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RRID:Addgene_41594 RRID Copied

Plasmid Information

Proper Citation: RRID:Addgene_41594

Insert Name: SA-GFP reporter

Organism: Mus musculus

Bacterial Resistance: Ampicillin

Defining Citation: PMID:15485900

Vector Backbone Description: Backbone Marker:Greg Donoho (Donoho, Jasin, & Berg, Mol Cell Biol. 1998) (PMID: 9632791); Backbone Size:2721; Vector Backbone:pGPD351 (modified from pBluescript); Vector Types:Mammalian Expression, Mouse Targeting, Other, DSB repair reporter substrate; Bacterial Resistance:Ampicillin

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.

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Plasmid Name: hprtSAGFP

Record Creation Time: 2022-04-22 03:22:15

Record Last Update: 2023-09-15 01:11:01

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