pQhsCDtRNA-
(Plasmid #86015)

• Purpose: Multi-gene expression system with human box C/D proteins with U14
• Depositing Lab: Hong Li
• Publication: Peng et al PLoS One. 2014 Jul 31;9(7):e103096. doi: 10.1371/journal.pone.0103096. eCollection 2014.

Backbone

• Vector backbone: pQLinkN (Addgene #13670)
• Backbone manufacturer: Konrad Buessow lab
• Vector type: Bacterial Expression ; Co-Expression; Purification

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: U14
• Species: H. sapiens (human)
• Entrez Gene: SNORD14A (a.k.a. RNU14, RNU14A, U14, U14-S13-5)

Gene/Insert 2

• Gene/Insert name: 15.5K
• Species: H. sapiens (human)
• Entrez Gene: SNU13 (a.k.a. 15.5K, FA-1, FA1, NHP2L1, NHPX, OTK27, SNRNP15-5, SPAG12, SSFA1)

Gene/Insert 3

• Gene/Insert name: fibrillarin
• Species: H. sapiens (human)
• Mutation: amino acids 83-316
• Entrez Gene: FBL (a.k.a. FIB, FLRN, Nop1, RNU3IP1)

Gene/Insert 4

• Gene/Insert name: NOP56
• Species: H. sapiens (human)
• Mutation: amino acids 1-411
• Entrez Gene: NOP56 (a.k.a. NOL5A, SCA36)
• Tag / Fusion Protein:
  • 6x-His (C terminal on insert)

Gene/Insert 5

• Gene/Insert name: NOP58
• Species: H. sapiens (human)
• Mutation: amino acids 1-401
• Entrez Gene: NOP58 (a.k.a. HSPC120, NOP5, NOP5/NOP58)

Terms and Licenses

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

Depositor Comments

Please see associated publication for additional graphics describing this plasmid.

Plasmid construction: To construct the human box C/D plasmid, pQhsCD, pQLinkN (Addgene #13670) was inserted with the coding sequences of Homo sapiens (Hs) NOP56 (amino acids 1 to 411), NOP58 (amino acids 1 to 401) and fibrillarin (amino acids 83 to 316), 15.5 K, and U14 snoRNA. This plasmid differs from #86014 in that U14 is not part of a tRNA-containing minigene.

Each protein-encoding gene was cloned into pQLink-N plasmid using BamHI and NotI sites. The plasmids containing two different protein-encoding genes were then digested by SwaI and PacI, respectively, treated with LIC qualified T4 polymerase (dCTP for Pac I digest and dGTP for Swa I digest) and then annealed at 70°C. Each clone was identified by digestion of the recombined plasmid with Pac I. This process was repeated for additional coding sequence until all were inserted.