URL: http://www.addgene.org/48696
Proper Citation: RRID:Addgene_48696
Insert Name: 8 copies of the amber suppressor tRNACUA-Leu
Organism: Other
Bacterial Resistance: Ampicillin
Defining Citation: PMID:23924161
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5400; Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: The amber suppressor tRNACUA-Leu was derived from E. coli tRNAUAA-Leu in which U35 and A36 are mutated to C and U, respectively. The gene lacking 3′-CCA but with a 3′-TTTTTT sequence, driven by the human H1 promoter was synthesized by IDT. This was PCR amplified, digested with BglII and BamHI and was inserted into the BglII site of pCDNA3.1. Additional copies of the tRNA expression cassette were introduced in the resulting plasmid by successive rounds of BglII digestion, ligation of the BglII/BamHI digested insert, and verification of the insert orientation in the resulting plasmid by DNA sequencing and digestion analysis. AnapRS was PCR amplified and was inserted into pCDNA3.1-8x H1-tRNACUA-EcLeu between HindIII and XhoI restriction sites to generate pAnap. Note that any small discrepancies between Addgene's quality control sequence and the assembled sequence from the depositor should not affect plasmid function.
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Source: Addgene