LOCUS       sequence-324677-        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 W1460A
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..55
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(75..152)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     358..377
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     461..480
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        461..479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    480..504
                     /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             519..541
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             560..577
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             584..1681
                     /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      1538..1547
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1655..1678
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1709..1732
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2546..2563
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2616..2634)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2630..2677
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2714..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2801..2820)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3011..3032
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3261..4076)
                     /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(3261..4076)
                     /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     3984..4003
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4068..4087
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4198..4786
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4687..4706
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4940..4957
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4972..5111
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5037..5056)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5197..5219
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5213..5404)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6213..7221,1..74))
                     /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 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
       61 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
      121 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
      181 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
      241 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
      301 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
      361 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
      421 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
      481 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      541 atataccatg ggcagcagcc atcatcatca tcatcacggt accaaaactg aagaaggtaa
      601 actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa
      661 attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa
      721 attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg
      781 ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca
      841 ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta
      901 cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc
      961 aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc
     1021 gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg
     1081 ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc
     1141 tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc
     1201 agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat
     1261 caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt
     1321 actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat
     1381 taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac
     1441 tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc
     1501 ttacgaggaa gagttggcga aagatccacg tattgccgcc accatggaaa acgcccagaa
     1561 aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc
     1621 ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac
     1681 tggtaccgat tacgatatcc caacgaccct ggaagtgctg ttccagggtc cgcatatggc
     1741 tagctccccc ttgggagtcc tcctgaaaat gatccataat gccctgcgct tcattcccgt
     1801 cacctctgtg gtgttgcttt accaccgcgt ccatcctgag gaagtcacct tccacctcta
     1861 cctgatccca agtgactgct ccattcggaa ggccatagat gatctagaaa tgaaattcca
     1921 gtttgtgcga atccacaagc cacccccgct gaccccactt tatatgggct gtcgttacac
     1981 tgtgtctggg tctggttcag ggatgttgga aatactcccc aaggaactgg agctctgcta
     2041 tcgaagccct ggagaagacc agctgttctc ggagttctac gttggccact tgggatcagg
     2101 gatcaggctg caagtgaaag acaagaaaga tgagactctg gtgtgggagg ccttggtgaa
     2161 accaggagat ctcatgcctg caactaccct gatccctcca gcccgcatag ccgtaccttc
     2221 acctctggat gccccgcagt tgctgcactt tgtggaccag tatcgagagc agctgatagc
     2281 ccgagtgaca tcggtggagg ttgtcttgga caaactgcat ggacaggtgc tgagccagga
     2341 gcagtacgag agggtgctgg ctgagaacac gaggcccagc cagatgcgga agctgttcag
     2401 cttgagccag tcctgggacc ggaagtgcaa agatggactc taccaagccc tgaaggagac
     2461 ccatcctcac ctcattatgg aactcgccga gaagggcagc aaaaagggac tcctgccact
     2521 cagcagctga gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2581 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2641 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2701 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2761 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2821 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2881 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2941 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3001 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3061 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3121 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3181 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3241 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     3301 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3361 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3421 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3481 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3541 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3601 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3661 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3721 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3781 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3841 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3901 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     3961 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     4021 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     4081 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     4141 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4201 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4261 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4321 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4381 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4441 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4501 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4561 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4621 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4681 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4741 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4801 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4861 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4921 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4981 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     5041 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5101 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5161 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5221 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5281 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     5341 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5401 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5461 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5521 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5581 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5641 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5701 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5761 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5821 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5881 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5941 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     6001 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     6061 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     6121 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     6181 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6241 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6301 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     6361 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     6421 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     6481 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6541 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     6601 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     6661 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     6721 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     6781 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     6841 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     6901 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     6961 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     7021 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     7081 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     7141 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     7201 gcagaaacgt ggctggcctg g
//
