Human GSDMD ORF clone (NM_024736) (Cat. No.: V020943)

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Basic Information

Note: pLVX-Puro-GSDMD(human)-AcGFP1 is a Lenti-X lentiviral vector expressing human gasdermin D (GSDMD), the pore-forming executor of pyroptosis, fused to AcGFP1, with puromycin resistance for selection. Used to study pyroptotic cell death, inflammasome signaling and GSDMD membrane insertion; AmpR backbone, Stbl3 propagation.

Name:: pLVX-Puro-GSDMD(human)-AcGFP1

Accession ID:: NM_024736.7

Antibiotic Resistance:: Ampicillin

Length:: 10215 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Lentivirus

Selection Marker:: Puro;AcGFP1

Promoter:: CMV

Fusion Tag:: AcGFP1

Expression Method:: Constiutive, Stable / Transient

Gene Synonyms:: DF5L; DFNA5L; FKSG10; GSDMDC1

Transcript Definition:: Homo sapiens gasdermin D (GSDMD), transcript variant 1, mRNA

Cat. No.: V020943 pLVX-Puro-GSDMD(human)-AcGFP1

$ 298.7

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Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Rathkey JK, Benson BL, Chirieleison SM, et al. Live-cell visualization of gasdermin D-driven pyroptotic cell death. Journal of Biological Chemistry. 2017;292(35):14649-14658. doi: 10.1074/jbc.m117.797217

Kuang S, Zheng J, Yang H, et al. Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis. Proceedings of the National Academy of Sciences. 2017;114(40):10642-10647. doi: 10.1073/pnas.1708194114.

Human GSDMD ORF clone (NM_024736) (Cat. No.: V020943) Sequence

LOCUS       V020943                10215 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V020943
VERSION     V020943
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             1..634
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     misc_feature    681..806
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1303..1536
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1721..1765
                     /codon_start=1
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
                     et al., 2013)"
                     /transl_table=1
                     /translation="KNEQELLELDKWASL"
                     /label="gp41 peptide"
     misc_feature    2028..2143
                     /note="central polypurine tract and central termination
                     sequence of HIV-1 (lacking the first T)"
                     /label="cPPT/CTS"
     enhancer        2201..2504
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2505..2708
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2807..3523
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="Aequorea coerulescens GFP"
                     /transl_table=1
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
                     /label="AcGFP1"
     CDS             3557..5008
                     /label="GSDMD(NM_024736)"
                     /note="GSDMD(NM_024736)"
                     /gene="GSDMD"
     promoter        5050..5549
                     /note="mouse phosphoglycerate kinase 1 promoter"
                     /label="PGK promoter"
     CDS             5570..6169
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /note="confers resistance to puromycin"
                     /product="puromycin N-acetyltransferase"
                     /transl_table=1
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
                     /label="PuroR"
     misc_feature    6183..6771
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6978..7611
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     primer_bind     complement(7740..7756)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7764..7780
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(7788..7818)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7833..7854
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8142..8730)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8901..9761)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(9762..9866)
                     /gene="bla"
                     /label="AmpR promoter"
     polyA_signal    9914..10048
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"