LOCUS       sequence-361198-        5932 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 5932)
  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 5932)
  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-IL18
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..161
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(155..346)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1155..2237)
                     /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     2199..2218
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2238..2315)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2521..2540
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2624..2643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2624..2642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2643..2667
                     /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             2682..2704
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2723..2740
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2750..2767
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2774..2794
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3420..3437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3490..3508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3504..3551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3588..4043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3675..3694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3885..3906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4135..4950)
                     /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(4135..4950)
                     /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     4858..4877
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4942..4961
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5072..5660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5561..5580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5814..5831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(5846..5932,1..53)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5911..5930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
       61 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
      121 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
      181 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
      241 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
      301 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
      361 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
      421 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
      481 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
      541 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
      601 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
      661 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
      721 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
      781 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
      841 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
      901 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
      961 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     1021 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     1081 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     1141 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1201 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     1261 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     1321 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     1381 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     1441 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     1501 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     1561 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     1621 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     1681 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     1741 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     1801 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     1861 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     1921 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     1981 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     2041 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     2101 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     2161 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     2221 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     2281 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     2341 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     2401 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     2461 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     2521 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     2581 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     2641 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     2701 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
     2761 cggcagccat atggaaaacc tgtattttca gggcggatcc atggctgcca tgtcagaaga
     2821 ctcttgcgtc aacttcaagg aaatgatgtt tattgacaac acgctttact ttatacctga
     2881 agaaaatgga gacctggaat cagacaactt tggccgactt cactgtacaa ccgcagtaat
     2941 acggaatata aatgaccaag ttctcttcgt tgacaaaaga cagcctgtgt tcgaggatat
     3001 gactgatatt gatcaaagtg ccagtgaacc ccagaccaga ctgataatat acatgtacaa
     3061 agacagtgaa gtaagaggac tggctgtgac cctctctgtg aaggatagta aaatgtctac
     3121 cctctcctgt aagaacaaga tcatttcctt tgaggaaatg gatccacctg aaaatattga
     3181 tgatatacaa agtgatctca tattctttca gaaacgtgtt ccaggacaca acaagatgga
     3241 gtttgaatct tcactgtatg aaggacactt tcttgcttgc caaaaggaag atgatgcttt
     3301 caaactcatt ctgaaaaaaa aggatgaaaa tggggataaa tctgtaatgt tcactctcac
     3361 taacttacat caaagttagg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc
     3421 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     3481 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3541 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     3601 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3661 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3721 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3781 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3841 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3901 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3961 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4021 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     4141 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     4201 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     4261 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     4321 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     4381 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     4441 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4501 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4561 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4621 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     4681 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4741 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4801 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     4861 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     4921 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     4981 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5041 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5101 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5161 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     5221 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5281 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5341 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5401 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5461 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5521 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5581 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5641 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5701 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5761 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5821 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5881 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg ca
//
