LOCUS       sequence-325079-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged CARD8 CARD (filment-deficient mutant and A452C 
            mutation for cysteine labelling applications).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  AUTHORS   Robert Hollingsworth L, David L, Li Y, Griswold AR, Ruan J, Sharif 
            H, Fontana P, Orth-He EL, Fu TM, Bachovchin DA, Wu H
  TITLE     Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 
            inflammasomes.
  JOURNAL   Nat Commun. 2021 Jan 8;12(1):189. doi: 10.1038/s41467-020-20320-y.
  PUBMED    33420033
REFERENCE   2  (bases 1 to 6714)
  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: "Nat 
            Commun."; date: "2021-01-8"; volume: "12(1):189. doi"; pages: " 
            10.1038/s41467-020-20320-y"
COMMENT     Sequence Label: pDB.His.MBP.3C CARD8 CARD A452C Y527A
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(331..408)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     614..633
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     717..736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        717..735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    736..760
                     /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             775..797
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             816..833
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             840..1937
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1794..1803
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1911..1934
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1965..1988
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2295..2312
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2365..2383)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2379..2426
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2463..2918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2550..2569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2760..2781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3010..3825)
                     /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(3010..3825)
                     /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     3733..3752
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3817..3836
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3947..4535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4436..4455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4689..4706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4721..4860
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4786..4805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4946..4968
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4962..5153)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5962..6714,1..330))
                     /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 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
       61 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
      121 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
      181 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
      241 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
      301 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
      361 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
      421 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
      481 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
      541 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
      601 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      661 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      721 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      781 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacggtacca
      841 aaactgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      901 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      961 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
     1021 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
     1081 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
     1141 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
     1201 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
     1261 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
     1321 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
     1381 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
     1441 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
     1501 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
     1561 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     1621 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     1681 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1741 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca
     1801 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1861 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1921 tgaaagacgc gcagactggt accgattacg atatcccaac gaccctggaa gtgctgttcc
     1981 agggtccgca tatggctagc cctcctttct caggtgcatg ctttgtgaag gagaaccacc
     2041 ggcaactcca agccaggatg ggggacctga aaggggtgct cgatgatctc caagacaatg
     2101 aggttcttac tgagaatgag aaggagctgg tggagcagga aaagacacgg cagagcaaga
     2161 atgaggcctt gctgagcatg gtggagaaga aaggggacct ggccctggac gtgctcttca
     2221 gaagcattag tgaaagggac cctgccctcg tgtcctatct tagacagcag aatttgtagg
     2281 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     2341 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2401 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2461 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2941 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3001 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3061 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3121 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3181 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3241 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     3301 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     3361 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     3421 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3481 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3541 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3601 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3661 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3721 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3781 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3841 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     3901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4081 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4561 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4621 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4681 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4741 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4801 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4861 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4921 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4981 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     5041 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     5101 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5161 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5221 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5281 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5341 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5401 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5461 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5521 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5581 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     5641 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     5701 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5761 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5821 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5881 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5941 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6001 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     6061 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     6121 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     6181 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     6241 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     6301 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     6361 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     6421 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     6481 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     6541 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     6601 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     6661 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcac
//
