Note: Descriptions are shown in the official language in which they were submitted.
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Novel genes encoding insecticidal proteins.
This application is a division of Canadian Application Serial No. 2,646,471
filed
March 16, 2007 (parent application).
It should be understood that the expression "the present invention" or the
like used
in this specification encompasses not only the subject matter of this
divisional application,
but that of the parent application also.
INTRODUCTION
The present invention relates to new gene sequences encoding insecticidal
proteins
produced by Bacillus thuringiensis strains. Particularly, new chimeric genes
encoding a
Cry1C protein are provided which are useful to protect plants from insect
damage. Also
included herein are plant cells or plants comprising such genes, and methods
of making
or using them, as well as plant cells or plants comprising such cry1C chimeric
gene and
at least one other gene encoding an insecticidal protein, such as new gene
sequences
encoding a Cry1B or Cry1D protein.
BACKGROUND OF THE INVENTION
Strain and proteins derived from Bacillus thuringiensis (abbreviated herein as
"Bt") are
well known for their specific toxicity to insect pests, and they have been
used since
almost a centiiry to control insect pests. Some transgenic plant species
expressing Bt
proteins are now available, and they successfully limit insect damage on
plants.
Despite the isolation of quite a number of insecticidal Bt proteins, only a
few Bt proteins,
have been expressed in transgenic plants that have been commercialized, and
this only
in some crops. Most commercialized transgenic Bt plants belong to the bigger
field
crops such as corn and cotton. In smaller market crops such as vegetables,
only a few
plant species have been transformed with Bt genes so as to render them
resistant to
major Lepidopteran insect pests, but to date no Lepidopteran-resistant
vegetable Bt-
plant or seed is deregulated and marketed. Zhao et al. (2003) have described
transgenic broccoli plants expressing a Cry1Ac or a Cry1C Bt toxin, as well as
crosses
between these plants so that both the Cry1Ac and Cry1C toxins are expressed in
the
same plants, but these plants have not been commercialized. NewLeaf T"
potatoes
comprising a Cry3A Coleopteran-active gene were briefly commercialized in
Northern
America, but have been withdrawn from the market in 2001.
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The current invention provides new genes encoding proteins of the Cry1C type
of Bt
proteins, which ideally are combiried with genes encoding proteins Of the
Cry18 or
Cry1D type Bt prOteins.
The DNA seqUericeb=Of the cry1C, cry1B or cry1 D genes Of the invention and of
the
modified transit peptide of the invention (shown in the enclosed. sequence
listing) are
artificial genes, not found in nature, and are, different from any known DNA
sequence.
Indeed, any one Of the DNA sequences of SEQ ID Nos. 1,3, 10, 14 or 16 shows at
most
76.6 % sequence identity with the closest known DNA sequences.
SUMMARY OF THE INVENTION
In the cUrrent invention; several new insect control genes derived from Bt are
provided
for Use In plants: -Specifically, such genes are useful in vegetableS
plantorops,
particularly Brassicaceae plants such as cauliflower, cabbage, Chinese
cabbage, turnip,
mustard, oilseed rape, kale, broccoli, Brussels sprouts, mustard spinach, and
the like.
Particularly, in one embodiment of this invention the following. Bressice
species plants
are protected from insects by-the new genes of the current Invention: B.
carinata, B.
elongate, B. fruticulosa, B. juncea, B. napus, B. narinosai B: nigra, B.
oleracea, B.
perviridis,B, raps, B. rUpestris, B. septieeps, B,.tournefortii, and the-like,
particularly
plants of the species BraSSica oleraceae or Brassica napus. The plants or-
seeds
comprising at least one of the new genes of the invention can be obtained by
transformation of plant cells and production of plants or seed therefrom
comprising the
genes of the invention. Also included herein are plants or seeds obtained by
crossing
with a plant transformed to contain at least one of the genes of the
invention, and by
application of routine breeding steps. Obviously, any plant species to be
protected from
insect species that are killed or controlled by the Bt proteins encoded by the
novel genes
of this invention can be transformed with the genes of the Invention to obtain
transgenic
plants and seeds with increased resistance to such insects.
In one embodiment, the current invention also provides.a
combination_ottechnologles to
allow for the most optimal product from a resistance management point of view.
Indeed,
in one embodiment of this invention the plants of the invention produce at
least 2
different Bt proteins and such proteins are encoded by the highly-expressed
cry genes of
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the invention which have been stably integrated, preferably at a single locus
in the
plant's genome. In one embodiment of the invention, such at least 2 Bt genes
include a
cry1C and a cry1B gene, a cry1C and a cry1D gene, or a combination of a cry1C,
a
cry1B and a cry1D gene of this invention. In one embodiment of the invention a
marker
gene allowing rapid identification of transgenic plants, preferably a
herbicide resistance
gene, is located in the same plant, particularly at the same locus in the
plant's genome
as a cry gene of the invention. In one embodiment of this invention, the
marker gene is
a gene encoding a phosphinothricin acetyltransferase or a glyphosate-
insensitive
EPSPS.
In the invention also novel cry1B and cry1D genes, particularly cry1B or cryl
D chimeric
genes, are provided, which can be expressed in plants at high levels, such as
the
cry1B1 and cry1B2 and the cry1D1 and cry1D2 genes. Also plants cells, plants
or seeds
comprising any of these genes and methods of producing or using them alone or
in
combination are provided herein.
Also, the current invention provides novel genes encoding an insecticidal
protein
comprising a functional plant intron in their coding sequence. The presence of
the intron
also secures that the gene does not express a functional protein when the gene
is in an
environment where the intron cannot be spliced, such as a bacteria or another
prokaryotic microorganism. The presence of this intron in the gene sequence
also
allows for high expression levels to be obtained in plants.
Also included herein are variants of the Cry1C protein of the invention
comprising the
sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position
627, but
wherein one, some or all of the following amino acids at the following
positions
compared to the positions in SEQ ID No. 2 are changed: the amino acid at
position 125
is Alanine, the amino acid at position 184 is Valine, the amino acid at
position 295 is
Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid
at position
593 is Arginine. Also provided herein are variants of the Cryl B protein of
the invention
comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648,
but
wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the
amino acid at
position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid
at
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position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in
SEQ ID
No. 11 is Arginine.
Also included in this invention is a novel DNA encoding a chloroplast transit
peptide,
particularly a DNA comprising the sequence of SEQ ID No. 16 from nucleotide
position 7
to nucleotide position 371, particularly the sequence of SEQ ID No. 16, as
well as such
DNA encoding a variant of the protein of SEQ ID No.17, such as a chloroplast
transit
peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to
amino
acid position 124, wherein the Cys amino acid at position 55 is replaced by
Tyr and/or
wherein a Gly amino acid is added after the Gly amino acid at position 51.
Specifically, the current invention provides a chimeric gene, comprising the
following
operably-linked sequences: a) a coding region encoding a Cryl C protein,
comprising the
DNA of any one of SEQ ID Nos. 1, 3, 4 or 6 or a variant thereof, and b) a
promoter
region capable of directing expression in plant cells. In one embodiment, such
promoter
comprises the sequence of SEQ ID No. 18 or 19. In another embodiment, the
chimeric
gene further comprises a 3' polyadenylation and transcript termination region,
particularly that of the NADP-malic enzyme gene from Flaveria bidentis. In
another
embodiment, the chimeric gene further comprises the leader sequence of the
tapetum
specific El gene of Oryza sativa between the promoter and the coding region.
The current invention also provides a DNA comprising any of the above chimeric
genes,
further comprising a second chimeric gene, said second chimeric gene
comprising the
following operably-linked sequences: a) a second coding region encoding a Cryl
B
protein comprising the DNA of SEQ ID No. 8 or 10, and b) a second promoter
region
capable of directing expression in plant cells; or a DNA comprising any of the
above
chimeric genes, further comprising a second chimeric gene, said second
chimeric gene
comprising the following operably-linked sequences: a) a coding region
encoding a
Cry 1 D protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter
region
capable of directing expression in plant cells. In one embodiment, the above
DNAs are
provided, wherein said second promoter region comprises the sequence of SEQ ID
No.
18 or 19 and is different from said first promoter region; or wherein said
second chimeric
gene further comprises a 3' polyadenylation and transcript termination region,
particularly of the NADP-malic enzyme gene from Flaveria bidentis. In one
embodiment,
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the second chimeric gene in these DNAs further comprises the leader sequence
of the
tapetum specific El gene of Oryza sativa between the promoter and the coding
region.
The current invention also provides the above DNAs, further comprising a third
chimeric
gene, said third chimeric gene comprising the following operably-linked
sequences: a) a
coding region encoding a Cryl D protein comprising the DNA of SEQ ID No. 12 or
14,
and
b) a promoter region capable of directing expression in plants.
Also included in the current invention are a transgenic plant cell or plant,
comprising any
of the above genes or DNAs stably incorporated in its genome, preferably when
the cell
or plant is a Brassica species plant or plant cell, particularly of the
species Brassica
oleraceae, more particularly cabbage or cauliflower.
Also included in this invention is the use of any of the above chimeric genes
or DNAs to
control insect pests, to obtain plant cells, plants or seeds with increased
resistance to
insects; the use of any of the above chimeric genes or DNAs to delay or
prevent insect
resistance development in transgenic plants expressing an insecticidal protein
by insects
attempting to feed on such plants; or the use of any of the above chimeric
genes or
DNAs to obtain cabbage, oilseed rape or cauliflower protected from Plutella
xylostella.
Also included herein are methods for controlling insects, comprising the step
of planting
or sowing in a field, plants comprising any of the above chimeric genes or
DNAs; as well
as methods of controlling insects in Brassica species plants, comprising the
step of
expressing any of the above chimeric genes or DNA in plants; or methods of
producing
plants or seeds resistant to insects, comprising the steps of: a) obtaining a
plant
transformed with the gene of any one of claims 1 to 5 or the DNA of any one of
claims 6
to 12, and b) selecting progeny of said plant or seeds thereof, containing
said gene or
DNA.
Also provided in accordance with this invention is a chimeric gene comprising
the
following operably-linked sequences: a) a first fragment of a coding sequence
encoding
an insecticidal protein, b) a plant intron sequence, c) a second fragment of
said coding
sequence, d) a promoter region capable of directing expression in plant cells,
and
wherein no insecticidal protein can be produced from such chimeric gene in a
given host
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cell wherein the intron is not spliced; particularly such chimeric gene
wherein such
intron is the second intron of the ST-LS1 gene of Solanum tube rosum.
Further provided herein is also a microorganism comprising any of the above
chimeric genes or DNAs, particularly when such microorganism is of the genus
Escherichia, Bacillus or Agrobacterium.
The current invention as claimed relates to:
- a chimeric nucleic acid comprising the following operably-linked
sequences: a) a
first fragment of a Bacillus thuringiensis coding sequence encoding an
insecticidal
protein, b) a plant intron sequence, c) a second fragment of said Bacillus
thuringiensis coding sequence, d) a promoter region capable of directing
expression
in plant cells, wherein no insecticidal protein can be produced from such gene
in a
given host cell wherein the intron is not spliced, and wherein said intron is
effectively
spliced in plant cells, wherein said effective splicing can be measured using
the
detection of a functional protein produced in plant cells and wherein the
plant intron
sequence is positioned between the first and second fragment;
- a transformed plant cell selected from the group consisting of: corn,
cotton,
watercress, horseradish, wasabi, arugula, cress, radish, canola, soybean,
vegetable
plants, Cruciferae plant species, cauliflower, cabbage, Chinese cabbage,
turnip,
mustard, oilseed rape, kale, broccoli, Brussels sprouts, and mustard spinach,
comprising the chimeric nucleic acid as described herein; and
- a transgenic plant cell, comprising the chimeric nucleic acid as
described herein
stably incorporated in its genome.
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DESCRIPTION
In accordance with this invention, a "nucleic acid sequence" refers to a DNA
or RNA
molecule in single or double stranded form, preferably a DNA molecule. An
"isolated
DNA", as used herein, refers to a DNA which is not naturally-occurring or no
longer in
the natural environment wherein it was originally present, e.g., a DNA coding
sequence
associated with other regulatory elements in a chimeric gene, a DNA
transferred into
another host cell, such as a plant cell, or an artificial, synthetically-made
DNA sequence
having a different nucleotide sequence compared to any naturally-occurring DNA
sequence.
In accordance with this invention, nucleic acid sequences, particularly DNA
sequences,
encoding Bt Cry toxins or variants thereof have been constructed. The new DNA
sequences are designated herein as cry1C1-4, co/1BI, cry1B2, cry1D1, and
cry1D2,
and their encoded proteins are designated herein as Cry1C (e.g., Cry1C1,
Cry1C3, and
Cry1C4), Cry1B (e.g., Cry1B1 and Cry1B2) and Cry1D (e.g., Cry1D1 and Cry1D2)
proteins. Also a new DNA sequence encoding a modified chloroplast transit
peptide is
provided herein, e.g., a DNA comprising the sequence of SEQ ID No. 16 from
nucleotide
position 7 to nucleotide position 371, particularly the sequence of SEQ ID No.
16, which
is designed for optimal expression in plants, particularly vegetables such as
Brassicaceae plants, especially cabbage and cauliflower.
In accordance with this invention "Cry1C protein" refers to any insecticidal
protein
comprising the smallest fragment of the amino acid sequence of SEQ ID No. 2
which
retains insecticidal activity (hereinafter referred to as "smallest toxic
fragment"),
particularly any protein comprising the amino acid sequence from the amino
acid at
position 29 to the amino acid at position 627 in SEQ ID No. 2, preferably any
insecticidal
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protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid
position 3
to amino acid position 627. Also included herein is an insecticidal protein
comprising the
amino acid sequence of SEQ ID No. 2 (also named Cry1C1 protein herein), SEQ ID
No.
(also named Cry1C3 protein herein) or SEQ ID No. 7 (also named Cry1C4 protein
herein).
A Cry1C protein comprising the amino acid sequence from the amino acid at
position 29
to the amino acid at position 627 in SEQ ID No. 2 retains all or most of the
insecticidal
activity of the entire protein as produced in nature, and addition of protein
sequences at
the N- or C-terminal part thereof do not disrupt this activity. Hence, any
protein
characterized by an amino acid sequence containing or including this region is
useful
and forms part of this invention. This includes insecticidal hybrid or
chimeric proteins
comprising the smallest toxic protein fragment of the protein of SEQ ID No. 2.
Also
included in this definition are variants of proteins comprising the amino acid
sequence
from the amino acid at position 29 to the amino acid at position 627 in SEQ ID
No. 2,
such as insecticidal proteins comprising a sequence having a sequence identity
of at
least 95 %, particularly at least 96 %, 97 %, 98 % or 99 % at the amino acid
sequence
level with this region of SEQ ID No.2, as determined using the Needleman-
Wunsch
global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum
alignment
over the entire length of the sequences, using default settings (gap opening
penalty 10,
gap extension penalty 0.5; for amino acid sequence comparisons, the EBLOSUM62
matrix is used), preferably proteins having some, preferably 5-10,
particularly less than
5, amino acids added, replaced or deleted without significantly changing,
preferably
without changing, the insecticidal activity of the protein. Preferred variants
of the Cry1C
protein of the invention include a protein comprising the sequence of SEQ ID
No. 2 from
amino acid position 29 to amino acid position 627, but wherein one, some or
all of the
following amino acids at the following positions compared to the positions in
SEQ ID No.
2 are changed: the amino acid at position 125 is Alanine, the amino acid at
position 184
is Valine, the amino acid at position 295 is Arginine, the amino acid at
position 454 is
Aspartic acid, or the amino acid at position 593 is Arginine. Also included
herein are any
Cry1C-based protein variants, hybrids or mutants retaining substantially the
same
insecticidal activity as that of the Cry1C protein of the invention defined
above.
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The terminology DNA or protein "comprising" a certain sequence X, as used
herein,
refers to a DNA or protein including or containing at least the sequence X, so
that other
nucleotide or amino acid sequences can be included at the 5' (or N-terminal)
and/or 3'
(or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker
protein as
disclosed in EP 0 193 259, (the nucleotide sequence of) a transit peptide,
and/or a 5' or
3' leader sequence.
For the purpose of this invention, the "sequence identity" of two related
nucleotide or
amino acid sequences, expressed as a percentage, refers to the number of
positions in
the two optimally aligned sequences which have identical residues (x100)
divided by the
number of positions compared. A gap, i.e., a position in an alignment where a
residue is
present in one sequence but not in the other, is regarded as a position with
non-identical
residues. The alignment of the two sequences is performed by the Needleman and
Wunsch algorithm (Needleman and Wunsch 1970) in EMBOSS (Rice et al., 2000) to
find
optimum alignment over the entire length of the sequences, using default
settings (gap
opening penalty 10, gap extension penalty 0.5).
The "smallest toxic fragment" of a Cry protein of the invention, as used
herein, is that
smallest fragment or portion of a Cry protein retaining insecticidal activity
that can be
obtained by enzymatic, such as trypsin or chymotrypsin, digestion of the full
length Cry
protein, or that smallest fragment or portion of a Cry protein retaining
insecticidal activity
that can be obtained by making nucleotide deletions in the DNA encoding a Cry
protein.
Such smallest toxic fragment can also be obtained by treatment of a Cry
protein with
insect gut juice, preferably midgut juice, from an insect species susceptible
to (i.e., killed
or otherwise negative affected in its growth or feeding by) such Cry protein.
In accordance with this invention, "Cry1D protein" refers to any insecticidal
protein
comprising the smallest toxic fragment of the amino acid sequence of SEQ ID
No. 15,
particularly any insecticidal protein comprising the amino acid sequence from
the amino
acid at position 21 or 29 to the amino acid at position 604 in SEQ ID No. 15,
preferably
any insecticidal protein comprising the amino acid sequence of SEQ ID No. 15
from
amino acid position 3 to amino acid position 604. Also included herein is an
insecticidal
protein comprising the amino acid sequence of SEQ ID No. 13 (also named Cry1D1
protein herein) or SEQ ID No. 15 (also named Cry1D2 protein herein). A Cryl D
protein
,
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= comprising the amino acid sequence from the amino acid at position 29 to
the amino
acid at position 604 in SEQ ID No. 15 retains all or most of the insecticidal
activity of the
entire protein as produced in nature, and addition of protein sequences at the
N- or C-
terminal part thereof do not disrupt this activity. Hence, any protein
characterized by an
amino acid sequence containing or including this region is useful and forms
part of this
invention. This includes insecticidal hybrid or chimeric proteins comprising
the smallest
toxic protein fragment of the protein of SEQ ID No. 15. Also included in this
definition
are protein variants differing in the amino acid sequence from the amino acid
at position
29 to the amino acid at position 604 in SEQ ID No. 15, such as proteins with a
sequence
identity of at least 95 %, particularly at least 97 %, at least 98 % or at
least 99 % at the
amino acid sequence level in this region of SEQ ID No. 15, as determined using
the
Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to
find
optimum alignment over the entire length of the sequences, using default
settings (gap
opening penalty 10, gap extension penalty 0.5, for amino acid sequence
comparisons,
the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-
10,
particularly less than 5, amino acids added, replaced or deleted in the region
from the
amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15
without
significantly changing, preferably without changing, the insecticidal activity
of the protein.
In accordance with this invention, "Cry1B protein" refers to any insecticidal
protein
comprising the smallest toxic fragment of the amino acid sequence of SEQ ID
No. 11,
particularly any insecticidal protein comprising the amino acid sequence from
the amino
acid at position 31 to the amino acid at position 648, in SEQ ID No. 11,
preferably any
insecticidal protein comprising the amino acid sequence of SEQ ID No. 11 from
amino
acid position 3 to amino acid position 648. Also included herein is any
insecticidal
protein comprising the amino acid sequence of SEQ ID No. 11 or SEQ ID No. 9. A
Cry1B protein comprising the amino acid sequence from the amino acid at
position 31 to
the amino acid at position 648 in SEQ ID No. 11 retains all or most of the
insecticidal
activity of the entire protein as produced in nature, and addition of protein
sequences at
the N- or C-terminal part thereof do not disrupt this activity. Hence, any
protein
characterized by an amino acid sequence containing or including this region is
useful
= and forms part of this invention. This includes insecticidal hybrids or
chimeric proteins
comprising the smallest toxic protein fragment of SEQ ID No. 11. Also included
in this
definition are insecticidal proteins comprising variants of the amino acid
sequence from
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the amino acid at position 31 to the amino acid at position 648 in SEQ ID No.
11, such
as insecticidal proteins having a sequence identity of at least 80 %,
particularly at least
85 %, 90 %, 95 /0, 96 %, 97 %, 98 %, or at least 99 % at the amino acid
sequence level
in this region of SEQ ID No. 11, as determined using pairwise alignments using
the
Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to
find
optimum alignment over the entire length of the sequences, using default
settings (gap
opening penalty 10, gap extension penalty 0.5, for amino acid sequence
comparisons,
the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-
10,
particularly less than 5, amino acids added, replaced or deleted in the amino
acid
sequence from the amino acid at position 31 to the amino acid at position 648
in SEQ ID
No. 11 without significantly changing, preferably without changing, the
insecticidal
activity of the protein. Preferred variants of the Cry1B protein of the
invention include an
insecticidal protein comprising the sequence of SEQ ID No. 11 from amino acid
position
31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is
Tyrosine or the
amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein
the amino
acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at
position 353 in
SEQ ID No. 11 is Arginine.
As used herein, the terms DNA or gene, as in "cry1C1 DNA", refers to any DNA
sequence encoding the Cry1C, Cry1B or Cry1D protein, respectively, as defined
above.
This includes naturally occurring, artificial or synthetic DNA sequences
encoding the
Cry1C, Cry1B or Cry1D proteins defined above such as any one of SEQ ID Nos. 2,
5, 7,
9, 11, 13, 15. Also included herein are DNA sequences encoding insecticidal
proteins
,vhich are similar enough to any one of the DNA sequences of SEQ ID No. 1, 3,
4, 6, 8,
10, 12, or 14 so that they can (i.e., have the ability to) hybridize to these
DNA sequences
under stringent hybridization conditions. Stringent hybridization conditions,
as used
herein, refers particularly to the following conditions: immobilizing the
relevant DNA
sequences on a filter, and prehybridizing the filters for either 1 to 2 hours
in 50 %
formamide, 5 % SSPE, 2x Denhardt's reagent and 0.1 % SDS at 42 C, or 1 to 2
hours
in 6x SSC, 2xDenhardt's reagent and 0.1 % SDS at 68 C. The denatured dig- or
radio-
labeled probe is then added directly to the prehybridization fluid and
incubation is carried
out for 16 to 24 hours at the appropriate temperature mentioned above. After
incubation, the filters are then washed for 30 minutes at room temperature in
2x SSC,
0.1 % SDS, followed by 2 washes of 30 minutes each at 68 C in 0.5 x SSC and
0.1 %
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SDS. An autoradiograph is established by exposing the filters for 24 to 48
hours to X-
ray film (Kodak XAR-2 or equivalent) at -70 C with an intensifying screen. Of
course,
equivalent conditions and parameters can be used in this process while still
retaining the
desired stringent hybridization conditions. Preferred variants of the cry1C,
cry1B or
ryylD DNA of this invention are n DNA encoding the insecticidal Cryl C, Cry1B
or Cry1D
= protein variants described above.
Also included herein as a Cry1C DNA or gene as defined herein are: a) a DNA
comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide position 85
to
nucleotide position 2073, b) a DNA comprising the nucleotide sequence of SEQ
ID No. 3
from nucleotide position 85 to nucleotide position 2073, c) a DNA comprising
the
nucleotide sequence of SEQ ID No. 1 from nucleotide position 85 to nucleotide
position
2073 fused to the DNA sequence of SEQ ID No. 16, d) a DNA comprising the
nucleotide
sequence of SEQ ID No. 4 from nucleotide position 7 to nucleotide position
2439, e) a
DNA comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide
position 85 to
nucleotide position 2073 fused to the DNA sequence of SEQ ID No. 16, or f) a
DNA
.=
comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 7
to
nucleotide position 2439.
Also included herein as a Cry1D DNA or gene as defined herein are: a) a DNA
comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide position
85 to
nucleotide position 1812, or b) a DNA comprising the nucleotide sequence of
SEQ ID
No. 12 from nucleotide position 7 to nucleotide position 2178.
Also included herein as a Cry1B DNA or gene as defined herein are: a) a DNA
comprising the nucleotide sequence of SEQ ID No. 8 from nucleotide position 7
to
nucleotide position 2310, or b) a DNA comprising the nucleotide sequence of
SEQ ID
=
No. 10 from nucleotide position 91 to nucleotide position 1944.
The DNA sequences of the cryl C, cryl B or cryl D genes of the invention (as
shown in
the sequence listing, without transit peptide sequence) show at most only 76.6
%
sequence identity with the closest previously known DNA sequences available in
databases. Available sequence databases were checked for the sequences with
closest
sequence identity using the well-known BLAST algorithm, and then the Needleman-
,
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Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) was used to
find the
optimum alignment between the closest sequences and the sequences of the
invention
(considering their entire length, using default settings (gap opening penalty
10, gap
extension penalty 0.5). For the Cry1D DNA, a fragment of the prior art
sequence (of
equal length) was selected to secure optimal alignment, but even then only
72.5 `)/0
sequence identity was the closest sequence identity with any known DNA
sequence
listed in the available databases.
Hence, also included herein as cry1C, cry1B or cry1D genes are DNA sequences
encoding an insecticidal protein with at least 80 %, 90 %, preferably at least
93 to 97 %,
pnrticulnrly f Q8 % rnt Ic.st 99 hi irmcvi irientity trI
nny nnP thP r-nriing
sequences of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 or DNA sequences encoding
an
insecticidal protein hybridizing to any one of SEQ ID No. 1, 3, 4, 6, 8, 10,
12, or 14 under
stringent hybridization conditions, preferably hybridizing stringently to that
part of the
DNA sequence of any one of SEQ ID No. 1, 3, 4,6, 8, 10, 12, or 14 which is
required to
encode the smallest toxic protein fragment of the proteins of this invention.
The DNA
sequence identities referred to herein are calculated using the Needleman-
Wunsch
global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum
alignment
over the entire length of the sequences, using default settings (gap opening
penalty 10,
gap extension penalty 0.5; for DNA sequence comparisons, the EDNAFULL matrix
is
used), the stringent hybridization conditions are as defined above.
"Insecticidal activity" of a protein, as used herein, means the capacity of a
protein to kill
insects, inhibit their growth or cause a reduction in insect feeding when such
protein is
ingested by insects, preferably by expression in a recombinant host such as a
plant cell.
It is understood that activity to insects of one insect species, preferably
the larvae
thereof, is sufficient for a protein to have insecticidal activity as used
herein, although
often insects of different insect species are affected by the proteins of the
invention. The
recombinant hosts expressing at least one of the Cry1C, Cry1B or Cry1D
proteins of the
invention are typically developed for or targeted to a specific major insect
pest species
for a certain crop or region where such insect species is a pest, e.g., the
diamondback
moth for Brassica plant species, but other insects will often also be
controlled by the
recombinant hosts of the invention, such as by the transgenic plant cells or
plants, e.g.,
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13
the exemplified transgenic Brassica cauliflower or cabbage plant cells or
plants of the
invention comprising the cry1C and/or cryl E3 gene in accordance with the
invention.
"(Insect-)controlling amounts" of or "control" by a protein, or a recombinant
host
expressing a protein of this invention, as used herein, refers to an amount of
protein
which is sufficient to limit damage to a plant by insects feeding on such
plant, e.g. by
killing the insects or by inhibiting the insect development, fertility or
growth in such a
manner that an insect species provides less damage to a plant. This does not
mean that
treatment of plants with chemical insecticides will no be longer necessary
(e.g., to
control insect species not affected by the proteins of the invention, such as
(secondary)
Coleopteran or Dipteran insect pests), but that treatment by chemical
insecticides for the
insects targeted by the proteins of the invention can be significantly reduced
or avoided,
while still obtaining acceptable plant performance in the field and acceptable
yield.
In accordance with this invention, insects susceptible to the new Cry proteins
of the
invention are contacted with these proteins in insect-controlling amounts,
preferably
insect-killing amounts. In one embodiment of this invention, recombinant hosts
of the
invention, such as transgenic plant cells or plants of the invention, express
a protein or a
combination of proteins of the invention at high levels, such that a "high
dose" level is
obtained. A "high dose level", "high dose insect resistance" or "high dose"
expression,
as used herein when referring to a recombinant plant cell or plant, refers to
a
concentration of the insecticidal protein in a plant cell or plant (measured
by ELISA as a
percentage of the total soluble protein, which total soluble protein is
measured after
extraction of soluble proteins in an extraction buffer (e.g., the extraction
buffer described
in Jansens et al., 1997) using Bradford analysis (Bio-Rad, Richmond, CA;
Bradford,
1976)) which kills a developmental stage of the target insect which is
significantly less
susceptible, preferably between 25 to 100 times less susceptible to the toxin
than the
first larval stage of the insect and can thus can be expected to ensure full
control of the
target insect. In one embodiment this refers to the obtaining of at least 97
percent,
preferably at least 99 percent, most preferably 100 percent, mortality for the
fourth larval
instar (for insects having 5 larval instars) or the last larval instar (for
insects having 4 or
less larval instars) of a target insect, as measured 10 to 14 days after
insect infestation
of such plant cells or plant in routine insect bioassays, preferably whole
plant bioassays,
using suitable controls. The existence of one target insect species (i.e., an
insect
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14
species, preferably the larvae thereof, which can cause significant damage to
a plant
species or variety, and which is typically an insect for which a transgenic Bt
plant is
designed and developed) for which transformed plant cells or plants according
to this
invention provide a "high dose" level insect resistance is sufficient for a
plant to be
designated as giving "high dose" expression in accordance with this invention.
Preferred
target insects for the proteins of this invention are economically damaging
insect pests of
plants.
The terms "Cry1 protein/DNA" or "Cry protein/DNA of this invention", as used
herein,
refer to any one of the Cry1C, Cry1B, or Cry1D proteins or any one of the
cry1C, cry1B
or cry1D DNA sequences as defined herein. A Cry or Cry1 protein, as used
herein, can
be a protein in the full length size, also named a protoxin, or can be in a
truncated form
as long as the insecticidal activity is retained, or can be a combination of
different
proteins in a hybrid or fusion protein. A "protoxin" refers to the full length
insecticidal
crystal protein as it is encoded by the naturally-occurring Bt DNA sequence, a
"toxin"
refers to an insecticidal fragment thereof, particularly the smallest toxic
fragment thereof,
typically in the molecular weight range of about 50-65 kD, particularly about
60 kD, as
determined by SDS-PAGE electrophoresis compared to routinely-used molecular
weight
standards.
A "chimeric gene", as used herein, is used to refer to a gene or DNA sequence
comprising at least two different DNA fragments (such as a promoter, 5'
untranslated
leader, coding region, intron, 3' untranslated trailer, and a 3' end
transcript formation and
polyadenylation region) which are not naturally associated with each other or
which
originate from different sources. Typically, a plant-expressible chimeric
gene, as used
herein, is a gene comprising a promoter region operably-linked to a synthetic,
man-made
coding sequence such as any of the cry1C, cry1B or cry1D genes of the
invention.
The DNA sequences encoding the Cry1 proteins of the invention can be
chemically
synthesized using routine techniques, and can be inserted in expression
vectors to
produce high amounts of Cry1 proteins. The Cryl proteins can be used to
prepare
specific monoclonal or polyclonal antibodies in a conventional manner (1-16fte
et al.,
1988) to develop immuno-assays (e.g., ELISA, Western blotting, antibody-coated
dip-
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sticks) to detect the presence of absence of these proteins in any material,
such as plant
material.
The tools developed to identify transgenic plant cells, plants, or plant
materials such as
leaves or seeds comprising any one of the cryl genes of the invention
integrated in their
= genome, or DNA-containing products which comprise or are derived from
plant material
comprising a cry1 gene of the invention are based on the specific sequence
characteristics of the novel genes of the invention, such as, a specific
restriction map of
= the genomic region comprising the introduced (foreign) cry1 gene,
molecular markers or
the sequence of the foreign DNA integrated in the plant's genome.
Once the sequence of a foreign DNA such as the cry1 genes of the invention is
known,
primers and probes can be developed which specifically recognize these
sequences in
the nucleic acid (DNA or RNA) of a sample by way of a molecular biological
technique.
For instance a PCR method can be developed to identify the genes of the
invention in
biological samples (such as samples of plants, plant material or products
comprising
plant material). Such a PCR is based on at least two specific "primers", e.g.,
one
=
recognizing a sequence within the cry1 gene and the other recognizing a
sequence
within the associated transit peptide sequence or within the regulatory
regions such as
= the promoter or 3' end of the chimeric gene comprising said cry1 gene of
the invention,
or both recognizing specifically the cry1 gene of the invention. The primers
preferably
have a sequence of between 15 and 35 nucleotides which under optimized PCR
conditions "specifically recognize" a sequence within the cry1 chimeric gene
of the
invention, so that a specific fragment ("integration fragment" or
discriminating amplicon)
is amplified from a nucleic acid sample comprising a cry1 gene of the
invention. This
means that only the targeted integration fragment, and no other sequence in
the plant
genome or foreign DNA, is amplified under optimized PCR conditions.
PCR primers suitable for the invention are oligonucleotides ranging in length
from 17
nucleotides to about 200 nucleotides, comprising a nucleotide sequence of at
least 17
consecutive nucleotides, preferably 20 consecutive nucleotides selected from
the cry1C,
=
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cry1B or cry1D chimeric gene sequence as transferred to plant cells or plants
of the
invention.
The primers may of course be longer than the mentioned 17 consecutive
nucleotides,
and may, e.g., be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even
longer. The
primers may entirely consist of nucleotide sequences selected from the cry1
nucleotide
sequences. However, the nucleotide sequence of the primers at their 5' end
(i.e.
outside of the 3'-located 17 consecutive nucleotides) is less critical. Thus,
the 5'
sequence of the primers may consist of a nucleotide sequence selected from the
cry1
chimeric gene sequence, as appropriate, but may contain several (e.g. 1, 2, 5,
10)
mismatches. The 5' sequence of the primers may even entirely consist of a
nucleotide
sequence unrelated to the cry1 genes of the invention, such as a nucleotide
sequence
representing one or more restriction enzyme recognition sites. Such unrelated
sequences or flanking DNA sequences with mismatches should preferably be no
longer
than 100, more preferably no longer than 50 or no longer than 25 nucleotides.
Moreover, suitable primers may comprise or consist of a nucleotide sequence at
their 3'
end spanning the joining region between the cry1 gene of the invention and the
associated transit peptide sequence or the regulatory elements in the cry1
chimeric gene
integrated in the plant DNA, such as a promoter sequence, a leader sequence, a
trailer
sequence or a 3' transcript termination and polyadenylation sequence. It will
also be
immediately clear to the skilled artisan that properly selected PCR primer
pairs should
also not comprise sequences complementary to each other.
The term "primer" as used herein encompasses any nucleic acid that is capable
of
priming the synthesis of a nascent nucleic acid in a template-dependent
process, such
as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but
longer
sequences can be employed. Primers may be provided in double-stranded form,
though
the single-stranded form is preferred. Probes can be used as primers, but are
designed
to bind to the target DNA or RNA and need not be used in an amplification
process.
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The term "recognizing" as used herein when referring to specific primers,
refers to the
fact that the specific primers specifically hybridize to a nucleic acid
sequence in the cry1
= genes of the invention under a standard PCR identification protocol,
whereby the
specificity is determined by the presence of positive and negative controls as
is well
= known in the art.
Also included herein is a kit to detect the cry1 genes of the invention in
biological
material, as well as the use of such kit to screen biological material. A
"kit" as used
herein refers to a set of reagents for the purpose of performing the
identification of the
oryl genes of the invention in biological samples. More particularly, a
preferred
=
= embodiment of the kit of the invention comprises at least one or two
specific primers, as
described above. Optionally, the kit can further comprise any other reagent
described
herein in the PCR identification protocol. Alternatively, according to another
embodiment of this invention, the kit can comprise a specific probe, as
described above,
which specifically hybridizes with nucleic acid of biological samples to
identify the
presence of the cry1 genes therein. Optionally, the kit can further comprise
any other
reagent (such as but not limited to hybridizing buffer, label) for
identification of the cry1
genes in biological samples, using the specific probe.
Standard PCR protocols are described in the art, such as in 'PCR Applications
Manual"
(Roche Molecular Biochemicals, 2nd Edition, 1999). The optimal conditions for
the
PCR, including the sequence of the specific primers, is specified in a PCR
identification
protocol for each cry1 gene-containing plant species. It is however understood
that a
= number of parameters in the PCR identification protocol may need to be
adjusted to
specific laboratory conditions, and may be modified slightly to obtain similar
results. For
instance, use of a different method for preparation of DNA may require
adjustment of, for
= instance, the amount of primers, polymerase and annealing conditions
used. Similarly,
the selection of other primers may dictate other optimal conditions for the
PCR
identification protocol. These adjustments will however be apparent to a
person skilled
in the art, and are furthermore detailed in current PCR application manuals
such as the
one cited above.
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Examples of suitable primer combinations in accordance with the invention are
(sequence 5' - 3') for the cry1B gene of the invention: P1B227 (TAC TTC GAA
CAG AM
GM CGA GM CGA G, SEQ ID No. 20) and P1B228 (GTC CAG CGA AAG GM CTC
CAA GAA, SEQ ID No. 21), and for the cryl C gene of the invention: P1C247 (MC
CTT
GAG GGA CU GGA MC, SEQ ID No. 22) and P1C252 (MG ATG AGG GTT TCT
GAT AGC AG, SEQ ID No. 23). Hence, any gene encoding an insecticidal Cry1B or
Cry1C protein and specifically recognized by these primers is included herein,
as well as
any method to detect such genes using such or other specific primers.
Also specific markers or labeled probes can be designed to detect the DNA
sequences
of this invention, and any use of specific markers or probes directed to any
of the cry1C,
cry1B or cry1D genes of the invention is included herein. In one embodiment of
this
invention, the specific markers, primers or labeled probes do not detect or
recognize any
plant, preferably any plant of the same species as the test plant, not
containing a cry1
DNA sequence of the invention, particularly any such markers, primers or
labeled probes
do not detect or recognize any plant expressing a Cryl C, Cry1D or Cry1B
protein
wherein such plant does not contain a DNA sequence of the invention (such as a
cry1C,
cry1D or cry1B DNA as defined herein, e.g., a DNA comprising the nucleotide
sequence
of any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14).
The DNA sequences of this invention can be slightly modified to allow for more
convenient restriction enzyme sites, or to make small changes without changing
the
efficacy and without significantly changing, preferably without changing, the
protein they
encode. Indeed, because of the degeneracy of the genetic code, it is well
known that
most amino acid codons can be replaced by others without changing the amino
acid
sequence of the protein. Furthermore, some amino acids can be substituted by
other
equivalent amino acids without significantly changing, preferably without
changing, the
insecticidal activity of the protein. Also, changes in amino acid sequence or
composition
in regions of the molecule, different from those responsible for binding or
pore formation
are less likely to cause a difference in insecticidal activity of the protein
(e.g., the C-
terminal part of the Cry1 protoxin can be removed or be replaced by another
amino acid
sequence without affecting the insecticidal activity of the Cry1 proteins of
the invention).
Equivalents of the DNA sequences of the invention include DNA sequences with
less
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than 20, preferably 5-10, nucleotide differences compared to the cry1 genes of
this
invention as defined herein, but which encode an insecticidal Cry1 protein of
the
invention, as defined herein.
Small modifications to a DNA sequence such as described above can be routinely
made, i.e., by PCR-mediated mutagenesis (Ho et al.,1989, White et al., 1989).
More
= profound modifications to a DNA sequence can be routinely done by de novo
DNA
synthesis of a desired coding region using available techniques.
The term "encoding", as used herein, when referring to a gene encoding a
protein, refers
to the capacity of such gene to produce a protein upon transcription and
translation of
the coding sequence contained in such gene in a target host cell. Hence, the
cry1C1
= chimeric gene of the invention encodes the Cry1C1 protein of the
invention, even though
this gene contains two coding sequences interrupted by a non-coding intron
sequence.
With the term "substantially the same", when referring to the amino acid
sequence of a
= Cryl protein of this invention, is meant to include an amino acid
sequence that differs in
= no more than 5 %, preferably no more than 2 %, to the amino acid sequence
of the
protein compared to; and when referring to toxicity of Cry protein, is meant
to include a
protein whose LC50 value obtained under the same conditions of bio-assay
(preferably
in the same bio-assay using insects from the same population and suitable
controls)
differs no more then 2 times, preferably no more than 50 %, of the LC50 value
obtained
for the protein compared to.
"Microorganism", as used herein, refers to any living organism that can be
observed only
with the aid of a microscope, such as bacteria, yeast cells, plant cells,
viruses, fungi.
This includes all generally unicellular organisms with dimensions beneath the
limits of
vision which can be propagated and manipulated in a laboratory, typically
prokaryotic or
unicellular eukaryotic life forms, including tissue cultures and plasmids.
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The cry1 DNA sequences of the invention, prepared from total DNA, can be
ligated in
suitable expression vectors and transformed in suitable host cells which can
then be
screened by conventional detection tools for presence and expression of the
toxin.
A database search with the genes of this invention indicates that the DNA
sequences of
the invention are significantly different from any previously described genes
or DNA
sequences encoding toxins with activity against Lepidoptera (see, e.g., the
January 26,
2006 version of DNA sequences described in patent applications (Geneseq
release
200602), Hofte and Whiteley, 1989; Crickmore et al., 1998; and the August 2,
2005
update on the Bt nomenclature website corresponding to the Crickmore et at,
(1998)
publication, found at:
http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/index.html).
The closest sequence identity at the DNA level (for the entire length of the
sequences of
the invention) in available DNA sequence databases (from patent or scientific
literature)
was 76.60 % for the cry1C DNA of SEQ ID No. 1 or 3, 73% for the cry1B DNA of
SEQ
ID No. 10, and 72.5 % for the cry1D DNA of SEQ ID No. 14, using the above
defined
Needleman-Wunsch default settings in EMBOSS. Hence, assuming the available DNA
sequence databases are representative of all known DNA sequences, the DNA
sequences of this invention differ in at least 23 % of their nucleotides from
any
previously known DNA sequence. Assuming the closest sequences are contained in
the
available databases, this reflects a difference in about 485 nucleotides for
the nucleotide
sequence of SEQ ID No. 1 or 3, a difference in about 524 nucleotides for the
nucleotide
sequence of SEQ ID No. 10, and a difference in about 498 nucleotides for the
nucleotide
sequence of SEQ ID No. 14 with their respective closest published DNA
sequence. This
difference will be even more pronounced for the DNA sequences of SEQ ID No. 4,
6, 8,
or 12, which encode a fusion protein with a transit peptide. Also the
optimized
chloroplast transit peptide DNA sequence of this invention (SEQ ID No. 16),
which was
adapted for expression in the target plants of the invention, was found to
have about
76.1 % sequence identity (for that part of equal length to the SEQ ID No. 16
sequence)
to the closest DNA sequence identified in available DNA sequence databases and
hence is very different.
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By an "insecticidally effective part (portion or fragment)" of DNA sequences
encoding a
Cry1 protein, also referred to herein as "truncated gene" or "truncated DNA",
is meant a
DNA sequence encoding a poiypeptide which has fewer amino acids than the Cryl
protein protoxin form but which is still insecticidal.
In order to express all or an insecticidally effective part of the DNA
sequence encoding a
= Cry protein of this invention in a recombinant host such as E. colt, in
other Bt strains or in
plants, suitable restriction sites can be introduced, flanking the DNA
sequence. This can
be done by site-directed mutagenesis, using well-known procedures (Stanssens
et al.,
1989; White et al., 1989). in order to obtain improved expression in plants,
the cry1
genes of the invention are artificial genes, wherein the sequence has been
adapted for
optimal expression by DNA synthesis. In such sequence, replacement of DNA
sequences inhibiting optimal expression is achieved by designing DNA sequences
comprising codons more preferred by plants, preferably the target plant genus
or
species.
For obtaining enhanced expression in plants or preventing expression of an
insecticidal
protein when not present in a plant host cell (such as in a bacterial host
cell), in one
embodiments of the invention a plant intron is inserted in the chimeric cry1
genes of the
= invention, preferably in the coding sequence of at least one of the cry1
genes of the
invention. Any of the known plant introns (e.g., Brown, 1986, Brown and
Simpson, 1998,
= Brown et a)., 1996) can be used herein as long as it is operably-linked
to the coding
sequence fragments so as to assure proper splicing. Operable linkage of the
intron and
the resulting proper splicing is conveniently checked in the target host plant
species or
cells thereof by RT-PCR or Northern blot or by any other means available in
the art. In
one embodiment an intron of a dicot plant gene is used in genes to be
expressed in dicot
plant cells, and a monocot intron is used in genes to be expressed in monocot
plants. In
one embodiment, the intron of the invention is the second intron of the light-
inducible
tissue-specific ST-LS1 gene of Solanum tuberosum (potato) as described by
Eckes et al.
(1986), e.g., the nucleotide sequence of SEQ ID No. 1 between nucleotide
position 672
and 862. In one embodiment of this invention a plant intron is introduced into
any Bt
insecticidal protein coding sequence, particularly the intron of SEQ ID No. 1
between
nucleotide position 672 and 862, so that it is effectively spliced in plant
cells. Effective
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splicing in plants cells can be measured using routine techniques, such as RT-
PCR,
Northern blotting, or the detection of a functional protein produced in plant
cells. Of
course, for effective splicing the intron needs to be inserted in the correct
position of the
coding sequence so that functional 5' and 3' splice sites are obtained in the
sequence.
The two cry genes of the invention, illustrated in SEQ ID Nos. 1 and 3, each
containing a
plant intron at a different location, were found by RT-PCR analysis to both be
effectively
spliced in Brassica oferaceae plant cells, and to produce an mRNA encoding the
expected Cry protein.
In accordance with one embodiment of this invention, the proteins are targeted
to
intracellular organelles such as plastids, preferably chloroplasts,
mitochondria, or are
secreted from the cell, potentially optimizing protein stability and/or
expression. For this
purpose, the chimeric genes of the invention comprise a coding region encoding
a signal
or target peptide, linked to the Cry protein coding region of the invention.
Particularly
preferred peptides to be included in the proteins of this invention are the
transit peptides
for chloroplast or other plastid targeting, especially duplicated transit
peptide regions
from plant genes whose gene product is targeted to the plastids, the optimized
transit
peptide described by Lebrun et al. (1996), or Capellades et al. (US Patent
5,635,618),
the transit peptide of ferredoxin-NADP+oxidoreductase from spinach (Oelmuller
et al.,
1993), the transit peptide described in Wong et al. (1992) and the targeting
peptides in
published PCT patent application WO 00/26371. In one embodiment of the
invention,
the chloroplast transit peptide comprises the sequence of SEQ ID No. 17 from
amino
acid position 3 to amino acid position 124 or variant thereof, such as a
chloroplast transit
peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to
amino
acid position 124, wherein the Cys amino acid at position 55 is replaced by
Tyr in SEQ
ID No. 17 and/or wherein a Gly amino acid is added after the Gly amino acid at
position
51 in SEQ ID No. 17. Also preferred are peptides signalling secretion of a
protein linked
to such peptide outside the cell, such as the secretion signal of the potato
proteinase
inhibitor II (Keil et al., 1986), the secretion signal of the alpha-amylase 3
gene of rice
(Sutliff et al., 1991) and the secretion signal of tobacco PR1 protein
(Cornelissen et at.,
1986).
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Particularly useful signal peptides in accordance with the invention include
the
chloroplast transit peptide (e.g., Van Den Broeck et al. (1985), or the
optimized
chloroplast transit peptide of US patent 5, 510,471 and US patent 5,635,618
causing
transport of the protein to the chloroplasts, a secretory signal peptide or a
peptide
targeting the protein to other plastids, mitochondria, the ER, or another
organelle. Signal
sequences for targeting to intracellular organelles or for secretion outside
the plant cell
or to the cell wall are found in naturally targeted or secreted proteins,
preferably those
described by Kletsgen et at. (1989), KlOsgen and Well (1991), Neuhaus & Rogers
(1998),
Bih et at. (1999), Morris et at. (1999), Hesse et al. (1989), Tavladoraki et
al. (1998),
Terashima et at. (1999), Park et al. (1997), Shcherban et at. (1995),
particularly the signal peptide sequences from
targeted or secreted proteins of Brassica plant species, corn, cotton, or
soybean. A
preferred DNA sequence encoding a transit peptide of the invention is a DNA
comprising
the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide
position 371,
particularly the sequence of SEQ ID No. 16.
Furthermore, for any target pest insect, the binding properties of the Cry
proteins of the
invention can be evaluated, using methods known in the art (e.g., Van Rie et
al., 1990),
to determine if the Cry1 proteins of the invention bind to sites on a target
insect midgut
that are not recognized (or competed for) by other Cry or non-Cry proteins.
Other Bt
toxins binding to different binding sites in relevant susceptible insects, or
other toxins
derived from Bt strains or other sources (such as VIP toxins or insect (gut)
proteinase
inhibitors) with a different mode of action are very valuable to also express
in a plant in
addition to any one of the cry1 genes herein, to prevent or delay the
development of
insect resistance to a plant expressing insecticidal toxins. Because of the
characteristics
of the new cry1 genes, they are extremely useful for transforming plants, e.g.
monocots
such as corn or wheat and dicots such as cotton, soybean and Brassica species
plants,
to protect these plants from insect damage.
Especially for insect resistance management purposes for a specific insect
pest, it is
preferred to combine a cry1C gene of this invention with another gene encoding
a
different insect control protein, particularly a Bt crystal protein, which
does not recognize
at least one binding site recognized by such Cry1C protein in a target insect.
Preferred
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insect control proteins to combine with the Cry1C proteins of this invention,
particularly
for simultaneous expression in plants, preferably Brassica species plants,
particularly
cabbage and cauliflower, include the Cry1B protein of this invention or the
Cry1D protein
of this invention, the VIP3Aa protein or a toxic fragment thereof as described
in Estruch
et al., 1996 and US Patent 6,291,156, or insecticidal proteins from
Xhenorhabdus,
Serratia or Photorhabdus species strains (e.g., Waterfield et al., 2001;
ffrench-Constant
and Bowen, 2000). In one embodiment, such co-expression is obtained by
transforming
a plant already expressing an insect control protein with a cry1 gene of this
invention, or
by crossing plants transformed with the insect control protein and plants
transformed
with the cry1 gene of this invention. For Brassica species plants, preferably
the cry1
gene is used as first gene and as second gene the Cry1B, Cry1D or VIP3Aa
protein or
variants or derivatives thereof are used. Methods for obtaining expression of
different Bt
(or similarly, for other insect control proteins) insecticidal proteins in the
same plant in an
effort to minimize or prevent resistance development to transgenic insect-
resistant plants
are described in EP patent 0 408 403. In one embodiment of the invention, the
cry IC
gene of the invention is located in one and the same locus as a second insect
control
gene, such as a Cry1B or Cry1D gene, in the transgenic plant cells or plants
of the
invention, so that these genes do not segregate in the progeny of such plant
cells or
plants.
Preferably, for selection purposes but also for increasing the weed control
options, the
transgenic plants of the invention are also transformed with a DNA encoding a
protein
inactivating a broad-spectrum herbicide or encoding a protein which is a
variant of the
protein target for the herbicide but which protein variant is insensitive to
such herbicide,
e.g., herbicides based on glufosinate or glyphosate.
The insecticidally effective cry1 gene, preferably the cry1 chimeric gene,
encoding an
insecticidally effective portion of the Cry protoxin, can be stably inserted
in a
conventional manner into the nuclear genome of a plant cell, and the so-
transformed
plant cell can be used in a conventional manner to produce a transformed plant
that is
insect-resistant. In this regard, a disarmed Ti-plasmid, containing the
insecticidally
effective cryl gene part, in Agrobacterium, e.g., Agrobacterium tumefaciens
can be used
to transform the plant cell, and thereafter, a transformed plant can be
regenerated from
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the transformed plant cell using the procedures described, for example, in EP
0 116 718,
EP 0 270 822, PCT publication WO 84/02913 and published European Patent
application ("EP") 0 242 246 and in De Block et al. (1989). Preferred Ti-
plastinid vectors
each contain the insecticidally effective cry gene part between the border
sequences, or
CL least IGGCLGA to the Iaff of the right border OGMUGUnG, of the T_MAA of
the Ti _ iC Jv
1%.
Of course, other types of vectors can be used to transform the plant cell,
using
procedures such as direct gene transfer (as described, for example in EP 0 233
247),
pollen mediated transformation (as described, for example in EP 0 270 356, PCT
publication WO 85/01856, and US Patent 4,684,611), plant RNA virus-mediated
transformation (as described, for example in EP 0 067 553 and US Patent
4,407,956),
liposome-mediated transformation (as described, for example in US Patent
4,536,475),
and other methods such as the methods for transforming certain lines of corn
(e.g., US
patent 6,140,553; Fromm et al., 1990; Gordon-Kamm et al., 1990) and the method
for
= transforming monocots generally (PCT publication WO 92/09696). For cotton
transformation, especially preferred is the method described in PCT patent
publication
WO 00/71733. For soybean transformation, reference is made to methods known in
the
art, e.g., Hinchee et al. (1988) and Christou et al. (1990) or the method of
WO 00/42207.
=
Also, besides transformation of the nuclear genome, also transformation of the
plastid
genome, preferably chloroplast genome, is included in the invention. Kota et
al. (1999)
have described a method to express a Cry2A protein in tobacco chloroplasts,
and Lin et
al. (2003) described expression of a cry1C gene in transplastomic tobacco
plants.
The resulting transformed plant can be used in a conventional plant breeding
scheme to
produce more transformed plants with the same characteristics or to introduce
the
insecticidally effective cry gene part in other varieties of the same or
related plant
species. Seeds, which are obtained from the transformed plants, contain the
insecticidally effective cry gene part as a stable genomic insert.
The insecticidally effective cry1 gene, preferably the sequence of SEQ ID No.
1, 3, 4 or
6, is inserted in a plant cell genome so that the inserted gene is downstream
(i.e., 3') of,
and under the control of, a promoter which can direct expression of the gene
in a plant
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26
cell (herein named a "plant-expressible promoter"). This is preferably
accomplished by
inserting the cry1 chimeric gene comprising a plant-expressible promoter in
the plant cell
genome, particularly in the nuclear or plastid (e.g., chloroplast) genome.
Preferred
plant-expressible promoters include: the strong constitutive 35S promoters
(the "35S
promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841
(Gardner et al.,
1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the
35S
promoter described by Odell et al. (1985), promoters from the ubiquitin family
(e.g., the
maize ubiquitin promoter of Christensen etal., 1992, see also Cornejo et at.,
1993), the
gos2 promoter (de Pater et at., 1992), the emu promoter (Last et al., 1990),
Arabidopsis
actin promoters such as the promoter described by An et al. (1996), rice actin
promoters
such as the promoter described by Zhang et al. (1991); promoters of the
Cassava vein
mosaic virus (WO 97/48819, Verdaguer et at. (1998)), the pPLEX series of
promoters
from Subterranean Clover Stunt Virus (WO 96/06932), particularly the
duplicated
promoter region derived from the subterranean clover stunt virus genome
segment 4 or
7 (referred to as the "S7S7" or "S4S4" promoters herein) described by Boevink
et at.
(1995) or Schunmann et at. (2003), an alcohol dehydrogenase promoter, e.g.,
pAdh1S
(GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2'
promoter (the "TR1' promoter" and "TR2' promoter", respectively) which drive
the
expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al.,
1984).
Alternatively, a promoter can be utilized which is not constitutive but rather
is specific for
one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby
the
inserted cry gene part is expressed only in cells of the specific tissue(s) or
organ(s). For
example, the insecticidally effective cry gene part could be selectively
expressed in the
leaves of a plant (e.g., corn, cotton) by placing the insecticidally effective
gene part
under the control of a light-inducible promoter such as the promoter of the
ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself
or of another
plant such as pea as disclosed in US Patent 5,254,799. Another alternative is
to use a
promoter whose expression is inducible, preferably by wounding such as insect
feeding,
e.g., the MPI promoter described by Cordera et at. (1994), or the
Agrobacterium TR2' or
mannopine synthase promoter (Velten et at., 1984) or a promoter inducible by
chemical
factors.
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The insecticidally effective cry gene part is preferably inserted in the plant
genome so
that the inserted gene part is upstream (i.e., 5') of suitable 3' end
transcription regulation
signals (i.e., transcript formation and polyadenylation signals). This is
preferably
accomplished by inserting the cry1 chimeric gene in the plant cell genome.
Preferred
polyadenylation and transcript formation signals include those of the 3'
untranslated
region of the NADP-malic enzyme gene from Flaveria bidentis (Marshall et al.,
1996),
nopaline synthase gene (Depicker et al., 1982), the octopine synthase gene
(Gielen et
al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-
untranslated
DNA sequences in transformed plant cells.
In one embodiment of this invention, at least one of the genes of the
invention,
preferably at least 2, are transformed into plants selected from the group
consisting of:
corn, cotton, watercress, horseradish, wasabi, arugula, cress, radish, canola,
soybean,
vegetable plants, Cruciferae plant species, Brassicaceae plant species such as
cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale,
broccoli,
Brussels sprouts, mustard spinach, and the like. Particularly, in one
embodiment of this
invention the following Brassica species plants are protected from insects by
the genes
of this invention: B. carinata, B. elongate, B. fruticulosa, B. juncea, B.
napus, B.
narinosa, B. hirta, B. rosularis, B. nigra, B. oleracea, B. perviridis, B.
rapa, B. rupestris,
B. septiceps, B. tournefortii, and the like, particularly plants of the
species Brassica
oleraceae (such as the subspecies botrytis and capitata) or Brassica napus, as
well as
plants of the following genus: Raphanus (such as R. sativus), Armoracia (such
as A.
rusticana), Wasabia (such as W. japonica), Eruca (such as E. vesicaria),
Nastrurtium
(such as N. officinale), and Lepidium (such as L. sativum).
The invention includes the above listed Brassica species plants transformed
with at least
one or two genes of the invention, such as the cry1B and cry1C genes of the
invention,
as well as plants obtained after crossing or breeding with related plants
(including plants
of a related plant species) that contain the genes of the invention. Such
crossing or
= breeding can be done using traditional breeding techniques known in the
art, but may
also include known in vitro work such as embryo rescue, protoplast fusion, and
the like.
The invention hence also relates to Brassicaceee plants such as B. napus, B.
rape, B.
juncea or B. carinata, that contain the gene or genes of the invention, such
as the cry1B
and cry1C genes of the invention, from crossings with a transformed B.
oleracea plant or
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28
the progeny thereof, or to B. oleracea plants that contain the gene or genes
of the
invention, such as the cry1B and cry1C genes of the invention, from crossings
with a
transformed B. napus plant, and to uses of such plants.
Transformation of plant cells can also be used to produce the proteins of the
invention in
large amounts in plant cell cultures, e.g., to produce a Cry1 protein that can
then be
applied onto crops after proper formulation. When reference to a transgenic
plant cell is
made herein, this refers to a plant cell (or also a plant protoplast) as such
in isolation or
in tissue culture, or to a plant cell (or protoplast) contained in a plant or
in a differentiated
organ or tissue, and both possibilities are specifically included herein.
Hence, a
reference to a plant cell in the description or claims is not meant to refer
only to isolated
cells in culture, but refers to any plant cell, wherever it may be located or
in whatever
type of plant tissue or organ it may be present.
All or part of the cryl genes of the invention, encoding an anti-lepidopteran
protein, can
also be used to transform bacteria, such as a B. thuringiensis which has
insecticidal
activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain
can be
produced which is useful for combatting a wide spectrum of lepidopteran and
coleopteran insect pests or for combatting additional lepidopteran insect
pests.
Transformation of bacteria, such as bacteria of the genus Pseudomonas,
Agrobacterium, Bacillus or Escherichia, with the cryl genes of this invention,
incorporated in a suitable cloning vehicle, can be carried out in a
conventional manner,
preferably using conventional electroporation techniques as described in
Mahillon et al.
(1989) and in PCT Patent publication WO 90/06999.
Transformed Bacillus species strains containing the cry gene of this invention
can be
fermented by conventional methods (DuImage, 1981; Bernhard and Utz, 1993) to
provide high yields of cells. Under appropriate conditions which are well
understood
(DuImage, 1981), these strains each sporulate to produce crystal proteins
containing the
Cry protoxin in high yields.
An insecticidal, particularly anti-lepidopteran, composition of this invention
can be
formulated in a conventional manner using the microorganisms transformed with
the cry
gene, or preferably their respective Cry proteins or the Cry protoxin, toxin
or
insecticidally effective protoxin portion as an active ingredient, together
with suitable
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29
carriers, diluents, emulsifiers and/or dispersants (e.g., as described by
Bernhard and
= Utz, 1993). This insecticide composition can be formulated as a wettable
powder,
pellets, granules or dust or as a liquid formulation with aqueous or non-
aqueous solvents
as a foam, gel, suspension, concentrate, etc.
A method for controlling insects, particularly Lepidoptera, in accordance with
this
invention can comprise applying (e.g., spraying), to a locus (area) to be
protected, an
insecticidal amount of the Cry proteins or host cells transformed with the cry
gene of this
invention. The locus to be protected can include, for example, the habitat of
the insect
pests or growing vegetation or an area where vegetation is to be grown.
In one embodiment of this invention, insects against which the cry1 genes or
Cry1
proteins of the invention can be used include insects selected from the group
consisting
of: Plutella xylostella, Spodoptera exigua, Spodoptera littoralis, Spodoptera
frugiperda,
Trichoplusia ni, Heliothis virescens, Mamestra brassicae, Pieris bra ssicae,
Manduca
sexta, Choristoneura fumiferana, Choristoneura occidentalis, Choristoneura
rosaceana,
Pandemis pyrusana, Platynota stultana, Lymantria dispar, Orgyia leucostigma,
Malacosoma disstria, Lambina fiscellaria, Chile suppressalis, Chile partellus,
Scirpophaga incertulas, Argyrotaenia citrana, Artogeia rapa, Chrysomela
scripta,
Ostrinia nubilalis, Pseudoplusia includens, and Thaumetopoea pityocampa. In
one
embodiment, Plutella xylostella (diamondback moth) is a preferred target
insect pest.
This is a cosmopolitan species that causes major losses in several Cruciferous
plants,
particularly Brassicacaea plants. The Cry1C, Cryl B and Cryl D proteins
encoded by the
= genes of this invention are particularly useful to control this insect,
e.g., by expression of
the genes of the invention in cells of a plant.
Such insects can be controlled by planting or growing plants comprising any
one of the
cryi G genes of the invention in a field, or by securing the presence of a
Cry1C protein
as defined herein in or on plants infested by such insects (e.g., by sowing or
planting a
Brassica species plant such as a cabbage or cauliflower plant transformed with
the
cry1C1 or cry1C2 gene of this invention, or spraying a composition containing
a Cry1C
protein of this invention). The invention also relates to the use of the cry1
genes of this
invention, at least the cry1C1 or cry1C2 genes, in plants to protect them
against
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Lepidopteran insect pests, preferably in combination with a cry1B or cry1D
gene of this
invention.
In the current invention, also a modified coding sequence encoding a
chloroplast transit
peptide is provided. Such coding sequence has a codon usage adapted for high
expression in plants, particularly Brassicaceae plants such as Brassica
oleracea or
Brassica napus, especially cabbage, cauliflower or oilseed rape (canola). In
one
embodiment of the invention, the modified transit peptide comprises the
nucleotide
sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position
371, -
particularly the sequence of SEQ ID No. 16. Also plant cells, plants or seeds
comprising
the modified transit peptide coding sequence of the invention, as well as the
use of this
transit peptide coding sequence for targeting any protein to the chloroplast,
particularly
to the chloroplast of vegetable plants, particularly Brassica species plants,
are included
in this invention.
These and/or other embodiments of this invention are reflected in the wordings
of the
claims, that form part of the description of the invention.
The following Examples illustrate the invention, and are not provided to limit
the
invention or the protection sought. Unless otherwise stated, all recombinant
DNA
techniques are carried out according to standard protocols as described in
Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994)
Current
Protocols in Molecular Biology, Current Protocols, USA. Standard materials and
methods for plant molecular work are described in Plant Molecular Biology
Labfax
(1993) by R.D.D. Croy published by BIOS Scientific Publications Ltd (UK) and
Blackwell
Scientific Publications, UK.
The enclosed sequence listing referred to in the Examples, the Claims and the
Description is as follows:
Sequence Listing:
SEQ ID No.1: optimized cry1C1 coding sequence comprising an intron at position
672
SEQ ID No.2: amino acid sequence of the Cry1C1 protein encoded by SEQ ID No. 1
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SEQ ID No.3: optimized cry1C2 coding sequence, comprising an intron at
position 489
SEQ ID No.4: optimized cry1C3 coding sequence, comprising the sequences of SEQ
ID
No. 1 and SEQ ID No. 16, encoding a fusion protein with a transit peptide
SEQ ID No.5: Cry1C3 protein encoded by SEQ ID No. 4
S'EQ ID 40 .Q. optimized cry 1C4 coding sequence, comprising the sequences of
SEQ ID
No. 3 and SEQ ID No. 16, encoding a fusion protein with a transit peptide
SEQ ID No.7: Cry1C4 protein encoded by SEQ ID No. 6
SEQ ID No.8: optimized cryl B1 coding sequence, including a transit peptide
coding
sequence
SEQ ID No.9: Cry1B1 protein encoded by the sequence of SEQ ID No. 8
SEQ ID No.10: optimized cry1B2 coding sequence
SEQ ID No.11: Cryl B2 protein encoded by the sequence of SEQ ID No. 10
SEQ ID No.12: optimized cry1D1 coding sequence, including a transit peptide
coding
sequence
SEQ ID No.13: Cry1D1 protein encoded by the sequence of SEQ ID No.12
SEQ ID No.14: optimized cry1D2 coding sequence
SEQ ID No.15: Cry1D2 protein encoded by the sequence of SEQ ID No. 14
SEQ ID No.16: coding sequence encoding an optimized chloroplast transit
peptide
SEQ ID No.17: chloroplast transit peptide encoded by the sequence of SEQ ID
No. 16
SEQ ID No.18: duplicated S7 subterranean clover stunt virus promoter sequence
(S7S7)
SEQ ID No.19: duplicated S4 subterranean clover stunt virus promoter sequence
(S4S4)
SEQ ID No. 20: cry1B gene primer P1B227
SEQ IDr:Io. 21: cry1B gene primer P1 B228
SEQ ID No. 22: cry1C gene primer P1C247
SEQ ID No. 23: cry1C gene primer P1C252
EXAMPLES
1.Construction of chimeric genes and transformation vectors.
Several cryl genes were designed and assembled using a combination of
technologies
to achieve genes with optimal performance in plant cells.
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The cry1C1 DNA which was designed for optimal expression in plant cells is
represented
in SEQ ID No. 1. This DNA encodes the insecticidal Cryl Cl protein of the
invention
(SEQ ID No. 2). For transformation of plants, a first chimeric gene (the cryl
CI chimeric
gene) is constructed comprising the following operably-linked elements (5' to
3'): a
promoter comprising the duplicated promoter region derived from the
subterranean
clover stunt virus genome segment 7 (S7S7 promoter, Boevink et al., 1995, SEQ
ID No.
18), the leader sequence of the tapetum-specific El gene (GE1) of Oryza sativa
(Michiels et al., 1992), the cryl Cl DNA comprising the second intron of the
light-
inducible tissue-specific ST-LS1 gene of Solanum tuberosum (Eckes et al.,
1986) at
position 672 (SEQ ID No. 1), and the sequence including the 3' untranslated
region of
the NADP-malic enzyme gene from Haveria bidentis (3' Mel, Marshall et al.,
1996).
A similar cryl C chimeric gene was made, wherein the ST-LS1 intron 2 is at
position 489
of the cryl C DNA (i.e., the cryl C2 DNA), this is the cryl C2 chimeric gene,
otherwise
constructed exactly like the cryl Cl chimeric gene.
To secure targeting of the Cryl C protein to the plant cell chloroplast,
variants of the
cryl Cl and cryl C2 chimeric genes are constructed which comprise a modified
sequence encoding an optimized transit peptide (SEQ ID No.16) as described by
Lebrun
et al. (1996) operably-linked to the cryl C coding region so that a transit
peptide fusion =
protein is expressed in plant cells. These are the cryl C3 and cryl C4
chimeric genes,
comprising the cryl C3 and cryl C4 coding sequences, respectively, which each
contain
the sequence of the modified chloroplast transit peptide of SEQ ID No.16. The
cryl C3
DNA sequence is shown in SEQ ID No. 4, it is a fusion of the cryl Cl sequence
of SEQ
ID No. 1 with the transit peptide coding sequence of SEQ ID No. 16. The cryl
C4 DNA
sequence is shown in SEQ ID No. 6, it is a fusion of the cryl C2 sequence of
SEQ ID No.
3 with the transit peptide coding sequence of SEQ ID No. 16.
The cryl B1 DNA which was designed for optimal expression in plant cells is
represented
in SEQ ID No. 8. This DNA encodes the insecticidal Cryl B1 protein of the
invention
(SEQ ID No. 9). For transformation of plants, a chimeric gene (the cryl 61
chimeric
gene) is constructed comprising the following operably-linked elements (5' to
3'): a
promoter comprising the duplicated promoter region derived from the
subterranean
clover stunt virus genome segment 4 (S4S4 promoter, Boevink et at., 1995, SEQ
ID No.
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19), the leader sequence of the El gene (GE1) of Otyza sativa (Michiels et
al., 1992),
the cryl B1 DNA comprising the sequence of the modified chloroplast transit
peptide of
SEQ ID No.16, and the sequence including the 3' untranslated region of the
NADP-malic
enzyme gene from Flaveria bidentis (3' Mel, Marshall et al., 1996).
A second form of the cry 1B chimeric gene was also made, using the cryl B2 DNA
(SEQ
ID No. 10), wherein no sequence encoding an optimized transit peptide is
contained, so
that cytoplasmic accumulation of the Cryl B protein occurs in plant cells.
This is the
Cryl B2 chimeric gene.
The cryl D1 DNA which was designed for optimal expression in plant cells is
represented
in SEQ ID No. 12. This DNA encodes the insecticidal Cry1D1 protein of the
invention
(SEQ ID No. 13). For transformation of plants, a chimeric gene (the cryl D1
chimeric
= gene) is cOnstructed comprising the following operably-linked elements
(5' to 3'): an
S4S4 promoter (SEQ ID No. 19), the leader sequence of the El gene (GE1) of
Oryza
sat iva (Michiels et al., 1992), the cryl D1 DNA comprising the sequence of
the modified
chloroplast transit peptide of SEQ ID No.16, and the sequence including the 3'
untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3'
Mel,
Marshall et al., 1996).
A second form of the cryl D chimeric gene was also made, using the cryl D2
DNA,
wherein no sequence encoding an optimized transit peptide is contained, so
that
cytoplasmic accumulation of the C.ryl n protein occurs in plant cells. This is
the rryi n9
chimeric gene.
= A DNA transformation vector (pT1C4B1) is made comprising between the T-
DNA
borders the cry1C4 chimeric gene and the cryl B1 chimeric gene in a head-to-
tail
orientation (3'Mel-cry1C4-GE1 leader-S7S7 ¨ 5454-GE1 leader-cry1B1-31viel), as
well
as a transfer vector (pT1C2B2) comprising between the T-DNA borders the cryl
C2
= chimeric gene and the cryl B2 chimeric gene in a head-to-tail orientation
(3'Mel-cryl C2-
GE1 leader-S7S7 ¨ S4S4-GE1 leader-cry1B2-3'Mel). In such manner, with both T-
DNA
vectors, the cryl C and cryl B genes of the invention will be co-transferred
to the plant
cell and will be located at one locus after successful transformation.
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Similar T-DNA vectors are constructed which contain the above cry1C chimeric
genes
but which contain as second chimeric gene the cry1D1 or cry1D2 chimeric genes
instead of the above cry1B chimeric genes. Also a triple cry gene
transformation vector
is constructed, comprising both the cryl C, crylD and cry1B genes (all either
with or
without modified transit peptide).
The transformation vectors containing the genes of the invention were derived
from
pGSC1700 (Cornelissen and Vandewiele, 1989). The vector backbone contains the
following genetic elements:
a) the plasmid core comprising the origin of replication from the plasmid
pBR322 (Bolivar
et al., 1977) for replication in Escherichia coil and a restriction fragment
comprising the
origin of replication from the Pseudomonas plasmid pVS1 (Itoh et al., 1984)
for
replication in Agrobacterium tumefaciens.
b) a selectable marker gene conferring resistance to streptomycin and
spectinomycin
(aadA) for propagation and selection of the plasmid in Escherichia coil and
Agrobacterium tumefaciens.
c) a DNA region consisting of a fragment of the neomycin phosphotransferase
coding
sequence of the nptl gene from transposon Tn903 (Oka et al., 1981).
The T-DNA region of each transformation vector also contains a chimeric bar
gene that
serves as selectable marker gene. Expression of the bar gene enables the
production of
an enzyme, phosphinothricin-acetyl transferase, that metabolizes the herbicide
glufosinate-ammonium, thus rendering it non-herbicidal in the plant. The
chimeric bar
gene comprises the 35S3 promoter region from the Cauliflower Mosaic Virus 35S
transcript (Odell et al.,1985), the bar coding sequence of the
phosphinothricin
acetyltransferase gene of Streptomyces hygroscopicus as described by Thompson
et al.
(1987), and a 3' transcript termination and polyadenylation sequence from the
3'
untranslated region of the nopaline synthase gene from the T-DNA of pTiT37
(Depicker
et al., 1982).
Similar transformation vectors as those described are also constructed,
wherein the
cry1C1 or cry1C3 chimeric genes are used (similar as the above cry1C genes but
having
the ST-LS1 intron at a position 489). Also these vectors contain the cry1B1 or
cry1B2
chimeric genes, or the cry1D1 or cry1D2 chimeric genes described above.
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All constructed plasmids are confirmed to be accurate by restriction enzyme
digest
analysis and by DNA sequencing, before they are used for plant transformation.
= 2.Plant transf"rm,tirµn
regenerntiNI.
The above transformation vectors pT1C4B1 and pT1C2B2 containing the cry1C and
cryl B genes of the invention are transferred into Agrobacterium tumefaciens
strains for
transformation in plants using routine methods.
Cauliflower and cabbage plants are transformed using Agrobacterium
transformation.
= Seeds of Brassica oleracea var. capitata (cabbage) or Brassica oleracea
var. botrytis
(cauliflower) are sterilized by dipping in 70% ethanol followed by submersion
in 6%
bleach. The seeds are then rinsed with sterile water and transferred to small
Petri-plates
containing MS based medium. The Petri-plates are placed in glass containers
and
incubated for 5-8 days at 24 C. Hypocotyl explants of 0.5-0.7 cm are cut and
placed in
liquid medium with appropriate hormones. Agrobacterium tumefaciens carrying
the
genes of interest are added to the medium to make a final concentration of
1x107
= bacteria/ml. After the co-cultivation period, the explants are washed in
liquid medium
with appropriate antibiotics and hormones and blotted dry on filter paper.
The explants are cultured for one week on callus induction medium with 5 mg/I
silver
= nitrate and 250 mg/I of both Triacillin and Carbenicillin and 10 mg/I
phosphinothricin for
selection of transformation events.
= Every two weeks explants are transferred to fresh medium. Every week
explants are
checked for callus formation. Celli are excised from the explants and
transferred to shoot
induction medium. Shoots are transferred to plastic containers with rooting
medium.
Shoots are kept on this medium until they are normalized or rooted. If they
are 3-10 cm
in size and have a nicely developed root system, they are transferred to the
greenhouse.
Oilseed rape plants are also transformed with the cry1C and cry1B genes using
Agrobacterium tumefaciens. Hypocotyl explants of Brassica napus are used in
routine
transformation and regeneration methods, e.g., the method described by De
Block et al.
(1989).
CA 02924415 2016-03-21
= WO 2007/107302
PCT/EP2007/002342
36
3. Analysis of transformants.
Once the transformed plants are regenerated, PCR and Southern analysis are
used to
confirm integration of the transgenes. Immunological analyses such as Cry1C-
and
Cry1B-specific ELISA assays or Western blots are used to select those
transformed
plants showing optimal expression levels of the Cry1C and Cry1B proteins.
RT-PCR experiments on RNA collected from cauliflower plants shown to be
transformed
with the cry1C genes of SEQ ID No. 1 or 3, comprising a plant intron at a
different
position, confirmed that splicing occurs correctly and that a functional Cry1C
protein is
produced in these plants. This is also confirmed by Northern blot analysis of
these
plants.
Also, insect assays using Plutella xylostella larvae under standard insect bio-
assay
conditions using proper controls with selected transformed cabbage,
cauliflower and
oilseed rape plants containing the Cry1C and Cry1B genes confirm the high
insecticidal
activity and the high dose of these protein expressed, in those transformed
plants
selected for optimal expression. Also, Plutella xylostella insects that have
been selected
for resistance to the Cry1C or Cry1B protein, are still effectively killed by
the plants of the
invention.
Progeny plants and seeds are also obtained from the transformed, selected
plants of the
invention, and the genes of the invention are shown to segregate in such
progeny in the
expected Mendelian fashion. Selection of the transgenic plants in the
greenhouse and
in the field at multiple locations will result in the identification of plant
lines which have
optimal stability and expression of the cry1 chimeric genes combined with
optimal
agronomical performance. Crossing of the selected best performing transgenic
plants
with several different commercial lines, and repeated backcrossing therewith,
result in
the presence of the (linked) cry1B and cry1C genes of the invention in
different cabbage,
cauliflower or oilseed rape genetic backgrounds, optimally adapted to
different areas or
climatic conditions.
= CA 02924415 2016-03-21
WO 2007/107302
PCT/EP2007/002342
37
Cited references:
= An et al. (1996) Plant J. 10, 107
Bernhard and Utz (1993) "Production of Bacillus thuringiensis insecticides for
experimental and commercial uses", In Bacillus thuringinsi.õAn
Environmental Biopesticide: Theory and Practice, pp.255-267, eds.
Entwistle, P.F., Cory, J.S., Bailey, M.J. and Higgs, S., John Wiley and
Sons, New York (1993).
Bih et al. (1999) J. Biol. Chem. 274, 22884-22894.
Boevink et al. (1995) Virology, 207, 354-361.
Bolivar et al. (1977) Gene, 2: 95-113.
Bradford et al. (1976) Anal. Biochem. 72, 248-254.
Brown (1986) Nucleic Acids Res. 1986 14, 9549-9559.
Brown and Simpson (1998) Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 77-95.
Brown et at (1996) Plant Mol Biol. 32, 531-535.
Christensen et al. (1992) Plant Mol. Biol. 18, 675-689.
Christou et al. (1990). Trends Biotechnology 8, 145.
Cordera et al. (1994) The Plant Journal 6, 141.
=
Cornejo et al. (1993) Plant Mol. Biol. 23, 567-581.
Cornelissen & Vandewiele (1989) Nucleic Acids Research, 17: 19-25.
Cornelissen et al. (1986) EMBO J. 5, 37-40.
Crickmore et al. (1998) Microbiol. Mol. Biol Rev. 62(3), 807-13.
natta et al. (1990) r,iofTechnology 8, 736-740.
De Block et al. (1989) Plant Physiol., 91: 694.
De Pater et al., 1992, Plant J. 2, 834-844.
Depicker et al. (1982) Journal of Molecular and Applied Genetics, 1: 561-573.
DuImage (1981), "Production of Bacteria for Biological Control of insects" in
Biological Control in Crop Production, Ed. Papanzas, D.C., Osmun
Publishers, Totowa, N.J., USA, pp. 129-141 (1981).
Eckes et al. (1986) Molecular and General Genetics, 205: 14-22.
Estruch et al., (1996), Proc Natl Acad Sci USA 93, 5389-94.
Ffrench-Constant and Bowen (2000) Cell Mol Life Sci 57, 828-33.
Franck et al. (1980) Cell 21, 285-294.
Fromm et al. (1990) BiofTechnology 8, 833-839.
CA 02924415 2016-03-21
- WO 2007/107302 PCT/EP2007/002342
38
Gardner et al. (1981) Nucleic Acids Research 9, 2871-2887.
Gielen et al. (1984) EMBO J 3, 835-845.
Gordon-Kamm et al. (1990) The Plant Cell 2, 603-618.
Hesse et al. (1989), EMBO J. 8 2453-2461.
Hinchee et al. (1988) BiofTechnology 6, 915.
Ho et al. (1989). Gene 77, 51-59.
Hate et al. (1988) Appl. and Environm. Microbiol. 54, 2010-2017.
Hate and Whiteley (1989) Microbiological Review 53, 242-255.
Hull and Howell (1987) Virology 86, 482-493.
ltoh et al. (1984) Plasmid, 11: 206-220.
Jansens et al. (1997) Crop Science 37, 1616-1624.
Keil et al. (1986), Nucl. Acids Res. 14, 5641-5650.
KlOsgen et al. (1989), Mol. Gen. Genet. 217, 155-161.
Klosgen and Weil (1991), Mol. Gen. Genet. 225, 297-304.
Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1840-1845.
Last et al. (1990) Theor. Appl. Genet. 81, 581-588.
Lebrun et at. (1996) US Patent 5,510,471.
Lin et al. (2003) Bot. Bull. Acad. Sin. 44:199-210.
Mahillon et al, FEMS Microbiol. Letters 60, 205-210 (1989).
Marshall et al. (1996) Plant Physiology, 111: 1251-1261.
Michiels et al. (1992) published PCT application W092/13956.
Morris et al. (1999), Biochem. Biophys. Res. Commun. 255, 328-333.
Needleman and Wunsch (1970) J. Mol. Biol., 48: 443-53.
Neuhaus & Rogers (1998), Plant Mol. Biol. 38, 127-144.
Odell et al. (1985) Nature, 313: 810-812.
Oelmuller et al., Mol. Gen. Genet. 237, 261-272 (1993).
Oka et al. (1981) Journal of Molecular Biology, 147: 217-226.
Park et al. (1997), J. Biol. Chem. 272, 6876-6881.
Rice et al. (2000) Trends in Genetics, 16: 276-277.
SchOnmann et al. (2003) Functional Plant Biology 30, 453 ¨ 460.
Shcherban et at. (1995) Proc. Natl. Acad. Sci USA 92, 9245-9249.
Shimamoto et al. (1989) Nature 338, 274-276.
Stanssens et al. (1989), Nucleic Acids Research 12, 4441-4454.
Sutliff et al. (1991) Plant Molec. Biol. 16, 579-591.
CA 02924415 2016-03-21
75749-47D1
39
Tavladoraki et at. (1998), FEBS Lett. 426, 62-66.
Terashima et at. (1999), Appl. Microbial. Biotechnol. 52, 516-523.
Thompson et at. (1987) The EMBO Journal, 6: 2519-2523.
Van Den Broeck at at., 1985, Nature 313, 358.
Van Rie et at. (1990) Science 247, 72.
Velten et at. (1984) J., EMBO J 3,2723-2730.
Velten and Schell (1985) Nucleic Acids Research 13, 6981-6998.
Verdaguer at at. (1998) Plant Mal. Biol. 37, 1055-1067.
VVaterfield et at. (2001) Trends Microbiol 9, 185-91.
White at at. (1989)Trends in Genet. 5, 185-189.
Wong et at. (1992) Plant Malec. Bio1.20, 81-93.
Zambryski (1988) Annual Review of Genetics, 22: .1-30.
Zhang et at. (1991) The Plant Cell 3,1155-1165.
Zhao at at. (2003) Nature Biotechnology, 21: 1493¨ 1497.
The citation of any of these references is not to be construed as an
acknowledgement of the
accuracy of every statement contained in such reference, nor as an
acknowledgement
that such reference is relevant prior art or part of the common general
knowledge in any
territory.
CA 02924415 2016-03-21
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a
sequence listing in electronic form in ASCII text format (file: 75749-47D1
Seq 16-MAR-16 v1.txt).
A copy of the sequence listing in electronic form is available from the
Canadian
Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the
following table.
SEQUENCE TABLE
<110> Bayer CropScience NV
<120> Novel genes encoding insecticidal proteins
<130> 75749-47D1
<140> Division of CA 2,646,471
<141> 2007-03-16
<160> 23
<170> PatentIn version 3.3
<210> 1
<211> 2076
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(672)
<220>
<221> Intron
<222> (673)..(861)
CA 02924415 2016-03-21
41
<220>
<221> CDS
<222> (862)..(2073)
<400> 1
atg gct gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48
Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
tog aac cct gag gag gtt ctt ctt gat gga gag aga atc tot act gga 96
Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
20 25 30
aac tot tot atc gat att tot ctt tot ctt gtt cag ttc ctt gtt tot 144
Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser
35 40 45
aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192
Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val
50 55 60
tgg gga atc gtt gga cct tot cag tgg gat got ttc ctt gtt cag atc 240
Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile
65 70 75 80
gag cag ctt atc aac gag aga atc got gag ttc got aga aac got got 288
Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95
atc got aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336
Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu
100 105 110
got ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384
Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr
115 120 125
aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432
Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp
130 135 140
att cct tot ttc aga atc tot gga ttc gag gtt cct ctt ctt tot gtt 480
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val
145 150 155 160
tac got cag got got aac ctt cat ctt got atc ctt aga gat tot gtt 528
Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175
atc ttc gga gag aga tgg gga ctt act act atc aac gtt aac gag aac 576
Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn
180 185 190
tac aac aga ctt atc aga cat atc gat gag tac got gat cat tgt got 624
Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala
195 200 205
CA 02924415 2016-03-21
,
, 42
aac act tac aac aga gga ctt aac aac ctt cct aag tot act tac cag
672
Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin
210 215 220
gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata
732
taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg
792
tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt
852
gatgtgcag gac tgg atc act tac aac aga ctt aga aga gat ctt act ctt
903
Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu
225 230 235
act gtt ctt gat att got got ttc ttc cot aac tac gat aac aga aga
951
Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg
240 245 250
tac cot atc cag cot gtt gga cag ctt act aga gag gtt tac act gat
999
Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp
255 260 265 270
cot ctt atc aac ttc aac cot cag ctt cag tot gtt got cag ctt cot
1047
Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro
275 280 285
act ttc aac gtt atg gag tot tot got atc aga aac cot cat ctt ttc
1095
Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe
290 295 300
gat att Ott aac aac ctt act atc ttc act gac tgg ttc tot gtt gga
1143
Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly
305 310 315
aga aac ttc tac tgg gga gga cat aga gtt atc tot tot ctt atc gga
1191
Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly
320 325 330
gga gga aac atc act tot cot atc tac gga aga gag got aac cag gag
1239
Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu
335 340 345 350
cot cot aga tot ttc act ttc aac gga cot gtt ttc aga act ctt tot
1287
Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser
355 360 365
aac cot act ctt aga ctt ctt cag cag cot tgg cot got cct cct ttc
1335
Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe
370 375 380
aac ctt aga gga gtt gag gga gtt gag ttc tot act cot act aac tot
1383
Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser
385 390 395
ttc act tac aga gga aga gga act gtt gat tot ctt act gag ctt cot
1431
Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro
400 405 410
CA 02924415 2016-03-21
43
cot gag gat aac tct gtt cot cot aga gag gga tac tct cat aga ctt 1479
Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu
415 420 425 430
tgt cat got act ttc gtt cag aga tct gga act cct ttc ctt act act 1527
Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr
435 440 445
gga gtt gtt ttc tct tgg act cat aga tct got act ctt act aac act 1575
Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr
450 455 460
atc gat cot gag agg atc aac cag atc cot ctt gtt aag gga ttc aga 1623
Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg
465 470 475
gtt tgg gga gga act tct gtt atc act gga cct gga ttc act gga gga 1671
Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly
480 485 490
gat att ctt aga aga aac act ttc gga gat ttc gtt tct ctt cag gtt 1719
Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val
495 500 505 510
aac atc aac tct cot atc act cag aga tac aga ctt aga ttc aga tac 1767
Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr
515 520 525
got tct tct aga gat got aga gtt atc gtt ctt act gga got got tct 1815
Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser
530 535 540
act gga gtt gga gga cag gtt tct gtt aac atg cot ctt cag aag act 1863
Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr
545 550 555
atg gag atc gga gag aac ctt act tct aga act ttc aga tac act gat 1911
Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp
560 565 570
ttc tct aac cot ttc tct ttc aga got aac cot gat att atc gga atc 1959
Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile
575 580 585 590
tct gag cag cct ctt ttc gga gct gga tct atc tct tct gga gag ctt 2007
Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu
595 600 605
tac atc gat aaa atc gag atc atc ctt got gat got act ttc gag got 2055
Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala
610 615 620
gag tct gat tta gag aga tga 2076
Glu Ser Asp Leu Glu Arg
625
CA 02924415 2016-03-21
44
<210> 2
<211> 628
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 2
Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
20 25 30
Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser
35 40 45
Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val
50 55 60
Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile
65 70 75 80
Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95
Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu
100 105 110
Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr
115 120 125
Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp
130 135 140
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175
Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn
180 185 190
Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala
195 200 205
Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin
210 215 220
Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro
245 250 255
Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu
260 265 270
Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe
275 280 285
Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile
290 295 300
Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn
305 310 315 320
Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335
Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro
340 345 350
Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro
355 360 365
CA 02924415 2016-03-21
Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu
370 375 380
Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr
385 390 395 400
Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu
405 410 415
Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His
420 425 430
Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val
435 440 445
Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp
450 455 460
Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp
465 470 475 480
Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
485 490 495
Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile
500 505 510
Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser
515 520 525
Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly
530 535 540
Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu
545 550 555 560
Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser
565 570 575
Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
580 585 590
Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile
595 600 605
Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser
610 615 620
Asp Leu Glu Arg
625
<210> 3
<211> 2076
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> exon
<222> (1)..(489)
<220>
<221> Intron
<222> (490)..(678)
<220>
<221> exon
<222> (679)..(2073)
CA 02924415 2016-03-21
46
<400> 3
atg got gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48
Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
tcg aac cot gag gag gtt ctt ctt gat gga gag aga atc tot act gga 96
Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
20 25 30
aac tot tot atc gat att tot ctt tot ctt gtt cag ttc ctt gtt tot 144
Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser
35 40 45
aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192
Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Lou Ile Asp Phe Val
50 55 60
tgg gga atc gtt gga cct tot cag tgg gat got ttc ctt gtt cag atc 240
Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile
65 70 75 80
gag cag ctt atc aac gag aga atc got gag ttc got aga aac got got 288
Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95
atc got aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336
Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu
100 105 110
got ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384
Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr
115 120 125
aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432
Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp
130 135 140
att cct tot ttc aga atc tot gga ttc gaa gtt cct ctt ctt tot gtt 480
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Lou Leu Ser Val
145 150 155 160
tac got cag gtaagtttct gcttctacct ttgatatata tataataatt 529
Tyr Ala Gin
atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt 589
atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta 649
atatatgacc aaaacatggt gatgtgcag got got aac ctt cat ctt got atc 702
Ala Ala Asn Lou His Leu Ala Ile
165 170
ctt aga gat tot gtt atc ttc gga gag aga tgg gga ctt act act atc 750
Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile
175 180 185
CA 02924415 2016-03-21
. .
,
47
aac gtt aac gag aac tac aac aga ctt atc aga cat atc gat gag tac
798
Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr
190 195 200
gct gat cat tgt gct aac act tac aac aga gga ctt aac aac ctt cot
846
Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro
205 210 215
aag tct act tac cag gac tgg atc act tac aac aga ctt aga aga gat
894
Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp
220 225 230 235
ctt act ctt act gtt ctt gat att gct gct ttc ttc cot aac tac gat
942
Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp
240 245 250
aac aga aga tac cct atc cag cot gtt gga cag ctt act aga gag gtt
990
Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val
255 260 265
tac act gat cot ctt atc aac ttc aac cot cag ctt cag tct gtt gct
1038
Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala
270 275 280
cag ctt cot act ttc aac gtt atg gag tct tct gct atc aga aac cot
1086
Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro
285 290 295
cat ctt ttc gat att ctt aac aac ctt act atc ttc act gac tgg ttc
1134
His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe
300 305 310 315
tct gtt gga aga aac ttc tac tgg gga gga cat aga gtt atc tct tct
1182
Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser
320 325 330
ctt atc gga gga gga aac atc act tct cot atc tac gga aga gag gct
1230
Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala
335 340 345
aac cag gag cot cot aga tct ttc act ttc aac gga cot gtt ttc aga
1278
Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg
350 355 360
act ctt tct aac cot act ctt aga ctt ctt cag cag cot tgg cot gct
1326
Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala
365 370 375
cot cot ttc aac ctt aga gga gtt gag gga gtt gag ttc tct act cot
1374
Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro
380 385 390 395
act aac tct ttc act tac aga gga aga gga act gtt gat tct ctt act
1422
Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr
400 405 410
CA 02924415 2016-03-21
48
gag ctt cot cot gag gat aac tot gtt cot cot aga gag gga tac tot 1470
Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser
415 420 425
cat aga ctt tgt cat got act ttc gtt cag aga tot gga act cot ttc 1518
His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe
430 435 440
ctt act act gga gtt gtt ttc tot tgg act cat aga tot got act ctt 1566
Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu
445 450 455
act aac act atc gat cot gag agg atc aac cag atc cot ctt gtt aag 1614
Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys
460 465 470 475
gga ttc aga gtt tgg gga gga act tot gtt atc act gga cot gga ttc 1662
Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe
480 485 490
act gga gga gat att ctt aga aga aac act ttc gga gat ttc gtt tot 1710
Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser
495 500 505
ctt cag gtt aac atc aac tot cot atc act cag aga tac aga ctt aga 1758
Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg
510 515 520
ttc aga tac got tot tot aga gat got aga gtt atc gtt ctt act gga 1806
Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly
525 530 535
got got tot act gga gtt gga gga cag gtt tot gtt aac atg cot ctt 1854
Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu
540 545 550 555
cag aag act atg gag atc gga gag aac ctt act tot aga act ttc aga 1902
Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg
560 565 570
tac act gat ttc tot aac cot ttc tot ttc aga got aac cot gat att 1950
Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile
575 580 585
atc gga atc tot gag cag cot ctt ttc gga got gga tot atc tot tot 1998
Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser
590 595 600
gga gag ctt tac etc gat aaa atc gag atc atc ctt got gat got act 2046
Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr
605 610 615
ttc gag got gag tot gat tta gag aga tga 2076
Phe Glu Ala Glu Ser Asp Leu Glu Arg
620 625
CA 02924415 2016-03-21
,
. .
,
49
<210> 4
<211> 2442
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(1038)
<220>
<221> Intron
<222> (1039)..(1227)
<220>
<221> CDS
<222> (1228)..(2439)
<400> 4
atg gct tot atc tot tct tot gtt gct act gtt tot aga act gct cct
48
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
gct cag gct aac atg gtt gct cot ttc act gga ctt aag tot aac gct
96
Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
gct ttc cot act act aag aag gct aac gat ttc tot act ctt cot tot
144
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
aac gga gga aga gtt cag tgt atg cag gtt tgg cot gct tac gga aac
192
Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn
50 55 60
aag aag ttc gag act ctt tot tac ctt cot cot ctt tot atg gct cot
240
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
act gtt atg atg gct tot tot gct act gct gtt gct cot ttc cag gga
288
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly
85 90 95
ctt aag tot act gct tot ctt cot gtt gct aga aga tot tot aga tot
336
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
ctt gga aac gtt tot aac gga gga aga atc aga tgt gag gag aac aac
384
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn
115 120 125
cag aac cag tgt atc cot tac aac tgt ctt tog aac cot gag gag gtt
432
Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val
130 135 140
CA 02924415 2016-03-21
ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile
145 150 155 160
tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528
Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly
165 170 175
gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cot 576
Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro
180 185 190
tct cag tgg gat gct ttc ctt gtt cag atc gag cag ctt atc aac gag 624
Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu
195 200 205
aga atc gct gag ttc gct aga aac gct gct atc gct aac ctt gag gga 672
Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly
210 215 220
ctt gga aac aac ttc aac atc tac gtt gag gct ttc aag gag tgg gag 720
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu
225 230 235 240
gag gat cot aac aac cot gag act aga act aga gtt atc gat aga ttc 768
Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe
245 250 255
aga atc ctt gat gga ctt ctt gag aga gat att cct tct ttc aga atc 816
Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile
260 265 270
tct gga ttc gag gtt cot ctt ctt tct gtt tac gct cag gct gct aac 864
Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn
275 280 285
ctt cat ctt gct atc ctt aga gat tct gtt atc ttc gga gag aga tgg 912
Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp
290 295 300
gga ctt act act atc aac gtt aac gag aac tac aac aga ctt atc aga 960
Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg
305 310 315 320
cat atc gat gag tac gct gat cat tgt gct aac act tac aac aga gga 1008
His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly
325 330 335
ctt aac aac ctt cot aag tct act tac cag gtaagtttct gcttctacct 1058
Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin
340 345
ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt 1118
tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat 1178
CA 02924415 2016-03-21
51
tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcag gac tgg atc 1236
Asp Trp Ile
act tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284
Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile
350 355 360 365
gct gct ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332
Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro
370 375 380
gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380
Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe
385 390 395
aac cct cag ctt cag tot gtt gct cag ctt cct act ttc aac gtt atg 1428
Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met
400 405 410
gag tot tot gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476
Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn
415 420 425
ctt act atc ttc act gac tgg ttc tot gtt gga aga aac ttc tac tgg 1524
Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp
430 435 440 445
gga gga cat aga gtt atc tot tot ctt atc gga gga gga aac atc act 1572
Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr
450 455 460
tot cct atc tac gga aga gag gct aac cag gag cct cct aga tot ttc 1620
Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe
465 470 475
act ttc aac gga cct gtt ttc aga act ctt tot aac cct act ctt aga 1668
Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg
480 485 490
ctt ctt cag cag cct tgg cct gct cct cct ttc aac ctt aga gga gtt 1716
Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val
495 500 505
gag gga gtt gag ttc tot act cct act aac tot ttc act tac aga gga 1764
Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly
510 515 520 525
aga gga act gtt gat tot ctt act gag ctt cct cct gag gat aac tot 1812
Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser
530 535 540
gtt cct cct aga gag gga tac tot cat aga ctt tgt cat gct act ttc 1860
Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe
545 550 555
CA 02924415 2016-03-21
52
gtt cag aga tct gga act cot ttc ctt act act gga gtt gtt ttc tct 1908
Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser
560 565 570
tgg act cat aga tct got act ctt act aac act atc gat cot gag agg 1956
Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg
575 580 585
atc aac cag atc cot ctt gtt aag gga ttc aga gtt tgg gga gga act 2004
Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr
590 595 600 605
tct gtt atc act gga cot gga ttc act gga gga gat att ctt aga aga 2052
Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg
610 615 620
aac act ttc gga gat ttc gtt tct ctt cag gtt aac atc aac tct cct 2100
Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro
625 630 635
atc act cag aga tac aga ctt aga ttc aga tac got tct tct aga gat 2148
Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp
640 645 650
got aga gtt atc gtt ctt act gga got got tct act gga gtt gga gga 2196
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly
655 660 665
cag gtt tct gtt aac atg cot ctt cag aag act atg gag atc gga gag 2244
Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu
670 675 680 685
aac ctt act tct aga act ttc aga tac act gat ttc tct aac cot ttc 2292
Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe
690 695 700
tct ttc aga gct aac cot gat att atc gga atc tct gag cag cot ctt 2340
Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu
705 710 715
ttc gga got gga tct atc tct tct gga gag ctt tac atc gat aaa atc 2388
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile
720 725 730
gag atc atc ctt got gat got act ttc gag got gag tct gat tta gag 2436
Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu
735 740 745
aga tga 2442
Arg
750
<210> 5
<211> 750
CA 02924415 2016-03-21
=
53
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 5
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn
115 120 125
Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val
130 135 140
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile
145 150 155 160
Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly
165 170 175
Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro
180 185 190
Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu
195 200 205
Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly
210 215 220
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu
225 230 235 240
Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe
245 250 255
Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile
260 265 270
Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn
275 280 285
Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp
290 295 300
Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg
305 310 315 320
His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly
325 330 335
Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn
340 345 350
Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe
355 360 365
Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin
370 375 380
CA 02924415 2016-03-21
54
Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin
385 390 395 400
Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser
405 410 415
Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile
420 425 430
Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His
435 440 445
Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile
450 455 460
Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn
465 470 475 480
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin
485 490 495
Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val
500 505 510
Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr
515 520 525
Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro
530 535 540
Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg
545 550 555 560
Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His
565 570 575
Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Tie Asn Gin
580 585 590
Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile
595 600 605
Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe
610 615 620
Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin
625 630 635 640
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val
645 650 655
Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser
660 665 670
Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr
675 680 685
Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg
690 695 700
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala
705 710 715 720
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile
725 730 735
Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg
740 745 750
<210> 6
<211> 2442
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
CA 02924415 2016-03-21
<220>
<221> CDS
<222> (1)..(855)
<220>
<221> Intron
<222> (856)..(1044)
<220>
<221> CDS
<222> (1045)..(2439)
<400> 6
atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192
Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
70 75 80
act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
ctt gga aac gtt tct aac gga gga aga atc aga tgt gag gag aac aac 384
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn
115 120 125
cag aac cag tgt atc cct tac aac tgt ctt tog aac cct gag gag gtt 432
Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val
130 135 140
ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile
145 150 155 160
tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528
Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly
165 170 175
CA 02924415 2016-03-21
56
gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cot 576
Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro
180 185 190
tct cag tgg gat got ttc ctt gtt cag atc gag cag ctt atc aac gag 624
Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu
195 200 205
aga atc got gag ttc got aga aac got got atc gct aac ctt gag gga 672
Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly
210 215 220
ctt gga aac aac ttc aac atc tac gtt gag got ttc aag gag tgg gag 720
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu
225 230 235 240
gag gat cct aac aac cct gag act aga act aga gtt atc gat aga ttc 768
Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe
245 250 255
aga atc ctt gat gga ctt ctt gag aga gat att cct tot ttc aga atc 816
Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile
260 265 270
tot gga ttc gaa gtt cct ctt ctt tot gtt tac got cag gtaagtttct 865
Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin
275 280 285
gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 925
aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 985
gtgtgtatat tttaatttat aacttttcta atatatgacc aaaacatggt gatgtgcag 1044
got got aac ctt cat ctt got atc ctt aga gat tot gtt atc ttc gga 1092
Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly
290 295 300
gag aga tgg gga ctt act act atc aac gtt aac gag aac tac aac aga 1140
Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg
305 310 315
ctt atc aga cat atc gat gag tac got gat cat tgt got aac act tac 1188
Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr
320 325 330
aac aga gga ctt aac aac ctt cct aag tot act tac cag gac tgg atc 1236
Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile
335 340 345
act tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284
Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile
350 355 360 365
got got ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332
Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro
370 375 380
CA 02924415 2016-03-21
57
gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380
Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe
385 390 395
aac cct cag ctt cag tct gtt gct cag ctt cct act ttc aac gtt atg 1428
Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met
400 405 410
gag tct tct gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476
Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn
415 420 425
ctt act atc ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524
Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp
430 435 440 445
gga gga cat aga gtt atc tct tct ctt atc gga gga gga aac atc act 1572
Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr
450 455 460
tct cct atc tac gga aga gag gct aac cag gag cct cct aga tct ttc 1620
Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe
465 470 475
act ttc aac gga cct gtt ttc aga act ctt tct aac cct act ctt aga 1668
Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg
480 485 490
ctt ctt cag cag cct tgg cct gct cct cct ttc aac ctt aga gga gtt 1716
Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val
495 500 505
gag gga gtt gag ttc tct act cct act aac tct ttc act tac aga gga 1764
Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly
510 515 520 525
aga gga act gtt gat tct ctt act gag ctt cct cct gag gat aac tct 1812
Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser
530 535 540
gtt cct cct aga gag gga tac tct cat aga ctt tgt cat gct act ttc 1860
Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe
545 550 555
gtt cag aga tct gga act cct ttc ctt act act gga gtt gtt ttc tct 1908
Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser
560 565 570
tgg act cat aga tct gct act ctt act aac act atc gat cct gag agg 1956
Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg
575 580 585
atc aac cag atc cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004
Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr
590 595 600 605
CA 02924415 2016-03-21
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tot gtt atc act gga cot gga ttc act gga gga gat att ctt aga aga
2052
Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg
610 615 620
aac act ttc gga gat ttc gtt tot ctt cag gtt aac atc aac tot cot
2100
Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro
625 630 635
atc act cag aga tac aga ctt aga ttc aga tac got tot tot aga gat
2148
Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp
640 645 650
got aga gtt atc gtt ctt act gga got got tot act gga gtt gga gga
2196
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly
655 660 665
cag gtt tot gtt aac atg cct ctt cag aag act atg gag atc gga gag
2244
Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu
670 675 680 685
aac ctt act tot aga act ttc aga tac act gat ttc tot aac cot ttc
2292
Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe
690 695 700
tot ttc aga got aac cot gat att atc gga atc tot gag cag cot ctt
2340
Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu
705 710 715
ttc gga got gga tot atc tot tot gga gag ctt tac atc gat aaa atc
2388
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile
720 725 730
gag atc atc ctt got gat got act ttc gag got gag tot gat tta gag
2436
Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu
735 740 745
aga tga
2442
Arg
750
<210> 7
<211> 750
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 7
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
CA 02924415 2016-03-21
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Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn
115 120 125
Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val
130 135 140
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile
145 150 155 160
Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly
165 170 175
Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro
180 185 190
Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu
195 200 205
Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly
210 215 220
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu
225 230 235 240
Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe
245 250 255
Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile
260 265 270
Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn
275 280 285
Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp
290 295 300
Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg
305 310 315 320
His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly
325 330 335
Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn
340 345 350
Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe
355 360 365
Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin
370 375 380
Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin
385 390 395 400
Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser
405 410 415
Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile
420 425 430
Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His
435 440 445
Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile
450 455 460
Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn
465 470 475 480
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin
485 490 495
CA 02924415 2016-03-21
Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val
500 505 510
Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr
515 520 525
Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro
530 535 540
Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg
545 550 555 560
Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His
565 570 575
Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gin
580 585 590
Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile
595 600 605
Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe
610 615 620
Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin
625 630 635 640
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val
645 650 655
Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser
660 665 670
Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr
675 680 685
Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg
690 695 700
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala
705 710 715 720
Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile
725 730 735
Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg
740 745 750
<210> 8
<211> 2313
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(2310)
<400> 8
atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96
Ala Gin Ala Asn Met Val Ala Pro She Thr Gly Leu Lys Ser Asn Ala
20 25 30
CA 02924415 2016-03-21
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gct ttc cot act act aag aag gct aac gat ttc tct act ctt cct tct
144
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
aac gga gga aga gtt cag tgt atg cag gtt tgg cot gct tac gga aac
192
Asn Gly Gly Arg Val Gln Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
aag aag ttc gag act ctt tct tac ctt cot cct ctt tct atg gct cct
240
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
act gtt atg atg gct tct tct gct act gct gtt gct cot ttc cag gga
288
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
ctt aag tct act gct tct ctt cot gtt gct aga aga tct tct aga tct
336
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
ctt gga aac gtt tct aac gga gga aga atc aga tgt act tog aac aga
384
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg
115 120 125
aag aac gag aac gag atc atc aac gct gtt tct aac cat tct gct cag
432
Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gin
130 135 140
atg gat ctt ctt cot gat gct aga atc gag gat tct ctt tgt atc gct
480
Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala
145 150 155 160
gag gga aac aac atc gat cot ttc gtt tct gct tct act gtt cag act
528
Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr
165 170 175
ggt atc aac atc gct gga aga att ctt gga gtt ctt gga gtt cot ttc
576
Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe
180 185 190
gct gga cag ctt gct tct ttc tac tct ttc ctt gtt gga gag ctt tgg
624
Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp
195 200 205
cot aga gga aga gat cag tgg gag atc ttc ctt gag cat gtt gag cag
672
Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe Leu Glu His Val Glu Gin
210 215 220
ctt atc aac cag cag atc act gag aac gct aga aac act gct ctt gct
720
Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala
225 230 235 240
aga ctt cag gga ctt gga gat tct ttc aga gct tac cag cag tct ctt
768
Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu
245 250 255
CA 02924415 2016-03-21
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gag gac tgg ctt gag aac aga gat gat gct aga act aga tot gtt ctt 816
Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu
260 265 270
cat act cag tac atc gct ctt gag ctt gat ttc ctt aac gct atg cct 864
His Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro
275 280 285
ctt ttc gct atc aga aac cag gag gtt cot ctt ctt atg gtt tac gct 912
Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu Leu Met Val Tyr Ala
290 295 300
cag gct gct aac ctt cat ctt ctt ctt ctt aga gat gct tot ctt ttc 960
Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
305 310 315 320
gga tot gag ttc gga ctt act tot cag gag atc cag aga tat tac gag 1008
Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu
325 330 335
aga cag gtt gag aga act aga gat tac tot gat tac tgt gtt gag tgg 1056
Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp
340 345 350
tac aac act gga ctt aac tot ctt aga gga act aac gct gct tot tgg 1104
Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp
355 360 365
gtt aga tac aac cag ttc aga aga gat ctt act ctt gga gtt ctt gat 1152
Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp
370 375 380
ctt gtt gct ctt ttc cot tot tac gac act aga act tac cct atc aac 1200
Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn
385 390 395 400
act tct gct cag ctt act aga gag gtt tac act gat gct atc gga gct 1248
Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala
405 410 415
act gga gtt aac atg gct tot atg aac tgg tac aac aac aac gct cot 1296
Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro
420 425 430
tot ttc tot gct atc gag gct gct gct atc aga tot cot cat ctt ctt 1344
Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu
435 440 445
gat ttc ctt gag cag ctt act atc ttc tot gct tot tot aga tgg tot 1392
Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser
450 455 460
aac act aga cac atg act tac tgg aga gga cat acc atc cag tot aga 1440
Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gln Ser Arg
465 470 475 480
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cct atc gga gga gga ctt aac act tot act cat gga gct act aac act 1488
Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr
485 490 495
tot atc aac cct gtt act ctt aga ttc got tot aga gat gtt tac aga 1536
Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg
500 505 510
act gag tot tac got gga gtt ctt ctt tgg gga atc tac ctt gag cot 1584
Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro
515 520 525
atc cac gga gtt cot act gtt aga ttc aac ttc act aac cot cag aac 1632
Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn
530 535 540
atc tot gat aga gga act got aac tac tot cag cot tac gag tot cct 1680
Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro
545 550 555 560
gga ctt cag ctt aag gat tot gag act gag ctt cot cot gag act act 1728
Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr
565 570 575
gag aga cot aac tac gag tot tac tot cat aga ctt tot cat atc gga 1776
Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly
580 585 590
atc atc ctt cag tct aga gtt aac gtt cot gtt tac tot tgg act cat 1824
Ile Ile Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His
595 600 605
aga tot got gat aga act aac act atc gga cot aac aga atc act cag 1872
Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gin
610 615 620
atc cot atg gtt aag got tot gag ctt cct cag gga act act gtt gtt 1920
Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val
625 630 635 640
aga gga cot gga ttc act gga gga gat atc ctt aga aga act aac act 1968
Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr
645 650 655
gga gga ttc gga cot atc aga gtt act gtt aac gga cot ctt act cag 2016
Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gin
660 665 670
aga tac aga atc gga ttc aga tac got tot act gtt gat ttc gat ttc 2064
Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe
675 680 685
ttc gtt tot aga gga gga act act gtt aac aac ttc aga ttc ctt aga 2112
Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg
690 695 700
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act atg aac tct gga gat gag ctt aag tac gga aac ttc gtt aga aga 2160
Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg
705 710 715 720
gct ttc act act cct ttc act ttc act cag atc cag gat atc atc aga 2208
Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin Ile Gin Asp Ile Ile Arg
725 730 735
act tct atc cag gga ctt tct gga aac gga gag gtt tac atc gat aaa 2256
Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys
740 745 750
atc gag atc atc cot gtt act got act ttc gag got gag tac gat tta 2304
Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu
755 760 765
gag aga tga 2313
Glu Arg
770
<210> 9
<211> 770
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 9
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
Asn Gly Gly Arg Val Gin Cys Met Gln Val Trp Pro Ala Tyr Gly Asn
50 55 60
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
Lou Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg
115 120 125
Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gin
130 135 140
Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala
145 150 155 160
Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr
165 170 175
Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe
180 185 190
Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Lou Trp
195 200 205
CA 02924415 2016-03-21
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Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe Leu Glu His Val Glu Gin
210 215 220
Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala
225 230 235 240
Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu
245 250 255
Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu
260 265 270
His Thr Gin Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro
275 280 285
Leu Phe Ala Ile Arg Asn Gin Glu Val Pro Leu Leu Met Val Tyr Ala
290 295 300
Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
305 310 315 320
Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu Ile Gin Arg Tyr Tyr Glu
325 330 335
Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp
340 345 350
Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp
355 360 365
Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp
370 375 380
Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn
385 390 395 400
Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala
405 410 415
Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro
420 425 430
Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu
435 440 445
Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser
450 455 460
Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gin Ser Arg
465 470 475 480
Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr
485 490 495
Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg
500 505 510
Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro
515 520 525
Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn
530 535 540
Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro
545 550 555 560
Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr
565 570 575
Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly
580 585 590
Ile Ile Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His
595 600 605
Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gin
610 615 620
Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val
625 630 635 640
Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr
645 650 655
CA 02924415 2016-03-21
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Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gin
660 665 670
Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe
675 680 685
Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg
690 695 700
Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg
705 710 715 720
Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin Ile Gin Asp Ile Ile Arg
725 730 735
Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys
740 745 750
Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu
755 760 765
Glu Arg
770
<210> 10
<211> 1947
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(1944)
<400> 10
atg gct act tcg aac aga aag aac gag aac gag atc atc aac gct gtt
48
Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val
1 5 10 15
tct aac cat tct gct cag atg gat ctt ctt cct gat gct aga atc gag
96
Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg Ile Glu
20 25 30
gat tct ctt tgt atc gct gag gga aac aac atc gat cct ttc gtt tct
144
Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser
35 40 45
gct tct act gtt cag act ggt atc aac atc gct gga aga att ctt gga
192
Ala Ser Thr Val Gin Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly
50 55 60
gtt ctt gga gtt cct ttc gct gga cag ctt gct tct ttc tac tct ttc
240
Val Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe
65 70 75 80
ctt gtt gga gag ctt tgg cct aga gga aga gat cag tgg gag atc ttc
288
Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe
85 90 95
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ctt gag cat gtt gag cag ctt atc aac cag cag atc act gag aac gct 336
Leu Glu His Val Glu Gin Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala
100 105 110
aga aac act gct ctt gct aga ctt cag gga ctt gga gat tct ttc aga 384
Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg
115 120 125
gct tac cag cag tct ctt gag gac tgg ctt gag aac aga gat gat gct 432
Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala
130 135 140
aga act aga tct gtt ctt cat act cag tac atc gct ctt gag ctt gat 480
Arg Thr Arg Ser Val Leu His Thr Gin Tyr Ile Ala Leu Glu Leu Asp
145 150 155 160
ttc ctt aac gct atg cot ctt ttc gct atc aga aac cag gag gtt cct 528
Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gin Glu Val Pro
165 170 175
ctt ctt atg gtt tac gct cag gct gct aac ctt cat ctt ctt ctt ctt 576
Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu
180 185 190
aga gat gct tct ctt ttc gga tct gag ttc gga ctt act tct cag gag 624
Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu
195 200 205
atc cag aga tat tac gag aga cag gtt gag aga act aga gat tac tct 672
Ile Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser
210 215 220
gat tac tgt gtt gag tgg tac aac act gga ctt aac tct ctt aga gga 720
Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly
225 230 235 240
act aac gct gct tct tgg gtt aga tac aac cag ttc aga aga gat ctt 768
Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu
245 250 255
act ctt gga gtt ctt gat ctt gtt gct ctt ttc cot tct tac gac act 816
Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr
260 265 270
aga act tac cot atc aac act tot gct cag ctt act aga gag gtt tac 864
Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr
275 280 285
act gat gct atc gga gct act gga gtt aac atg gct tct atg aac tgg 912
Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp
290 295 300
tac aac aac aac gct cot tct ttc tct gct atc gag gct gct gct atc 960
Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile
305 310 315 320
CA 02924415 2016-03-21
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aga tot cot cat ctt ctt gat ttc ctt gag cag ctt act atc ttc tot 1008
Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser
325 330 335
got tot tot aga tgg tot aac act aga cac atg act tac tgg aga gga 1056
Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly
340 345 350
cat acc atc cag tot aga cot atc gga gga gga ctt aac act tot act 1104
His Thr Ile Gin Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr
355 360 365
cat gga got act aac act tot atc aac cot gtt act ctt aga ttc got 1152
His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala
370 375 380
tot aga gat gtt tac aga act gag tot tac got gga gtt ctt ctt tgg 1200
Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp
385 390 395 400
gga atc tac ctt gag cot atc cac gga gtt cot act gtt aga ttc aac 1248
Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn
405 410 415
ttc act aac cot cag aac atc tot gat aga gga act got aac tac tot 1296
Phe Thr Asn Pro Gin Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser
420 425 430
cag cot tac gag tot cot gga ctt cag ctt aag gat tot gag act gag 1344
Gin Pro Tyr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu
435 440 445
ctt cot cot gag act act gag aga cot aac tac gag tot tac tot cat 1392
Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His
450 455 460
aga ctt tot cat atc gga atc atc ctt cag tot aga gtt aac gtt cct 1440
Arg Leu Ser His Ile Gly Ile Ile Leu Gin Ser Arg Val Asn Val Pro
465 470 475 480
gtt tac tot tgg act cat aga tot got gat aga act aac act atc gga 1488
Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly
485 490 495
cot aac aga atc act cag atc cot atg gtt aag got tot gag ctt cot 1536
Pro Asn Arg Ile Thr Gin Ile Pro Met Val Lys Ala Ser Glu Leu Pro
500 505 510
cag gga act act gtt gtt aga gga cot gga ttc act gga gga gat atc 1584
Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile
515 520 525
ctt aga aga act aac act gga gga ttc gga cot atc aga gtt act gtt 1632
Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val
530 535 540
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aac gga cct ctt act cag aga tac aga atc gga ttc aga tac gct tot 1680
Asn Gly Pro Leu Thr Gin Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser
545 550 555 560
act gtt gat ttc gat ttc ttc gtt tot aga gga gga act act gtt aac 1728
Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn
565 570 575
aac ttc aga ttc ctt aga act atg aac tot gga gat gag ctt aag tac 1776
Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr
580 585 590
gga aac ttc gtt aga aga got ttc act act cot ttc act ttc act cag 1824
Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin
595 600 605
atc cag gat atc atc aga act tot atc cag gga ctt tot gga aac gga 1872
Ile Gin Asp Ile Ile Arg Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly
610 615 620
gag gtt tac atc gat aaa atc gag atc atc cot gtt act got act ttc 1920
Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe
625 630 635 640
gag got gag tac gat tta gag aga tga 1947
Glu Ala Glu Tyr Asp Leu Glu Arg
645
<210> 11
<211> 648
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 11
Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val
1 5 10 15
Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg Ile Glu
20 25 30
Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser
35 40 45
Ala Ser Thr Val Gin Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly
50 55 60
Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe
65 70 75 80
Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe
85 90 95
Leu Glu His Val Glu Gin Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala
100 105 110
Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg
115 120 125
Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala
130 135 140
CA 02924415 2016-03-21
Arg Thr Arg Ser Val Leu His Thr Gin Tyr Ile Ala Leu Glu Leu Asp
145 150 155 160
Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gin Glu Val Pro
165 170 175
Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu
180 185 190
Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu
195 200 205
Ile Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser
210 215 220
Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly
225 230 235 240
Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu
245 250 255
Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr
260 265 270
Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr
275 280 285
Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp
290 295 300
Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile
305 310 315 320
Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser
325 330 335
Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly
340 345 350
His Thr Ile Gin Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr
355 360 365
His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala
370 375 380
Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp
385 390 395 400
Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn
405 410 415
Phe Thr Asn Pro Gin Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser
420 425 430
Gin Pro Tyr Glu Ser Pro Gly Lou Gin Leu Lys Asp Ser Glu Thr Glu
435 440 445
Lou Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His
450 455 460
Arg Leu Ser His Ile Gly Ile Ile Leu Gin Ser Arg Val Asn Val Pro
465 470 475 480
Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly
485 490 495
Pro Asn Arg Ile Thr Gin Ile Pro Met Val Lys Ala Ser Glu Leu Pro
500 505 510
Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile
515 520 525
Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val
530 535 540
Asn Gly Pro Leu Thr Gin Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser
545 550 555 560
Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn
565 570 575
Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr
580 585 590
CA 02924415 2016-03-21
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Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln
595 600 605
Ile Gin Asp Ile Ile Arg Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly
610 615 620
Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe
625 630 635 640
Glu Ala Glu Tyr Asp Leu Glu Arg
645
<210> 12
<211> 2181
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(2178)
<400> 12
atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct
48
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct
96
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct
144
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac
192
Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct
240
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga
288
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct
336
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
ctt gga aac gtt tct aac gga gga aga atc aga tgt gag atc aac aac
384
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn
115 120 125
CA 02924415 2016-03-21
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72
cag aac cag tgt gtt Oct tac aac tgt ctt tot aac cct aag gag atc
432
Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile
130 135 140
atc ctt gga gag gag aga ctt gag act gga aac act gtt got gat atc
480
Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile
145 150 155 160
tot ctt gga ctt atc aac ttc ctt tac tot aac ttc gtt cot gga ggt
528
Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly
165 170 175
gga ttc atc gtt gga ctt ctt gag ctt atc tgg gga ttc atc gga cot
576
Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro
180 185 190
tot cag tgg gat atc ttc ctt got cag atc gag cag ctt atc tot cag
624
Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile Glu Gin Leu Ile Ser Gin
195 200 205
aga atc gag gag ttc got aga aac cag got atc tot aga ctt gag gga
672
Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly
210 215 220
ctt tot aat ctt tac aaa gtt tac gtt aga got ttc tot gac tgg gag
720
Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu
225 230 235 240
aag gat cot act aac cct got ctt aga gag gag atg aga atc cag ttc
768
Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gin Phe
245 250 255
aac gat atg aac tot got ctt atc act got atc cot ctt ttc aga gtt
816
Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val
260 265 270
cag aac tac gag gtt got ctt ctt tot gtt tac gtt cag got gat aac
864
Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn
275 280 285
ctt cat ctt tot atc ctt aga gat gtt tot gtt ttc gga gag aga tgg
912
Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp
290 295 300
gga tac gat act got act atc aac aac aga tac tot gat ctt act tot
960
Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser
305 310 315 320
ctt atc cat gtt tac act aac cat tgt gtt gat act tac aac cag gga
1008
Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly
325 330 335
ctt aga aga ctt gag gga aga ttc ctt tot gac tgg atc gtt tac aac
1056
Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn
340 345 350
CA 02924415 2016-03-21
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aga ttc aga aga cag ctt act atc tot gtt ctt gat atc gtt got ttc 1104
Arg Phe Arg Arg Gin Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe
355 360 365
ttc cot aac tac gat atc aga act tac cct atc cag act got act cag 1152
Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gin Thr Ala Thr Gin
370 375 380
ctt act aga gag gtt tac ctt gat ctt cot ttc atc aac gag aac ctt 1200
Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu
385 390 395 400
tot cot got got tot tac cot act ttc tot got got gag tot got atc 1248
Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile
405 410 415
atc aga tot cot cat ctt gtt gat ttc ctt aac tot ttc act atc tac 1296
Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr
420 425 430
act gat tot ctt got aga tac got tac tgg gga gga cat ctt gtt aac 1344
Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn
435 440 445
tot ttc aga act gga act aca act aac ctt atc aga tot cot ctt tac 1392
Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr
450 455 460
gga aga gag gga aac act gag aga cot gtt act atc act got tot cot 1440
Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro
465 470 475 480
tot gtt cot atc ttc aga act ctt tot tac atc act gga ctt gat aac 1488
Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn
485 490 495
tot aac cot gtt got gga atc gag gga gtt gag ttc cag aac act atc 1536
Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gin Asn Thr Ile
500 505 510
tot aga tot atc tac aga aag tot gga cot atc gat tot ttc tot gag 1584
Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu
515 520 525
ctt cct cct cag gat got tot gtt tot cot got atc gga tac tot cat 1632
Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His
530 535 540
aga ctt tgt cat got act ttc ctt gag aga atc tot gga cot aga atc 1680
Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile
545 550 555 560
got gga act gtt ttc tot tgg act cat aga tot got tot cot act aac 1728
Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn
565 570 575
CA 02924415 2016-03-21
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, 74
gag gtt tct cct tot aga atc act cag atc cct tgg gtt aag got cat
1776
Glu Val Ser Pro Ser Arg Ile Thr Gin Ile Pro Trp Val Lys Ala His
580 585 590
act ctt got tot gga got tot gtt atc aag gga cct gga ttc act gga
1824
Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly
595 600 605
gga gat atc ctt act aga aac tot atg gga gag ctt gga act ctt aga
1872
Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg
610 615 620
gtt act ttc act gga aga ctt cct cag tot tac tac atc aga ttc aga
1920
Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr Ile Arg Phe Arg
625 630 635 640
tac got tot gtt got aac aga tot gga act ttc aga tac tot cag cct
1968
Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro
645 650 655
cct tot tac gga atc tot ttc cct aag act atg gat got gga gag cct
2016
Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro
660 665 670
ctt act tot aga tot ttc got cat aca act ctt ttc act cct atc act
2064
Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr
675 680 685
ttc tot aga got cag gag gag ttc gat cta tac atc cag tot gga gtt
2112
Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr Ile Gin Ser Gly Val
690 695 700
tac atc gat aga atc gag ttc atc cct gtt act got act ttc gag got
2160
Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala
705 710 715 720
gag tac gat tta gag aga tga
2181
Glu Tyr Asp Leu Glu Arg
725
<210> 13
<211> 726
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 13
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
CA 02924415 2016-03-21
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Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn
115 120 125
Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile
130 135 140
Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile
145 150 155 160
Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly
165 170 175
Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro
180 185 190
Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile Glu Gin Leu Ile Ser Gin
195 200 205
Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly
210 215 220
Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu
225 230 235 240
Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gin Phe
245 250 255
Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val
260 265 270
Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn
275 280 285
Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp
290 295 300
Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser
305 310 315 320
Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly
325 330 335
Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn
340 345 350
Arg Phe Arg Arg Gin Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe
355 360 365
Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gin Thr Ala Thr Gin
370 375 380
Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu
385 390 395 400
Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile
405 410 415
Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr
420 425 430
Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn
435 440 445
Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr
450 455 460
Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro
465 470 475 480
Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn
485 490 495
CA 02924415 2016-03-21
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Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gin Asn Thr Ile
500 505 510
Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu
515 520 525
Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His
530 535 540
Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile
545 550 555 560
Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn
565 570 575
Glu Val Ser Pro Ser Arg Ile Thr Gin Ile Pro Trp Val Lys Ala His
580 585 590
Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly
595 600 605
Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg
610 615 620
Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr Ile Arg Phe Arg
625 630 635 640
Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro
645 650 655
Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro
660 665 670
Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr
675 680 685
Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr Ile Gin Ser Gly Val
690 695 700
Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala
705 710 715 720
Glu Tyr Asp Leu Glu Arg
725
<210> 14
<211> 1815
<212> DNA
<213> Artificial Sequence
<220>
<223> modified sequence
<220>
<221> CDS
<222> (1)..(1812)
<400> 14
atg gct gag atc aac aac cag aac cag tgt gtt cct tac aac tgt ctt
48
Met Ala Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu
1 5 10 15
tct aac cct aag gag atc atc ctt gga gag gag aga ctt gag act gga
96
Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly
20 25 30
aac act gtt gct gat atc tct ctt gga ctt atc aac ttc ctt tac tct
144
Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser
35 40 45
CA 02924415 2016-03-21
77
aac ttc gtt cot gga ggt gga ttc atc gtt gga ctt ctt gag ctt atc 192
Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile
50 55 60
tgg gga ttc atc gga cot tot cag tgg gat atc ttc ctt got cag atc 240
Trp Gly Phe Ile Gly Pro Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile
65 70 75 80
gag cag ctt atc tot cag aga atc gag gag ttc got aga aac cag got 288
Glu Gin Leu Ile Ser Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala
85 90 95
atc tot aga ctt gag gga ctt tot aat ctt tac aaa gtt tac gtt aga 336
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg
100 105 110
got ttc tot gac tgg gag aag gat cot act aac cot got ctt aga gag 384
Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
gag atg aga atc cag ttc aac gat atg aac tot got ctt atc act got 432
Glu Met Arg Ile Gin Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala
130 135 140
atc cot ctt ttc aga gtt cag aac tac gag gtt got ctt ctt tot gtt 480
Ile Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Lou Leu Ser Val
145 150 155 160
tac gtt cag got got aac ctt cat ctt tot atc ctt aga gat gtt tot 528
Tyr Val Gin Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser
165 170 175
gtt ttc gga gag aga tgg gga tac gat act got act atc aac aac aga 576
Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg
180 185 190
tac tot gat ctt act tot ctt atc cat gtt tac act aac cat tgt gtt 624
Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val
195 200 205
gat act tac aac cag gga ctt aga aga ctt gag gga aga ttc ctt tot 672
Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser
210 215 220
gac tgg atc gtt tac aac aga ttc aga aga cag ctt act atc tot gtt 720
Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr Ile Ser Val
225 230 235 240
ctt gat atc gtt got ttc ttc cot aac tac gat atc aga act tac cot 768
Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro
245 250 255
atc cag act got act cag ctt act aga gag gtt tac ctt gat ctt cct 816
Ile Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro
260 265 270
CA 02924415 2016-03-21
. ' .
, 78
ttc atc aac gag aac ctt tct cot gct gct tct tac cct act ttc tct
864
Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser
275 280 285
gct gct gag tct gct atc atc aga tct cct cat ctt gtt gat ttc ctt
912
Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu
290 295 300
aac tct ttc act atc tac act gat tct ctt gct aga tac gct tac tgg
960
Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp
305 310 315 320
gga gga cat ctt gtt aac tct ttc aga act gga act aca act aac ctt
1008
Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu
325 330 335
atc aga tct cot ctt tac gga aga gag gga aac act gag aga cct gtt
1056
Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val
340 345 350
act atc act gct tct cot tct gtt cot atc ttc aga act ctt tct tac
1104
Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr
355 360 365
atc act gga ctt gat aac tct aac cot gtt gct gga atc gag gga gtt
1152
Ile Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val
370 375 380
gag ttc cag aac act atc tct aga tct atc tac aga aag tct gga cot
1200
Glu Phe Gin Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro
385 390 395 400
atc gat tct ttc tct gag ctt cot cot cag gat gct tct gtt tct cct
1248
Ile Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro
405 410 415
gct atc gga tac tct cat aga ctt tgt cat gct act ttc ctt gag aga
1296
Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg
420 425 430
atc tct gga cot aga atc gct gga act gtt ttc tct tgg act cat aga
1344
Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg
435 440 445
tct gct tct cot act aac gag gtt tct cot tct aga atc act cag atc
1392
Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gin Ile
450 455 460
cot tgg gtt aag gct cat act ctt gct tct gga gct tct gtt atc aag
1440
Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys
465 470 475 480
gga cot gga ttc act gga gga gat atc ctt act aga aac tct atg gga
1488
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly
485 490 495
CA 02924415 2016-03-21
79
gag ctt gga act ctt aga gtt act ttc act gga aga ctt cct cag tct 1536
Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser
500 505 510
tac tac atc aga ttc aga tac gct tct gtt gct aac aga tct gga act 1584
Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr
515 520 525
ttc aga tac tct cag cct cot tct tac gga atc tct ttc cct aag act 1632
Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr
530 535 540
atg gat gct gga gag cot ctt act tct aga tct ttc gct cat aca act 1680
Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr
545 550 555 560
ctt ttc act cct atc act ttc tct aga gct cag gag gag ttc gat cta 1728
Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu
565 570 575
tac atc cag tct gga gtt tac atc gat aga atc gag ttc atc cot gtt 1776
Tyr Ile Gin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val
580 585 590
act gct act ttc gag gct gag tac gat tta gag aga tga 1815
Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg
595 600
<210> 15
<211> 604
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 15
Met Ala Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly
20 25 30
Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser
35 40 45
Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile
50 55 60
Trp Gly Phe Ile Gly Pro Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile
65 70 75 80
Glu Gin Leu Ile Ser Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg
100 105 110
Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala
130 135 140
CA 02924415 2016-03-21
, == 80
Ile Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Leu Leu Ser Val
145 150 155 160
Tyr Val Gin Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg
180 185 190
Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val
195 200 205
Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser
210 215 220
Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr Ile Ser Val
225 230 235 240
Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro
245 250 255
Ile Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro
260 265 270
Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser
275 280 285
Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu
290 295 300
Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp
305 310 315 320
Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu
325 330 335
Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val
340 345 350
Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr
355 360 365
Ile Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val
370 375 380
Glu Phe Gin Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro
385 390 395 400
Ile Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro
405 410 415
Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg
420 425 430
Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg
435 440 445
Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gin Ile
450 455 460
Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys
465 470 475 480
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly
485 490 495
Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser
500 505 510
Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr
515 520 525
Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr
530 535 540
Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr
545 550 555 560
Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu
565 570 575
CA 02924415 2016-03-21
81
Tyr Ile Gin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val
580 585 590
Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg
595 600
<210> 16
<211> 372
<212> DNA
<213> Artificial Sequence
<220>
<223> modified transit peptide
<220>
<221> CDS
<222> (1)..(372)
<400> 16
atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96
Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192
Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn
50 55 60
aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly
85 90 95
ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
ctt gga aac gtt tct aac gga gga aga atc aga tgt 372
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys
115 120
<210> 17
<211> 124
<212> PRT
<213> Artificial Sequence
CA 02924415 2016-03-21
82
<220>
<223> Synthetic Construct
<400> 17
Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro
1 5 10 15
Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala
20 25 30
Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser
35 40 45
Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn
50 55 60
Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro
65 70 75 80
Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly
85 90 95
Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser
100 105 110
Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys
115 120
<210> 18
<211> 988
<212> DNA
<213> Subterranean clover stunt virus
<400> 18
ctagataatt gttattatca ataaaagaat ttttattgtt attgtgttat ttggtaattt 60
atgcttataa gtaattctat gattaattgt gaattaataa gactaatgag gataataatt 120
gaatttgatt aaattaactc tgcgaagcca tatgtctttc acgtgagagt cacgtgatgt 180
ctccgcgaca ggctggcacg gggcttagta ttacccccgt gccgggatca gagacatttg 240
actaaatgtt gacttggaat aatagccctt ggattagatg acacgtggac gctcaggatc 300
tgtgatgcta gtgaagcgct taagctgaac gaatctgacg gaagagcgga caaacgcaca 360
tggactatgg cccactgctt tattaaagaa gtgaatgaca gctgtctttg cttcaagacg 420
aagtaaagaa tagtggaaaa cgcgttaatt gttattatca ataaaagaat ttttattgtt 480
attgtgttat ttggtaattt atgcttataa gtaattctat gattaattgt gaattaataa 540
gactaatgag gataataatt gaatttgatt aaattaactc tgcgaagcta tatgtctttc 600
acgtgagagt cacgtgatgt ctccgcgaca ggctggcacg gggcttagta ttacccccgt 660
gccgggatca gagacatttg actaaatgtt gacttggaat aatagccctt ggattagatg 720
acacgtggac gctcaggatc tgtgatgcta gtgaagcgct taagctgaac gaatctgacg 780
gaagagcgga caaacgcaca tggactatgg cccactgctt tattaaagaa gtgaatgaca 840
gctgtctttg cttcaagacg aagtaaagaa tagtggaaaa cgcgtaaaga ataagcgtac 900
tcagtacgct tcgtggcttt ataaatagtg cttcgtctta ttcttcgttg tatcatcaac 960
gaagaagtta agctttgttc tgcgtttc 988
<210> 19
<211> 1042
<212> DNA
<213> Subterranean clover stunt virus
<400> 19
taattaatag taattatgat taattatgag ataagagttg ttattaatgc ttatgaggaa 60
taaagaatga ttaatattgt ttaattttat tccgcgaagc ggtgtgttat gtttttgttg 120
gagacatcac gtgactctca cgtgatgtct ccgcgacagg ctggcacggg gcttagtatt 180
CA 02924415 2016-03-21
= , 83
acccccgtgc cgggatcaga gacatttgac taaatattga cttggaataa tagcccttgg
240
attagatgac acgtggacgc tcaggatctg tgatgctagt gaagcgctta agctgaacga
300
atctgacgga agagcggaca tacgcacatg gattatggcc cacatgtcta aagtgtatct
360
ctttacagct atatcgatgt gacgtaagat gctttacttc gcttcgaagt aaagtaggaa
420
attgctcgct aagttattct tttctgaaag aaattaattt aattctaatt aaattaaatg
480
agtggcctgc agtaattaat agtaattatg attaattatg agataagagt tgttattaat
540
gcttatgagg aataaagaat gattaatatt gtttaatttt attccgcgaa gcggtgtgtt
600
atgtttttgt tggagacatc acgtgactct cacgtgatgt ctccgcgaca ggctggcacg
660
gggcttagta ttacccccgt gccgggatca gagacatttg actaaatatt gacttggaat
720
aatagccctt ggattagatg acacgtggac gctcaggatc tgtgatgcta gtgaagcgct
780
taagctgaac gaatctgacg gaagagcgga catacgcaca tggattatgg cccacatgtc
840
taaagtgtat ctctttacag ctatatcgat gtgacgtaag atgctttact tcgcttcgaa
900
gtaaagtagg aaattgctcg ctaagttatt cttttctgaa agaaattaat ttaattctaa
960
attaaattaa atgagtggct ataaatagtg tcgatgctac ctcacatcgt attcttcttc
1020
gcatcgtctg ttctggtttt aa
1042
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 20
tacttcgaac agaaagaacg agaacgag
28
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 21
gtccagcgaa aggaactcca agaa
24
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 22
aaccttgagg gacttggaaa c
21
<210> 23
<211> 23
CA 02924415 2016-03-21
84
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 23
aagatgaggg tttctgatag cag 23
