Basic Parts

BBa_K2605000- mCherry + BGH

mCherry red fluorescent protein (RFP) codon-optimized for expression in human/mammalian cells with a BGH terminator attached. The addition of BGH makes this part an improvement of the original mCherry part,BBa_J176005.

BBa_K2605001- CMV with 5' SalI restriction site

Cytomegalovirus (CMV) promoter with an additional 5' SalI site, which allows this promoter to be used within our Multiple-Cloning Site flanked by FRT sites. This is an improvement of part  BBa_K747096 and you can learn more about its characterization and improvement here.

BBa_K2605002- Multiple cloning site with flanking FRT sites

Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites. The MCS carries sites for an arbitrary construct to be directionally cloned. The MCS was designed for our first iteration of targeted gene integration, which involved recombinase-mediated casette exchange (RMCE), thus it was flanked by FRT sites.

BBa_K2605003- A2UCOE

BBa_K2605004- Chicken beta-globin insulator

BBa_K2605005- FRT2

A FLP recombinase recognition target (FRT) site.

BBa_K2605006- Hybrid FlpO-beta resolvase

Composite Parts

BBa_K2605007- CMV with 5' SalI restriction site + monomeric RFP optimized for bacteria

BBa_K2605008- CMV + monomeric RFP optimized for bacteria

BBa_K2605009- CMV with 5' SalI restriction site + mCherry + BGH

BBa_K26050010- CMV + mCherry + BGH

Parts Collection

Parts BBa_K2605000 to BBa_K2605004 and BBa_K2605009 comprise the first iteration of a novel toolkit for targeted gene integration and maintenance of expression in eukaryotic chassis. The collection contains chromatin modifying elements that limit silencing of the integrated gene and minimize neighborhood effects. These elements, as well as the mCherry-BGH reporter gene with our improved CMV promoter, have restriction sites for directional cloning into part BBa_K2605002, which is a Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites. The MCS carries sites for an arbitrary construct to be directionally cloned.

In the first iteration of our gene integration strategy, the FRT sites are leveraged for recombinase-mediated casette exchange (RMCE). Representing an experimental second design,BBa_K2605006 is a prototype part intended to test our novel FLP-Beta strategy. For either RMCE or FLP-Beta, the toolkit is intended to leverage genome-integrated recombinase target sites.