BB44_pmc.DonorR5.TS
(Plasmid #199223)

• Purpose: Donor plasmid with CCR5 target sites for ITPN or HMEJ knock-in at the human CCR5 safe harbour locus
• Depositing Lab: Manuel Goncalves
• Publication: Wang et al Nucleic Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165.

Backbone

• Vector backbone: pBR322
• Backbone size w/o insert (bp): 2700
• Total vector size (bp): 6832
• Vector type: Mammalian Expression ; Gene targeting donor plasmid

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: Expression unit for mCherry - monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)
• Species: Synthetic
• Insert Size (bp): 1490
• Promoter: Human PGK1 gene promoter

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: M13 rev
• 3′ sequencing primer: M13 fwd

Resource Information

• Supplemental Documents:
  • BB44_pmc.DonorR5.TS.gbk
• A portion of this plasmid was derived from a plasmid made by: Details on mCherry in: Shaner et al. Nat Biotechnol. 2004 22:1567-72.

Terms and Licenses

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

Depositor Comments

This target site-containing donor plasmid can be used to insert a transgene at the human CCR5 safe harbor locus when delivered together with gRNA expression plasmid AY22_pgRNA.R5.1 (Addgene plasmid #100294) and a SpCas9 nuclease (HMEJ-based gene targeting) or a SpCas9.D10A nickase (ITPN-based gene targeting).

Note: to reduce the chances for off-targeting donor DNA insertions when applying HMEJ-based genome editing it is preferable using AW01_pU.CAG.eSpCas9(1.1).rBGpA (Addgene plasmid #199255) or a similar construct, i.e., expressing a high specificity nuclease. Combining this donor with a S. pyogenes Cas9 nickase (e.g., Addgene plasmids #199256, #199253, #199254 or #199252) leads to seamless ITPN-mediated gene targeting.