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From 2006.igem.org
Mutant loxP sites can be created using PCR with partially overlapping primers. The mutant loxP sites formed are lox66 and lox71. The last five nucleotides of lox66 are CGGTA instead of TACCG found in the wild type loxP. The first five nucleotides of lox71 are TACCG instead of ATAAC found in the wild type loxP. When CRE is allowed to act on these sites in the forward reaction a wild type and a double mutant lox site result. The double mutated loxP site is not recognized by CRE therefore after the initial inverstion there are not any subsequent inversions or excisions. So we are able to flip one pancake and put the spatula down.
CRE recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein http://nar.oxfordjournals.org/cgi/content/full/30/17/e90#GNF089F2
