Table 4.
Sequences to add BsaI or SapI sites to the PCR products prior to Golden Gate Cloning for a two-part (promoter plus reporter) scar-less assembly into the destination vector.
| Primer | Recognition/Overhang | Sequence to add |
|---|---|---|
| BsaI forward promoter | 5′-GGTCTCNAGTA- | Starting from the first bp of your promoter |
| BsaI reverse promoter | 5′-GGTCTCNTCAT- 1 | Reverse complement of your promoter starting from the last bp |
| BsaI forward reporter | 5′-GGTCTCNATGA- 1 | Starting from the 5th bp of your reporter |
| BsaI reverse reporter | 5′-GGTCTCNTCCA- | Reverse complement of your reporter starting from the last bp |
| SapI forward promoter | 5′-GCTCTTCNAGT- | Starting from the first bp of your promoter |
| SapI reverse promoter | 5′-GCTCTTCNCAT- 2 | Reverse complement of your promoter starting from the last bp |
| SapI forward reporter | 5′-GCTCTTCNATG- 2 | Starting from the 4th bp of your reporter |
| SapI reverse reporter | 5′-GCTCTTCNCCA- | Reverse complement of your reporter starting from the last bp |
1
Assumes the first 4 bps of your reporter is 5′-ATGA-3′.
2
Assumes the first 3 bps of your reporter is 5′-ATG-3′.
BsaI primers apply to the pGoldenGate-SE7 and pGoldenGate-SE9 plasmids, whereas SapI primers apply to the pGoldenGate-MCY2 plasmid. Overhangs are shown in bold.