|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||32115||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
Backbone manufacturerNew England Biolabs
- Backbone size w/o insert (bp) 6700
Modifications to backboneAlready has two copies of MT inserted to begin with. This parent plasmid is available as Addgene plasmid 32089.
Vector typeBacterial Expression
Growth in Bacteria
Bacterial Resistance(s)Ampicillin, 100 μg/mL
Copy numberHigh Copy
Gene/Insert nameMT (3x)
SpeciesM. musculus (mouse)
Entrez GeneMt1 (a.k.a. MT-I, Mt-1)
- Promoter P-lac
/ Fusion Protein
- MBP (N terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site XmnI (not destroyed)
- 3′ cloning site AflIII/NcoI (destroyed during cloning)
- 5′ sequencing primer MBP_F (Common Sequencing Primers)
A portion of this plasmid was derived from a plasmid made byThe metallothionein (MT) gene was amplified from pET-3d-MT (a gift from the Winge laboratory, University of Utah), which contains the MT gene within the pET-3d vector (Novagen, Madison, WI).
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
In this construct, a translational modification was made that changes an alanine to an aspartic acid at the junction between MT genes. The third copy of the MT gene was inserted into the pMAL-c2x-MT2 plasmid (Addgene plasmids 32102) before the existing MT copies. The inserted MT gene was amplified from pET-3d-MT using the forward primer (50-CTCGGGATCGAGGGAAGGATTTCAAGATATACCATGGACCCC-30), which codes for the MBP linker region containing an XmnI and NcoI site fused in frame to the MT gene, and the reverse primer (50-GTGACCACATGTCACAGCACGTGCACTTGTCC-3 0 ), which contains an AflIII site at the MT–MT junction. The pMAL-c2x-MT2 plasmid was prepared by digesting with XmnI and NcoI, and the PCR product was constructed with XmnI and AflIII. Since NcoI and AflIII produced equivalent DNA ends, the second copy of MT was inserted with removal of both restriction sites at the ligation junction. This process can be reproduced iteratively to introduce as many copies of MT in frame into the construct as desired. In addition to this plasmid Addgene offers versions with 1 copy (32101), 2 copies (32102) and 4 copies (32100) of MT.
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:pMAL-c2x-MT3 was a gift from David DeRosier & Chris Mercogliano & Cristina Risco Ortiz (Addgene plasmid # 32115 ; http://n2t.net/addgene:32115 ; RRID:Addgene_32115)
For your References section:Visualization of proteins in intact cells with a clonable tag for electron microscopy. Diestra E, Fontana J, Guichard P, Marco S, Risco C. J Struct Biol. 2009 Mar;165(3):157-68. Epub 2008 Dec 10. 10.1016/j.jsb.2008.11.009 PubMed 19114107