- Backbone size (bp) 5725
Vector typeMammalian Expression ; zebrafish expression
Growth in Bacteria
Copy numberHigh Copy
/ Fusion Protein
- Flag (N terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site XbaI (not destroyed)
- 3′ cloning site BamHI (not destroyed)
- 5′ sequencing primer CMV-F (Common Sequencing Primers)
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)
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pMLM292 was a gift from Keith Joung (Addgene plasmid # 21873)
Map generated by Addgene from full sequence.