Appeal No. 2003-0936
Application No. 09/532,806
about the 400 most 3' based pairs with an ABA-inducible
genomic clone reported by Gomez et al. (1988)(GenBank
Accession Number X12564).
Example 2 characterizes the ZMGRP promoter constructs used
in subsequent examples of transient expression analyses and
transformations therewith. Example 2 in its entirety reads
(Spec., pp. 112-113)(emphasis added):
Sequence characterization of the ZMGRP promoter-
containing plasmid revealed that the ~ 4.0 kb SacI insert
contained a HindIII site 97 bp from the 5' end of the
insert and approximately 360 bp of ZMGRP coding sequence
3' of the ATG start codon. Restriction enzyme analysis
determined that there was a unique XhoI site approximately
400bp 5' of the ATG start site. The sequence around the
XhoI site was determined and used to design a 5' PCR
primer. A 3' PCR primer was designed to change the
sequence around the ATG start site to create an NcoI site
and to introduce a SmaI site 4 bp 5' of the ATG start
codon. These primers were used to PCR amplify the DNA
at the 3' end of the promoter from the XhoI site to the
newly created NcoI site. The PCR fragment was used in a
three way ligation, employing a HindIII to XhoI fragment
containing the 5' ~3.2kbp part of the ZMGRP promoter
region, the XhoI to NcoI fragment containing the 3' 0.4 kbp
part of the ZMGRP promoter region, and the gus-nos sequence
containing vector pGN73, which had been digested with
HindIII and NcoI. The resulting construct was designated
pZMGRP-GN73 (Fig. 2, SEQ ID NO:2)5. A construct designated
pZMGRP-Act2-int-GN73 was made by replacing the SmaI - NcoI
region of the ZMGRP promoter with a PvuII - NcoI restriction
fragment from pDPG836 containing a rice Act2 intron 1
deletion derivative (Act2-int)(Fig. 1, SEQ ID NO:3)6.
5 SEQ ID NO:2 includes 8076 nucleotides (Raw Sequence
Listing (Paper No. 4)).
6 SEQ ID NO:3 includes 9002 nucleotides (Raw Sequence
Listing (Paper No. 4)).
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