AD59_pEP.DonorCLYBL.TS
(Plasmid #199227)

• Purpose: Donor plasmid with CLYBL target sites for ITPN or HMEJ knock-in at the human CLYBL safe harbor 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): 2850
• Total vector size (bp): 5899
• Vector type: Mammalian Expression ; Gene targeting donor plasmid
• Selectable markers: Puromycin

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 PuroR.T2A.EGFP selectable and reporter markers
• Species: Synthetic
• Insert Size (bp): 2181
• Promoter: Human PGK1 gene promoter

Cloning Information

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

Resource Information

• Supplemental Documents:
  • AD59_pEP.DonorCLYBL.TS.gbk

Terms and Licenses

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

Depositor Comments

This target site-containing donor plasmid can be used to insert a transgene(s) at the human CLYBL safe harbor locus when delivered together with Sp-gRNA expression plasmid AY27_pU6.gRNA.CLYBL (Addgene plasmid #199238) or Sa-gRNA expression plasmid AM77_pU6.Sa-gRNA.CLYBL (Addgene plasmid #199237).

Combining this donor with a S. pyogenes Cas9 or a S. aureus Cas9 nuclease allows for HMEJ-mediated gene targeting.

Note: to reduce the chances for off-targeting donor DNA insertions when applying the Sp-gRNA for 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) or a S. aureus Cas9 nickase (Addgene plasmid #199251) leads to seamless ITPN-mediated gene targeting.