pEVOL-pAzF[TAG]
(Plasmid #164579)

• Purpose: Machinery plasmid containing two copies of the Mj pCNFRS and cognate tRNA for incorporation of pAzF, pCNF, or pENF at TAG codons
• Depositing Lab: Ryan Mehl
• Publication: Bednar et al ACS Chem Biol. 2021 Nov 19;16(11):2612-2622. doi: 10.1021/acschembio.1c00649. Epub 2021 Sep 30.

Backbone

• Vector backbone: pEVOL
• Backbone size w/o insert (bp): 4179
• Total vector size (bp): 6104
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: Mj pCNFRS[TAG] (aaRS1)
• Alt name: pAzFRS (1)
• Species: Methanocaldococcus jannaschii (engineered)
• Insert Size (bp): 924
• Mutation: Y32L L65V F108W Q109M D158G I159P
• Promoter: araBAD

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ATGCCATAGCATTTTTATCC
• 3′ sequencing primer: CTTCTGCGTTCTGATTTAATCTG

Gene/Insert 2

• Gene/Insert name: Mj pCNFRS[TAG] (aaRS2)
• Alt name: pAzFRS (2)
• Species: Methanocaldococcus jannaschii (engineered)
• Insert Size (bp): 924
• Mutation: Y32L L65V F108W Q109M D158G I159P
• Promoter: glnS

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: TCCCGGTCATCAATCATCC
• 3′ sequencing primer: AACTCAATATATTGCAGAGATCATG

Gene/Insert 3

• Gene/Insert name: Mj tRNA[TAG]
• Alt name: Mj tRNA[CTA]
• Species: Methanocaldococcus jannaschii
• Insert Size (bp): 77
• Promoter: proK

Cloning Information for Gene/Insert 3

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CCTGCAGGTAATTCCGCTTCGCAACATGTGAGCACCGG
• 3′ sequencing primer: AATAAATCCATGGCAAATTCGACCCTGAGCTGCTCGAGCATGC

Terms and Licenses

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

Depositor Comments

Machinery plasmid for incorporation of para-Azidophenylalanine (pAzF), para-cyanophenylalanine (pCNF), and para-ethynylphenylalanine (pENF) at TAG codons.

Please visit https://doi.org/10.1101/2021.04.12.439361 for bioRxiv preprint.