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

A methylation-free CpG island that provides stable and localization of integration-independent transgene expression when fused to a eukaryotic promoter. This part contains a 5' MfeI restriction site and 3' NsiI restriction site so it can be used within our Multiple-Cloning Site flanked by FRT sites (Bba_K2605002).

BBa_K2605004- Chicken beta-globin insulator

DNAse‐I hypersensitive CpG‐rich structure that provides stable and localization of integration-independent transgene expression and prevents unrelated enhancer-promoter interactions when flanking a transgene. This part contains a 5' BmtI restriction site and 3' BamHI restriction site so it can be used within our Multiple-Cloning Site flanked by FRT sites (Bba_K2605002).

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

Part used to characterize and validate the improvement of part BBa_K2605001. Read more about its characterization here.

BBa_K2605008- CMV + monomeric RFP optimized for bacteria

Part used to characterize and validate the improvement of part BBa_K2605001. Read more about its characterization here.

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

Composite part of BBa_K2605001 (promoter) fused to BBa_K2605000 (reporter gene). The part contains a SalI restriction site on the 5' end and a BmtI restriction site on the 3' end to allow for this part to be used within our Multiple-Cloning Site flanked by FRT sites (Bba_K2605002).

BBa_K2605010- CMV + mCherry + BGH

Composite part of BBa_K747096 (promoter) fused to BBa_K2605000 (reporter gene). The part contains a SalI restriction site on the 5' end.

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.

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.

Each part in this collection (except Bba_K2605002 and Bba_K2605006) is designed with two different restriction sites on either end to allow for simple bi-directional cloning. The entire MCS is flanked by SapI restriction sites so that the entire cloning site and FRT sites can be transferred to different plasmids. AflII restriction sites flank the MCS interior of the FRT sites so that only the MCS can be transferred to different plasmids. A schematic of our MCS (Bba_K2605002) can be seen below.

The figure below shows an example of how chromatin-modifying elements and a reporter gene can be used in our system. The restriction sites for bi-directional cloning of each part is shown.