LOCUS       sequence-361196-        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_mouse-Caspase-1-H340D_enzymatic-domain
FEATURES             Location/Qualifiers
     source          1..6175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..54
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(48..239)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1048..2130)
                     /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     2092..2111
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2131..2208)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2414..2433
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2517..2536
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2517..2535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2536..2560
                     /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             2575..2597
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2616..2633
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2643..2660
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2667..2687
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3556..3573
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3626..3644)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3640..3687
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3724..4179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3811..3830)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4021..4042
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4271..5086)
                     /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(4271..5086)
                     /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     4994..5013
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5078..5097
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5208..5796
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5697..5716
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5950..5967
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5982..6121
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6047..6066)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
       61 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
      121 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
      181 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
      241 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
      301 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
      361 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
      421 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
      481 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
      541 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
      601 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
      661 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
      721 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
      781 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
      841 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
      901 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
      961 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     1021 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1081 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1141 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     1201 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     1261 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     1321 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     1381 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     1441 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     1501 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     1561 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     1621 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     1681 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     1741 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     1801 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     1861 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     1921 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     1981 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     2041 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     2101 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     2161 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     2221 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     2281 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     2341 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     2401 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     2461 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     2521 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     2581 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
     2641 gcctggtgcc gcgcggcagc catatggaaa acctgtattt tcagggcgga tccgggaccc
     2701 tcaagttttg ccctttagaa aaagcccaga agttatggaa agaaaatcct tcagagattt
     2761 atccaataat gaatacaacc actcgtacac gtcttgccct cattatctgc aacacagagt
     2821 ttcaacatct ttctccgagg gttggagctc aagttgacct cagagaaatg aagttgctgc
     2881 tggaggatct ggggtatacc gtgaaagtga aagaaaatct cacagctctg gagatggtga
     2941 aagaggtgaa agaatttgct gcctgcccag agcacaagac ttctgacagt actttccttg
     3001 tattcatgtc tcatggtatc caggagggaa tatgtgggac cacatactct aatgaagttt
     3061 cagatatttt aaaggttgac acaatctttc agatgatgaa cactttgaag tgcccaagct
     3121 tgaaagacaa gcccaaggtg atcattattc aggcatgccg tggagagaaa caaggagtgg
     3181 tgttgttaaa agattcagta agagactctg aagaggattt cttaacggat gcaatttttg
     3241 aagatgatgg cattaagaag gcccatatag agaaagattt tattgctttc tgctcttcaa
     3301 caccagataa tgtgtcttgg agagatcctg tcaggggctc acttttcatt gagtcactca
     3361 tcaaacacat gaaagaatat gcctggtctt gtgacttgga ggacattttc agaaaggttc
     3421 gattttcatt tgaacaacca gaatttaggc tacagatgcc cactgctgat agggtgaccc
     3481 tgacaaaacg tttctacctc ttcccgggac attaagaatt cgagctccgt cgacaagctt
     3541 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     3601 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3661 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     3721 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3781 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3841 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3901 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3961 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4021 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4081 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4141 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     4201 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4261 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     4321 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     4381 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     4441 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     4501 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     4561 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     4621 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     4681 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     4741 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     4801 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     4861 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     4921 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     4981 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     5041 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     5101 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     5161 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5221 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5281 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5341 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5401 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5461 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5521 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5581 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5641 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5701 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5761 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5821 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5881 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5941 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     6001 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     6061 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     6121 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccg
//
