Western blot analysis of Cre reconstitution by intein‐mediated trans‐splicing. Brackets indicate reconstituted full‐length Cre protein, and asterisks denote near complete conversion of C‐terminal effector fragment using Npu‐based split‐Cre pairs.
Reconstitution of full‐length tTA protein by intein‐mediated trans‐splicing.
PCR‐based analysis of Cre‐mediated DNA recombination.
Quantification of tTA activity by dual luciferase assays in the presence of doxycycline (dox). Data depicted as mean ± SD, n = 3.
Reconstitution of biologically active tTA protein at different ratios of N‐ and C‐terminal fragments. A constant amount of Npu tTA N was co‐transfected with varying amounts of Npu tTA C to mimic potential differences in expression levels. tTA activity was quantified by dual luciferase assays. Data depicted as mean ± SD, n = 3.
In vitro testing of split‐iCre reconstitution used for insertion into endogenous Ccsp and Spc gene loci. Phage P1‐derived Cre fragments within the Npu N1/C1 split‐pair were replaced by equivalent sequences from a codon‐improved Cre recombinase (iCre). Cre activity was quantified by dual luciferase assays. Data depicted as mean ± SD, n = 3.
.; CDS, coding sequence.
