LOCUS       sequence-423185-        5932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein overexpression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5932)
  AUTHORS   Devant P, Dong Y, Mintseris J, Ma W, Gygi SP, Wu H, Kagan JC
  TITLE     Structural insights into cytokine cleavage by inflammatory 
            caspase-4.
  JOURNAL   Nature. 2023 Dec;624(7991):451-459. doi: 10.1038/s41586-023-06751-9.
            Epub 2023 Nov 22.
  PUBMED    37993712
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"; doi: 
            "10.1038/s41586-023-06751-9"; journalName: "Nature"; date: 
            "2023-12"; volume: "624"; issue: "7991"; pages: "451-459"
COMMENT     Sequence Label: pET28a_mouse-Pro-IL-18_humanized-triple-mutant
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(783..803)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(810..827)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(837..854)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             873..895
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    910..934
                     /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)."
     primer_bind     complement(934..953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(935..953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1037..1056)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1262..1339
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1340..2422
                     /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     complement(1359..1378)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3231..3422
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3416..3438)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3524..3663
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3579..3598
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3678..3695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3849..4437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3929..3948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4548..4567)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4559..5374
                     /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             4559..5374
                     /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     complement(4632..4651)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5466..5921)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5603..5624)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5815..5834
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattccta actttgatgt aagttagtga gagtgaacat tacagattta
      241 tccccatttt catccttttt tttcagaatg agtttgaaag catcatcttc cttttggcaa
      301 gcaagaaagt gtccttcata cagtgaagat tcaaactcca tcttgttgtg tcctggaaca
      361 cgtttctgaa agaatatgag atcactttgt atatcatcaa tattttcagg tggatccatt
      421 tcctcaaagg aaatgatctt gttcttacag gagagggtag acattttact atccttcaca
      481 gagagggtca cagccagtcc tcttacttca ctgtctttgt acatgtatat tatcagtctg
      541 gtctggggtt cactggcact ttgatcaata tcagtcatat cctcgaacac aggctgtctt
      601 ttgtcaacga agagaacttg gtcatttata ttccgtatta ctgcggttgt acactcaagt
      661 cggccaaagt agtctgattc caggttttcg tcgtcttcag gtataaagta aagcgtgttg
      721 tcaataaaca tcatttcctt gaagttgacg caagagtctt ctgacatggc agccatggat
      781 ccgccctgaa aatacaggtt ttccatatgg ctgccgcgcg gcaccaggcc gctgctgtga
      841 tgatgatgat gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat
      901 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
      961 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1021 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1081 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     1141 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     1201 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     1261 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     1321 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1381 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1441 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1501 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1561 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1621 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1681 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1741 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1801 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1861 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1921 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1981 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     2041 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     2101 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     2161 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     2221 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     2281 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     2341 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2401 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2461 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2521 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2581 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2641 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2701 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2761 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     2821 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     2881 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     2941 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3001 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3061 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3121 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     3181 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     3241 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     3301 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3361 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3421 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3481 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3541 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3601 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3661 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3721 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3781 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4261 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4501 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4561 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4621 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4681 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4741 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4801 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4861 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     4921 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     4981 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5041 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5101 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5161 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5221 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5281 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5341 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5461 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5521 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5581 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5641 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5701 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5761 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5821 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5881 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
