TABLE 1.
A. FLP recombinase expression constructs | |||
---|---|---|---|
Plasmid | Promoter | Genea | unc-119(+)b |
pGC94 | hsp-16.2 | FLP | No |
pGC95 | hsp-16.41 | FLP | No |
pGC133 | hsp-16.41 | FLP | Yes |
pGC146 | hsp-16.2 | FLP | Yes |
pGC158 | lag-2 | FLP | No |
B. FLP recombinase Gatewayd destination vectors to generate expression constructs in the form [promoter-FLP recombinase] | |||||
Plasmid | attd | attd | iAc | Genea | unc-119(+)b |
pGC180 | R1 | R2 | None | FLP | Yes |
pGC181 | R2 | R1 | None | FLP | Yes |
pGC267 | R1 | R2 | Yes | FLP | Yes |
C. FRT-containing full FLP-out target cassettes in the form [promoter<GFP<gene/reporter/hairpin] | ||||||||||||||
Plasmid | att | Promoter | att | FRT | iAc | Genea | unc-119(+)b | FRT | iAc | att | Gene | att | 3′end | unc-119(+)b |
pGC185 | B1 | rpl-28 | B2 | Yes | No | 4xNLS-GFP | Yes | Yes | No | No | 4xNLS-tdimer2(12) | No | let-858 | No |
pGC200 | B1 | pro-1 | B2 | Yes | No | 4xNLS-GFP | Yes | Yes | Yes | No | 1xNLS-lacZ | No | unc-54 | No |
pGC219 | B1 | hlh-12 | B2 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | B4 | hlh-12(R15K) | B3 | let-858 | Yes |
pGC220 | B1 | hlh-12 | B2 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | B4 | hlh-12(R25K) | B3 | let-858 | Yes |
pGC240 | B1 | lim-7 | B2 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | No | 4xNLS-tdimer2(12) | No | let-858 | Yes |
pGC452 | B4 | hlh-12 | B3 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | B1 | hairpin_hlh-12 | B1 | let-858 | Yes |
D. FRT-containing Gateway destination vectors in the form [Gatewayd (R1-R2)<reporter<reporter] for insertion of promoter upstream of FLP-out cassette containing a reporter in the cassette and a reporter downstream of the cassette | |||||||||||||
Plasmid | attd | attd | FRT | iA | Genea | unc-119(+)b | FRT | iA | att | Gene | att | 3′end | unc-119(+)b |
pGC93 | R1 | R2 | Yes | No | 4xNLS-GFP | Yes | Yes | No | No | 1xNLS-lacZ | No | unc-54 | No |
pGC97 | R1 | R2 | Yes | No | 4xNLS-GFP | Yes | Yes | No | No | tdimer2(12) | No | let-858 | No |
pGC183 | R1 | R2 | Yes | No | 4xNLS-GFP | Yes | Yes | No | No | 4xNLS-tdimer2(12) | No | let-858 | No |
pGC238 | R1 | R2 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | No | 4xNLS-tdimer2(12) | No | let-858 | Yes |
pGC160 | R1 | R2 | Yes | No | tdimer2(12) | No | Yes | No | No | 4xNLS-GFP | No | let-858 | Yes |
E. FRT-containing Gateway destination vectors in the form [Gatewayd (R1-R2)<reporter<Gatewayd (R4-R3)] for insertion of promoter upstream of FLP-out cassette and gene of interest downstream of FLP-out cassette | ||||||||||||
Plasmid | attd | attd | FRT | iA | Genea | unc-119(+)b | FRT | iA | attd | attd | 3′end | unc-119(+)b |
pGC162 | R1 | R2 | Yes | No | 4xNLS-GFP | Yes | Yes | No | R4 | R3 | let-858 | No |
pGC247 | R1 | R2 | Yes | Yes | 4xNLS-GFP | No | Yes | No | R4 | R3 | let-858 | Yes |
pGC163 | R1 | R2 | Yes | No | tdimer2(12) | No | Yes | No | R4 | R3 | let-858 | Yes |
pGC225 | R1 | R2 | Yes | No | 4xNLS-tdimer2(12) | No | Yes | No | R4 | R3 | let-858 | Yes |
pGC276 | R1 | R2 | Yes | Yes | 4xNLS-tdimer2(12) | No | Yes | Yes | R4 | R3 | let-858 | Yes |
F. FRT-containing Gateway destination vectors in the form [Gatewayd (R4-R3)<reporter<Wormgatee] for insertion of promoter upstream of FLP-out cassette and RNAi hairpin downstream of FLP-out cassette | |||||||||||||
Plasmid | attd | attd | FRT | iA | Genea | unc-119(+)b | FRT | iA | att | att | 3′end | unc-119(+)b | |
pGC245 | R4 | R3 | Yes | Yes | 4xNLS-GFP | No | Yes | Yes | R1 | Wormgatee | R1 | let-858 | Yes |
pGC320 | R4 | R3 | Yes | Yes | 4xNLS-mCherry | No | Yes | Yes | R1 | Wormgatee | R1 | let-858 | No |
G. Donor vector for Gateway(R4-R3) cassette |
Plasmid |
pGC188f |
See materials and methods or supplemental Methods for plasmid construction details. See also supplemental Toolkit Documentation and supplemental table for strategies and additional constructs to facilitate construction of FLP- and FRT-containing plasmids.
All plasmids contain the let-858 3′end after the FLP recombinase and all reporters [GFP, tdimer2(12), or mCherry]. This sequence adds the let-858 3′-UTR onto the transcript.
C. briggsae unc-119(+); see text for details.
Intron A from Fire vectors.
Whole Gateway cassettes (with ccdB gene and chloramphenicol resistance gene; Invitrogen) are between attR sites unless otherwise indicated.
Wormgate (Johnson et al. 2005) consists of two reciprocally oriented Gateway cassettes flanking an intron. This system accepts cDNA clones directly from the ORFeome library.
See materials and methods and supplemental Toolkit Documentation for details on pGC188.