Subject: Re: pAct5CSRS
Hi Lynn,
It took a while, but I found my notebook entries on the construction of
pACT5CSRS (March 1987).  I'm not even sure how you tracked me down since
the Han et al paper referenced K. Burgess.
The genesis of the vector was as follows:
The pUC18 vector was modified to remove the EcoRI site by end filling, so
the new polylinker sequence around teh Ri site would be "GAATTAATTC".
The 5' fragment of Act5C was obtained by digesting pUChsneo Act5C (obtained
from Howard Lipshitz) by Bgl II + BamH I, and cloning the ~2.6 kb fragment
obtained into the BamH I site of Larry Marsh's vector pIC20R (Reference:
Gene  32:481-485  (1984)). The orientation of this clone was such that the 5'
end of this fragment (relative to the direction of Act5C transcription) was
adjacent to the Sal I site in the pIC20R vector, and the 3' end adjacent to
the EcoRI site.  The resulting subclone was then digested by Sal I and
EcoRI to obtain an ~ 2.6 kb Sal-RI fragment.
The 3' fragment of Act5C was obtained by digesting pUChsneo Act5C (obtained
from Howard Lipshitz) by Sal I, and cloning the ~1.1 kb fragment into
pIC20R digested with Sal I and Xho I.  The orientation of this clone was
such that the 3' end of this fragment (relative to the direction of Act5C
transcription) remained a Sal I site. This subclone was then digested with
EcoRI plus Sal I to obtain an ~ 1.1 kb RI-Sal fragment.
These two fragments were mixed and ligated and then digested with Sal I.
The fusion fragment (Sal-5'Act5C-RI-3'Act5C-Sal) was then cloned into the
modified pUC18 vector with filled-in EcoRI site. The orientation of the
insert relative to the pUC18 polylinker is
HIII-PstI-SalI-5'Act5C-3'Act5C-SalI-XbaI-BamHI-SacI-EcoRI(deleted).
This created the pACT5CSRS vector, which I would estimate to be about 6.4
kb (not 10363 bp as listed in the Flybase entry).
Also, the report on the vector in the link on the transposon search page
contains one incorrect reference to a paper from Holmgren's lab - this
involved a completely different Act 5C vector.
If you'ld like, you could list K. Burtis, personal communication as a
reference since I never published the vector.
Hope this was helpful.
Best regards,
Ken
>Hi, Ken,
>
>FlyBase is compiling data and maps on some of the more useful
>P transformation and cell culture vectors.  We will be publishing
>them very shortly in DIS.  pAct5CSRS is on the list, and I would
>appreciate some input from you if you can fill in some holes and/or
>verify that what I've been able to piece together so far is correct.
>
>I've put together an annotated map and a fairly complete sequence
>based on info in Han et al., 1989.  You can access them through
>TransposonSearch (at the top of the page is a "New - annotated maps"
>button).  If you would prefer, I can e-mail the sequence to you.
>
>If you spot any errors in any of the other vectors in the annotated
>maps list, that information would also be most appreciated.
>
>Hope to hear from you soon,
>
>--Lynn
>
>Lynn Crosby
>FlyBase
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