pMLM802
(Plasmid #27203)

• Depositing Lab: Keith Joung
• Publication: Miller et al Nat Biotechnol. 2007 Jul . 25(7):778-85.

Backbone

• Vector backbone: pMLM802
• Backbone size w/o insert (bp): 5727
• Vector type: Mammalian Expression ; zebrafish expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Growth instructions: Grow in XL1 Blue
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: FokI endonuclease
• Tag / Fusion Protein:
  • FLAG (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: CMV-F

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor 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.