LOCUS       sequence-362584-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of catalytic domain of inflammatory caspase in 
            bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6163)
  AUTHORS   Devant P, Cao A, Kagan JC
  TITLE     Evolution-inspired redesign of the LPS receptor caspase-4 into an 
            interleukin-1beta converting enzyme.
  JOURNAL   Sci Immunol. 2021 Aug 10;6(62). doi: 10.1126/sciimmunol.abh3567.
  PUBMED    34734155
REFERENCE   2  (bases 1 to 6163)
  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: "Sci 
            Immunol."; date: "2021-08-10"; volume: "6(62). doi"; pages: " 
            10.1126/sciimmunol.abh3567"
COMMENT     Sequence Label: pET28_mouse-pro-IL1B
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1366..1557
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1551..1573)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1659..1798
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1714..1733
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1813..1830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1984..2572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2064..2083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2683..2702)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2694..3509
                     /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             2694..3509
                     /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(2767..2786)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3601..4056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3738..3759)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3950..3969
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4093..4140)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4136..4154
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4207..4224)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5081..5101)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5108..5125)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5135..5152)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5171..5193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5208..5232
                     /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(5232..5251)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5233..5251)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5335..5354)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5560..5637
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5638..6163,1..557)
                     /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(5657..5676)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
       61 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
      121 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
      181 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
      241 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
      301 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
      361 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
      421 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
      481 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      541 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
      601 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
      661 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
      721 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
      781 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
      841 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
      901 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
      961 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     1021 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     1081 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     1141 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     1201 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     1261 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     1321 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     1381 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     1441 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     1501 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     1561 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     1621 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     1681 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     1741 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     1801 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1861 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     1921 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1981 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2041 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2101 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2161 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2221 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2281 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2341 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2401 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     2461 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2521 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2581 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2641 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     2701 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     2761 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     2821 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     2881 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     2941 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     3001 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     3061 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     3121 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     3181 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     3241 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     3301 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     3361 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     3421 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     3481 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     3541 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3601 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3661 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3721 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3781 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3841 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3901 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3961 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4021 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt
     4081 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
     4141 ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg
     4201 atctcagtgg tggtggtggt ggtgctcgag tgcggccgca agcttgtcga cggagctcga
     4261 attcttagga agacacggat tccatggtga agtcaattat gtcctgacca ctgttgtttc
     4321 ccaggaagac aggcttgtgc tctgcttgtg aggtgctgat gtaccagttg gggaactctg
     4381 cagactcaaa ctccactttg ctcttgactt ctatcttgtt gaagacaaac cgcttttcca
     4441 tcttcttctt tgggtattgc ttgggatcca cactctccag ctgcagggtg ggtgtgccgt
     4501 ctttcattac acaggacagg tatagattct ttcctttgag gcccaaggcc acaggtattt
     4561 tgtcgttgct tggttctcct tgtacaaagc tcatggagaa tatcacttgt tggttgatat
     4621 tctgtccatt gaggtggaga gctttcagct catatgggtc cgacagcacg aggctttttt
     4681 gttgttcatc tcggagcctg tagtgcagct gtctaatggg aacgtcacac accagcaggt
     4741 tatcatcatc atcccatgag tcacagagga tgggctcttc ttcaaagatg aaggaaaaga
     4801 aggtgctcat gtcctcatcc tggaaggtcc acgggaaaga cacaggtagc tgccacagct
     4861 tctccacagc cacaatgagt gatactgcct gcctgaagct cttgttgatg tgctgctgtg
     4921 agatttgaag ctggatgctc tcatctggac agcccaggtc aaaggtttgg aagcagccct
     4981 tcatcttttg gggtccgtca acttcaaaga acaggtcatt ctcatcactg tcaaaaggtg
     5041 gcatttcaca gttgagttca ggaacagttg ccatagatcc gccctgaaaa tacaggtttt
     5101 ccatatggct gccgcgcggc accaggccgc tgctgtgatg atgatgatga tggctgctgc
     5161 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     5221 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
     5281 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     5341 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     5401 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     5461 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     5521 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     5581 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     5641 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     5701 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     5761 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     5821 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     5881 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     5941 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     6001 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     6061 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     6121 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcg
//
