pFGC-I2Cas9
(Plasmid #173158)

• Purpose: Expresses Cas9 in dividing Arabidopsis cells. Contains dsRED and Basta for fast transformant selection.
• Depositing Lab: Piotr Ziolkowski
• Publication: Bieluszewski et al Nat Commun. 2022 Jan 12;13(1):277. doi: 10.1038/s41467-021-27882-5.

Backbone

• Vector backbone: pFGC-RCS
• Backbone size w/o insert (bp): 6615
• Total vector size (bp): 16301
• Modifications to backbone: Cloned dsRED cassette under napin promoter Cloned ICU2 promoter to control Cas9 expression
• Vector type: Plant Expression
• Selectable markers: Basta ; Transgenic seeds display DsRed fluorescence.

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For growing in Agrobacterium GV3101 strain, use kanamycin, gentamycin and rifampicin.
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: hSpCas9
• Alt name: human codon optimized Streptococcus pyogenes Cas9
• Species: Streptococcus pyogenes
• Insert Size (bp): 4101
• Mutation: codon usage optimized for expression in human
• Tags / Fusion Proteins:
  • 3xFLAG-NLS (N terminal on backbone)
  • NLS (C terminal on backbone)

Gene/Insert 2

• Gene/Insert name: pNAP:dsRED:tNOS
• Alt name: DsRed
• Species: Brassica napus, Discosoma sp., Agrobacterium tumefaciens
• Insert Size (bp): 2060
• Mutation: Contains a D169N mutation with no visible effect on fluorescence.

Gene/Insert 3

• Gene/Insert name: pMAS:BAR:tMAS
• Alt name: Basta
• Species: Agrobacterium tumefaciens, Streptomyces hygroscopicus
• Insert Size (bp): 1200

Gene/Insert 4

• Gene/Insert name: ICU2 upstream regulatory region
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1810

Resource Information

• Supplemental Documents:
  • pFGC-I2Cas9.gb
• A portion of this plasmid was derived from a plasmid made by: The 3×Flag-NLS-hSpCas9-NLS::NOSt cassette was a gift from Dr. Jian-Kang Zhu.
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This vector was designed and constructed by Dr. Tomasz Bieluszewski.

Detailed protocol for CRISPR/Cas9-based mutagenesis using this system in:
Bieluszewski T, Szymanska-Lejman M, Dziegielewski W, Zhu L, Ziolkowski PA. Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions, in: Plant Gametogenesis: Methods and Protocols (ed. Christophe Lambing), Methods in Molecular Biology, vol. 2484.
https://doi.org/10.1007/978-1-0716-2253-7_13
https://pubmed.ncbi.nlm.nih.gov/35461452/