LOCUS       sequence-423144-        5935 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 5935)
  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 5935)
  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_dog
FEATURES             Location/Qualifiers
     source          1..5935
                     /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(786..806)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(813..830)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(840..857)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             876..898
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    913..937
                     /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(937..956)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(938..956)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1040..1059)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1265..1342
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1343..2425
                     /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(1362..1381)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3234..3425
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3419..3441)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3527..3666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3582..3601
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3681..3698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3852..4440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3932..3951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4551..4570)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4562..5377
                     /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             4562..5377
                     /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(4635..4654)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5469..5924)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5606..5627)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5818..5837
                     /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 gctcttgttt tgaacagtga acattatgga tttatcccca
      241 ttttcatcct tgtctttcaa aatgagtttg aaaagatcgt tctctttttt acaagctaga
      301 aagtgtcctt tgtacaatga agactcaaat tgtatcttat catcatggcc tggaacactt
      361 ctctgaaaga atatgatgtc atttccttca tcattgatac tatccggagg actcattttc
      421 tgaaaggaaa tagttttgtt cttacaggag agagtagaca ttgtcttata cttcacagag
      481 atagttactg ccagacctct agtgaggcta tctttataca tatagatgat aaatatggta
      541 tggggtgcat tatctgtaca gtcagaatcg ggcatatcct caaatacagg ttgatttccc
      601 tcgttaacga agaggacttg gtcgttcaaa tttcgtatga ttgagagttt aggttcaagc
      661 ttgccaaagt aatctgattc caggccttca tcactttccg ctttaaagta cagtgtattg
      721 ttaacaaatt tcattttcac aaggttgatg caattgtctt ctattaggtt agcagccatg
      781 gatccgccct gaaaatacag gttttccata tggctgccgc gcggcaccag gccgctgctg
      841 tgatgatgat gatgatggct gctgcccatg gtatatctcc ttcttaaagt taaacaaaat
      901 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
      961 cgcgggatcg agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     1021 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     1081 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     1141 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     1201 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
     1261 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     1321 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     1381 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     1441 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     1501 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     1561 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     1621 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     1681 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     1741 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     1801 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     1861 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     1921 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     1981 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     2041 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     2101 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     2161 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     2221 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     2281 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     2341 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2401 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     2461 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     2521 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     2581 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     2641 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     2701 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     2761 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     2821 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     2881 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     2941 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     3001 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     3061 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     3121 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     3181 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     3241 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     3301 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     3361 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     3421 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     3481 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     3541 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     3601 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3661 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3721 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3781 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3961 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4021 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4081 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4141 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4201 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4261 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     4321 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4381 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4441 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4501 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     4561 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     4621 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     4681 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     4741 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     4801 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     4861 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     4921 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     4981 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     5041 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     5101 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     5161 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     5221 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     5281 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     5341 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     5401 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5461 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5521 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5581 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5641 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5701 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5761 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5821 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5881 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
