pMLM36 (aka pBR-UV5-GP-FD2)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||21871||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size (bp) 3508
Vector typeBacterial Expression
Growth in Bacteria
Growth Strain(s)XL1 Blue
Growth instructionsGrow in liquid LB supplemented with 50 µg/ml carbenicillin or on LB plates supplemented with 100 µg/ml carbenicillin. Due to toxicity associated with high expression from the lacUV5 promoter, this plasmid must be propagated in a lacIq strain (e.g. XL-1 Blue).
Copy numberLow Copy
- Cloning method Restriction Enzyme
- 5′ sequencing primer lac promoter (Common Sequencing Primers)
Combinatorial libraries of zinc finger arrays made by OPEN are cloned into this plasmid (see Maeder et al., Mol Cel 2008, PMID 18657511). Ligate zinc finger array fragments with BbsI-digested pBR-UV5-GP-FD2 vector backbone to create plasmids that express the zinc finger array as a FLAG-tagged Gal11P fusion in the B2H system.
The lacUV5 promoter which drives expression of the Gal11P fusion protein contains a lac operator and therefore can be repressed by lac repressor.
This plasmid is a low copy number plasmid and therefore we recommend that minipreps be performed from a 4 to 10 ml culture.
Annotations for pMLM36:
12-17: lacUV5 promoter -35 box / 36-41: lacUV5 promoter -10 box / 48: lacUV5 promoter transcription start point / 86-88: Start codon for Gal11P protein fragment / 89-358: Coding sequence for Gal11P a.a. 263-352 (with additional translationally silent mutations) / 407-412: BbsI site #1 / 433-438: BbsI site #2 / 568-1024: f1 phage origin of replication / 1156-2013: beta-lactamase gene cassette (confers resistance to ampicillin and carbenicillin) / 2016-2956: ColE1 origin of replication (plasmid origin) / 3203-3390: rop gene (associated with regulation of copy number)
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:pMLM36 (aka pBR-UV5-GP-FD2) was a gift from Keith Joung (Addgene plasmid # 21871 ; http://n2t.net/addgene:21871 ; RRID:Addgene_21871)