LOCUS       sequence-324630-        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 M1457A
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..29
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     113..132
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        113..131
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    132..156
                     /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             171..193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             212..229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             236..1333
                     /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      1190..1199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1307..1330
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1361..1384
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2198..2215
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2268..2286)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2282..2329
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2366..2821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2453..2472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2663..2684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2913..3728)
                     /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(2913..3728)
                     /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     3636..3655
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3720..3739
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3850..4438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4339..4358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4592..4609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4624..4763
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4689..4708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4849..4871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4865..5056)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5865..6947)
                     /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     6909..6928
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6948..7025)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
       61 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
      121 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
      181 tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcacg gtaccaaaac
      241 tgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga
      301 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
      361 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
      421 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
      481 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
      541 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
      601 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
      661 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
      721 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
      781 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
      841 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
      901 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
      961 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     1021 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     1081 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     1141 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccaccatgga
     1201 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     1261 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     1321 agacgcgcag actggtaccg attacgatat cccaacgacc ctggaagtgc tgttccaggg
     1381 tccgcatatg gctagctccc ccttgggagt cctcctgaaa atgatccata atgccctgcg
     1441 cttcattccc gtcacctctg tggtgttgct ttaccaccgc gtccatcctg aggaagtcac
     1501 cttccacctc tacctgatcc caagtgactg ctccattcgg aaggccatag atgatctaga
     1561 aatgaaattc cagtttgtgc gaatccacaa gccacccccg ctgaccccac tttatatggg
     1621 ctgtcgttac actgtgtctg ggtctggttc agggatgttg gaaatactcc ccaaggaact
     1681 ggagctctgc tatcgaagcc ctggagaaga ccagctgttc tcggagttct acgttggcca
     1741 cttgggatca gggatcaggc tgcaagtgaa agacaagaaa gatgagactc tggtgtggga
     1801 ggccttggtg aaaccaggag atctcatgcc tgcaactacc ctgatccctc cagcccgcat
     1861 agccgtacct tcacctctgg atgccccgca gttgctgcac tttgtggacc agtatcgaga
     1921 gcagctgata gcccgagtga catcggtgga ggttgtcttg gacaaactgc atggacaggt
     1981 gctgagccag gagcagtacg agagggtgct ggctgagaac acgaggccca gccagatgcg
     2041 gaagctgttc agcttgagcc agtcctggga ccggaagtgc aaagatggac tctaccaagc
     2101 cctgaaggag acccatcctc acctcattgc cgaactctgg gagaagggca gcaaaaaggg
     2161 actcctgcca ctcagcagct gagcggccgc actcgagcac caccaccacc accactgaga
     2221 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2281 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2341 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2401 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2461 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2521 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2581 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2641 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2701 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2761 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2821 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2881 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     2941 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     3001 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     3061 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     3121 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     3181 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     3241 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     3301 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     3361 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     3421 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     3481 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     3541 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     3601 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     3661 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     3721 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     3781 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3841 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3901 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3961 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4021 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4081 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4141 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4201 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4261 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4321 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4381 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4441 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4501 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4561 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4621 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4681 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     4741 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4801 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4861 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4921 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4981 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5041 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5101 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5161 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5221 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5281 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5341 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5401 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5461 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     5521 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5581 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5641 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     5701 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5761 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5821 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5881 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5941 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     6001 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     6061 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     6121 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     6181 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     6241 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     6301 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     6361 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6421 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6481 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6541 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6601 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6661 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6721 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6781 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6841 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6901 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6961 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     7021 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     7081 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     7141 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     7201 cccgaagtgg cgagcccgat c
//
