I’m sharing the following details that may be useful to you:

(1) Plasmid Full Name:

pTRE3GBi_EF1A_Tet3G_IRES_Puro-VHH hFBXW11-mCherry

or pTRE-BI-hGRAD

> Bacterial Resistance: Ampicillin.

> Addgene Link: https://www.addgene.org/207837/

(2) Reference Paper:

https://rupress.org/jcb/article/223/2/e202304030/276463/hGRAD-A-versatile-one-fits-all-system-to-acutely

(3) Plasmid Map: Attached.

(4) Plasmid Concentration:

The plasmid yield can be low with Miniprep, so I recommend using a Midiprep for better results.

(5) Stable Integration:

> Linearization of the hGRAD plasmid is recommended for stable integration. Use the AseI restriction enzyme to cut in the ampicillin resistance gene of the plasmid to linearize it.

> Due to low plasmid yield during purification, digest 1–1.5 µg of plasmid in a 20 µL reaction, inactivate the enzyme, and directly use this reaction mix for transfection into cells.

> For selection, use Puromycin (2 µg/ml). Once stable cell lines are generated, induce hGRAD expression with Doxycycline (Dox, 1 µg/ml), and perform FACS sorting by gating for low GFP and high mCherry (reporter) to sort cells with functional hGRAD.

> Maintain the cell line under puromycin selection pressure.

(6) Linker we use for EGFP knock-in:

I’ve attached the HDR template map for Srrm2-EGFP Knockin, which includes the "linker" used for the C-terminal EGFP knock-in.

(7) The sequencing file for insert: "Fbox-GFP nanobody" is attached.

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