hprtSAGFP
(Plasmid #41594)

• Purpose: In vivo single-strand annealing substrate composed of two truncated GFP genes, separated by a drug selection marker, and flanked by mouse hprt targeting sequences
• Depositing Lab: Maria Jasin
• Publication: Stark et al Mol Cell Biol. 2004 Nov;24(21):9305-16.

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, 100 μg/mL
• 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

• Supplemental Documents:
  • hprtSAGFP annotated sequence
• Articles Citing this Plasmid:
  • 6 References

Terms and Licenses

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

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.