Employing site-specific integrases from various phages to insert a DNA at a single locus of the genome
Phage-derived att integrases employ a site specific recombinase to insert a DNA at a single locus of the genome. Section divider: att Integrases. Employing site-specific integrases from various phages to insert a DNA at a single locus of the genome.φ80 is the one in your example files
Genomic CRIM after flp.seq · drawn schematically, not to scale
And this is what the locus looks like at the end. One FRT site, the gene, and nothing else you added. Compare it with the map two slides ago and the difference is the marker and the origin, both gone. The same genomic locus after Flp excision, with a single FRT site remaining.Why can’t you use a normal pUC plasmid for doing CRIM integration?
Because it would replicate — so every transformant keeps the marker whether or not it integrated, and there is nothing to select.
A pUC origin replicates a plasmid constitutively. So, when you transform in the plasmid to integrate it, it will stably replicate as a plasmid and there will be no way to select for the integrants. You will just get a lawn of transformed cells. That is what the R6K origin buys you: in a strain without pir it cannot replicate at all, so the only cells that keep the marker are the ones that put it in the genome. A question for the room, marked with the deck's thinking figure: why can't you use a normal pUC plasmid for doing CRIM integration? The answer is not on the slide.