RMHE vectors

I have developed several RMHE-compatible RMCE (figure 1) and rRMCE (figure 2) vectors which are available upon request while the technique remains unpublished.

Figure 1. Overview of the available RMHE compatible RMCE vectors. The Multiple Cloning Site (MCS) contains numerous common restrictions sites as well as SapI sites for Golden Gate Cloning. The unique sites in the MCS differ slightly from vector to vector due to MCS orientation and sites present in attB and attP. Some vectors are available with the MCS SapI sites arranged <SapI (TGG) SapI> (GTA) and in the opposite orientation <SapI (TAC) SapI>(CCA). These are denoted with a terminal ‘r’ below. Some vectors contain ‘full-sized’ attB and attP sites (Thorpe et al. 1998) and other have minimally defined sites (Groth et. al. 2000). The red and yellow lines in the att sites define the sites of phiC31 mediated recombination. Additional vectors are also available which swap the position of the attB and attP site relative to the MCS.

The original RMCE vector which does not contain phiC31 att sites for recombination.

This RMCE integration vector contains a ‘full-sized’ phiC31 attB site on the 3′ end of the insertion domain. It also contains synthetic sgRNA sites both on the 5′ and 3′ end of the insertion domain and a rps-7 3′ UTR region on the 5′ end of the insertion domain that was included to reduce potential read through from sequences adjacent to the insert.

Identical to pRMHEB except with a minimal attB.

This RMCE integration vector contains a ‘full-sized’ phiC31 attP site on the 5′ end of the insertion domain. It also contains synthetic sgRNA sites both on the 5′ and 3′ end of the insertion domain and an rps-17 3′ UTR on the 5′ of the insertion domain that was included to reduce potential read through from sequences 5′ of the insert.

Identical to pRMHEP except with a minimal attP.

Figure 2. Overview of the available RMHE compatible rRMCE vectors. The Multiple Cloning Site (MCS) contains numerous common restrictions sites as well as SapI sites for Golden Gate Cloning. The unique sites in the MCS differ slightly from vector to vector due to MCS orientation and sites present in attB and attP. These vectors contain ‘full-sized’ attB and attP sites (Thorpe et al. 1998). The red and yellow lines in the att sites define the sites of phiC31 mediated recombination.

The original rRMCE vectors which do not contain phiC31 att sites for recombination.

These rRMCE integration vectors contains a ‘full-sized’ phiC31 attB site on the 3′ end of the insertion domain. One will yield GFP-C1 cis-marked transgenes, and the other nls-Scarlet marked transgense. The vectors also contain synthetic sgRNA sites both on the 5′ and 3′ end of the insertion domain and a rps-7 3′ UTR region on the 5′ end of the insertion domain that was included to reduce potential read through from sequences adjacent to the insert.

These rRMCE integration vectors contains a ‘full-sized’ phiC31 attP site on the 5′ end of the insertion domain. One will yield GFP-C1 cis-marked transgenes, and the other nls-Scarlet marked transgense. The vectors also contain synthetic sgRNA sites both on the 5′ and 3′ end of the insertion domain and an rps-17 3′ UTR on the 5′ of the insertion domain that was included to reduce potential read through from sequences 5′ of the insert.

References

  1. Groth, A. C., Olivares, E. C., Thyagarajan, B. & Calos, M. P. A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A 97, 5995-6000 (2000). PMID 10801973.
  2. Thorpe, H. M. & Smith, M. C. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95, 5505-5510 (1998). PMID 9576912.

Last updated 12-1-2023