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. 2012 Feb 29;7(2):e32646. doi: 10.1371/journal.pone.0032646

Figure 1. FASTarget rational and experimental outline.

Figure 1

(A) Targeting strategy using ATG-less (ORF-trap) EYFP cassette. Genomic structure of the CENP-A locus is outlined. Coding exons in light blue, UTRs in dark blue. Targeting construct is placed within rAAV inverted terminal repeats (ITR) required for transduction. Regions of homology are indicated in beige. Upon targeting the ATG-less EYFP is placed in frame with the last codon of the CENP-A gene resulting in a CENP-A-EYFP knockin driving EYFP fluorescence from the endogenous CENP-A promoter. Random integration will likely result in a failure to express EYFP due to the lack of a nearby promoter or out of frame fusion. (B) Schematic and time-line for rAAV production, transduction, enrichment and purification of targeted clones. Following initial enrichment and expansion cells can be screened as a polyclonal pool by PCR or microscopy to verify (*) presence of targeted cells.