LOCUS       sequence-361195-        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_enzymatic-domain
FEATURES             Location/Qualifiers
     source          1..6175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(959..2041)
                     /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     2003..2022
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2042..2119)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2325..2344
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2428..2447
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2428..2446
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2447..2471
                     /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             2486..2508
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2527..2544
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2554..2571
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2578..2598
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3467..3484
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3537..3555)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3551..3598
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3635..4090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3722..3741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3932..3953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4182..4997)
                     /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(4182..4997)
                     /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     4905..4924
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4989..5008
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5119..5707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5608..5627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5861..5878
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5893..6032
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5958..5977)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6118..6140
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6134..6175,1..150))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
       61 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
      121 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
      181 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
      241 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
      301 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
      361 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
      421 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
      481 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
      541 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
      601 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
      661 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
      721 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
      781 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
      841 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
      901 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
      961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1021 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     1081 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     1141 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     1201 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     1261 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     1321 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     1381 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     1441 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     1501 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     1561 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     1621 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     1681 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     1741 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     1801 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     1861 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1921 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1981 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     2041 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     2101 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     2161 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     2221 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     2281 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     2341 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     2401 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     2461 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     2521 agcagccatc atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggaa
     2581 aacctgtatt ttcagggcgg atccgggacc ctcaagtttt gccctttaga aaaagcccag
     2641 aagttatgga aagaaaatcc ttcagagatt tatccaataa tgaatacaac cactcgtaca
     2701 cgtcttgccc tcattatctg caacacagag tttcaacatc tttctccgag ggttggagct
     2761 caagttgacc tcagagaaat gaagttgctg ctggaggatc tggggtatac cgtgaaagtg
     2821 aaagaaaatc tcacagctct ggagatggtg aaagaggtga aagaatttgc tgcctgccca
     2881 gagcacaaga cttctgacag tactttcctt gtattcatgt ctcatggtat ccaggaggga
     2941 atatgtggga ccacatactc taatgaagtt tcagatattt taaaggttga cacaatcttt
     3001 cagatgatga acactttgaa gtgcccaagc ttgaaagaca agcccaaggt gatcattatt
     3061 caggcatgcc gtggagagaa acaaggagtg gtgttgttaa aagattcagt aagagactct
     3121 gaagaggatt tcttaacgga tgcaattttt gaagatgatg gcattaagaa ggcccatata
     3181 gagaaagatt ttattgcttt ctgctcttca acaccagata atgtgtcttg gagacatcct
     3241 gtcaggggct cacttttcat tgagtcactc atcaaacaca tgaaagaata tgcctggtct
     3301 tgtgacttgg aggacatttt cagaaaggtt cgattttcat ttgaacaacc agaatttagg
     3361 ctacagatgc ccactgctga tagggtgacc ctgacaaaac gtttctacct cttcccggga
     3421 cattaagaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     3481 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     3541 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     3601 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3661 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3721 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3781 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3841 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3901 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3961 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4021 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4081 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4141 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     4201 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     4261 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     4321 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     4381 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     4441 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     4501 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     4561 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     4621 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     4681 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     4741 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     4801 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     4861 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     4921 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     4981 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     5041 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5101 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5161 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5221 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5281 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5341 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5401 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5461 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5521 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5581 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5641 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5701 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5761 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5821 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5881 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5941 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6001 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6061 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6121 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagct
//
