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    Plasmid map of pRL-TK_CXCR4_2x

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    pRL-TK CXCR4 2x
    Gene/insert name: 2 bulged bind sites for CXCR4 siRNA antisense
    Insert size (bp): 69
    Fusion proteins or tags: Rr-luc
    Terminal: N terminal on backbone
    Vector backbone: pRL-TK (Search Vector Database)
    Backbone manufacturer: Promega
    Type of vector: Luciferase
    Backbone size (bp): 4118
    Cloning site 5': XbaI
    Site destroyed during cloning: No
    Cloning site 3': ApaI
    Site destroyed during cloning: No
    5' Sequencing primer: See map (List of Sequencing Primers)
    3' Sequencing primer: EBV-rev
    Bacteria resistance: Ampicillin
    High or low copy: High Copy
    Grow in standard E. coli @ 37C: Yes
    Principal Investigator: Phil Sharp
    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.
    Article: siRNAs can function as miRNAs. Doench JG et al. (Genes Dev. 2003 Feb 15. 17(4):438-42. Pubmed)



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