GeneBLAzer™ N-terminal Gateway™ Fusion Kit, for in vitro detection - FAQs

Can I use E. coli containing an F' plasmid, such as Top 10F', to propagate a vector with the ccdB gene?

While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.

For propagation of Gateway vectors containing ccdB, we recommend the One Shot ccdB Survival 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.

Is the sequence of the CMV promoter in pcDNA3.1 vector complete, or is it only the core CMV promoter?

In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".

Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.