pRL-TK CXCR4 1P
(Plasmid #11309)

• Depositing Lab: Phil Sharp
• Publication: Doench et al Genes Dev. 2003 Feb 15. 17(4):438-42.

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: 1 perfect bind site for CXCR4 siRNA antisense
• Insert Size (bp): 46
• 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
• 3′ sequencing primer: EBV-rev

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Renilla Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The end of the ORF and the beginning of the 3' UTR is (with the TAA
stop codon, followed by T, then the XbaI site TCTAGA):
AAATGAACAA TAA T TCTAGA GCGGCCGCT.

We digested the plasmids with XbaI, and ligated in the following:
ctaga ttccgagatatcggtaat gggcc.

This produced a modified vector, still containing an XbaI site, but
now also containing an ApaI site (GGGCCC), to allow for directional
cloning of 3' UTR inserts. The final vector contains, starting with
the stop codon TAA: TAA T Tctaga ttccgagatatcggtaat gggccC TAGA.

All UTR inserts were then constructed in the following format:
TCTAGA CTCGAGCCGG[binding site]ATCGC GGGCCC. A single perfect site
sequence is AAGTTTTCACTCCAGCTAACACCGG.

CXCR4 siRNA used is GUUUUCACUCCAGCUAACACATTCAAAAGUGAGGUCGAUUGU.