pNTI194
(Plasmid #84266)

• Purpose: Expresses Avi-tagged RPL1 and humanized birA
• Depositing Lab: Nicholas Ingolia
• Publication: Ingolia et al Cell Rep. 2014 Sep 11;8(5):1365-79. doi: 10.1016/j.celrep.2014.07.045. Epub 2014 Aug 21.

Backbone

• Vector backbone: pcDNA5/FRT/TO
• Backbone manufacturer: Invitrogen
• Vector type: Mammalian Expression
• Selectable markers: Hygromycin

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: RPL1
• Alt name: RPL10A
• Alt name: ribosomal protein L10a
• Species: H. sapiens (human); (*see below)
• GenBank ID: NM_007104.4 NP_009035.3
• Entrez Gene: RPL10A (a.k.a. CSA19, Csa-19, L10A, NEDD6)
• Promoter: CMV
• Tags / Fusion Proteins:
  • Gly/Ser linker (C terminal on insert)
  • TEV site (C terminal on insert)
  • Avi tag (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: none

Gene/Insert 2

• Gene/Insert name: humanized birA
• Species: H. sapiens (human); E. coli
• Mutation: R33A, A34E (*see below)
• Promoter: P2A peptide
• Tag / Fusion Protein:
  • Flag (C terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: none
• 3′ sequencing primer: BGH-rev

Resource Information

• Supplemental Documents:
  • Supporting Information for article
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

*Two mutations, R33A and A34E, were introduced into the biotin ligase order to eliminate an unwanted DNA-binding activity without affecting biotinylation, as described in Buoncristiani et al., 1986.

Note that the RPL10A protein sequences are identical in human, mouse, and rat, but the human DNA sequence is used in this plasmid.