FRT-gb2-cm-FRT

Prokaryotic Chloramphenicol Selection Cassette flanked by FRT-sites

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Description

The FRT-cm-FRT cassette is designed to allow chloramphenicol selection in prokaryotic cells. The prokaryotic promoter gb2 driving the gene for chloramphenicol resistance is a slightly modified version of the Em7 promoter. It mediates higher transcription efficiency than the generally used Tn5 promoter. A synthetic polyadenylation signal terminates the chloramphenicol expression. The cassette is flanked by FRT sites for later excision by Flp Recombinase. Unique NotI and XhoI sites flank the cassette for convenient cloning with restriction sites. Using the provided PCR template one can easily create a FRT-cm-FRT cassette flanked by any other restriction sites to clone the cassette into the vector of choice. The restriction sites can be introduced by adding the corresponding sequence in the PCR primer. The template can easily be used to engineer the E.coli genome by Red/ET Recombination. The FRT-cm-FRT cassette is not linear but plasmid based (3302 bp in size). Due to its R6K origin the plasmid cannot replicate in most E.coli strains. The PCR product can therefore be used directly for downstream applications without any further purification. At least 20 PCR reactions can be performed using 1 ul per reaction as template.

Contents

• FRT-cm-FRT: PCR template (50 ng/ul, 20ul)
• Manual

Sequences

FRT-gb2-cm-FRT