pUASTLOTattB_vhhGFP4::Vkg::mCherry
(Plasmid #163929)

• Purpose: Expression of GrabFP-ECM under control of UAS or LOP enhancer
• Depositing Lab: Markus Affolter
• Publication: Harmansa et al Elife. 2017 Apr 11;6. doi: 10.7554/eLife.22549.

Backbone

• Vector backbone: pUASTLOTattB
• Backbone manufacturer: Kanca, O. et.al., Raeppli: a whole-tissue labeling tool for live imaging of Drosophila development. Development (2014)
• Backbone size w/o insert (bp): 8900
• Total vector size (bp): 15500
• Vector type: Insect Expression, Cre/Lox

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: Fusion of vhhGPF4 nanobody with Viking (Collagen IV) protein and mCherry fluorophor
• Alt name: GrabFP-ECM
• Species: D. melanogaster (fly)
• Mutation: G1767D
• Tag / Fusion Protein:
  • mCherry (N terminal on insert)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Addgene NGS result had G1767D mutation towards the end of the Vkg, depositor confirms that this mutation does not affect the plasmid function. vhhGFP4 and mCherry coding sequences, separated by a short linker region, were inserted between the first and second exon in the Vkg full-length plasmid (obtained from Wang et al., 2008). This insertion site was chosen, since a viable Vkg GFP-trap line exists which carries an exogenous GFP exon at this position (Morin et al., 2001). Finally the vhhGFP4::Vkg::mCherry construct was inserted into the MCS of the pUASTLOTattB vector (Kanca et al., 2014).

pUASTLOTattB vector:
Kanca, O., Caussinus, E., Denes, A. S., Percival-Smith, A. & Affolter, M. Raeppli: a
whole-tissue labeling tool for live imaging of Drosophila development.
Development 141, 472–480 (2014).

vhhGFP4 plasmid:
Saerens D, Pellis M, Loris R, Pardon E, Dumoulin M, Matagne A, Wyns L, Muyldermans S, Conrath K
J Mol Biol. 2005 Sep 23; 352(3):597-607.

Vkg plasmid:
Type IV collagens regulate BMP signalling in Drosophila. Wang X, Harris RE, Bayston LJ, Ashe HL Nature. 2008 Sep 4; 455(7209):72-7.

mCherry: Clonetech