pHEE2E-TRI
(Plasmid #71288)

• Purpose: Egg cell-specific promoter-controlled expression 3×FLAG-NLS-zCas9-NLS and two sgRNAs targeting three genes: ETC2, TRY, and CPC
• Depositing Lab: Qi-Jun Chen
• Publication: Wang et al Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0.

Backbone

• Vector backbone: pCambia
• Vector type: Plant Expression, CRISPR ; Plant binary vector
• Selectable markers: Hygromycin

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: sgRNA targeting TRY and CPC genes
• Species: Synthetic
• Promoter: U6-26p Arabidopsis U6 gene promoter

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: M13-fwd

Gene/Insert 2

• Gene/Insert name: sgRNA targeting ETC2 gene
• Species: Synthetic
• Promoter: U6-26p Arabidopsis U6 gene promoter

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: M13-fwd

Gene/Insert 3

• Gene/Insert name: zCas9
• Alt name: 3xFLAG-NLS-zCas9-NLS
• Species: S. pyogenes
• Mutation: Zea mays codon-optimized Cas9
• Promoter: EC1.2 enhancer fused to EC1.1 promoter
• Tags / Fusion Proteins:
  • 3x FLAG (N terminal on insert)
  • NLS (N terminal on insert)
  • NLS (C terminal on insert)

Cloning Information for Gene/Insert 3

• Cloning method: Unknown
• 5′ sequencing primer: n/a
• 3′ sequencing primer: M13-rev

Resource Information

• Supplemental Documents:
  • Annotated GenBank file
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

TRY & CPC sgRNA target sequence is: AATATCTCTCTATCTCCTC

ETC2 sgRNA target sequence is: GAAGTGAGTAGCATCGAAT

See Figure 1B of the associated publication for more details.