Plasmid 21873: pMLM292

• Gene/insert name: None
• Fusion protein or tag: Flag
• Terminal: N terminal on backbone
• Vector backbone: pMLM292
  (Search Vector Database)
• Vector type: Mammalian Expression ; zebrafish expression
• Backbone size (bp): 5725
• Cloning site 5': XbaI
• Site destroyed during cloning: No
• Cloning site 3': BamHI
• Site destroyed during cloning: No
• 5' sequencing primer: CMV-F
• Bacterial resistance(s) Ampicillin
• Growth strain(s) DH5alpha
• Growth temperature (℃): 37
• High or low copy: High Copy
• Sequence: View sequences (3)
• Principal Investigator: Keith Joung
• Terms and Licenses: MTA

Comments: 

Zinc fingers are cloned in between the unique XbaI and BamHI sites. This results in an expression vector which encodes a ZFN with the OPEN zinc finger array fused to the mutant FokI nuclease domain.

pMLM292 is identical to pST1374 except that it harbors two mutations in the FokI nuclease domain (Q486E, I499L; aka the “-“ mutation see Miller et al., Nat. Biotech 2007, PMID 17603475) which confers heterodimeric behavior on these domains

235- 822: CMV promoter / 771-775: CAAT box / 804-808: TATA box / 819: 3' end of hCMV / 904-906: ZFN start codon / 910-930: SV40 nuclease localization signal (NLS) / 940- 963: FLAG tag / 964- 969: XbaI site / 980- 985: BamHI site / 986-1573: FokI nuclease domain / 1292&1294: Q486E heterodimer FokI mutation / 1331: I499L heterodimer FokI mutation / 1574-1576: ZFN stop codon / 1686-1910: BGH polyadenylation sequence / 1956-2384: f1 origin / 2411-2720: SV40 early promoter and origin / 2775-2841: EM-7 Promoter / 2842-3240: Blasticidin resistance gene / 3398-3528: SV40 late polyA signal / 3955-4574: pUC origin / 4729-5589: ampicillin (bla) resistance gene (complementary strand) / 5590-5688: bla promoter (complementary strand)