pcDNA3.1(+)_4x(U6 tRNA M15)_CMV NESPylRS(AF)
(Plasmid #182652)

• Purpose: Encodes codon-optimized AF mutant of M. mazei pyrrolysine (Pyl) tRNA synthetase fused to a nuclear export signal (NESPylRSAF) & 4x M15 tRNACUA used for amber codon suppression in mammalian cells
• Depositing Lab: Ivana Nikić-Spiegel
• Publication: Arsic et al Nat Commun. 2022 Jan 14;13(1):314. doi: 10.1038/s41467-022-27956-y.

Backbone

• Vector backbone: pcDNA3.1(+)
• Vector type: Mammalian Expression
• Selectable markers: Zeocin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: codon-optimized Y306A/Y384F (AF) double mutant of Methanosarcina mazei-derived pyrrolysine (Pyl) tRNA synthetase
• Alt name: NESPylRS(AF)
• Species: Methanosarcina mazei
• Mutation: Y306A/Y384F (AF) double mutant of Methanosarcina mazei-derived pyrrolysine (Pyl) tRNA synthetase
• Promoter: CMV
• Tag / Fusion Protein:
  • nuclear export signal (NES) (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: CMVfw
• 3′ sequencing primer: NA

Gene/Insert 2

• Gene/Insert name: 4xU6-tRNAM15
• Species: Synthetic; Methanosarcina mazei
• Mutation: synthetic M15 tRNA(amber) variant
• Promoter: U6

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: NA
• 3′ sequencing primer: NA

Resource Information

• Supplemental Documents:
  • 454_pcDNA3.1(+)_4x(U6 tRNA M15)_CMV NESPylRS(AF).gb
• A portion of this plasmid was derived from a plasmid made by: The codon-optimized gene of PylRS(AF) was custom synthesized by GeneScript. 4xU6-M15 tRNA cassette is derived from the following plasmid https://www.addgene.org/105830/

Terms and Licenses

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