LOCUS       sequence-437100-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of a mutant bacterial gasdermin (bGSDM) encoded by a 
            Nostoc species with a WELQut protease cleavage site for controlled 
            activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  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 6765)
  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_Nostoc-bGSDM-WELQ
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2079..2098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2239..2261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2255..2446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4801..4820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4840..4917)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5123..5142
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5226..5245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5226..5244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5245..5269
                     /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             5285..5307
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5327..5344
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6332..6343
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(6492..6510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6506..6553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6728..6733,6734..6750,6751..6756))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     2821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     2881 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     2941 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3001 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3061 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     3121 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     3181 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     3241 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     3301 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     3361 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     3421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3661 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3721 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3781 aaactgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3901 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3961 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4021 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4081 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4141 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4201 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4261 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4321 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4381 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4441 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4501 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4561 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4621 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4681 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4741 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4801 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4861 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4921 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4981 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5041 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5101 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5161 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5221 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagagag
     5281 ctcttttgtt taactttaag aaggagatat acatatgggt tcttctcacc atcaccatca
     5341 ccatggttca ggagtcaaga ctgagaacaa cgatcatatt aatttgaagg tggcggggca
     5401 ggatggttct gtggtgcagt ttaagattaa gaggcataca ccacttagta aactaatgaa
     5461 agcctattgt gaacgacagg gattgtcaat gaggcagatc agattccgat ttgacgggca
     5521 accaatcaat gaaacagaca cacctgcaca gttggaaatg gaggatgaag atacaattga
     5581 tgtgtttcaa cagcagacgg gtggatccgc aacctgtacc gataaagcaa ttgatcatct
     5641 gaatcagctg ggctataacg caattaaact gcctcgtgaa ggtattacac cgctgaccgt
     5701 tctgagccgt gatccgaaaa ccagcattag caccatttat ggtaatctgc gtgatattgt
     5761 tgcagaaccg gtgccgagcc tgccgcaggt ttttgaaaat ctgcaggcag gcaccattag
     5821 cggtctgaaa accaataaac tggaactgaa atttggcatc agcatcctga atgatctgct
     5881 gagcgcatta ggtggtaaaa gcgcaggtat tgaaagcgtt tttcgtaaag caagcaccat
     5941 cgaatttgag tatgaaaacg tgcagtatga taccgttaca ccggcaagcg ttaccaaatt
     6001 tctgcgtggt agcaaaccga gcattgatga tgattttgtt gcccagttta atgaacaggg
     6061 tgaagcctat attatcatcg atgtgctgaa aagcaacagc tttggtattc gtgtttatca
     6121 ggatagcgat cgcggtatcg atattaacat tgatgccatc aaaaacgtga ttggcctgaa
     6181 cagcaaaatt agcgttgaaa agaaagtgga caccaaaatc agctttaatg gtagccaggc
     6241 attaggcttt ggttttaaag catgtccgat ttgggttgaa accgttggtg gtgttagccg
     6301 ttttaaactg aatccgaata caaatcagag ctgggaactg cagggtagca ccgaaagcag
     6361 caccgaaccg acactggatg atgaagatgg tctgacaccg gtgctgattg caccgaatca
     6421 actgattgaa ctggtttgat aagcggccgc tgctaacaaa gcccgaaagg aagctgagtt
     6481 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     6541 gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt
     6601 accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg
     6661 agtgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa
     6721 ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaa
//
