pRL-TK CXCR4 6x
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||11308||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4045
Growth in Bacteria
Copy numberHigh Copy
Gene/Insert name6 bulged bind sites for CXCR4 siRNA antisense
Insert Size (bp)165
/ Fusion Protein
- Rr-luc (N terminal on backbone)
- 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 (Common Sequencing Primers)
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pRL-TK CXCR4 6x was a gift from Phil Sharp (Addgene plasmid # 11308 ; http://n2t.net/addgene:11308 ; RRID:Addgene_11308)
For your References section:siRNAs can function as miRNAs. Doench JG, Petersen CP, Sharp PA. Genes Dev. 2003 Feb 15. 17(4):438-42. 10.1101/gad.1064703 PubMed 12600936