pET21-10XHis-GST-HRV-Her1-dL5-Her1
(Plasmid #73217)

• Purpose: Bacterial expression vector for Z1907-dL5(E52D)-Z1907 affibody fusion protein (AFA) with N-terminal His10, GST and HRV3C cleavage site (MBIC5, dL5**, FAP, AffiFAP)
• Depositing Lab: Marcel Bruchez
• Publication: Wang et al Bioconjug Chem. 2015 Jan 21;26(1):137-44. doi: 10.1021/bc500525b. Epub 2014 Dec 19.

Backbone

• Vector backbone: pET21a
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5387
• Total vector size (bp): 7238
• Modifications to backbone: none
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For expression use a strain that contains T7 RNA polymerase and trxB/gor double mutant such as Rosetta-gami(DE3)
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: 10XHis-GST-HRV-Her1-dL5-Her1
• Alt name: HLH
• Alt name: AFA
• Species: H. sapiens (human), Synthetic; Schistosoma japonicum
• Insert Size (bp): 1851
• Mutation: The dL5** FAP is E50D, L89S, also known as E52D/L91S and NP138
• Entrez Gene: EGFR (a.k.a. ERBB, ERBB1, ERRP, HER1, NISBD2, NNCIS, PIG61, mENA)
• Promoter: T7
• Tags / Fusion Proteins:
  • 10XHis-GST-HRV (N terminal on insert)
  • The Her1 Affibody and dL5 FAP are fused with 8 and 6 amino acids between

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI and HindIII for Affibody-FAP-Affibody (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: T7 and GSTSeqF
• 3′ sequencing primer: T7term

Terms and Licenses

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

Depositor Comments

Please visit the Molecular Biosensor and Imaging Center website for current protocols and information about dyes to be used in combination with the proteins expressed from this plasmid.

http://pathways.mbic.cmu.edu/

Note: Compared to the depositor's provided genbank file, Addgene's quality control sequencing finds one single nucleotide mismatch, G->A at bp 6867, which causes a G to R amino acid residue substitution. This discrepancy falls between the functional domains, and although it destroys a cloning site (BamHI) from the original clone construction, it should not otherwise alter plasmid function.