Notes on constructs used in generating CPTI protein trap lines
N. Lowe Nov2009

The progenitors of the constructs used in the Cambridge Protein Trap Screen:

pGA, pGB, pGC made by Xavier Morin for the screen  described in Morin et al 2001 These provided the white gene and the artificial exon consisting of a 6His-EGFP flanked by MCH splice donor and acceptor sequences. EGFP flanked by NcoI and SalI sites. Variable length linkers outside the NcoI and SalI sites create the different splice frames (Morin et al 2001).

p3E2.1 Original piggyBac transposon plasmid which provided the piggyBac sequences (Fraser et al 1995).  Sequences required for traping constructs were cloned into a unique HpaI site lying within the piggyBac orf.

pP{SVS-A} N473. P element construct based on the original Morin construct pGA. StrepTagII affinity tags added at each end of YFP venus by PCR and cloned back into NcoI/SalI sites in pGA.

Notes on Nomenclature - PBac - These constructs only contain piggyBac transposon sequences.  - PIGP - These constructs  have P element  sequences, as found in the original  Morin constructs,  inserted at a unique HpaI site in the piggyBac orf. - Letters within the curled brackets  e.g. {SVS-} denote the type of affinity tag incorporated;  F= 3xFLAG, S= StrepTagII, V= Venus YFP - Splice frames are usually termed 0, 1, 2 and these designations are included in the names of the constructs. In the Morin et al 2001 paper these were designated C, A, B respectively.

Constructs used in the screen:

544 - PBac544{SVS-1} - Protein trapping construct based on piggyBac vector p3E1.2. White gene and Strep-tagged VenusYFP from pP{SVS-A}473 inserted into unique HpaI site in piggyBac vector p3E1.2. Only one splice frame was made of this type of vector. Used in small scale screen to produce NPSV protein trap collection (CPTI-100034 to 100066).

PBac544{SVS-1} Genbank format

 

 

 

566 - PIGP566{SVS-1} - Hybrid piggyBac/P element vector using p3E1.2 based piggyBac plasmid as vector. Used in small scale screen to produce PPSV protein trap collection (CPTI-100000 to 100032).

PIGP566{SVS-1} Genbank format

 

 

602 - PIGP602{SVS-1} - Hybrid piggyBac/P element trapping construct Identical to 566 but several potential cryptic splice sites in piggyBac sequences have been mutated. This mutated backbone was used in all subsequent  PIGP constructs Unknown whether these potential cryptic sites had any effect on trapping effiency. Used in high throughput screen with embryo sorter

PIGP602{SVS-1} Genbank format

 

 

681 - PIGP681{FSVS-1} - Hybrid piggyBac/P-element protein trapping vector containing 3xFLAG-StrepTag-Venus-StrepTag. Splice frame 1

PIGP681{FSVS-1} Genbank format

 

 

754 - PIGP754{FSVS-0} - Hybrid piggyBac/P element protein trapping vector contining 3xFLAG-StrepTag-venus-StrepTag

PIGP754{FSVS-0} Genbank format

 

 

768 - PBac768{FSVS-0} - piggyBac protein trapping vector with 3xFLAG-StrepTag-Venus-StrepTag. Splice frame 0. Trapping exon identical to 754 (Note no P element sequences in this construct)

PBac768{FSVS-0} Genbank format

 

 

769 ­- PBac769{FSVS-1} - piggyBac protein trap vector with 3xFLAG-Strep-Venus-Strep. Splice frame 1. Identical to 768 but splice frame shifted by one base. (NOT USED?)

PBac769{FSVS-1} Genbank format

 

 

802 - PIGP802{SVS-2} - Hybrid piggyBac/P element protein trapping construct with StrepTag-Venus-StrepTag in splice frame 2. This construct was made with a mutated ATG in the P element transposase to prevent the construct acting as a promoter trap.

PIGP802{SVS-2} Genbank format

 

 

803 - PIGP803{SVS-0} - Hybrid piggyBac/P element protein trapping construct with StrepTag-Venus-StrepTag in splice frame 0. Identical to 802 but  splice frame 0

PIGP803{SVS-0} Genbank file

 

 

806 - PBac806{LOX-SVS-2} - piggyBac protein trap construct without P elemet. This was used since it was known that P elemet traps in this splice frame were prone to act as enhancer traps giving in-frame fusions to P element ORF. Lox sites were incorporated to allow exchange of internal sequences by RMCE.

PBLOX806{SVS-2} Genbank file

 

 

810 - PIGP810{FSVS-2} - Hybrid piggyBac/P element protein trapping construct with StrepTag-Venus-StrepTag in splice frame 2. This construct was made with a mutated ATG in the P element transposase to prevent the construct acting as a promoter trap.

PIGP810{FSVS-2} Genbank file

 

 

 

References.

Fraser et al. Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA. Virology (1995) vol. 211 (2) pp. 397-407

Morin et al. A protein trap strategy to detect  GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci USA (2001) vol. 98 (26) pp. 15050-5