LOCUS       sequence-440273-        6732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of a mutant bacterial gasdermin (bGSDM) encoded by a 
            Bacteroidetes species with a TEV protease cleavage site for 
            controlled activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6732)
  AUTHORS   Johnson AG, Mayer ML, Schaefer SL, McNamara-Bordewick NK, Hummer G, 
            Kranzusch PJ
  TITLE     Structure and assembly of a bacterial gasdermin pore.
  JOURNAL   Nature. 2024 Apr;628(8008):657-663. doi: 10.1038/s41586-024-07216-3.
            Epub 2024 Mar 20.
  PUBMED    38509367
REFERENCE   2  (bases 1 to 6732)
  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/s41586-024-07216-3"; journalName: "Nature"; date: 
            "2024-04"; volume: "628"; issue: "8008"; pages: "657-663"
COMMENT     Sequence Label: pETSUMO2_Bacteroidetes-bGSDM-TEV
FEATURES             Location/Qualifiers
     source          1..6732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..35)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        103..207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(208..276,277..1068)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(426..445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1239..1827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1728..1747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1981..1998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2013..2152
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2078..2097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2238..2260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2254..2445)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4800..4819
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4839..4916)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5122..5141
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5225..5244
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5225..5243
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5244..5268
                     /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             5284..5306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5326..5343
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6328..6348
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(6458..6476)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6472..6519
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6694..6699,6700..6716,6717..6722))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
       61 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      121 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      181 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
      241 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      301 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      361 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
      421 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
      481 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      541 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
      601 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      661 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      721 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
      781 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
      841 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
      901 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
      961 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1021 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1081 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1141 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1201 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1261 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1381 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     1861 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     1921 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     1981 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2041 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2101 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2161 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2221 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2281 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2341 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2401 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2461 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2521 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2581 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2641 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2701 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     2761 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     2821 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     2881 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     2941 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     3001 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
     3061 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
     3121 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
     3181 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg
     3241 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
     3301 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
     3361 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
     3421 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3481 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3541 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3601 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     3661 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3721 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3781 aactgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3901 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3961 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4021 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4081 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4141 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4201 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4261 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4321 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4381 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4441 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4501 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4561 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4621 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4681 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4741 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4801 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4861 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4921 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4981 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5041 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5101 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5161 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     5221 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagagagc
     5281 tcttttgttt aactttaaga aggagatata catatgggtt cttctcacca tcaccatcac
     5341 catggttcag gagtcaagac tgagaacaac gatcatatta atttgaaggt ggcggggcag
     5401 gatggttctg tggtgcagtt taagattaag aggcatacac cacttagtaa actaatgaaa
     5461 gcctattgtg aacgacaggg attgtcaatg aggcagatca gattccgatt tgacgggcaa
     5521 ccaatcaatg aaacagacac acctgcacag ttggaaatgg aggatgaaga tacaattgat
     5581 gtgtttcaac agcagacggg tggatccatc aaatatctgc agagccatct ggaagaacag
     5641 ggttacctgt ttgttaccct gccgaaaccg gatctggcac cgctgcagct gctgaccgaa
     5701 tataaaggtc acctggaaga atatgatggt agcctgctgg acctgtttga accggatggt
     5761 agtccgtttc cgattcgtga tcgtcagctg ccgaatttta gtggtcagca actgctgcag
     5821 accgattgga gtgccggtgc cgatctgctg catggtctgt ttaaactgtt tcagcagaaa
     5881 gaggataaac tgaaagcaag cctgagcggt atgaaaggtc tggttctgag ctttgcctat
     5941 gaaaacattg aagaagaacg cgttagcgaa caggcactgg ataattttct ggcaggcgca
     6001 atgccgaaga aagaaggttt tcagcgtagc gttgaacgtc tgcaggatgg tgaactgtat
     6061 gttctgacca gcgttatgcg tagcaatcag tttaccgtta ccattgattg tcagcgtgaa
     6121 gatcagggta aactggaagc agccgttgca gaaattgttg atgcacatgc aagcattgaa
     6181 cgcaaacaga gcaatagctt tagcctgcag acagaaggtg aacaggcatt tgtttttgca
     6241 tgtcgtgcag cacaggttct gtataataag aaacagtggt ttcagttctg gaaaaaggat
     6301 aaagatggct tccgtatcga aaaacgcgaa aacctgtatt ttcagggcga agaagatttt
     6361 agcgttcagc cgctgcaggc accgagtggt ttactgaaac tgtgataagc ggccgctgct
     6421 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     6481 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     6541 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     6601 gggtgacggt gccgaggatg acgatgagtg cattgttaga tttcatacac ggtgcctgac
     6661 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     6721 aaacatgaga at
//
