pANAP
(Plasmid #48696)

• Purpose: allows co-translational incorporation of a fluorescent AA directly into the target protein; expresses amber suppressor tRNA and the synthetase AnapRS
• Depositing Lab: Peter Schultz
• Publication: Chatterjee et al J Am Chem Soc. 2013 Aug 28;135(34):12540-3. doi: 10.1021/ja4059553. Epub 2013 Aug 14.

Backbone

• Vector backbone: pcDNA3.1(+)
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 5400
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: 8 copies of the amber suppressor tRNACUA-Leu
• Species: E.coli
• Promoter: human H1 promoter

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BglII (not destroyed)
• 3′ cloning site: BglII (destroyed during cloning)
• 5′ sequencing primer: NA

Gene/Insert 2

• Gene/Insert name: AnapRS
• Species: E.coli
• Mutation: mutant E.coli leucyl synthetase specific for Anap (U35 and A36 are mutated to C and U, respectively)
• Promoter: CMV

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (not destroyed)
• 3′ cloning site: XbaI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: BGH-Rev

Resource Information

• Articles Citing this Plasmid:
  • 9 References

Terms and Licenses

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

Depositor 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.