pET GFP domain 11 LIC cloning vector (GFP C-11)
(Plasmid #30183)

• Purpose: (Empty Backbone)
• Depositing Lab: Scott Gradia
• Publication: C-terminal fluorescent fusion vectors for E. coli expression (unpublished)

Backbone

• Vector backbone: pET
• Backbone size (bp): 5422
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: None
• Tags / Fusion Proteins:
  • GFP domain 11 (C terminal on backbone)
  • Biotin-H6-TEV recognition (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: LIC site vGFP (destroyed during cloning)
• 3′ cloning site: LIC site vGFP (destroyed during cloning)
• 5′ sequencing primer: T7 forward
• 3′ sequencing primer: T7 reverse

Terms and Licenses

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

Depositor Comments

This plasmid is an empty vector. Your gene can be inserted with a LIC cloning protocol.

This vector adds a short GFP domain 11 tag to the C-terminus of your protein. After expression and purification, your protein can be mixed with a "biosensor" molecule (GFP domains 1-10), and fluorescence will develop. This technique can be useful if directly fusing the entire GFP molecule interferes with the function of your protein.

To clone into this vector, add LIC tags to the 5' end of your PCR primers.

Forward - 5' TACTTCCAATCCAATGCA3'

Reverse - 5' CTCCCACTACCAATGCC 3'

Do NOT include a stop codon with your reverse primer.

Linearize the plasmid with SspI, then gel purify.

When digesting the DNA with T4 polymerase, use dCTP for the insert and dGTP for your linearized vector.

More information on this vector can be found through http://qb3.berkeley.edu/qb3/macrolab/