LOCUS       sequence-423146-        5962 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 5962)
  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 5962)
  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_Pro-IL-18_rabbit
FEATURES             Location/Qualifiers
     source          1..5962
                     /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(201..230)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(813..833)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(840..857)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(867..884)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             903..925
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    940..964
                     /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(964..983)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(965..983)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1067..1086)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1292..1369
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1370..2452
                     /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(1389..1408)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3261..3452
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3446..3468)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3554..3693
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3609..3628
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3708..3725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3879..4467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3959..3978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4578..4597)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4589..5404
                     /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             4589..5404
                     /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(4662..4681)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5496..5951)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5633..5654)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5845..5864
                     /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 cagatcctct tctgagatga gtttttgttc attcttgttt
      241 tggacactga aaattacaga tttatcgttt ttgtcatctt tacgtttcaa aacaagtttg
      301 aaaagatctt tctcctttcc acaagctaga aagtatccgt catacaatga agactcaaac
      361 tgcatcttat ctttctgtcc tgcgagatgt ttctgaaaga atatgatgtc actttttgta
      421 tcgttgatgt cattaggagg agtgagttcc ttaaaggaaa taattttgtt ctcacaggag
      481 agtgtaaaaa ttttcttaca cttcacagag aggcttacag ccatgcctct agtattgctg
      541 tctttataca tatatattat aaaaatggtc tggggtgcat tttctataca gtcagaatcg
      601 ggcatatcct caaacacagg ttgctgtcct tggtccatga agagaacttg gtcattcagg
      661 tttcgtatga tggagatttt cggttcaagc ttgccaaagt aatctgattc caggttctca
      721 tcgttttcag ttataaagta aagtgtgttg tcaataaatt tcattcccac aaagttgatg
      781 caagcctctt ctggttcagc agccatggat ccgccctgaa aatacaggtt ttccatatgg
      841 ctgccgcgcg gcaccaggcc gctgctgtga tgatgatgat gatggctgct gcccatggta
      901 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
      961 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
     1021 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1081 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     1141 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     1201 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     1261 gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg
     1321 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     1381 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     1441 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     1501 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     1561 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     1621 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     1681 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     1741 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     1801 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     1861 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     1921 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     1981 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     2041 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     2101 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     2161 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     2221 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     2281 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     2341 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     2401 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     2461 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
     2521 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2581 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     2641 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     2701 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     2761 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
     2821 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     2881 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     2941 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3001 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3061 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3121 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3181 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     3241 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     3301 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     3361 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     3421 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3481 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3541 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     3601 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     3661 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     4321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc
     4561 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc
     4621 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     4681 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     4741 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     4801 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     4861 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     4921 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     4981 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     5041 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     5101 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     5161 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     5221 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     5281 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     5341 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     5401 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5461 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     5521 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5581 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     5641 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5701 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     5761 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     5821 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     5881 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     5941 acagggcgcg tcccattcgc ca
//
