LOCUS       sequence-361186-        6208 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 6208)
  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 6208)
  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_human-Caspase-4_enzymatic-domain
FEATURES             Location/Qualifiers
     source          1..6208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             463..480
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(533..551)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      547..594
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      631..1086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(718..737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     928..949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1178..1993)
                     /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(1178..1993)
                     /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     1901..1920
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1985..2004
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2115..2703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2604..2623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2857..2874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2889..3028
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2954..2973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3114..3136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3130..3321)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4130..5212)
                     /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     5174..5193
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5213..5290)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5496..5515
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5599..5618
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5599..5617
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5618..5642
                     /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             5657..5679
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5698..5715
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5725..5742
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5749..5769
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 tcaacaaccg caactgcctc agtctgaagg acaaacccaa ggtcatcatt gtccaggcct
       61 gcagaggtgc aaaccgtggg gaactgtggg tcagagactc tccagcatcc ttggaagtgg
      121 cctcttcaca gtcatctgag aacctggagg aagatgctgt ttacaagacc cacgtggaga
      181 aggacttcat tgctttctgc tcttcaacgc cacacaacgt gtcctggaga gacagcacaa
      241 tgggctctat cttcatcaca caactcatca catgcttcca gaaatattct tggtgctgcc
      301 acctagagga agtatttcgg aaggtacagc aatcatttga aactccaagg gccaaagctc
      361 aaatgcccac catagaacga ctgtccatga caagatattt ctacctcttt cctggcaatt
      421 gagaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac
      481 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      541 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      601 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      661 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      721 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      781 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      841 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      901 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      961 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1021 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1081 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1141 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1201 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1261 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1321 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1381 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1441 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1501 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1561 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1621 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1681 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1741 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1801 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1861 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1921 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     1981 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2041 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2101 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2161 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2221 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2281 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2341 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2401 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2461 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2521 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2581 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2641 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2701 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2761 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2821 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2881 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     2941 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3001 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3061 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3121 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3181 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3241 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3301 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3361 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3421 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3481 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     3541 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     3601 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     3661 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     3721 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     3781 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     3841 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     3901 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3961 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4021 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4081 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4141 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4201 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4261 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4321 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4381 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4441 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4501 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4561 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4621 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4681 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4741 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4801 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4861 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4921 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4981 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5041 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5101 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5161 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5221 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5281 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5341 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5401 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5461 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5521 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     5581 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     5641 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
     5701 catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatgga aaacctgtat
     5761 tttcagggcg gatccgctgg accacctgag tcaggagaat ctacagatgc cctcaagctt
     5821 tgtcctcatg aagaattcct gagactatgt aaagaaagag ctgaagagat ctatccaata
     5881 aaggagagaa acaaccgcac acgcctggct ctcatcatat gcaatacaga gtttgaccat
     5941 ctgcctccga ggaatggagc tgactttgac atcacaggga tgaaggagct acttgagggt
     6001 ctggactata gtgtagatgt agaagagaat ctgacagcca gggatatgga gtcagcgctg
     6061 agggcatttg ctaccagacc agagcacaag tcctctgaca gcacattctt ggtactcatg
     6121 tctcatggca tcctggaggg aatctgcgga actgtgcatg atgagaaaaa accagatgtg
     6181 ctgctttatg acaccatctt ccagatat
//
