LOCUS       sequence-423170-        6178 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 6178)
  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 6178)
  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-11_humanized-mutant
FEATURES             Location/Qualifiers
     source          1..6178
                     /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(1029..1049)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1056..1073)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1083..1100)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1119..1141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1156..1180
                     /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(1180..1199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1181..1199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1283..1302)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1508..1585
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1586..2668
                     /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(1605..1624)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3477..3668
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3662..3684)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3770..3909
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3825..3844
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3924..3941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4095..4683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4175..4194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4794..4813)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4805..5620
                     /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             4805..5620
                     /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(4878..4897)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5712..6167)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5849..5870)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6061..6080
                     /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 gttgccagga aagaggtaga aatatcttgt caaggttgcc
      241 cgatcaatgg tgggcatctg agctttggcc cttggagttt caaatgattg ttgcaccttc
      301 aggaatatat caaagagatg acaagagcaa gcatgtttcc ggaagcagga aatgagtcta
      361 gtgatgaagt aagagcctcc tgttttgtct cggtaggaca agtgatgtgg ggttgtagag
      421 tagaaggcaa tgaagtcctt ctccacgtgg gttttataga cagcatcagc ttccatattc
      481 ctaggtagat ctacacctct gcacaactgg ggttttgaag actctctgat ccacatttct
      541 ccagagttcc cacctctgca ggcctgcaca atgatgactt tgggtttgtc tcgtagacct
      601 ggacagtggc aattgttgaa tatctgatag atggtatcat actgtagcac atctggagtt
      661 ttttcactgt gcattgttcc acaaatgcca tgcagtgtgc catgagacat tagcaccagg
      721 aatgtgctgt ctgatgtctg gtgttctgag agtgcagcaa agtctttcat ctctgactcc
      781 atgccctctg ctgtaagctc ctctttcacc accacatcgt agcctaagtc ttcaagaagg
      841 cctttcatac caatgatgtc aaatttagcc ccatacctca gtgagagatg tttgaactct
      901 gtattgcata tgataagagc ctttcgtgta cggccattgg cctcctttat tgggtaaatt
      961 tcttgtgtct tttctctgca aagccttgtg aactcttcag gggaacaaag cttgagagtg
     1021 ttggatccgc cctgaaaata caggttttcc atatggctgc cgcgcggcac caggccgctg
     1081 ctgtgatgat gatgatgatg gctgctgccc atggtatatc tccttcttaa agttaaacaa
     1141 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
     1201 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1261 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1321 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1381 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1441 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1501 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1561 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1621 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1681 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1741 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1801 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1861 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     1921 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     1981 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2041 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2101 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2161 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2221 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2281 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2341 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     2401 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2461 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2521 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2581 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2641 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     2701 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2761 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     2821 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     2881 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     2941 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3001 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3061 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3121 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3181 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3241 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3301 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3361 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3421 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3481 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     3541 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3601 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3661 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3721 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3781 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     3841 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3901 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3961 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4021 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4081 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4141 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4201 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4261 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4321 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4381 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4441 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4501 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4561 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4621 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4681 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4741 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     4801 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     4861 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     4921 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     4981 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     5041 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     5101 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     5161 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     5221 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     5281 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     5341 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     5401 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     5461 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     5521 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     5581 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat
     5641 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5701 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     5761 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     5821 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     5881 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     5941 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6001 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     6061 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     6121 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
