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
• Depositing Organization: Massachusetts Institute of Technology
  Located in the United States of America
• 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.

DNA (Catalog # 73145-D50)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 50 μg
• Concentration: 1 μg/μL
• Pricing: $495 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.

DNA (Catalog # 73145-D100)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 100 μg
• Concentration: 1 μg/μL
• Pricing: $585 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.