( expression of gene fusions wit...)New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

expression of gene fusions with the GAL4 DNA binding domain and the NF-kB transcriptional activation domain, respectively (Figure 2). The pCMV-BD vector is designed to construct bait plasmids. This vector contains DNA encoding amino acids 1 to 147 of the GAL4 gene (DNA binding domain) and unique 3 cloning sites. The pCMV-AD vector is used to construct target plasmids. This vector contains a DNA encoding nuclear localization sequence from the SV40 large T-antigen (amino acid PKKKRKV), amino acids 364 to 550 of the mouse NF-kB gene (transcriptional activation domain), and unique 3 cloning sites. Both the pCMV-BD and pCMV-AD vectors contain the ColE1 origin for replication in E. coli. The pCMV-BD vector contains the kanamycin-resistant gene and the pCMV-AD vector contains the ampicillin-resistant gene. The CMV promoter^### governs the expression of the bait and target proteins in both the pCMV-BD and pCMV-AD vectors. The SV40 poly (A) signal provides the signals necessary for transcriptional termination and polyadenylation of the bait and target genes in mammalian cells.

Stratagene offers this system as a vector kit containing both the bait vector, target (prey) vector, the pFR-luc reporter vector, and three control vectors. All plasmid vectors are available separately.

Characterization of the NF-kB p65 Transcriptional Activation Domain

The transcription activation domain we used for pCMV-AD is derived from the carboxyl terminus of the mouse NF-kB p65 protein.⁹ The p65 activation domain has been reported to be significantly more potent than that derived fr
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