LOCUS       sequence-324680-        6705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged NLRP1 CARD .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  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 6705)
  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 NLRP1 CARD WT
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..1098
                     /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      955..964
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1072..1095
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1126..1146
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1447..1464
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1517..1535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1531..1578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1615..2070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1702..1721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1912..1933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2162..2977)
                     /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(2162..2977)
                     /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     2885..2904
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2969..2988
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3099..3687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3588..3607
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3841..3858
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3873..4012
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3938..3957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4098..4120
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4114..4305)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5114..6196)
                     /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     6158..6177
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6197..6274)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6480..6499
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6583..6602
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6583..6601
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6602..6626
                     /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             6641..6663
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6682..6699
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 aaaactgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
       61 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
      121 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
      181 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
      241 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
      301 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
      361 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
      421 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
      481 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
      541 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
      601 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
      661 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
      721 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
      781 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
      841 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
      901 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc
      961 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     1021 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     1081 ctgaaagacg cgcagactgg taccgattac gatatcccaa cgaccgaaaa cctttacttc
     1141 cagggccata tgctgcactt tgtggaccag tatcgagagc agctgatagc ccgagtgaca
     1201 tcggtggagg ttgtcttgga caaactgcat ggacaggtgc tgagccagga gcagtacgag
     1261 agggtgctgg ctgagaacac gaggcccagc cagatgcgga agctgttcag cttgagccag
     1321 tcctgggacc ggaagtgcaa agatggactc taccaagccc tgaaggagac ccatcctcac
     1381 ctcattatgg aactctggga gaagggcagc aaaaagggac tcctgccact cagcagctga
     1441 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1501 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1561 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1621 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1681 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1741 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1801 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1861 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1921 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1981 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2041 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     2101 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     2161 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2221 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2281 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2341 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2401 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     2461 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2521 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2581 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2641 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2701 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2761 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2821 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2881 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2941 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3001 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3061 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3121 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3181 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3241 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3301 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3361 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3421 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3481 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3541 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3601 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3661 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3721 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3781 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3841 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3901 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3961 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4021 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4081 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4141 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4201 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4261 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4321 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4381 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4441 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4501 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4561 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4621 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4681 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4741 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     4801 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     4861 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     4921 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4981 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5041 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5101 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5161 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5221 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5281 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     5341 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     5401 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     5461 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     5521 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     5581 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     5641 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     5701 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     5761 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     5821 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     5881 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     5941 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6001 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6061 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6121 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6181 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6241 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6301 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     6361 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     6421 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     6481 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     6541 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     6601 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     6661 agatatacca tgggcagcag ccatcatcat catcatcacg gtacc
//
