LOCUS       sequence-209497-        7107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mouse GSDMD in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7107)
  AUTHORS   Kambara H, Liu F, Zhang X, Liu P, Bajrami B, Teng Y, Zhao L, Zhou S,
            Yu H, Zhou W, Silberstein LE, Cheng T, Han M, Xu Y, Luo HR
  TITLE     Gasdermin D Exerts Anti-inflammatory Effects by Promoting Neutrophil
            Death.
  JOURNAL   Cell Rep. 2018 Mar 13;22(11):2924-2936. doi: 
            10.1016/j.celrep.2018.02.067.
  PUBMED    29539421
REFERENCE   2  (bases 1 to 7107)
  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: 
            "10.1016/j.celrep.2018.02"; journalName: "Cell Rep"; date: 
            "2018-03-13- 13"; volume: "22"; issue: "11"; pages: "2924-2936"
COMMENT     Sequence Label: pET-SUMO-mGSDMD
FEATURES             Location/Qualifiers
     source          1..7107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             309..326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             336..353
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             360..653
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2180..2197
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2250..2268)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2264..2311
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2348..2803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2435..2454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2645..2666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2895..3710)
                     /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             complement(2895..3710)
                     /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     3618..3637
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3702..3721
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3832..4420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4321..4340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4574..4591
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4606..4745
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4671..4690)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4831..4853
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4847..5038)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5847..6929)
                     /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     6891..6910
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6930..7007)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 gcagcagcca tcatcatcat catcacggca gcggcctggt gccgcgcggc agcgctagca
      361 tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg
      421 agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa
      481 agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg
      541 actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc cctgaagatt
      601 tggacatgga ggataacgat attattgagg ctcacagaga acagattggt ggtatgccat
      661 cggcctttga gaaagtggtc aagaatgtga tcaaggaggt aagcggcagc agaggcgatc
      721 tcattccggt ggacagcctg cggaactcca ccagcttcag gccctactgc cttctgaaca
      781 ggaaattttc aagctcaagg ttctggaaac cccgttattc atgtgtcaac ctgtcaatca
      841 aggacatcct ggagcccagt gctccagaac cagaaccgga gtgttttggc tccttcaaag
      901 tctctgatgt cgtcgatggg aacattcagg gcagagtgat gttgtcaggc atgggagaag
      961 ggaaaatttc tggtggggct gcagtgtctg acagttccag tgcctccatg aatgtgtgta
     1021 tactgcgtgt gactcagaag acctgggaga ccatgcagca tgaaaggcac cttcagcagc
     1081 ctgagaacaa aatcctgcaa cagcttcgga gtcgtgggga tgacctgttt gtggtgaccg
     1141 aggtgctgca gacaaaggag gaagtgcaga tcactgaggt ccacagccaa gagggctcag
     1201 gccagtttac gctgcctgga gctttatgct tgaagggtga aggcaagggc caccaaagcc
     1261 ggaagaagat ggtgaccatt cctgcaggca gcatcctggc attccgagtg gcccaactgc
     1321 ttattggctc taaatgggat atccttctcg tctcagatga gaaacagagg acctttgagc
     1381 cctcctcagg tgacagaaaa gcagtgggcc agaggcacca tggcctcaat gtgcttgctg
     1441 cgctttgttc catcggaaag cagctcagtc tcctgtcaga tgggattgat gaggaggaat
     1501 taattgaggc ggcagacttc cagggcctgt atgctgaggt gaaggcttgc tcctcagaac
     1561 tggagagctt ggaaatggag ttgagacaac agatactggt gaacatcgga aagattttac
     1621 aggaccagcc cagcatggaa gccttagagg cctcactagg gcagggcctg tgcagtggcg
     1681 gccaggtgga gcctctggac ggcccagctg gctgcatcct tgagtgtctg gtgcttgact
     1741 ctggagaact ggtgccggaa ctcgcagccc ctatcttcta cctgctggga gcactggctg
     1801 tgctgagtga aacccagcag cagctgctag ctaaggctct ggagacaacg gtgctgtcaa
     1861 agcagctgga gttggtgaag cacgtcttgg aacagagcac cccgtggcag gagcagagtt
     1921 ctgtgtccct gcccaccgtg ctccttgggg actgctggga tgaaaagaat cccacctggg
     1981 tcttgctaga agaatgtggc ctaaggctgc aggtagaatc cccccaggtg cactgggaac
     2041 caacgtctct gatccccaca agtgcgctct atgcctccct gttcctattg tcaagtctag
     2101 gccagaaacc ttgttagaga caagcttagg tatttattcg gcgcaaagtg cgtcgggtga
     2161 tgctgccaac ttagtcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     2221 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     2281 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     2341 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2401 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2461 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2521 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2581 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2641 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2701 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2761 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2821 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2881 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2941 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     3001 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     3061 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     3121 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     3181 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     3241 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     3301 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     3361 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     3421 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     3481 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     3541 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     3601 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     3661 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     3721 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     3781 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3841 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3901 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3961 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4021 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4081 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4141 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4201 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4261 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4321 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4381 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4441 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4501 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4561 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4621 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     4681 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4741 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4801 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4861 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4921 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4981 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     5041 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     5101 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     5161 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     5221 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     5281 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     5341 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     5401 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     5461 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     5521 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     5581 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     5641 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     5701 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     5761 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5821 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5881 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     5941 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     6001 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     6061 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     6121 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     6181 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     6241 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     6301 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     6361 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     6421 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     6481 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     6541 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     6601 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     6661 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6721 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     6781 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     6841 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     6901 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     6961 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     7021 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     7081 caccgccgcc gcaaggaatg gtgcatg
//
