pPB-tetO(hCMV1)-HA-Tet1mHxD(201R2)-IV
(Plasmid #102422)

• Purpose: Inducible expression of siRNA resistant mouse Tet1-201 (ENSMUST00000050826.13) with mutated catalytic domain (H1620Y & D1622A), HA-tag and IRES-Venus in piggyBac (PB) transposon vector
• Depositing Lab: Kian Peng Koh
• Publication: Khoueiry et al Nat Genet. 2017 Jul;49(7):1061-1072. doi: 10.1038/ng.3868. Epub 2017 May 15.

Backbone

• Vector backbone: pPB-hCMV1-hNANOG-IV
• Backbone manufacturer: Austin Smith (Addgene plasmid# 60432)
• Modifications to backbone: human NANOG removed to insert Tet1 (201R2)
• Vector type: Mammalian Expression
• Selectable markers: mVenus

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Tet1 catalytic domain mutant
• Species: M. musculus (mouse)
• Mutation: H1620Y and D1622A mutations in Tet1 catalytic domain; Modified at the Dharmacon SMARTpool siRNA #2 target site to make it siRNA-resistant
• GenBank ID: NM_027384.1
• Entrez Gene: Tet1 (a.k.a. 2510010B09Rik, Cxxc6, D10Ertd17e, LCX, mKIAA1676)
• Promoter: TetO-CMV
• Tag / Fusion Protein:
  • HA (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: blunt end (destroyed during cloning)
• 3′ cloning site: NotI (unknown if destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: IRES-R

Resource Information

• Supplemental Documents:
  • 102422.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • piggyBac Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The full length transcript 201 refers to the coding sequence (CDS) of Ensembl transcript Tet1-201 (transcript ID ENSMUST00000050826.13) or NCBI RefSeq NM_027384.1. This variant (isoform 2) lacks an alternate in-frame exon in the central coding region, compared to variant 1.