Plasmid Backbones
This page displays descriptions of the various plasmid backbones that are used in the EUCOMM pipeline.
L3L4_pZero_DTA_kan
Plasmid backbone containing a L3/L4 Gateway exchange cassette including the ccdB (bacterial negative selection) and chlR (linked positive selection) gene which serves as an acceptor for the transfer of the entire targeting region of the intermediate vector (area between R3/R4 sites). Appropriate for use with L1L2 targeting cassettes which are NOT KanR (gt and st series promoterless L1L2 cassettes).
| ES Cell Negative Selection | Phosphoglycerate Kinase 1 (PGK) promoter, Diptheria Toxin A (DTA) cassette: PGK promoter / DTA-A / BGH pA |
| Plasmid | High copy number pZero based backbone, Kan resistant (25µg/ml). Must also be grown in the presence of 17µg/ml chloramphenicol as well as Kan (25µg/ml) to guard against deletions of the ccdB negative selection cassette. Must be grown in a ccdB resistant E. Coli host strain such as DB3.1 (available from Invitrogen 11782-018). |
L3L4_pD223_DTA_T_spec
Plasmid backbone containing a L3/L4 Gateway exchange cassette including the ccdB (bacterial negative selection) and chlR (linked positive selection) gene which serves as an acceptor for the transfer of the entire targeting region of the intermediate vector (area between R3/R4 sites). Appropriate for use with L1L2 targeting cassettes which are KanR (most promoter containing targeting cassettes).
| ES Cell Negative Selection | Phosphoglycerate Kinase 1 (PGK) promoter, Diptheria Toxin A (DTA) cassette: PGK promoter / DTA-A / BGH pA |
| Plasmid | High copy number p223 based backbone, Spectinomycin resistant (50µg/ml). Must also be grown in the presence of 17µg/ml chloramphenicol as well as Spectinomycin to guard against deletions of the ccdB negative selection cassette. Must be grown in a ccdB resistant E. Coli host strain such as DB3.1 (available from Invitrogen 11782-018). |
L4L3_DTA_spec
Plasmid backbone containing a L4/L3 Gateway exchange cassette including the ccdB (bacterial negative selection) and chlR (linked positive selection) gene which serves as an acceptor for the transfer of the entire targeting region of the intermediate vector (area between R3/R4 sites). Appropriate for use with L1L2 targeting cassettes which are KanR (most promoter containing targeting cassettes).
| ES Cell Negative Selection | Phosphoglycerate Kinase 1 (PGK) promoter, Diptheria Toxin A (DTA) cassette: PGK promoter / DTA-A / BGH pA |
| Plasmid | High copy number p223 based backbone, Spectinomycin resistant (50µg/ml). Must also be grown in the presence of 17µg/ml chloramphenicol as well as Spectinomycin to insure against deletions of the ccdB negative selection cassette. Must be grown in a ccdB resistant E. Coli host strain such as DB3.1 (available from Invitrogen 11782-018). |
R3R4_pBR_amp
This backbone is found on intermediate vectors which have undergone targeting cassette insertion via the R1/R2 Gateway reaction but not plasmid backbone replacement via the R3/R4 Gateway reaction. It is the original pBR322 based backbone of the intermediate vector. No negative selection cassette is present.
| Plasmid | Medium copy, pBR322, Amp (50µg/ml) resistant. |
R3R4_pBR_DTA+_Bsd_amp
These vectors contain DTA negative selection and are constructed by adding an additional recombineering step after gap repair to introduce a DTA negative selection cassette and a blasticidin(bsd)selection marker into the intermediate vector pBR322 R3R4 plasmid backbone. The final vector is then assembled by a two-way gateway reaction between L1L2 targeting cassette and the R1R2 Gateway insertion on the intermediate vector plasmid.
| ES Cell Negative Selection | Phosphoglycerate Kinase 1 (PGK) promoter, Diptheria Toxin A (DTA) cassette: PGK promoter / DTA-A / BGH pA |
| Plasmid | Medium copy, pBR322, Amp(50µg/ml) and Bsd(60µg/ml) resistant. |