CAG sHRPa-FRB-pre mGRASP
(Plasmid #73145)

• Purpose: Large 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.
• Depositing Lab: Alice Ting
• Publication: Martell et al Nat Biotechnol. 2016 May 30. doi: 10.1038/nbt.3563.

Backbone

• Vector backbone: pCAG
• Backbone size w/o insert (bp): 4247
• Total vector size (bp): 6173
• Vector type: Mammalian Expression

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: sHRPa-FRB-pre mGRASP
• Species: Synthetic
• Insert Size (bp): 1926
• Promoter: CAG
• Tag / Fusion Protein:
  • Flag tag

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: gctaaccatgttcatgccttc

Terms and Licenses

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

Depositor Comments

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

CAG promoter
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.