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URL: http://www.addgene.org/32115

Proper Citation: RRID:Addgene_32115

Insert Name: MT (3x)

Organism: Mus musculus

Bacterial Resistance: Ampicillin

Defining Citation: PMID:19114107

Vector Backbone Description: Backbone Marker:New England Biolabs; Backbone Size:6700; Vector Backbone:pMAL-c2x; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

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.

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