Abbina S, Rut W, Vederas JC, Szekely L, Szakos A, Drag M, 
            Kizhakkedathu JN, Mossman K, Hirota JA, Jan E, Luo H, Banerjee A, 
            Overall CM
  TITLE     Mechanistic insights into COVID-19 by global analysis of the 
            SARS-CoV-2 3CL(pro) substrate degradome.
  JOURNAL   Cell Rep. 2021 Oct 26;37(4):109892. doi: 
            10.1016/j.celrep.2021.109892. Epub 2021 Oct 9.
  PUBMED    34672947
REFERENCE   2  (bases 1 to 6480)
  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: "Cell Rep.";
            date: "2021-10-26"; volume: "37(4):109892. doi"; pages: " 
            10.1016/j.celrep.2021.109892. Epub 2021 Oct 9"
COMMENT     Sequence Label: pET21b(+)_SARS-CoV-2_3CLpro-Q306A
FEATURES             Location/Qualifiers
     source          1..6480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1286..1477
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1471..1493)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1579..1718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1634..1653
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1733..1750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1904..2492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1984..2003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2663..3454,3455..3523))
                     /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     3286..3305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3524..3628)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3654..4109)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3791..3812)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4003..4022
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4146..4193)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4189..4207
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4260..4277)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4327..4344)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4351..4380)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(4387..4452)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4459..4470)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             5408..5430
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5445..5469
                     /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(5469..5488)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5470..5488)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5572..5591)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5797..5874
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5875..6480,1..477)
                     /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(5894..5913)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
       61 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
      121 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
      181 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
      241 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
      301 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
      361 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      421 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      481 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
      541 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
      601 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
      661 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
      721 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
      781 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
      841 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
      901 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
      961 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     1021 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     1081 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     1141 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     1201 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     1261 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     1321 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     1381 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     1441 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     1501 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     1561 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     1621 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     1681 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     1741 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1801 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1861 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1921 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1981 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2041 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2101 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2161 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2221 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2281 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2341 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2401 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2461 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2521 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2581 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2641 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2701 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2761 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2821 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2881 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2941 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     3001 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3061 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3121 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3181 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3241 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3301 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3361 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3421 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3481 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3541 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3601 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     3661 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3721 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3781 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3841 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3901 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3961 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4021 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4081 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct
     4141 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
     4201 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatctcag
     4261 tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt cgacggagct cgaattcgga
     4321 tcctcaatga tgatgatgat gatggccgcc cagatcctct tctgagatga gtttttgttc
     4381 gccgcccttg tcatcgtcat ccttgtaatc gatgtcatga tctttataat caccgtcatg
     4441 gtctttgtag tcgccgccac gtccctcgat gccgccgcct gcgaaagtaa cacctgagca
     4501 ttgtctaaca acatcaaaag gtgtaaattc atcttctaat aaagcactac ccaatatggt
     4561 acgtccattc ataccatttt gcagtaattc ttttaatgaa gcacacatat ctaaaacggc
     4621 aattccagtt tgagcagaaa gaggtcctag tatgtcaaca tggtcttgtg ttagaggttc
     4681 ataattgtac ttcatagcca caaggttaaa gtcattaaga gttgtggtaa atcgattgag
     4741 aaaccacctg tctccattta taacagcagc gtacaaccaa gctaaaacat taactgtaat
     4801 agttgtgtcc gtaccagctg cttgtgctgt ttgcctgtca acaaaaggtc cataaaagtt
     4861 accttctaag tctgtgccag catgaactcc agttggtaat tccatgtggt gcatgtaaca
     4921 aaaagagaca cagtcataat ctatgttaaa accaacacta ccacatgaac cattaaggaa
     4981 tgaaccctta atagtgaaat tgggcctcat agcacattgg taaacaccag atggtgaacc
     5041 attgtaacaa gctaacactg aaaaagtctg tcctggttga atgcgaacaa acttatactt
     5101 aggtgtctta ggattggctg tatcaacctt aagcttaagt acacaatttt gcatagaatg
     5161 tccaataacc ctgagttgaa cattaccagc ctgtaccaag aaattatgat tagacttacg
     5221 aatgagtaaa tcttcataat tagggttaag catgtcttca gaggtgcaga tcacatgtct
     5281 tggacagtaa actacgtcat caagccaaag accgttaagt gtagttgtac cacaagttac
     5341 ttgtaccata caaccctcaa ctttaccaga tgggaatgcc atttttctaa aaccactcat
     5401 atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     5461 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     5521 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     5581 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     5641 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     5701 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     5761 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     5821 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     5881 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     5941 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     6001 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     6061 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     6121 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     6181 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     6241 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     6301 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     6361 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     6421 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
//
