LOCUS       sequence-324629-        7221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged NLRP1 UPA-CARD (dimer interface mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  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 7221)
  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 UPA-CARD Y1445A
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..108
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             139..162
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             976..993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1046..1064)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1060..1107
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1144..1599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1231..1250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1441..1462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1691..2506)
                     /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(1691..2506)
                     /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     2414..2433
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2498..2517
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2628..3216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3117..3136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3370..3387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3402..3541
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3467..3486)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3627..3649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3643..3834)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4643..5725)
                     /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     5687..5706
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5726..5803)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6009..6028
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6112..6131
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6112..6130
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6131..6155
                     /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             6170..6192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6211..6228
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(6235..7221,1..111)
                     /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      7189..7198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
       61 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac tggtaccgat
      121 tacgatatcc caacgaccct ggaagtgctg ttccagggtc cgcatatggc tagctccccc
      181 ttgggagtcc tcctgaaaat gatccataat gccctgcgct tcattcccgt cacctctgtg
      241 gtgttgcttt accaccgcgt ccatcctgag gaagtcacct tccacctcta cctgatccca
      301 agtgactgct ccattcggaa ggccatagat gatctagaaa tgaaattcca gtttgtgcga
      361 atccacaagc cacccccgct gaccccactt tatatgggct gtcgttacac tgtgtctggg
      421 tctggttcag ggatgttgga aatactcccc aaggaactgg agctctgcta tcgaagccct
      481 ggagaagacc agctgttctc ggagttctac gttggccact tgggatcagg gatcaggctg
      541 caagtgaaag acaagaaaga tgagactctg gtgtgggagg ccttggtgaa accaggagat
      601 ctcatgcctg caactaccct gatccctcca gcccgcatag ccgtaccttc acctctggat
      661 gccccgcagt tgctgcactt tgtggaccag tatcgagagc agctgatagc ccgagtgaca
      721 tcggtggagg ttgtcttgga caaactgcat ggacaggtgc tgagccagga gcagtacgag
      781 agggtgctgg ctgagaacac gaggcccagc cagatgcgga agctgttcag cttgagccag
      841 tcctgggacc ggaagtgcaa agatggactc gcccaagccc tgaaggagac ccatcctcac
      901 ctcattatgg aactctggga gaagggcagc aaaaagggac tcctgccact cagcagctga
      961 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1021 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1081 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1141 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1201 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1261 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1321 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1381 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1441 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1501 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1561 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1621 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1681 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1741 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1801 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1861 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1921 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1981 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2041 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2101 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2161 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2221 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2281 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2341 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2401 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2461 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2521 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2761 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3241 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3301 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3361 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3421 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3481 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3541 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3601 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3661 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3721 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3781 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3841 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3901 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3961 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4021 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4081 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4141 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4201 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4261 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4321 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4381 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4441 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4501 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4561 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4621 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4681 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4741 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4801 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4861 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4921 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4981 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5041 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5101 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5161 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5221 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5281 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5341 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5401 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5461 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5521 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5581 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5641 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5701 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5761 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5821 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5881 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5941 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6001 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6061 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     6121 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     6181 ttaagaagga gatataccat gggcagcagc catcatcatc atcatcacgg taccaaaact
     6241 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     6301 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     6361 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     6421 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     6481 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     6541 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     6601 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     6661 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     6721 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     6781 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     6841 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     6901 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     6961 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     7021 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     7081 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     7141 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc caccatggaa
     7201 aacgcccaga aaggtgaaat c
//
