A series of over a dozen KanR vectors designed to simplify creating SapI Golden Gate entry plasmids for the assembly of targeting plasmids for C. elegans genome manipulation. The vectors are compatible with inserting sequences using many different approaches including traditional restriction enzyme cloning, Gibson assembly and BsaI Golden Gate Cloning (Figure 1). A detailed manual that describes the methods is available.
Six alternative methods for creating Golden Gate entry vectors. A) a BsaI Golden Gate approach, B) a SapI cloning approach, C) a Gibson assembly cloning approach, D) a traditional double sticky-end cloning approach E) a multi-fragment BsaI Golden Gate approach, and F) a multi-fragment Gibson assembly cloning approach. Restrictions sites: B= BsaI, R=EcoRI, H=HindIII, S= SapI. Inserts are shown in red and the vector in black.
Below is a list of the available plasmids:
NM # | Name | SapI Slot (caps) & BsaI overlap | .gb file [all seqs] |
3855 | DR274 sgRNA – BsaI | gAAC-CAAc | [seq] |
3854 | DR274 U6p -BsaI | gCAA-TGGc | [seq] |
3467 | DR274 5’ arm – BsaI | gTGG-GCGa | [seq] |
3468 | DR274 CT – BsaI | gGCG-ATGa | [seq] |
3469 | DR274 FP – BsaI | gATG-AAGg | [seq] |
3707 | DR274 SEC- BsaI | gAAG-GGTc | [seq] |
3456 | DR274 NT – BsaI | gGGT-ACGc | [seq] |
3470 | DR274 3’ arm – BsaI | gACG-GTAg | [seq] |
3967† | pET28- FP -BsaI | gATG-AAGg | [seq] |
3844 | DR274 sgRNA-U6p – BsaI | gAAC-TGGc | [seq] |
3698 | DR274 5’ arm-CT – BsaI | gTGG-ATGa | [seq] |
3701 | DR274 5′ arm- SEC -BsaI | gTGG-GGTa | [seq] |
3773 | DR274 CT-NT – BsaI | gGCG-ACGc | [seq] |
4617 | DR274 FP-SEC -BsaI | gATG-GGTa | [seq] |
3869 | DR274 FP-NT – BsaI | gATG-ACGc | [seq] |
3843 | DR274 SEC-NT – BsaI | gAAG-ACGc | [seq] |
3702 | DR274 SEC-3’ arm – Bsa | gAAG-GTAg | [seq] |
Last updated 7-29-2023