pFGC-pcoCas9
(Plasmid #52256)

• Purpose: A binary plasmid containing 35SPPDK:pcoCas9:NOS cassette for plant expression of Cas9 and multiple cloning sites for insertion of guide RNA
• Depositing Lab: Jen Sheen
• Publication: Sheen lab CRISPR plasmids (unpublished)

Backbone

• Vector backbone: pFGC-RCS
• Modifications to backbone: expanded multiple cloning sites
• Vector type: CRISPR ; Plant expression
• Selectable markers: Basta

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: 35SPPDK:pcoCas9:NOS cassette
• Alt name: Cas9
• Alt name: plant codon optimized Cas9
• Species: Synthetic
• Promoter: constitutive 35SPPDK promoter

Cloning Information

• 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’

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 13 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.