pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS
(Plasmid #89508)

• Purpose: Expression of Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS in bacterial cells.
• Depositing Lab: Jin-Soo Kim
• Publication: Kim et al Nat Biotechnol. 2017 Feb 27. doi: 10.1038/nbt.3816.

Backbone

• Vector backbone: pET28a
• Backbone size w/o insert (bp): 5379
• Total vector size (bp): 10528
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For protein expression, transform into Rosetta (DE3) Competent cells. Induce at 0.6 OD with 1 mM IPTG final concentration. Growing overnight (12~14 hours) at 18 C.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: rAPOBEC1-nCas9 (D10A)-UGI-NLS
• Alt name: rAPOBEC1-nCas9-UGI-NLS
• Species: R. norvegicus (rat); Streptococcus pyogenes (bacteria)
• Insert Size (bp): 5149
• Mutation: D10A mutation of Cas9
• Promoter: T7
• Tags / Fusion Proteins:
  • rAPOBEC-XTEN (N terminal on insert)
  • UGI (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• A portion of this plasmid was derived from a plasmid made by: rAPOBEC-XTEN and UGI were excised from the pCMV-BE3 Vector (Addgene, 73021) and then placed into the pET28a-nCas9(D10A)-NLS Vector.

Terms and Licenses

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