LOCUS       sequence-325087-        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 E490R
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..22
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(42..119)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     325..344
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     428..447
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        428..446
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    447..471
                     /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             486..508
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             527..544
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             551..1648
                     /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      1505..1514
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1622..1645
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1676..1699
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2006..2023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2076..2094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2090..2137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2174..2629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2261..2280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2471..2492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2721..3536)
                     /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(2721..3536)
                     /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     3444..3463
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3528..3547
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3658..4246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4147..4166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4400..4417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4432..4571
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4497..4516)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4657..4679
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4673..4864)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5673..6714,1..41))
                     /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 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
       61 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
      121 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
      181 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
      241 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
      301 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
      361 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
      421 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
      481 ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca
      541 tcacggtacc aaaactgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta
      601 taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
      661 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
      721 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc
      781 tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
      841 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta
      901 taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa
      961 agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
     1021 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
     1081 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
     1141 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
     1201 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
     1261 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
     1321 gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa
     1381 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     1441 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat
     1501 tgccgccacc atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     1561 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     1621 cgatgaagcc ctgaaagacg cgcagactgg taccgattac gatatcccaa cgaccctgga
     1681 agtgctgttc cagggtccgc atatggctag ccctcctttc tcaggtgcat gctttgtgaa
     1741 ggagaaccac cggcaactcc aagccaggat gggggacctg aaaggggtgc tcgatgatct
     1801 ccaagacaat gaggttctta ctgagaatga gaaggagctg gtgcggcagg aaaagacacg
     1861 gcagagcaag aatgaggcct tgctgagcat ggtggagaag aaaggggacc tggccctgga
     1921 cgtgctcttc agaagcatta gtgaaaggga cccttacctc gtgtcctatc ttagacagca
     1981 gaatttgtag gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2041 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2101 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2161 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2221 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2281 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2341 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2401 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2461 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2521 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2581 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2641 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2701 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2761 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     2821 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     2881 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     2941 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3001 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3061 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3121 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3181 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3241 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3301 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3361 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     3421 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     3481 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     3541 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     3601 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3661 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3721 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3781 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3841 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3901 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3961 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4021 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4081 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4141 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4201 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4261 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4321 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4381 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4441 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4501 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4561 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4621 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4681 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4741 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4801 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4861 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4921 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4981 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5041 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5101 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5161 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5221 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5281 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5341 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5401 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5461 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5521 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5581 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5641 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5701 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5761 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5821 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5881 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5941 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6001 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     6061 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     6121 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     6181 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     6241 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     6301 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     6361 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     6421 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     6481 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     6541 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6601 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6661 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gaca
//
