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.

pMLM290 is identical to pST1374 except that it harbors two mutations in the FokI nuclease domain (E490K and I538K; 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 / 1304: E490K heterodimer FokI mutation / 1449-1450: I538K 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)

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.