LOCUS       sequence-361213-        6139 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 6139)
  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 6139)
  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-IL1B
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     138..160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(154..345)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1130..2212)
                     /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     2174..2193
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2213..2290)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2496..2515
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2599..2618
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2599..2617
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2618..2642
                     /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             2657..2679
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2698..2715
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2725..2742
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2749..2769
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3626..3643
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3696..3714)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3710..3757
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3794..4249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3881..3900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4091..4112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4341..5156)
                     /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(4341..5156)
                     /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     5064..5083
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5148..5167
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5278..5866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5767..5786
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6020..6037
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6052..6139,1..52)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6117..6136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
       61 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      121 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      181 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
      241 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
      301 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
      361 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
      421 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
      481 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
      541 tacagatgta ggtgttccac agggtagccg gaacataatg gtgcagggcg ctgacttccg
      601 cgtttccaga ctttacgaaa cacggaaacc gaagnccatt catgttgttg ctcaggtcgc
      661 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
      721 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
      781 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
      841 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
      901 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
      961 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     1021 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     1081 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     1141 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1201 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1261 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1321 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1381 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1441 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1501 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1561 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1621 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1681 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1741 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1801 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1861 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1921 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1981 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2041 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     2101 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     2161 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     2221 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     2281 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     2341 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     2401 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     2461 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     2521 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     2581 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     2641 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
     2701 catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatgga aaacctgtat
     2761 tttcagggcg gatctatggc aactgttcct gaactcaact gtgaaatgcc accttttgac
     2821 agtgatgaga atgacctgtt ctttgaagtt gacggacccc aaaagatgaa gggctgcttc
     2881 caaacctttg acctgggctg tccagatgag agcatccagc ttcaaatctc acagcagcac
     2941 atcaacaaga gcttcaggca ggcagtatca ctcattgtgg ctgtggagaa gctgtggcag
     3001 ctacctgtgt ctttcccgtg gaccttccag gatgaggaca tgagcacctt cttttccttc
     3061 atctttgaag aagagcccat cctctgtgac tcatgggatg atgatgataa cctgctggtg
     3121 tgtgacgttc ccattagaca gctgcactac aggctccgag atgaacaaca aaaaagcctc
     3181 gtgctgtcgg acccatatga gctgaaagct ctccacctca atggacagaa tatcaaccaa
     3241 caagtgatat tctccatgag ctttgtacaa ggagaaccaa gcaacgacaa aatacctgtg
     3301 gccttgggcc tcaaaggaaa gaatctatac ctgtcctgtg taatgaaaga cggcacaccc
     3361 accctgcagc tggagagtgt ggatcccaag caatacccaa agaagaagat ggaaaagcgg
     3421 tttgtcttca acaagataga agtcaagagc aaagtggagt ttgagtctgc agagttcccc
     3481 aactggtaca tcagcacctc acaagcagag cacaagcctg tcttcctggg aaacaacagt
     3541 ggtcaggaca taattgactt caccatggaa tccgtgtctt cctaagaatt cgagctccgt
     3601 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     3661 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     3721 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     3781 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3841 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3901 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3961 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4021 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4081 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4141 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4201 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4261 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4321 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     4381 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     4441 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     4501 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     4561 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     4621 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     4681 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     4741 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     4801 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     4861 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     4921 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     4981 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     5041 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     5101 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     5161 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     5221 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5281 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5341 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5401 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5461 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5521 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5581 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5641 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5701 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5761 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5821 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5881 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5941 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6001 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     6061 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     6121 atctgctctg atgccgcat
//
