LOCUS       sequence-423151-        5862 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 5862)
  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 5862)
  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_p20_C258A
FEATURES             Location/Qualifiers
     source          1..5862
                     /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(713..733)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(740..757)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(767..784)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             803..825
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    840..864
                     /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(864..883)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(865..883)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(967..986)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1192..1269
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1270..2352
                     /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(1289..1308)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3161..3352
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3346..3368)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3454..3593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3509..3528
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3608..3625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3779..4367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3859..3878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4478..4497)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4489..5304
                     /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             4489..5304
                     /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(4562..4581)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5396..5851)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5533..5554)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5745..5764
                     /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 gctcagtctc
      181 tgacccacag ttccccacgg tttgcacctc ttgcggcctg gacaatgatg accttgggtt
      241 tgtccttcag actgaggcag ttgcggttgt tgaatatctg gaagatggtg tcataaagca
      301 gcacatctgg ttttttctca tcatgcacag ttccgcagat tccctccagg atgccatgag
      361 acatgagtac caagaatgtg ctgtcagagg acttgtgctc tggtctggta gcaaatgccc
      421 tcagcgctga ctccatatcc ctggctgtca gattctcttc tacatctaca ctatagtcca
      481 gaccctcaag tagctccttc atccctgtga tgtcaaagtc agctccattc ctcggaggca
      541 gatggtcaaa ctctgtattg catatgatga gagccaggcg tgtgcggttg tttctctcct
      601 ttattggata gatctcttca gctctttctt tacatagtct caggaattct tcatgaggac
      661 aaagcttgag ggcatctgta gattctcctg actcaggtgg tccagcggat ccgccctgaa
      721 aatacaggtt ttccatatgg ctgccgcgcg gcaccaggcc gctgctgtga tgatgatgat
      781 gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg
      841 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
      901 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
      961 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1021 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1081 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1141 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     1201 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1261 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     1321 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     1381 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     1441 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     1501 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     1561 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     1621 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     1681 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     1741 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     1801 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     1861 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     1921 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     1981 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     2041 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     2101 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     2161 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     2221 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     2281 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2341 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     2401 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     2461 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     2521 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     2581 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     2641 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     2701 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     2761 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     2821 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     2881 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     2941 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     3001 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     3061 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     3121 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     3181 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     3241 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     3301 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3361 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3421 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     3481 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     3541 cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     3601 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3661 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3721 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3781 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3841 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3901 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3961 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4021 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4081 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4141 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4201 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4261 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4321 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4381 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4441 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     4501 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     4561 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     4621 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     4681 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     4741 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     4801 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     4861 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     4921 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     4981 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     5041 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     5101 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     5161 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     5221 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     5281 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     5341 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     5401 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5461 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5521 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5581 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5641 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5701 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5761 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5821 gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
