LOCUS       sequence-325076-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding a HRV 3C-cleavable 
            His-MBP-tagged CARD8 UPA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7161)
  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 7161)
  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 CARD8 UPA-STOP
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(81..158)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     364..383
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     467..486
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        467..485
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    486..510
                     /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             525..547
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             566..583
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             590..1687
                     /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      1544..1553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1661..1684
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1715..1738
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2492..2509
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2562..2580)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2576..2623
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2660..3115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2747..2766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2957..2978
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3207..4022)
                     /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(3207..4022)
                     /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     3930..3949
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4014..4033
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4144..4732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4633..4652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4886..4903
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4918..5057
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4983..5002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5143..5165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5159..5350)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6159..7161,1..80))
                     /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 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
       61 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
      121 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
      181 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
      241 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
      301 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
      361 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      421 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
      481 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
      541 aaggagatat accatgggca gcagccatca tcatcatcat cacggtacca aaactgaaga
      601 aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg
      661 taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga
      721 agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca
      781 cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc
      841 gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat
      901 tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa
      961 cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa
     1021 gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga
     1081 cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga
     1141 taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat
     1201 gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat
     1261 gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt
     1321 aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc
     1381 aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct
     1441 gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct
     1501 gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca tggaaaacgc
     1561 ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg
     1621 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
     1681 gcagactggt accgattacg atatcccaac gaccctggaa gtgctgttcc agggtccgca
     1741 tatggctagc tctctgatgg gcatcctgct gcggatcgcc agtgggactc gcctctccat
     1801 ccccatcact tccaacacat tgatctatta tcacccccac cccgaagata ttaagttcca
     1861 cttgtacctt gtccccagcg acgccttgct aacaaaggcg atagatgatg aggaagatcg
     1921 cttccatggt gtgcgcctgc agacttcgcc cccaatggaa cccctgaact ttggttccag
     1981 ttatattgtg tctaattctg ctaacctgaa agtaatgccc aaggagttga aattgtccta
     2041 caggagccct ggagaaattc agcacttctc aaaattctat gctgggcaga tgaaggaacc
     2101 cattcaactt gagattactg aaaaaagaca tgggactttg gtgtgggata ctgaggtgaa
     2161 gccagtggat ctccagcttg tagctgcatc agcccctcct taattctcag gtgcagcctt
     2221 tgtgaaggag aaccaccggc aactccaagc caggatgggg gacctgaaag gggtgctcga
     2281 tgatctccaa gacaatgagg ttcttactga gaatgagaag gagctggtgg agcaggaaaa
     2341 gacacggcag agcaagaatg aggccttgct gagcatggtg gagaagaaag gggacctggc
     2401 cctggacgtg ctcttcagaa gcattagtga aagggaccct tacctcgtgt cctatcttag
     2461 acagcagaat ttgtaggcgg ccgcactcga gcaccaccac caccaccact gagatccggc
     2521 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     2581 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     2641 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2701 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2761 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2821 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2881 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2941 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3001 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3061 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     3121 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3181 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     3241 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     3301 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     3361 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     3421 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     3481 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     3541 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     3601 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     3661 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     3721 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3781 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3841 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3901 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3961 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     4021 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     4081 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4141 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4201 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4261 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4321 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4381 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4441 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4501 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4561 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4621 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4681 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4741 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4801 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4861 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4921 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4981 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     5041 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5101 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5161 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     5221 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     5281 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     5341 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     5401 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     5461 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     5521 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     5581 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     5641 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     5701 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     5761 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     5821 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     5881 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5941 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     6001 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     6061 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     6121 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6181 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6241 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     6301 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     6361 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     6421 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     6481 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     6541 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     6601 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     6661 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     6721 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     6781 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6841 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6901 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6961 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     7021 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     7081 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     7141 gttttcgcag aaacgtggct g
//
