pRL-TK 4x mut G
(Plasmid #11320)

• Depositing Lab: Phil Sharp
• Publication: Doench et al Genes Dev. 2004 Mar 1. 18(5):504-11.

Backbone

• Vector backbone: pRL-TK
• Backbone manufacturer: Promega
• Backbone size w/o insert (bp): 4045
• Vector type: Luciferase

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: 4 bulged binding sites for CXCR4 siRNA antisense
• Mutation: Mutation G (see paper) on inner 2 binding sites.
• Tag / Fusion Protein:
  • Rr-luc (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: ApaI (not destroyed)
• 5′ sequencing primer: See map

Terms and Licenses

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

Depositor Comments

Two identical mutant binding sites, separated by 4 nt, were flanked by two of the orginal bulged binding sites, each 11 nt away.

Two original CXCR4 sites, with XhoI and SpeI restriction sites between them, were inserted into the XbalI site in the 3' UTR of the pRL-TK plasmid. The mutant binding sites were then inserted by ligating annealed oligos into the XhoI and SpeI sites.

See Addgene plasmid 11307 for cloning of binding sites into the backbone, used in Doench, Sharp 2003 paper.