DNA recombinases are used to manipulate the genomic 
structure and to control the genetic expression in all living 
organisms. Cre is a P1 bacteriophage-derived tyrosine 
recombinase mediating the site-specific recombination 
between two loxP DNA recognition sites. Cre/loxP system 
allows generation of tissue specific mutations and is widely 
used in bioengineering and in mouse genetics. However, 
various factors limit its applicability such as lack of 
temporal control over its recombination activity, presence of 
cellular compensatory mechanisms, loss of site-specificity 
at high expression levels and its limited use for conditional 
recombination in certain brain structures due to a lack of 
sufficiently selective promoters. One approach used to 
overcome these drawbacks is the so-called split-iCRE 
technique that employs complementation of split-iCRE 
fragments via artificial FKBP12-rapamycin or ɑ-helix 
interactions. Rapamycin is a pharmacologically active 
substance while leucine zipper-mediated complementation 
so far only insufficiently restores recombinase activity. Here 
we introduce a system for controlling ɑ-complementation of 
two independent iCRE fragments under the control of  two different promoters to reconstitute recombinase 
activity. Unsplit-iCRE was split between Lys130 and   
Asp132. Fragments were complemented restoring ~ 95% of 
the recombinase activity with very little background 
activity. Adding an external nuclear localization signal to 
the C-terminal fragment resulted in even higher enzymatic 
activity. Using an extended rigid linker between the 
polypeptide and the yeast GCN4-coil/coil leucine zipper 
domains was more efficient than a semi-flexible separator. 
The system was validated by knocking out the essential 
circadian clock component Bmal1 (Arntl) in MEF cells 
resulting in a loss of clock function in MEF cells.