Plasmid 27203: pMLM802

• Gene/insert name: FokI endonuclease
• Fusion protein or tag: FLAG
• Terminal: N terminal on insert
• Vector backbone: pMLM802
  (Search Vector Database)
• Vector type: Mammalian Expression ; zebrafish expression
• Backbone size w/o insert (bp): 5727
• Cloning site 5': XbaI
• Site destroyed during cloning: No
• Cloning site 3': NotI
• Site destroyed during cloning: No
• 5' sequencing primer: CMV-F
• Bacterial resistance(s) Ampicillin
• Growth strain(s) XL1 Blue
• Growth temperature (℃): 37
• Growth instructions: Grow in XL1 Blue
• High or low copy: High Copy
• Sequence: View sequences (2)
• Principal Investigator: Keith Joung
• Terms and Licenses: MTA

Comments: 

For expression of a "-" heterodimeric FokI domain. (Miller et al., Nat Biotech 2007) in mammalian cells or zebrafish for ZFN targets with a 7bp spacer (Handel et al 2008, Mol Ther). To clone a zinc finger array into this plasmid, perform the following steps: (1) amplify the zinc finger array coding sequence from a B2H expression vector by PCR using primers OMM429 (5’-GATGACAAATCTAGACCCGGGGAGCG-3’) and OMM430 (5’-CTGCGGCCGCACCGGGTCCTGTGTGGGTTTTTAGGTG-3’), (2) digest the resulting PCR fragment with XbaI and NotI, and (3) clone the digested fragment into MLM802 vector backbone that has been digested with XbaI and NotI. The resulting plasmid will encode a ZFN that can be paired with a ZFN cloned into expression vector MLM800 to target a ZFN target site with a 7 bp spacer (Handel et al. 2008, Mol Ther.).

The difference between pMLM800/802 and pMLM290/292 is the length of the “spacer” sequence in the full ZFN target site. If the spacer is 7 bp, scientists should use pMLM800/802. If the spacer is 5 or 6 bps, scientists should use pMLM290/292.

Article: An improved zinc-finger nuclease architecture for highly specific genome editing. Miller et al (Nat Biotechnol. 2007 Jul . 25(7):778-85. PubMed)