Last data update: 31 October 2020 04:21 CET
Plasmid name: pCAGGS-E-mBNIP3 (LMBP 4773)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Mouse Bcl2/adenovirus E1B 19kDa interacting protein 3 cDNA (mBNIP3)
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Further information:||The plasmid was constructed by inserting a KpnI-BglII PCR fragment, containing the mouse Bcl2/adenovirus E1B 19kDa interacting protein 3 coding sequence (mBNIP3), into a KpnI-BglII opened pCAGGS-E-ABIN vector.
mBNIP3 is a member of the Bcl2/adenovirus E1B 19 kd-interacting protein (BNIP) family. It interacts with the E1B 19 kDa protein which is responsible for the protection of virally-induced cell death, as well as with E1B 19 kDa-like sequences of Bcl2, also an apoptotic protector. mBNIP3 contains a BH3 domain and a transmembrane domain, which have been associated with pro-apoptotic function. The dimeric mitochondrial protein encoded by this gene is known to induce apoptosis, even in the presence of Bcl2. It is toxic in human embryonic kidney (HEK) cells.
pCAGGS-E-mBNIP3 is useful for highly efficient expression of mouse BNIP3 under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.
The AG promoter sequence consists of the chicken β-actin promoter, the first exon and part of the first intron (that seems to have a strong enhancer-like activity) linked to a rabbit β-globin fragment, consisting of a 3' part of the second intron (inclusive a branch point which is required for normal splicing reactions) and a 5' part of the third exon.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727034.1.
|EMBL Accession number:||LT727034.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||19/11/2003|
Nucleotide sequence at the start of the fusion gene: ---------------------- E-tag ---------------------> 5' ... CCTCGAG.ATG.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAT.CCG.CTG.GAA.CCG.CGT.GAA. AvaI Met Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala XhoI *** BamHI EcoRI --------------------- mBNIP3 ----------------------> TTC.GGT.ACC.ATG.TCG.CAG.AGC.GGG.GAG.GAG.AAC.CTG.CAG.GGC.TCC.TGG ... 3' Phe Gly Thr Met Ser Gln Ser Gly Glu Glu Asn Leu Gln Gly Ser Trp KpnI *** PstI RsaI Nucleotide sequence at the end of the fusion gene: ------------------ mBNIP3 -----------------> -- ß-globin polyA -> 5' ... AGG.CGT.CTG.ACA.ACT.TCC.ACT.AGC.ACC.TTC.TGA.AGATCTTTTTCCCTCTGCC ... 3' Arg Arg Leu Thr Thr Ser Thr Ser Thr Phe +++ BglII ***: Start codon. +++: Termination codon. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BamHI, BglII/NcoI, KpnI, PstI and TaqI.|
|History of deposit:||This plasmid was deposited by Dr X. Saelens(1) (2) and Prof. Dr P. Vandenabeele (1) (2) and constructed by G. van Loo(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061|
|Host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Related host reference:||Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGS-E-mBNIP3 (LMBP 4773) is available at BCCM/GeneCorner. This plasmid was deposited by Dr X. Saelens and Prof. Dr P. Vandenabeele and constructed by G. van Loo .|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.