LOCUS       sequence-325117-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a 5GSS-linked 
            ASC(CARD)-CASP1(CARD) construct to express an octamer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  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 7008)
  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: ASC-CASP1 Octamer
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             30..52
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    67..91
                     /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)."
     primer_bind     complement(91..110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(92..110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(194..213)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        419..496
                     /gene="lacI"
                     /label=lacI promoter
     CDS             497..1579
                     /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     complement(516..535)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2388..2579
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2573..2595)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2681..2820
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2736..2755
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2835..2852)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3006..3594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3086..3105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3705..3724)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3716..4531
                     /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             3716..4531
                     /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     complement(3789..3808)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4623..5078)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4760..4781)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4972..4991
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5115..5162)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5158..5176
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5229..5246)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5850..5870)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5898..6995)
                     /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"
     primer_bind     complement(5901..5924)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      6032..6041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(7002..7008,1..11))
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 tgatgatgat ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc
       61 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
      121 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
      181 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
      241 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
      301 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
      361 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
      421 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
      481 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
      541 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
      601 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
      661 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
      721 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
      781 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
      841 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
      901 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
      961 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     1021 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     1081 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     1141 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     1201 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     1261 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     1321 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     1381 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     1441 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     1501 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     1561 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     1621 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     1681 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     1741 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     1801 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     1861 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc
     1921 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     1981 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     2041 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     2101 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     2161 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     2221 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     2281 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     2341 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     2401 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     2461 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     2521 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     2581 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     2641 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     2701 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     2761 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     2821 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2881 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2941 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3001 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3061 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3121 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3181 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3241 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3301 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3361 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3421 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     3481 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3541 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3601 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3661 ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     3721 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
     3781 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     3841 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     3901 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     3961 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     4021 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     4081 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     4141 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     4201 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     4261 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     4321 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     4381 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     4441 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     4501 gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg
     4561 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4621 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     4681 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4741 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4801 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4861 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4921 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4981 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5041 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa tccggatata
     5101 gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct
     5161 agttattgct cagcggtggc agcagccaac tcagcttcct ttcgggcttt gttagcagcc
     5221 ggatctcagt ggtggtggtg gtggtgctcg agttatgctg agagtcccag cgtccctgcc
     5281 aggtaactgt cttcttcaca aatgtatgtg atgcaaattt ggcatgcctg tgcccctttc
     5341 ggaataacgg agtcaatcaa agctcgggtc ttatccataa ctgtagcatt ttcacgtttt
     5401 actttctcca tctcttcctt gttcagcacc cttgtctgta ataattcatc cagtaagcca
     5461 tttattgttt tttcacccat ggaacggata aacagctttc tcttctcctt caggaccttg
     5521 tcggccatag aagaaccaga agaaccagag gatccagaag aaccagaaga accgctccgc
     5581 tccaggtcct ccaccaggta ggactgggac tcccttaggg cctggaggag caagtccttg
     5641 caggtccagt tgccggctgg tgtgaaactg aagagcttcc gcatcttgct tgggttggtg
     5701 ggctcggccc gcactgcctg gtactgctca tccgtcagga ccttcccgta cagagcatcc
     5761 agcagccact caacgtttgt gaccctcgcg ataagcgcag cccggtgctg gtctataaag
     5821 tgcaggcctg gcttggctgc cgacatatgg ccctggaagt aaaggttttc ggtcgttggg
     5881 atatcgtaat cggtaccagt ctgcgcgtct ttcagggctt catcgacagt ctgacgaccg
     5941 ctggcggcgt tgatcaccgc agtacgcacg gcataccaga aagcggacat ctgcgggatg
     6001 ttcggcatga tttcaccttt ctgggcgttt tccatggtgg cggcaatacg tggatctttc
     6061 gccaactctt cctcgtaaga cttcagcgct acggcaccca gcggtttgtc tttattaacc
     6121 gcttccagac cttcatcagt cagcagatag ttttcgagga actcttttgc cagctctttg
     6181 ttcggactgg cggcgttaat acctgcgctc agcacgccaa cgaacggttt ggatggttga
     6241 cccttgaagg tcggcagtac cgttacacca taattcactt tgctggtgtc gatgttggac
     6301 catgcccacg ggccgttgat ggtcatcgct gtttcgcctt tattaaaggc agcttctgcg
     6361 atggagtaat cggtgtctgc attcatgtgt ttgtttttaa tcaggtcaac caggaaggtc
     6421 agacccgctt tcgcgccagc gttatccacg cccacgtctt taatgtcgta cttgccgttt
     6481 tcatacttga acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct
     6541 tgcaggttga acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg
     6601 atctcttccc aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac
     6661 gcttcaacag cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta
     6721 aacggataca gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat
     6781 tgagcgtagc caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt
     6841 gccgcaacct gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt
     6901 ccggtatctt tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg
     6961 ttaatccaga ttaccagttt accttcttca gttttggtac cgtgatga
//
