Human AAVS1 inducible gene expression donor vectors are meticulously designed to target the human AAVS1 "safe harbor" site for an introduce a inducible gene expression. AAVS1 safe harbor site is located within intron 1 of the PPP1R12C gene (protein phosphatase 1 regulatory subunit 12C). This specific locus is labeled a "safe harbor" due to its minimal impact on cellular functions. It excels in facilitating strong transcription, serving as an optimal location to uphold the expression of an externally introduced gene while reducing the chance of unintended integration in other regions. Our latest-generation inducible gene expression cassette is based on incorporate a multiple cloning site (MCS) strategically positioned downstream of the CAG promoter and upstream of a poly-A tail. The well-known TeTOn gene inducible system is a molecular biology tool that allows precise control of gene expression. It responds to specific signals, tetracycline, enabling researchers to turn genes on or off when needed. This system is essential for studying gene function, creating conditional knockout models, and regulating transgene expression in various cells. In this donor vector, the gene of interest (GOI) is situated downstream of the Tet Response Element (TRE) and upstream of the polyA signal. Moreover, a CAG promoter is utilized for expressing the reverse tetracycline-controlled transactivator (rtTA), which, when combined with tetracycline, activates the TRE function and ensures the effective operation of the TRE element. This arrangement ensures a streamlined and straightforward path to control enduring and strong expression of the Gene of Interest (GOI) by Tetracycline. 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 expression can only occur when the construct integrates within an intron. Consequently, puromycin expression becomes intricately linked to the PPP1R12C transcript, significantly reducing the risk of unintended off-target integrations when employing Neomycin selection. When this vector used in conjunction with the pSpCAS9 nuclease & AAVS1 gRNA vector, it enables the seamless integration of the GOI into the AAVS1 safe harbor site, simplifying the inducible expression of GOI by TetOn system.

Vector Information

This AAVS1 inducible gene expression donor vector contains the necessary elements for introducing an inducible GOI (gene of interest) to the AAVS1 Safe Harbor site. A ubiquitous SA-T2A-Puro cassette ensures the integration of the GOI in the AAVS1 safe harbor site by antibiotic selection. The GOI is situated downstream of the Tet Response Element (TRE) and upstream of the polyA signal. Moreover, a CAG promoter is utilized for expressing the reverse tetracycline-controlled transactivator (rtTA), ensuring the TRE's proper activation when combined with doxycycline and the overall effectiveness of the TRE element.  

IMPORTANT NOTICE

Store the vial at -20°C immediately upon receipt.