pTO_CHMP3_LAP
(Plasmid #101849)

• Purpose: Tet-inducible expression of human CHMP3 with a C-terminal GFP and long linker (LAP tag), compatible with Flp-In recombination for stable integration
• Depositing Lab: Daniel Gerlich
• Publication: Mierzwa et al Nat Cell Biol. 2017 Jul;19(7):787-798. doi: 10.1038/ncb3559. Epub 2017 Jun 12.

Backbone

• Vector backbone: pTO_LAP_N-DEST
• Backbone size w/o insert (bp): 6098
• Total vector size (bp): 6766
• Modifications to backbone: Cloning procedure: 1. Starting vector: pcDNA5/FRT/TO (Invitrogen) 2. Cloning of LAP tag with Gateway cloning sites: starting point mCherry_N_dest 2.1. inserted a point mutation in XbaI site to remove Dam methylation site so it can be used for cloning (from GATC to GAAG) -> pmCherry_N_dest 2.2.exchanged mCherry with GFP using KpnI and XbaI --> pLAP_N_dest 3. Inserted LAP tag and Gateway cloning site from pLAP_N_dest into pcDNA5/FRT/TO -> pTO_LAP_N-DEST
• Vector type: Mammalian Expression, Bacterial Expression ; Gateway cloning, Flp-FRT recombination, Tet-inducible expression
• Selectable markers: Hygromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Charged Multivesicular Body Protein 3
• Species: H. sapiens (human)
• Insert Size (bp): 668
• GenBank ID: NM_016079.3
• Entrez Gene: CHMP3 (a.k.a. CGI-149, NEDF, VPS24)
• Promoter: CMV
• Tag / Fusion Protein:
  • Localization and Affinity Purification (LAP) tag (C terminal on backbone)

Cloning Information

• Cloning method: Gateway Cloning
• 5′ sequencing primer: TACGGTGGGAGGTCTAT
• 3′ sequencing primer: TAGAAGGCACAGTCGAGG

Resource Information

• Supplemental Documents:
  • 0733_TO_CHMP3_LAP.gb
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Part of the plasmid was derived from pmCherry_N_dest from the Helenius Lab (ETH Zuerich)