LOCUS       sequence-426860-        6183 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 6183)
  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 6183)
  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_Caspase-4_sheep_catalytic domain
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(20..67)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     63..81
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(134..151)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1028..1048)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1055..1072)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1082..1099)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1118..1140
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1155..1179
                     /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(1179..1198)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1180..1198)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1282..1301)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1507..1584
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1585..2667
                     /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(1604..1623)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3476..3667
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3661..3683)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3769..3908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3824..3843
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3923..3940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4094..4682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4174..4193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4793..4812)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4804..5619
                     /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             4804..5619
                     /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(4877..4896)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5711..6166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5848..5869)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6060..6079
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt
       61 atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag
      121 cagccggatc tcagtggtgg tggtggtggt gctcgagtgc ggccgctcag ttgccaggaa
      181 agaggtagaa atatcttgtc atagagagtc tttcaacagt gggcatctgg gctttaacat
      241 ttggtttttc aaatgattgt tgtaccttcc taaatacttc ctcaaggtga cagcaccaag
      301 catatttttg gaagcatgtg atgagtcttg taatgaagag agaacctttt ttgatgtctc
      361 tccaggacac attatgtgga gttgaggaac agaaagcaat gaagtccttc tccacgtggg
      421 tcttgtaaac agcatcctct tccaggttct cgggtgactg tgaaaagctg tctgccaagg
      481 ctgctggaga gtcactgacc cacaattccc cacgatttgc acctctgcag gcctggacaa
      541 tgatgacttt aggtttgtcc tttagactga ggcaattacg gttgttgaat gtccggaaga
      601 tggtgtcata aggtaataca tctggttcct catctctgtg catagtccca cagatcccat
      661 ccaggatgcc atgagacatg aataccaaga atgtactgtc tgaggatttg tgctcttcac
      721 gtgcagcaaa tttccacagc actgattcca tgtctctggc tgtgagtttc tcttccactt
      781 ccacagtgta gccaagaccc tcaagcagct gcttcattcc aagaatgtca aggtcagccc
      841 cattcctggg aggcagatga tcaaactctg tattgcatat gatgagagcc agacgagtgc
      901 ggtcctttct ttcttttatt ggatatatct ctccagccct ttctttacac agtttcagga
      961 attcttcatg agggcaaagc ttgagggtag cggcagatcc tgctgactca tctggtccag
     1021 cggatccgcc ctgaaaatac aggttttcca tatggctgcc gcgcggcacc aggccgctgc
     1081 tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa
     1141 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     1201 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     1261 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     1321 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     1381 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     1441 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     1501 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     1561 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     1621 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     1681 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     1741 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     1801 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     1861 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     1921 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     1981 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     2041 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     2101 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     2161 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     2221 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     2281 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     2341 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     2401 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     2461 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     2521 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     2581 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2641 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     2701 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     2761 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2821 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     2881 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     2941 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     3001 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     3061 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     3121 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     3181 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     3241 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     3301 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     3361 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     3421 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     3481 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     3541 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     3601 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3661 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3721 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3781 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3841 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3901 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3961 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4021 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4081 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4141 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4201 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4261 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4321 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4381 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4441 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4501 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4561 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4621 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4681 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4741 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     4801 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     4861 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     4921 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     4981 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     5041 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     5101 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     5161 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     5221 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     5281 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     5341 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     5401 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     5461 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     5521 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     5581 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     5641 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5701 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5761 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5821 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5881 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5941 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6001 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     6061 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     6121 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc
     6181 cgg
//
