LOCUS       sequence-361192-        6175 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 6175)
  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 6175)
  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_canine-Caspase-1/4_enzymatic-domain
FEATURES             Location/Qualifiers
     source          1..6175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..42
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    57..196
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(122..141)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     282..304
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(298..489)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1298..2380)
                     /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     2342..2361
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2381..2458)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2664..2683
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2767..2786
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2767..2785
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2786..2810
                     /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             2825..2847
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2866..2883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2893..2910
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2917..2937
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3806..3823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3876..3894)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3890..3937
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3974..4429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4061..4080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4271..4292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4521..5336)
                     /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(4521..5336)
                     /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     5244..5263
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5328..5347
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5458..6046
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5947..5966
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
       61 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
      121 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
      181 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      241 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      301 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
      361 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
      421 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
      481 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
      541 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
      601 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
      661 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
      721 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
      781 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
      841 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
      901 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
      961 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     1021 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     1081 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     1141 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     1201 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     1261 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1321 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1381 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1441 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1501 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1561 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1621 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1681 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1741 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1801 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1861 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1921 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1981 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     2041 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     2101 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     2161 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2221 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2281 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2341 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2401 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     2461 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     2521 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     2581 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     2641 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     2701 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     2761 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     2821 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     2881 cacagcagcg gcctggtgcc gcgcggcagc catatggaaa acctgtattt tcagggcgga
     2941 tccactctga agttatgccc acccgagacg tttgtaaaga tgtataaaga gaaggctgag
     3001 gagatttacc ctatcaagga gcgcaaggac cgcacacgtt tagcattgat tatttgtaac
     3061 atcgaatttg atcatctgag tacccgtgat ggcgctgaat tagacattgc tgggatggag
     3121 tccctgcttg aggggttagg gtattcggtt gtggtcaagc gtaaattaac tgccaaggga
     3181 atggaatccg tcttacgtga gtttgctgcg cgtccggagc ataaatcatc agactcaacc
     3241 tttctggtac tgatgtctca cggaatttta aatggtatct gcgggactgc gcattcagtt
     3301 gagaacccag atgtccttgc ttacgataca atcttccaaa tctttaataa ccgtcattgt
     3361 ttgaatttaa aggataaacc gaaggttatc attattcagg cttgccgtgg agagaaccct
     3421 ggggaattat gggtttctga ttcgccaaag gcgagcacag actcctggac ccaccaacct
     3481 cttatgttac aatcagacgc aattcataag gttcacgtcg aaaaggattt cattgcgttc
     3541 tgttcctcca cgcctcataa cgtaagctgg cgtcacatca ccaaaggatc tttgttcatc
     3601 gcgcagctta ttacttgttt tcaaaagtac tcatggtgtt gccatcttga aggcgttttt
     3661 cgcaaggtgc aacagtcttt cgagaagcct gacgtaaaag cccagatgcc cacaatcgag
     3721 cgtgttagca tgacacgtta cttctacttg tttccaggtc actaagaatt cgagctccgt
     3781 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     3841 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     3901 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     3961 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4021 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4081 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4141 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4201 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4261 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4321 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4381 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4441 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4501 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     4561 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     4621 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     4681 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     4741 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     4801 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     4861 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     4921 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     4981 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     5041 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     5101 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     5161 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     5221 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     5281 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     5341 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     5401 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5461 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5521 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5581 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5641 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5701 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5761 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5821 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5881 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5941 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6001 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6061 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6121 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgct
//
