Subject: Help FlyBase - P{Ub<y+>GAL4}
Hi,
I am curating a paper of yours for FlyBase:
Aza-Blanc et al., 1997, Cell 89(7): 1043-1053
in which you use the construct P{Ub<y+>GAL4}.
We do not have this construct in FlyBase so could you please provide
minimal details of its construction and references if you have them.
Please include details of:
Which gene the promoter fragment is taken from
Length and ends of promoter fragment
Length and ends of yellow fragment, cNDA or genomic DNA
Origin of the stop signal within the FRT cassette.
Many thanks,
Eleanor Whitfield
FlyBase
>
Subject: pUb>y+>GAL4
regarding the plasmid pUb>y+>GAL4 (Aza-Blanc et al., 1997):
made on W8 vector
contains: poly-ubiquitin promoter; frt-y+-frt-ires cassette; GAL4 gene;
tubulin3' utr
Promoter used: poly-ubiquitin promoter, taken from pCa4GFPh27 (built by
Ilan Davis). A Eco RI site was introduced by PCR substituting the first
codon, and then the promoter was cut out with EcoRI. Approx. size: 2000bp
y+-frt cassette: taken from pKB700 (from K. Basler). contains an approx.
7500bp genomic fragment including promoter and open reading frame of y+
flanked by FRT sequences (approx. 75bp each) It also contains an IRES
(internal ribosomal entry site) after the second FRT. the cassette was
taken out from pkb700 by cutting with KpnI (other KpnI sites within the y+
region have been destroyed, see pkb700)
GAL4 was obtained from pGAWB, by cutting with NotI/partial HindIII;(there
is another HindIII site right after the 5' NotI site, so GAL4 can be
excised out with HindIII alone.
Tubulin 3' UTR: from pKB700, by excision with EcoRI/XbaI; Approximate
size: 800bp
			Pedro Aza-Blanc