Just remember: no SalI, no EcoRV, and always verify your map. When in doubt, a quick virtual digest takes 30 seconds and saves three days of failed ligations.
Happy cloning! Have you found a rare unique site in PGPS3 that I missed? Or a tricky methylation issue? Drop a comment below or tag me on Twitter @PlantBiotechLab – let’s build a better restriction map together. pgps3 unique restriction sites
If you work with plant transformation, particularly in Arabidopsis thaliana or related species, you’ve likely encountered the PGPS3 vector. Derived from the pGreen series, PGPS3 is a compact, high-copy-number binary vector known for its small size (approximately 3.2 kb), making it ideal for golden gate cloning, site-directed mutagenesis, and efficient transformation into Agrobacterium tumefaciens . Just remember: no SalI, no EcoRV, and always verify your map
| Restriction Enzyme | Position (approx.) | Recognition Sequence | Best Use Case | |-------------------|-------------------|----------------------|----------------| | | 1,234 | G^AATTC | Insert cloning (5' end) | | BamHI | 1,567 | G^GATCC | Insert cloning (3' end) | | HindIII | 2,345 | A^AGCTT | Backbone linearization | | XbaI | 789 | T^CTAGA | Subcloning from other vectors | | SacI | 2,890 | GAGCT^C | Terminator swaps | | PstI | 3,101 | CTGCA^G | Rare cutter for large inserts | | KpnI | 456 | GGTAC^C | Directional cloning with SacI | | NotI | 2,567 | GC^GGCCGC | For GC-rich inserts (rare cutter) | Have you found a rare unique site in PGPS3 that I missed
| Enzyme | Problem | |--------|---------| | | Cuts twice (once in MCS, once in the pSa origin of replication). | | EcoRV | Cuts three times (unexpectedly in the LacZ alpha fragment and the kanamycin resistance gene). | | ClaI | Cuts twice (in the MCS and the ColE1 origin). |
However, successful cloning depends entirely on one thing: knowing which restriction enzymes cut once —and only once—in your plasmid.