LOCUS       sequence-266229-        7066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Gsdmd-NT mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7066)
  AUTHORS   Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J
  TITLE     Inflammasome-activated gasdermin D causes pyroptosis by forming 
            membrane pores.
  JOURNAL   Nature. 2016 Jul 6;535(7610):153-8. doi: 10.1038/nature18629.
  PUBMED    27383986
REFERENCE   2  (bases 1 to 7066)
  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"; journalName: 
            "Nature"; date: "2016-07-6- 6"; volume: "535"; issue: "7610"; pages:
            "153-8"
COMMENT     Sequence Label: Flag-Gsdmd-NT C122A
FEATURES             Location/Qualifiers
     source          1..7066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..49
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     41..58
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     42..58
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        219..598
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        599..802
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     753..773
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             846..869
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1726..2348
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1925..1944)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2372..2392)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2390..2687
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2741..3535
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2795..2814)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3405..3424
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4187..4321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4224..4243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4278..4297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4326..4345)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4327..4345)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4359..4375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4359..4375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4372..4394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4383..4399
                     /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)."
     promoter        complement(join(4407..4413,4414..4431,4432..4437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4452..4473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4590..4607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4761..5349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4841..4860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5520..6311,6312..6380))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6143..6162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6381..6485)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6512..6967
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6599..6618)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6809..6830
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc
       61 caagctgatc tatacattga atcaatattg gcaattagcc atattagtca ttggttatat
      121 agcataaatc aatattggct attggccatt gcatacgttg tatctatatc ataatatgta
      181 catttatatt ggctcatgtc caatatgacc gccatgttga cattgattat tgactagtta
      241 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      301 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      361 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      421 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc
      481 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      541 cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      601 gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc
      661 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      721 tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg
      781 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag aattaattca gatcgatcta
      841 ccatggacta caaagacgat gacgacaagc ttgcggccgc aatgccatcg gcctttgaga
      901 aagtggtcaa gaatgtgatc aaggaggtaa gcggcagcag aggcgatctc attccggtgg
      961 acagcctgcg gaactccacc agcttcaggc cctactgcct tctgaacagg aaattttcaa
     1021 gctcaaggtt ctggaaaccc cgttattcat gtgtcaacct gtcaatcaag gacatcctgg
     1081 agcccagtgc tccagaacca gaaccggagt gttttggctc cttcaaagtc tctgatgtcg
     1141 tcgatgggaa cattcagggc agagtgatgt tgtcaggcat gggagaaggg aaaatttctg
     1201 gtggggctgc agtgtctgac agttccagtg cctccatgaa tgtggctata ctgcgtgtga
     1261 ctcagaagac ctgggagacc atgcagcatg aaaggcacct tcagcagcct gagaacaaaa
     1321 tcctgcaaca gcttcggagt cgtggggatg acctgtttgt ggtgaccgag gtgctgcaga
     1381 caaaggagga agtgcagatc actgaggtcc acagccaaga gggctcaggc cagtttacgc
     1441 tgcctggagc tttatgcttg aagggtgaag gcaagggcca ccaaagccgg aagaagatgg
     1501 tgaccattcc tgcaggcagc atcctggcat tccgagtggc ccaactgctt attggctcta
     1561 aatgggatat ccttctcgtc tcagatgaga aacagaggac ctttgagccc tcctcaggtg
     1621 acagaaaagc agtgggccag aggcaccatg gcctcaatgt gcttgctgcg ctttgttcca
     1681 tcggaaagca gctcagtctc ctgtcagatt agtctagagg atcccgggtg gcatccctgt
     1741 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     1801 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     1861 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     1921 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     1981 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     2041 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     2101 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     2161 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt aaaataacta
     2221 taccagcagg aggacgtcca gacacagcat aggctacctg gccatgccca accggtggga
     2281 catttgagtt gcttgcttgg cactgtcctc tcatgcgttg ggtccactca gtagatgcct
     2341 gttgaattgg gtacgcggcc agcttggctg tggaatgtgt gtcagttagg gtgtggaaag
     2401 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2461 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2521 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2581 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2641 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg aattgatcag
     2701 cttgggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2761 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     2821 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     2881 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     2941 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3001 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3061 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3121 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3181 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3241 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3301 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3361 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3421 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3481 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3541 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3601 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3661 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccgg gctcgatccc
     3721 ctcgcgagtt ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag
     3781 tgcaaatccg tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa
     3841 ctgcaggagt ggggaggcac gatggccgct ttggtcgacc cggacgggac gctcctgcgc
     3901 ctgatacaga acgaattgct tgcaggcatc tcatgagtgt gtcttcccgt tttccgcctg
     3961 aggtcactgc gtggatggag cgctggcgcc tgctgcgcga cggcgagctg ctcaccaccc
     4021 actcgccaag ctggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4081 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4141 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     4201 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4261 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     4321 caattcccta tagtgagtcg tattaaattc gtaatcatgt catagctgtt tcctgtgtga
     4381 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4441 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4501 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4561 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4621 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4681 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4741 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4801 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4861 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4921 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4981 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5041 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5101 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5161 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5221 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5281 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5341 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5401 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5461 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5521 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5581 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5641 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5701 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5761 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5821 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5881 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5941 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6001 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6061 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6481 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     6541 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6601 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6661 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6721 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6781 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6841 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     6901 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     6961 acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     7021 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcg
//
