LOCUS       sequence-423168-        6208 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 6208)
  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 6208)
  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_Caspase-4_W267N
FEATURES             Location/Qualifiers
     source          1..6208
                     /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(1059..1079)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1086..1103)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1113..1130)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1149..1171
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1186..1210
                     /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(1210..1229)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1211..1229)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1313..1332)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1538..1615
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1616..2698
                     /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(1635..1654)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3507..3698
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3692..3714)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3800..3939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3855..3874
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3954..3971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4125..4713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4205..4224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4824..4843)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4835..5650
                     /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             4835..5650
                     /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(4908..4927)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5742..6197)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5879..5900)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6091..6110
                     /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 cgaattctca attgccagga aagaggtaga aatatcttgt catggacagt
      241 cgttctatgg tgggcatttg agctttggcc cttggagttt caaatgattg ctgtaccttc
      301 cgaaatactt cctctaggtg gcagcaccaa gaatatttct ggaagcatgt gatgagttgt
      361 gtgatgaaga tagagcccat tgtgctgtct ctccaggaca cgttgtgtgg cgttgaagag
      421 cagaaagcaa tgaagtcctt ctccacgtgg gtcttgtaaa cagcatcttc ctccaggttc
      481 tcagatgact gtgaagaggc cacttccaag gatgctggag agtctctgac gttcagttcc
      541 ccacggtttg cacctctgca ggcctggaca atgatgacct tgggtttgtc cttcagactg
      601 aggcagttgc ggttgttgaa tatctggaag atggtgtcat aaagcagcac atctggtttt
      661 ttctcatcat gcacagttcc gcagattccc tccaggatgc catgagacat gagtaccaag
      721 aatgtgctgt cagaggactt gtgctctggt ctggtagcaa atgccctcag cgctgactcc
      781 atatccctgg ctgtcagatt ctcttctaca tctacactat agtccagacc ctcaagtagc
      841 tccttcatcc ctgtgatgtc aaagtcagct ccattcctcg gaggcagatg gtcaaactct
      901 gtattgcata tgatgagagc caggcgtgtg cggttgtttc tctcctttat tggatagatc
      961 tcttcagctc tttctttaca tagtctcagg aattcttcat gaggacaaag cttgagggca
     1021 tctgtagatt ctcctgactc aggtggtcca gcggatccgc cctgaaaata caggttttcc
     1081 atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg gctgctgccc
     1141 atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc
     1201 tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac
     1261 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
     1321 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
     1381 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
     1441 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     1501 atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac
     1561 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     1621 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     1681 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     1741 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     1801 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     1861 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     1921 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     1981 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     2041 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     2101 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     2161 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     2221 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     2281 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     2341 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     2401 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     2461 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     2521 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     2581 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     2641 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     2701 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     2761 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     2821 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     2881 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     2941 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     3001 tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc
     3061 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat
     3121 caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa
     3181 caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc
     3241 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac
     3301 ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc
     3361 gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat
     3421 cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca
     3481 gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg
     3541 cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg
     3601 atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct
     3661 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     3721 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     3781 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     3841 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa
     3901 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3961 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4021 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4081 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4141 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4201 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4261 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4321 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4381 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4441 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4501 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4561 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4621 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4681 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4741 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     4801 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     4861 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4921 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     4981 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5041 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     5101 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     5161 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     5221 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     5281 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     5341 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     5401 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     5461 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     5521 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5581 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5641 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     5701 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     5761 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5821 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     5881 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5941 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6001 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6061 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6121 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6181 gccgctacag ggcgcgtccc attcgcca
//
