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EUCOMMTOOLS Gene Targeting Vector Plasmid Backbones



Custom recombineering cassettes for the construction of Gateway-adapted intermediate plasmids.




The R1/R2 pheS:zeo recombineering plasmid (top) contains the R6K origin of replication and the bacterial EM7 promoter driving the zeo and pheS genes. The pheS gene3 serves as a negative selection marker for the Gateway exchange reaction. The attR1 and attR2 sites are each flanked by 20 bp of unique sequence for PCR amplification (arrows). The R3/R4 plasmid backbone for gap repair (bottom) is a modified pBR322 plasmid (AmpR, TetS) containing attR3 and attR4 sites flanking a bacterial positive-negative selection cassette (cat-ccdB). The R3 and R4 sites are each flanked with 20 bp of unique sequence for PCR amplification (arrows). A very rare AsiSI site next to the R3 site is used to linearize the final targeting construct.

Custom Gateway-adapted targeting elements for the construction of targeted vectors.




The promoter-driven targeting cassette (pL1L2_BactP) contains IRES:lacZ flanked by the En2 splice acceptor sequence and SV40 polyA (pA) signal. A floxed human β-actin promoter (Bact) drives the expression of neo in ES cells. FRT sites are included on either side of the targeting cassette. Promoterless targeting cassettes (pL1L2_GT) contain an in-frame fusion of lacZ and neo flanked by the En2 splice acceptor sequence and SV40 polyA (pA) signal. T2A peptide sequences are included to allow expression of lacZ and neo as independent polypeptides. These vectors have been engineered in each of the three possible reading frames (0, 1, 2) and with a Kozak ATG (K). FRT sites are included on either side of the cassette along with a loxP site at the 3’ end of the vector. Secretory trap versions of the promoterless targeting cassettes (pL1L2_ST) contain the rat CD4 transmembrane domain (TM) fused to the N-terminus of lacZ. The negative selection plasmid backbone (pL3L4-DTA) contains L3 and L4 sites flanking the bacterial ccdB and cat genes followed by a diphtheria toxin A-chain (DTA) expression cassette. DTA is expressed from the mouse phosphoglycerate kinase 1 (PGK) promoter.