CAG sHRPa-FRB-pre mGRASP
PurposeLarge fragment of split HRP and FRB fused to the extracellular terminus of the "pre mGRASP" scaffold published by Magee, in which the extracellular domain of NRX is replaced by portions of CD4.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||73145||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4247
- Total vector size (bp) 6173
Vector typeMammalian Expression
Growth in Bacteria
Bacterial Resistance(s)Ampicillin, 100 μg/mL
Copy numberHigh Copy
Gene/Insert namesHRPa-FRB-pre mGRASP
Insert Size (bp)1926
- Promoter CAG
/ Fusion Protein
- Flag tag
- Cloning method Restriction Enzyme
- 5′ cloning site EcoRI (not destroyed)
- 3′ cloning site HindIII (not destroyed)
- 5′ sequencing primer gctaaccatgttcatgccttc (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
sHRPa is the large split HRP fragment. It consists of amino acids 1-213 of horseradish peroxidase (HRP) with the following 4 mutations: T21I, P78S, R93G, N175S.
The insert contains the following features:
EcoRI-β integrin ss-AgeI-sHRPa-10 aa linker-XhoI-FRB-MfeI-flag-CD4-2(25-242 aa)-NRX1β(414-468 aa)-NotI-stop-HindIII
10 aa linker: KGSGSTSGSG
FRB: we used a 94 aa sequence that matches residues 5-98 of chain A in PDB 1AUE
Nematode β integrin ss: MPPSTSLLLLAALLPFALPASDWKTGEVTAS
This construct is identical to the previously reported “pre-mGRASP” (Addgene ID 34910) except AgeI and NotI restriction sites are added here, and the 5 aa linker here is shorter than the previously reported 10 aa linker.
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:CAG sHRPa-FRB-pre mGRASP was a gift from Alice Ting (Addgene plasmid # 73145 ; http://n2t.net/addgene:73145 ; RRID:Addgene_73145)
For your References section:A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses. Martell JD, Yamagata M, Deerinck TJ, Phan S, Kwa CG, Ellisman MH, Sanes JR, Ting AY. Nat Biotechnol. 2016 May 30. doi: 10.1038/nbt.3563. 10.1038/nbt.3563 PubMed 27240195