LOCUS       sequence-361212-        5935 bp    DNA     circular SYN 14-APR-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 5935)
  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 5935)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-pro-IL18
FEATURES             Location/Qualifiers
     source          1..5935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(934..2016)
                     /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     1978..1997
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2017..2094)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2300..2319
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2403..2422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2403..2421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2422..2446
                     /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             2461..2483
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2502..2519
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2529..2546
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2553..2573
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3202..3219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3272..3290)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3286..3333
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3370..3825
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3457..3476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3667..3688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3917..4732)
                     /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(3917..4732)
                     /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     4640..4659
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4724..4743
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4854..5442
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5343..5362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5596..5613
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5628..5767
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5693..5712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5853..5875
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(5869..5935,1..125))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
       61 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
      121 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
      181 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
      241 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
      301 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
      361 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
      421 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
      481 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
      541 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
      601 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
      661 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
      721 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
      781 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      841 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
      901 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
      961 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1021 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     1081 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     1141 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     1201 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     1261 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     1321 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     1381 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     1441 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     1501 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     1561 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     1621 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     1681 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     1741 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     1801 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     1861 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     1921 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     1981 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     2041 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     2101 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     2161 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     2221 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     2281 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     2341 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     2401 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     2461 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
     2521 gcagcggcct ggtgccgcgc ggcagccata tggaaaacct gtattttcag ggcggatcca
     2581 tggctgctga accagtagaa gacaattgca tcaactttgt ggcaatgaaa tttattgaca
     2641 atacgcttta ctttatagct gaagatgatg aaaacctgga atcagattac tttggcaagc
     2701 ttgaatctaa attatcagtc ataagaaatt tgaatgacca agttctcttc attgaccaag
     2761 gaaatcggcc tctatttgaa gatatgactg attctgactg tagagataat gcaccccgga
     2821 ccatatttat tataagtatg tataaagata gccagcctag aggtatggct gtaactatct
     2881 ctgtgaagtg tgagaaaatt tcaactctct cctgtgagaa caaaattatt tcctttaagg
     2941 aaatgaatcc tcctgataac atcaaggata caaaaagtga catcatattc tttcagagaa
     3001 gtgtcccagg acatgataat aagatgcaat ttgaatcttc atcatacgaa ggatactttc
     3061 tagcttgtga aaaagagaga gaccttttta aactcatttt gaaaaaagag gatgaattgg
     3121 gggatagatc tataatgttc actgttcaaa acgaagacta ggaattcgag ctccgtcgac
     3181 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     3241 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     3301 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     3361 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3421 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3481 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3541 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3601 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3661 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3721 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3781 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3841 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3901 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     3961 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     4021 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     4081 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     4141 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     4201 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     4261 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     4321 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     4381 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     4441 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     4501 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     4561 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     4621 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     4681 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     4741 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     4801 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4861 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4921 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4981 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5041 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5101 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5161 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5221 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5281 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5341 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5401 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5461 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5521 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5581 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     5641 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     5701 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     5761 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5821 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5881 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcg
//
