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hprtSAGFP
(Plasmid #41594)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 41594 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pGPD351 (modified from pBluescript)
  • Backbone manufacturer
    Greg Donoho (Donoho, Jasin, & Berg, Mol Cell Biol. 1998) (PMID: 9632791)
  • Backbone size w/o insert (bp) 2721
  • Total vector size (bp) 11342
  • Modifications to backbone
    From pBluescript: Targeting DNA consisting of a 5.5-kb DNA fragment of the hprt gene on the mouse X chromosome inserted
  • Vector type
    Mammalian Expression, Mouse Targeting ; DSB repair reporter substrate
  • Selectable markers
    Puromycin

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    SA-GFP reporter
  • Alt name
    hprt-SAGFP
  • Alt name
    GFP
  • Species
    M. musculus (mouse), Synthetic
  • Insert Size (bp)
    8621

Resource Information

Depositor Comments

This construct encodes a novel chromosomal reporter substrate, SA-GFP, which that allow the introduction of a double-strand break (DSB) and which assays single-strand annealing (SSA). The DSB is generated by the rare-cutting endonuclease I-SceI, whose 18-bp recognition sequence has been integrated within the green fluorescent protein gene (GFP) in such a way that it disrupts the gene. The SA-GFP reporter consists of two tandem GFP gene fragments: 5'GFP and SceGFP3'. Repair of the I-SceI-generated DSB in SceGFP'3 by SSA results in a functional GFP gene when a DNA strand from SceGFP3' is annealed to the complementary strand of 5GFP', followed by appropriate DNA-processing steps. As a result, SSA between the homologous sequences in the GFP gene fragments produces a 2.7-kb deletion in the chromosome. The SA-GFP reporter can also be repaired by HDR, but this repair does not restore a functional GFP gene.

The hprtSAGFP construct has homology to the hypoxanthine phosphoribosyl transferase (hprt) locus and contains the selectable puromycin resistance gene (puroR) as well as the SSA reporter substrate SA-GFP. Cells that integrate this fragment at a random genomic location are resistant to puromycin, whereas cells in which the reporter is successfully targeted to the hprt locus are additionally resistant to 6-thioguanine (6-TG).

Green fluorescent protein (GFP) gene fragments for SA-GFP were derived from pEGFP-N1 (Clonetech). The SA-GFP reporter was cloned into the mouse hprt targeting vector pGPD351 (a kind gift of Greg Donoho; Donoho, Jasin, & Berg, Mol Cell Biol. 1998). The direction of transcription of the SceGFP gene is the same as that for hprt.

A complete annotated sequence is available for download in the supplementary information above.

Please Note: Addgene NGS unable to fully resolve CAG promoter sequence. Please see depositor sequence for that section of the plasmid.

How to cite this plasmid ( Back to top)

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:

    hprtSAGFP was a gift from Maria Jasin (Addgene plasmid # 41594 ; http://n2t.net/addgene:41594 ; RRID:Addgene_41594)
  • For your References section:

    Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M. Mol Cell Biol. 2004 Nov;24(21):9305-16. 10.1128/MCB.24.21.9305-9316.2004 PubMed 15485900