Last data update: 06 May 2021 08:34 CEST
Plasmid name: pCAGGS-E-mABIN-2mAQAA (LMBP 4386)
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|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
Mouse A20-binding inhibitor of NF-κB activation 2 cDNA (ABIN-2, TNIP2); mutated sequence|
|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 NotI-BglII fragment of the mutated mouse A20-binding inhibitor of NF-κB activation 2 cDNA (mABIN-2), into the NotI-BglII opened pCAGGS-E-mABIN-2 vector. As compared to the wt mABIn-2 cDNA, codonsubstitutions were introduced at position 260 (Lys -> ALa), 261 (Glu -> Gln), 264 (Arg -> Ala) and 265 (Leu -> Ala).
pCAGGS-E-mABIN-2mAQAA is useful for highly efficient expression of mutated mABIN-2 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 the mABIN-2 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AJ304865.1.
Other name of the plasmid is pCAGGS-E-mABIN-2(KE260AQ,RL264AA).
|EMBL Accession number:||AJ304865.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||12/07/2001|
Fusion PCR was used on the pCAGGS-E-mABIN-2 vector to generate the insert : Primers PCR1 : Forward: 5' TGAATTCGGGATCCGTATGTCGTC 3' Reverse: 5' CTGTCTGTTAGCTGCGGAAATCTGCGCTTTCATCAG 3' Primers PCR2 : Forward: 5' CCGCAGCTAACAGACAGTTGGAGGAGA 3' Reverse: 5' GGAGATCTTCATTGGCAGCACTCAGACAG 3' Nucleotide sequence of the mutated mABIN-2 cDNA: wild type : 5' GAG.CTG.ATG.AAG.AAG.GAG.ATT.TCC.CGA.CTT.AAC.AGA.CAG.TTG.GAG 3' Glu Leu Met Lys Lys Glu Ile Ser Arg Leu Asn Arg Gln Phe Glu mutated : 5' GAG.CTG.ATG.AAA.GCG.CAG.ATT.TCC.GCA.GCT.AAC.AGA.CAG.TTG.GAG 3' Glu Leu Met Lys Ala Gln Ile Ser Ala Ala Asn Arg Gln Phe Glu
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by S. Van Huffel(1) (2) and Prof. Dr R. Beyaert(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Van Huffel et al., J. Biol. Chem. 276 (2001), 30216-30223 [PMID: 11390377]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGS-E-mABIN-2mAQAA (LMBP 4386) is available at BCCM/GeneCorner. This plasmid was deposited by S. Van Huffel and Prof. Dr R. Beyaert .|
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.