Description:

Human AAVS1 donor vectors are meticulously designed to target the human AAVS1 "safe harbor" site nestled within intron 1 of the PPP1R12C gene (protein phosphatase 1 regulatory subunit 12C). This specific locus is aptly called a "safe harbor" because it exerts minimal influence on cellular functions. It excels in supporting robust transcription, making it an ideal site for maintaining the expression of an externally introduced gene while mitigating the risk of unintentional integration elsewhere. Our cutting-edge trackable donor vectors feature a multiple cloning site (MCS) strategically placed downstream of an SFFV promoter and upstream of a poly-A tail in reverse orientation. This setup guarantees a simplified and efficient pathway to achieve robust and lasting expression of the Gene of Interest (GOI). Additionally, an EGFP driven by a CAG promoter can serve as a convenient transfection indicator. Furthermore, leveraging the intronic location of the AAVS1 safe harbor locus, the Puromycin selection marker is thoughtfully integrated with a splice acceptor (SA) site, devoid of its own promoter. This design guarantees that puromycin-resistant gene expression can only occur when the construct integrates within an intron. Consequently, puromycin-resistant gene expression becomes intricately linked to the PPP1R12C transcript, significantly reducing the risk of unintended off-target integrations when employing puromycin selection. This trackable donor vector plays a pivotal role in the ALSTEM pAAVS1/Cas9 Knock-In vector system. When used in conjunction with the SpCas9 nuclease & AAVS1 gRNA expression vector, it enables the seamless integration of the GOI into the AAVS1 safe harbor site with an EGFP indicator, simplifying the process of enhancing GOI expression.

Specifications: