pJHG003
(Plasmid #126454)

• Purpose: Expresses Cas9-XTEN-dTdT in mammalian cells.
• Depositing Lab: Chang Liu
• Publication: Loveless et al Nat Chem Biol. 2021 Jun;17(6):739-747. doi: 10.1038/s41589-021-00769-8. Epub 2021 Mar 22.

Backbone

• Vector backbone: pcDNA3.1
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 5523
• Total vector size (bp): 11238
• Modifications to backbone: Added a Kozak consensus sequence downstream of the NheI site. Switched neomycin resistance to hygromycin.
• Vector type: Mammalian Expression
• Selectable markers: Hygromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Cas9-XTEN-dTdT
• Alt name: terminal deoxynucleotidyl transferase
• Alt name: DNA nucleotidylexotransferase
• Alt name: Cas9
• Species: H. sapiens (human); Streptococcus pyogenes
• Insert Size (bp): 5715
• Mutation: Added D343E and D345E to inactivate TdT.
• GenBank ID: NM_004088.3
• Entrez Gene: DNTT (a.k.a. TDT)
• Promoter: CMV

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CMV Forward
• 3′ sequencing primer: BGH Reverse

Resource Information

• A portion of this plasmid was derived from a plasmid made by: George Church; Cas9 is from Addgene plasmid 41815.

Terms and Licenses

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

Depositor Comments

XTEN was used as a linker in a Cas9 fusion protein by Komor et al., 2016: doi 10.1038/nature17946.

The inactivating TdT mutations D343E and D345E were identified by Yang et al., 1994, J Biol Chem. 1994 Apr 22;269(16):11859-68 and combined by Repasky et al., 2004, doi: 10.4049/jimmunol.172.9.5478. Please visit https://doi.org/10.1101/639120 for bioRxiv preprint.