pUASTLOTattB_vhhGFP4::T48-Baz::mCherry
(Plasmid #163927)

• Purpose: Expression of GrabFP-A-extracellular 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): 13500
• 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 (extracellular) nanobody with T48 protein, Bazooka minimal localization sequence and mCherry fluorophor
• Alt name: GrabFP-A extracellular
• Species: D. melanogaster (fly)
• Tag / Fusion Protein:
  • mCherry (C 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

The HA-tag was replaced by vhhGFP4 in the T48-HA plasmid (obtained from Kölsch et al. [2007]). mCherry was inserted at the C-terminal end of vhhGFP4::T48. In addition, the 2316 base pair minimal apical localization sequence of Bazooka (obtained from Krahn et al., 2010) was attached C-terminally to mCherry. A Drosophila Kozak sequence (CAAA) was added when inserting vhhGFP4::T48-Baz::mCherry 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.

T48 plasmid:
Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2. Kölsch V, Seher T, Fernandez-Ballester GJ, Serrano L, Leptin M, Science. 2007 Jan 19; 315(5810):384-6.

Baz minimal localization sequence:
Membrane targeting of Bazooka/PAR-3 is mediated by direct binding to phosphoinositide lipids. Krahn MP, Klopfenstein DR, Fischer N, Wodarz A, Curr Biol. 2010 Apr 13; 20(7):636-42.

mCherry: Clonetech