|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||52256||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Modifications to backboneexpanded multiple cloning sites
Vector typeCRISPR ; Plant expression
Growth in Bacteria
Gene/Insert name35SPPDK:pcoCas9:NOS cassette
Alt nameplant codon optimized Cas9
- Promoter constitutive 35SPPDK promoter
- Cloning method Restriction Enzyme
- 5′ cloning site StuI (not destroyed)
- 3′ cloning site StuI (not destroyed)
- 5′ sequencing primer Sequencing primer for multiple cloning sites (from somewhere close to the EcoRI site): 5’ AATAAAAACTGACTCGGA 3’
- 3′ sequencing primer Sequencing primer for 35SPPDK:pcoCas9:NOS (from somewhere close to the 35SPPDK promoter): 5’ AGTTGGGTAACGCCAGGG 3’ (Common Sequencing Primers)
All labeled restriction sites (RS) in red on the map of the binary plasmid pFGC-pcoCas9 is single cut except StuI, which was used to insert the 35SPPDK:pcoCas9:NOS expression cassette. Among those RSs, EcoRI, XhoI, AscI, PacI, AsiSI, SbfI, and XmaI/SmaI can be used to insert single or multiple guide RNA expression cassettes (AscI, PacI, and SbfI are strongly recommended).
The pUC119-gRNA (Addgene plasmid #52255) construct is used as the PCR template to assemble a new guide RNA expression cassette flanked by desired restriction site(s) according to Fig. S5 in Li et al. Nat. Biotechnol. 31: 688-691.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pFGC-pcoCas9 was a gift from Jen Sheen (Addgene plasmid # 52256 ; http://n2t.net/addgene:52256 ; RRID:Addgene_52256)