LOCUS       sequence-361217-        6300 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 6300)
  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 6300)
  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_redesigned_Caspase-4_ANR261ENP_D315H_M318K_T352K_enzymatic 
            domain
FEATURES             Location/Qualifiers
     source          1..6300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(993..2075)
                     /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     2037..2056
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2076..2153)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2359..2378
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2462..2481
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2462..2480
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2481..2505
                     /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             2520..2542
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2561..2578
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2588..2605
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2612..2632
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3534..3551
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3604..3622)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3618..3665
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3702..4157
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3789..3808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3999..4020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4249..5064)
                     /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(4249..5064)
                     /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     4972..4991
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5056..5075
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5186..5774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5675..5694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5928..5945
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5960..6099
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6025..6044)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6185..6207
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
       61 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
      121 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
      181 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
      241 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
      301 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
      361 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
      421 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
      481 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
      541 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
      601 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
      661 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
      721 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
      781 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
      841 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
      901 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
      961 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1021 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1081 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     1141 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     1201 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     1261 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     1321 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     1381 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     1441 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     1501 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     1561 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     1621 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     1681 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     1741 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     1801 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     1861 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     1921 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     1981 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     2041 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     2101 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     2161 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     2221 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     2281 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     2341 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     2401 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     2461 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
     2521 ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc atcatcacag
     2581 cagcggcctg gtgccgcgcg gcagccatat ggaaaacctg tattttcagg gcggatccgc
     2641 tggaccacct gagtcaggag aatctacaga tgccctcaag ctttgtcctc atgaagaatt
     2701 cctgagacta tgtaaagaaa gagctgaaga gatctatcca ataaaggaga gaaacaaccg
     2761 cacacgcctg gctctcatca tatgcaatac agagtttgac catctgcctc cgaggaatgg
     2821 agctgacttt gacatcacag ggatgaagga gctacttgag ggtctggact atagtgtaga
     2881 tgtagaagag aatctgacag ccagggatat ggagtcagcg ctgagggcat ttgctaccag
     2941 accagagcac aagtcctctg acagcacatt cttggtactc atgtctcatg gcatcctgga
     3001 gggaatctgc ggaactgtgc atgatgagaa aaaaccagat gtgctgcttt atgacaccat
     3061 cttccagata ttcaacaacc gcaactgcct cagtctgaag gacaaaccca aggtcatcat
     3121 tgtccaggcc tgcagaggtg aaaacccggg ggaactgtgg gtcagagact ctccagcatc
     3181 cttggaagtg gcctcttcac agtcatctga gaacctggag gaagatgctg tttacaagac
     3241 ccacgtggag aaggacttca ttgctttctg ctcttcaacg ccacacaacg tgtcctggag
     3301 acacagcaca aaaggctcta tcttcatcac acaactcatc acatgcttcc agaaatattc
     3361 ttggtgctgc cacctagagg aagtatttcg gaaggtacag caatcatttg aaaaaccaag
     3421 ggccaaagct caaatgccca ccatagaacg actgtccatg acaagatatt tctacctctt
     3481 tcctggcaat tgagaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     3541 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     3601 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3661 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     3721 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3781 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3841 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3901 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3961 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4021 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4081 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4141 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4201 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     4261 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     4321 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     4381 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     4441 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     4501 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     4561 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     4621 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     4681 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     4741 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     4801 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     4861 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     4921 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     4981 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     5041 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     5101 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5161 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5221 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5281 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5341 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5401 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5461 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5521 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5581 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5641 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5701 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5761 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5821 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5881 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5941 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     6001 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     6061 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     6121 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     6181 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     6241 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
//
