LOCUS       sequence-324682-        6879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged NLRP1 UPA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6879)
  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 6879)
  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 WT
FEATURES             Location/Qualifiers
     source          1..6879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(209..1024)
                     /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(209..1024)
                     /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     932..951
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1016..1035
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1146..1734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1635..1654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1888..1905
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1920..2059
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1985..2004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2145..2167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2161..2352)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3161..4243)
                     /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     4205..4224
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4244..4321)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4527..4546
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4630..4649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4630..4648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4649..4673
                     /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             4688..4710
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4729..4746
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4753..5850
                     /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      5707..5716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5824..5847
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5878..5901
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6373..6390
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6443..6461)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6457..6504
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6541..6879,1..117)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6628..6647)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6838..6859
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
       61 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
      121 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      181 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
      241 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
      301 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
      361 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
      421 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
      481 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
      541 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
      601 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
      661 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
      721 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
      781 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
      841 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
      901 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
      961 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     1021 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     1081 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1141 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1201 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1261 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1321 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1381 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1441 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1501 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1561 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1621 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1681 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1741 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     1801 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     1861 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     1921 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     1981 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2041 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2101 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2161 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     2221 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     2281 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     2341 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     2401 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     2461 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     2521 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     2581 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     2641 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     2701 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     2761 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     2821 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     2881 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     2941 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3001 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3061 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3121 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3181 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3241 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3301 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3361 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3421 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     3481 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3541 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     3601 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     3661 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     3721 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     3781 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     3841 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     3901 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     3961 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4021 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4081 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4141 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4201 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4261 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4321 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4381 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     4441 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     4501 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     4561 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     4621 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     4681 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     4741 catcacggta ccaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
     4801 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
     4861 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
     4921 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
     4981 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
     5041 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
     5101 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
     5161 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
     5221 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
     5281 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
     5341 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     5401 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     5461 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     5521 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     5581 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     5641 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     5701 attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     5761 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     5821 gtcgatgaag ccctgaaaga cgcgcagact ggtaccgatt acgatatccc aacgaccctg
     5881 gaagtgctgt tccagggtcc gcatatgtcc cccttgggag tcctcctgaa aatgatccat
     5941 aatgccctgc gcttcattcc cgtcacctct gtggtgttgc tttaccaccg cgtccatcct
     6001 gaggaagtca ccttccacct ctacctgatc ccaagtgact gctccattcg gaaggccata
     6061 gatgatctag aaatgaaatt ccagtttgtg cgaatccaca agccaccccc gctgacccca
     6121 ctttatatgg gctgtcgtta cactgtgtct gggtctggtt cagggatgct ggaaatactc
     6181 cccaaggaac tggagctctg ctatcgaagc cctggagaag accagctgtt ctcggagttc
     6241 tacgttggcc acttgggatc agggatcagg ctgcaagtga aagacaagaa agatgagact
     6301 ctggtgtggg aggccttggt gaaaccagga gatctcatgc ctgcaactac tctgatccct
     6361 ccatagctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     6421 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     6481 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     6541 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6601 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6661 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6721 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     6781 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6841 gactcttgtt ccaaactgga acaacactca accctatct
//
