LOCUS       sequence-325098-        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 E485R
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..33
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    48..187
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(113..132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     273..295
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(289..480)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1289..2371)
                     /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     2333..2352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2372..2449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2655..2674
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2758..2777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2758..2776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2777..2801
                     /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             2816..2838
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2857..2874
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2881..3978
                     /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      3835..3844
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3952..3975
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4006..4029
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4336..4353
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4406..4424)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4420..4467
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4504..4959
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4591..4610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4801..4822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5051..5866)
                     /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(5051..5866)
                     /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     5774..5793
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5858..5877
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5988..6576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6477..6496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
       61 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
      121 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
      181 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      241 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      301 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      361 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      421 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      481 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      541 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      601 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      661 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      721 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      781 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      841 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      901 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
      961 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     1021 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     1081 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     1141 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     1201 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     1261 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1321 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1381 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1441 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1501 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1561 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1621 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1681 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1741 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1801 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     1861 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     1921 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     1981 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2041 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2101 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2161 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2221 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2281 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2341 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2401 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     2461 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     2521 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     2581 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     2641 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     2701 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     2761 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     2821 ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacggtacc
     2881 aaaactgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
     2941 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
     3001 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
     3061 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
     3121 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
     3181 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
     3241 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
     3301 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
     3361 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
     3421 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
     3481 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
     3541 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
     3601 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
     3661 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
     3721 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
     3781 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc
     3841 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     3901 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     3961 ctgaaagacg cgcagactgg taccgattac gatatcccaa cgaccctgga agtgctgttc
     4021 cagggtccgc atatggctag ccctcctttc tcaggtgcat gctttgtgaa ggagaaccac
     4081 cggcaactcc aagccaggat gggggacctg aaaggggtgc tcgatgatct ccaagacaat
     4141 gaggttctta ctgagaatcg gaaggagctg gtggagcagg aaaagacacg gcagagcaag
     4201 aatgaggcct tgctgagcat ggtggagaag aaaggggacc tggccctgga cgtgctcttc
     4261 agaagcatta gtgaaaggga cccttacctc gtgtcctatc ttagacagca gaatttgtag
     4321 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     4381 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     4441 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     4501 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4561 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4621 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4681 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4741 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4801 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4861 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4921 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     4981 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5041 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     5101 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     5161 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     5221 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     5281 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     5341 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     5401 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     5461 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     5521 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     5581 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     5641 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     5701 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     5761 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     5821 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     5881 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     5941 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6001 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6061 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6121 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6181 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6241 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6301 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6361 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6421 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6481 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6541 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6601 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6661 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagc
//
