pMAL-c2x-MT3
(Plasmid #32115)

• Depositing Labs: David DeRosier, Chris Mercogliano, Cristina Risco Ortiz
• Publication: Diestra et al J Struct Biol. 2009 Mar;165(3):157-68. Epub 2008 Dec 10.

Backbone

• Vector backbone: pMAL-c2x
• Backbone manufacturer: New England Biolabs
• Backbone size w/o insert (bp): 6700
• Modifications to backbone: Already has two copies of MT inserted to begin with. This parent plasmid is available as Addgene plasmid 32089.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: MT (3x)
• Species: M. musculus (mouse)
• Entrez Gene: Mt1 (a.k.a. MT-I, Mt-1)
• Promoter: P-lac
• Tag / Fusion Protein:
  • MBP (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XmnI (not destroyed)
• 3′ cloning site: AflIII/NcoI (destroyed during cloning)
• 5′ sequencing primer: MBP_F

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The 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

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.