LOCUS       sequence-437094-        6732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of a mutant bacterial gasdermin (bGSDM) encoded by a 
            Vitiosangium species with a thrombin 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_Vitiosangium-bGSDM-thrombin
FEATURES             Location/Qualifiers
     source          1..6732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(753..770)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             790..812
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    828..852
                     /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(852..871)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(853..871)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(955..974)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1180..1257
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1277..1296)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3651..3842
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3836..3858)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3944..4083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3999..4018
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4098..4115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4269..4857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4349..4368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5028..5819,5820..5888))
                     /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     5651..5670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5889..5993)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6061..6079
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6106..6111,6112..6128,6129..6134)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(6309..6356)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6352..6370
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 aattcgttgc ttttaatcac ctgggtaata acatacaggc gaccattacc taacacatac
       61 tgtttcagaa ccggattatc tgcatcaact tcggccattt tcagaaactg accaactgcc
      121 agcggttcaa cgctatcggc cagaacattg ctaaattcaa aacgcacttt acgtgcctga
      181 ttatactgtg cttcaatacc tgcaccggca ccaaactgtg ccagaacgct tttcagaatg
      241 ttgatgccaa tgctaaagct cagattttcg gttttctgac cattaatacc tgctgctgcc
      301 tgatcacggg taattgcagg cggttcgctc ggaggctggg taatcagttt ttccaggcta
      361 cccagatatt caacggtgcc acgctgctga cccagcagat gcagcggctg aataccttca
      421 cgaggcagac gaacaacgtt ataacccaga cgtttcagat aggtaattgc cggatcgcta
      481 cacagaccgg atccacccgt ctgctgttga aacacatcaa ttgtatcttc atcctccatt
      541 tccaactgtg caggtgtgtc tgtttcattg attggttgcc cgtcaaatcg gaatctgatc
      601 tgcctcattg acaatccctg tcgttcacaa taggctttca ttagtttact aagtggtgta
      661 tgcctcttaa tcttaaactg caccacagaa ccatcctgcc ccgccacctt caaattaata
      721 tgatcgttgt tctcagtctt gactcctgaa ccatggtgat ggtgatggtg agaagaaccc
      781 atatgtatat ctccttctta aagttaaaca aaagagctct ctagagggga attgttatcc
      841 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
      901 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
      961 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     1021 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     1081 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     1141 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     1201 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     1261 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     1321 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     1381 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1441 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1501 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1561 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1621 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1681 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     1741 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     1801 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     1861 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     1921 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     1981 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     2041 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     2101 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     2161 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     2221 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     2281 ttggccgatt cattaatgca gctggcacga cagtttcccg actggaaagc gggcagtgag
     2341 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     2401 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     2461 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     2521 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     2581 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     2641 ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg ctacgtcttg
     2701 ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct cgcttccggc
     2761 ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga cgaccatcag
     2821 ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat tggaccgctg
     2881 atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc atggattgta
     2941 ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc
     3001 acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg
     3061 gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg cagaacatat
     3121 ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc gttgggtcct
     3181 ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3241 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3301 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3361 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3421 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3481 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3541 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     3601 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     3661 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     3721 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3781 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3841 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3901 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3961 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     4021 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4081 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4681 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4921 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4981 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5041 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5101 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5161 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5221 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5281 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5341 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5401 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5461 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5521 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5581 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5641 tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5701 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5761 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5821 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5881 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5941 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6001 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6061 aataggcgta tcacgaggcc ctttcgtctt caagaattct catgtttgac agcttatcat
     6121 cgataagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc aggcaccgtg
     6181 tatgaaatct aacaatgcac tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg
     6241 cataggcttg gttatgccgg tactgccggg cctcttgcgg gatatccgga tatagttcct
     6301 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
     6361 tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc agcggccgct
     6421 tatcaaaaac cttccagatc cagcagacca ccttcatcca gcagtgcgct atcgctcggc
     6481 ataacaccac cggctgcatc cagtgcgctt gcacgcggaa ccagctggct tgcaaacagg
     6541 gtaatttcgc cgtttttcac accaatttca aagcatttga agccaaacac cagctgtttt
     6601 tcacctttat aggtaacggt gctctggctg ctaacgcttg cttcaacttt cagtttacca
     6661 ccaacaactt tctgaatttc cggaacatcc agctgaatgc taccaccacc tgatttttcg
     6721 gctgcaacgg ta
//
