LOCUS       sequence-348601-        6480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of inactive SARS-CoV-2 3C-like-C145A 
            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-C145A-Q306A
FEATURES             Location/Qualifiers
     source          1..6480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1313..1504
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1498..1520)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1606..1745
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1661..1680
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1760..1777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1931..2519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2011..2030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2690..3481,3482..3550))
                     /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     3313..3332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3551..3655)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3681..4136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3818..3839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4030..4049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4173..4220)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4216..4234
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4287..4304)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4354..4371)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4378..4407)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(4414..4479)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(4486..4497)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             5435..5457
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5472..5496
                     /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(5496..5515)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5497..5515)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5599..5618)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5824..5901
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5902..6480,1..504)
                     /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(5921..5940)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
       61 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
      121 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
      181 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
      241 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
      301 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
      361 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
      421 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      481 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
      541 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
      601 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
      661 aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc
      721 gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt
      781 cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc
      841 ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
      901 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
      961 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     1021 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     1081 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     1141 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     1201 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     1261 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     1321 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     1381 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     1441 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     1501 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     1561 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     1621 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     1681 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     1741 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1801 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1861 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1921 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2101 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2341 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     2401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2521 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2581 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2641 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2701 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2761 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2821 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2881 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2941 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3001 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3061 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3121 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3181 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3241 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3301 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3361 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3421 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3481 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3541 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3601 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3661 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3721 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3781 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3841 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     3901 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3961 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4021 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4081 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     4141 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     4201 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     4261 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag tgcggccgca
     4321 agcttgtcga cggagctcga attcggatcc tcaatgatga tgatgatgat ggccgcccag
     4381 atcctcttct gagatgagtt tttgttcgcc gcccttgtca tcgtcatcct tgtaatcgat
     4441 gtcatgatct ttataatcac cgtcatggtc tttgtagtcg ccgccacgtc cctcgatgcc
     4501 gccgcctgcg aaagtaacac ctgagcattg tctaacaaca tcaaaaggtg taaattcatc
     4561 ttctaataaa gcactaccca atatggtacg tccattcata ccattttgca gtaattcttt
     4621 taatgaagca cacatatcta aaacggcaat tccagtttga gcagaaagag gtcctagtat
     4681 gtcaacatgg tcttgtgtta gaggttcata attgtacttc atagccacaa ggttaaagtc
     4741 attaagagtt gtggtaaatc gattgagaaa ccacctgtct ccatttataa cagcagcgta
     4801 caaccaagct aaaacattaa ctgtaatagt tgtgtccgta ccagctgctt gtgctgtttg
     4861 cctgtcaaca aaaggtccat aaaagttacc ttctaagtct gtgccagcat gaactccagt
     4921 tggtaattcc atgtggtgca tgtaacaaaa agagacacag tcataatcta tgttaaaacc
     4981 aacactaccc gctgaaccat taaggaatga acccttaata gtgaaattgg gcctcatagc
     5041 acattggtaa acaccagatg gtgaaccatt gtaacaagct aacactgaaa aagtctgtcc
     5101 tggttgaatg cgaacaaact tatacttagg tgtcttagga ttggctgtat caaccttaag
     5161 cttaagtaca caattttgca tagaatgtcc aataaccctg agttgaacat taccagcctg
     5221 taccaagaaa ttatgattag acttacgaat gagtaaatct tcataattag ggttaagcat
     5281 gtcttcagag gtgcagatca catgtcttgg acagtaaact acgtcatcaa gccaaagacc
     5341 gttaagtgta gttgtaccac aagttacttg taccatacaa ccctcaactt taccagatgg
     5401 gaatgccatt tttctaaaac cactcatatg tatatctcct tcttaaagtt aaacaaaatt
     5461 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     5521 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     5581 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     5641 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     5701 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     5761 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     5821 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     5881 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     5941 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     6001 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     6061 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     6121 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     6181 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     6241 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     6301 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     6361 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     6421 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
//
