LOCUS       sequence-423175-        5863 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 5863)
  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 5863)
  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: pET28_human-pro-IL-18_DeltaN24
FEATURES             Location/Qualifiers
     source          1..5863
                     /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(714..734)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(741..758)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(768..785)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             804..826
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    841..865
                     /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(865..884)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(866..884)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(968..987)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1193..1270
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1271..2353
                     /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(1290..1309)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3162..3353
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3347..3369)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3455..3594
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3510..3529
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3609..3626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3780..4368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3860..3879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4479..4498)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4490..5305
                     /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             4490..5305
                     /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(4563..4582)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5397..5852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5534..5555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5746..5765
                     /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 gtcttcgttt tgaacagtga acattataga tctatccccc
      241 aattcatcct cttttttcaa aatgagttta aaaaggtctc tctctttttc acaagctaga
      301 aagtatcctt cgtatgatga agattcaaat tgcatcttat tatcatgtcc tgggacactt
      361 ctctgaaaga atatgatgtc actttttgta tccttgatgt tatcaggagg attcatttcc
      421 ttaaaggaaa taattttgtt ctcacaggag agagttgaaa ttttctcaca cttcacagag
      481 atagttacag ccatacctct aggctggcta tctttataca tacttataat aaatatggtc
      541 cggggtgcat tatctctaca gtcagaatca gtcatatctt caaatagagg ccgatttcct
      601 tggtcaatga agagaacttg gtcattcaaa tttcttatga ctgataattt agattcaagc
      661 ttgccaaagt aatctgattc caggttttca tcatcttcag ctataaagga tccgccctga
      721 aaatacaggt tttccatatg gctgccgcgc ggcaccaggc cgctgctgtg atgatgatga
      781 tgatggctgc tgcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg
      841 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
      901 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
      961 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1021 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1081 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     1141 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     1201 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     1261 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     1321 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1381 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1441 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1501 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1561 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1621 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     1681 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     1741 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     1801 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     1861 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     1921 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     1981 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     2041 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     2101 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     2161 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     2221 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     2281 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2341 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     2401 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2461 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     2521 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     2581 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     2641 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     2701 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     2761 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     2821 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     2881 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     2941 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     3001 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     3061 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     3121 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     3181 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     3241 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     3301 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3361 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3421 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3481 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3541 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3601 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3661 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3721 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3781 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3841 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3901 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3961 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4021 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4081 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4141 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4201 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4261 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4321 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4381 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4441 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     4501 tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata
     4561 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     4621 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     4681 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     4741 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     4801 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     4861 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     4921 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     4981 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     5041 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     5101 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     5161 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     5221 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     5281 tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta
     5341 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     5401 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5461 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5521 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5581 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5641 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5701 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5761 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5821 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
