LOCUS       sequence-324681-        6705 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged NLRP1 CARD G1467C.
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 Apr 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 G1467C
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             31..1128
                     /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      985..994
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1102..1125
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1156..1176
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1477..1494
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1547..1565)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1561..1608
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1645..2100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1732..1751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1942..1963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2192..3007)
                     /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(2192..3007)
                     /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     2915..2934
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2999..3018
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3129..3717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3618..3637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3871..3888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3903..4042
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3968..3987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4128..4150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4144..4335)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5144..6226)
                     /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     6188..6207
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6227..6304)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6510..6529
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6613..6632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6613..6631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6632..6656
                     /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             6671..6693
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 agcagccatc atcatcatca tcacggtacc aaaactgaag aaggtaaact ggtaatctgg
       61 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
      121 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      181 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      241 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      301 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      361 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
      421 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
      481 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      541 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
      601 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
      661 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
      721 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
      781 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
      841 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
      901 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
      961 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg
     1021 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1081 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactgg taccgattac
     1141 gatatcccaa cgaccgaaaa cctttacttc cagggccata tgctgcactt tgtggaccag
     1201 tatcgagagc agctgatagc ccgagtgaca tcggtggagg ttgtcttgga caaactgcat
     1261 ggacaggtgc tgagccagga gcagtacgag agggtgctgg ctgagaacac gaggcccagc
     1321 cagatgcgga agctgttcag cttgagccag tcctgggacc ggaagtgcaa agatggactc
     1381 taccaagccc tgaaggagac ccatcctcac ctcattatgg aactctggga gaagggcagc
     1441 aaaaagtgcc tcctgccact cagcagctga ctcgagcacc accaccacca ccactgagat
     1501 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1561 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1621 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1681 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1741 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1801 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1861 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1921 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1981 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2041 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2101 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2161 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2221 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2281 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2341 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2401 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2461 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2521 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2581 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2641 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2701 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2761 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2821 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2881 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2941 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3001 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3061 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3121 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3181 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3241 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3301 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3361 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3421 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3481 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3541 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3601 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3661 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3721 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3781 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3841 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3901 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3961 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4021 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4081 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4141 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4201 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4261 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4321 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4381 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4441 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4501 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4561 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4621 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4681 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4741 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4801 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4861 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4921 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4981 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5041 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5101 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5161 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5221 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5281 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5341 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5401 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     5461 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5521 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5581 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5641 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5701 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5761 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5821 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5881 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5941 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6001 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6061 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6121 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6181 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6241 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6301 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6361 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     6421 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     6481 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     6541 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     6601 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     6661 tagaaataat tttgtttaac tttaagaagg agatatacca tgggc
//
