LbCas12a-DR-GC-swap-cr in pBCSK+
(Plasmid #126643)

• Purpose: Cloning vector LbCas12a-DR-GC-swap-crRNA for expression in mammalian cells
• Depositing Lab: Scot Wolfe
• Publication: Liu et al Nucleic Acids Res. 2019 Mar 20. pii: 5403491. doi: 10.1093/nar/gkz184.

Backbone

• Vector backbone: pBCSK+
• Backbone size w/o insert (bp): 3232
• Total vector size (bp): 3267
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: LbCas12a Full Length Direct Repeat GC Swap crRNA
• Species: Synthetic
• Insert Size (bp): 35
• Promoter: U6 Promoter

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BbsI (destroyed during cloning)
• 3′ cloning site: BbsI (destroyed during cloning)
• 5′ sequencing primer: U6
• 3′ sequencing primer: T3

Resource Information

• Supplemental Documents:
  • LbCas12a-DR-GC-swap-cr in pBCSK+.gb

Terms and Licenses

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

Depositor Comments

For cloning crRNA guide sequences into the GC swap frameworks: The top oligo should contain a GTGG overhang on its 5' end and the bottom oligo should contain AAAA overhang on its 5' end to match the overhangs that are produced by BbsI. In addition, for these constructs the guide oligos should encode the final two bases [GT] of the constant region of the crRNA prior to the spacer [targeting] sequence and then two additional Ts following the spacer sequence to ensure robust RNA Polymerase III termination.