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URL: http://www.addgene.org/48323

Proper Citation: RRID:Addgene_48323

Bacterial Resistance: Spectinomycin

Vector Backbone Description: Backbone Size:3566; Vector Backbone:pET; Vector Types:Bacterial Expression; Bacterial Resistance:Spectinomycin

Comments: This plasmid is an empty vector to be used with a LIC cloning protocol. It has a Spec resistance. To clone into this vector, add LICv2 fusion tags to the 5' end of your PCR primers. LICv2 Forward - 5'TTTAAGAAGGAGATATAGATC3' LICv2 Reverse - 5'TTATGGAGTTGGGATCTTATTA3' Linearize the plasmid with EcoRV and gel purify. When digesting the DNA with T4 polymerase for LIC, use dCTP for insert and dGTP for vector. The 13-series vectors were designed to enable rapid cloning for a co-expression system. Vectors are based on Novagen's Duet system. Each gene is expressed on its own vector, which has been optimized so that each gene expresses at approximately equal levels. The 13-series vectors are all compatible with 2-series transfer vectors, so if cotransformation fails, there is a readily available backup in polycistronic expression. 13S CDF origin SpecR13K ColA origin KanR2-series transfer ColE1 origin AmpR13S vectors must be cotransformed with a 2-series vector for optimal expression (that is, the presence of an empty 2A-T vector enhances expression of a gene in 13S). For triple expressions, the proteins all seem to express at approximately equal levels. Genes in the CDF vector consistently express at very slightly lower levels, so if you're trying to pull down stoichiometric complexes, it's probably best to put His6-fusion protein in this vector. More information on this vector can be found through http://qb3.berkeley.edu/qb3/macrolab/

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