If you want to replace the 35S promoter with a tissue-specific promoter (e.g., RBCS or AtUBQ10 ):
If you are inserting a gene of interest:
Today, we’re going to map out the and discuss how to leverage them for seamless construct assembly. pgps3 unique restriction sites
| 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) |
) marker. This marker can be removed or replaced using unique restriction sites that flank the gene, or by using or EcoRV , which also interact with the Transprimer region. If you want to replace the 35S promoter
PGPS3 does NOT have unique sites for common enzymes like NcoI , NdeI , SmaI , or XhoI . Do not rely on these for linearization—they either cut zero times or multiple times.
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. PGPS3 does NOT have unique sites for common
If you received PGPS3 from a collaborator or bought a modified version (e.g., PGPS3-EGFP), do this: