U.S. patent application number 13/178292 was filed with the patent office on 2011-11-03 for mutant delta-8 desaturase genes engineered by targeted mutagensis and their use in making polyunsaturated fatty acids.
Invention is credited to HOWARD GLENN DAMUDE, HONGXIAN HE, DER-ING LIAO, QUINN QUN ZHU.
Application Number | 20110268864 13/178292 |
Document ID | / |
Family ID | 39402637 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110268864 |
Kind Code |
A1 |
DAMUDE; HOWARD GLENN ; et
al. |
November 3, 2011 |
Mutant Delta-8 Desaturase Genes Engineered By Targeted Mutagensis
and Their Use In Making Polyunsaturated Fatty Acids
Abstract
The present invention relates to mutant delta-8 desaturase
genes, which have the ability to convert eicosadienoic acid [20:2
omega-6, EDA] to dihomo-.gamma.-linolenic acid [20:3, DGLA] and/or
eicosatrienoic acid [20:3 omega-3, ETrA] to eicosatetraenoic acid
[20:3 omega-3, ETA]. Isolated nucleic acid fragments and
recombinant constructs comprising such fragments encoding delta-8
desaturase along with methods of making long-chain polyunsaturated
fatty acids (PUFAs) using these mutant delta-8 desaturases in
plants and oleaginous yeast are disclosed.
Inventors: |
DAMUDE; HOWARD GLENN;
(HOCKESSIN, DE) ; HE; HONGXIAN; (DREXEL HILL,
PA) ; LIAO; DER-ING; (WILMINGTON, DE) ; ZHU;
QUINN QUN; (WEST CHESTER, PA) |
Family ID: |
39402637 |
Appl. No.: |
13/178292 |
Filed: |
July 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11951697 |
Dec 6, 2007 |
8017838 |
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13178292 |
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60868953 |
Dec 7, 2006 |
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Current U.S.
Class: |
426/601 ;
554/230 |
Current CPC
Class: |
C12N 9/0083 20130101;
C12N 15/8247 20130101 |
Class at
Publication: |
426/601 ;
554/230 |
International
Class: |
C07C 57/03 20060101
C07C057/03; C07C 57/12 20060101 C07C057/12; A23K 1/00 20060101
A23K001/00; A23D 9/00 20060101 A23D009/00; A23D 7/00 20060101
A23D007/00 |
Claims
1-22. (canceled)
23. Oil obtained from a transgenic seed obtained from an oilseed
plant comprising a recombinant construct comprising at least one
regulatory sequence operably linked to a polynucleotide comprising:
(a) a nucleotide sequence encoding a polypeptide having delta-8
desaturase activity wherein the polypeptide has an amino acid
sequence as set forth in SEQ ID NO:2, wherein SEQ ID NO:2 comprises
at least one mutation selected from the group consisting of S4A,
K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F,
L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A,
L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q,
A418G, G419A, and L422Q, wherein the mutations are defined with
respect to SEQ ID NO:10; (b) a nucleotide sequence encoding a
polypeptide having delta-8 desaturase activity wherein the
polypeptide has an amino acid sequence as set forth in SEQ ID
NO:198, wherein SEQ ID NO:198 comprises at least one mutation
selected from the group consisting of S4A, K5S, L9V, T12V, T16K,
T17V, D19E, V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A,
E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S,
Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V,
T279L, L280T, L293M, F297V, V298L, 1309V, V310I, I346V, I347L,
T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein
the mutations are defined with respect to SEQ ID NO:10; or (c) a
complement of the nucleotide sequence of part (a) or (b), wherein
the complement and the nucleotide sequence consist of the same
number of nucleotides and are 100% complementary.
24. The oil of claim 23, wherein the oilseed plant is selected from
the group consisting of soybean, Brassica species, sunflower,
maize, cotton, flax, and safflower.
25. Oil obtained from a transgenic seed obtained from an oilseed
plant comprising: (a) a first recombinant construct comprising at
least one regulatory sequence operably linked to a polynucleotide
comprising a nucleotide sequence encoding a polypeptide having
delta-8 desaturase activity wherein the polypeptide has an amino
acid sequence as set forth in (i) SEQ ID NO:2, wherein SEQ ID NO:2
comprises at least one mutation selected from the group consisting
of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L,
G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V,
V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S,
A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are
defined with respect to SEQ ID NO:10; or (ii) SEQ ID NO:198,
wherein SEQ ID NO:198 comprises at least one mutation selected from
the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E,
V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V,
A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V1225, Q125H, M126L,
Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T,
L293M, F297V, V298L, I309V, V310I, I346V, I347L, T348S, A407S,
V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations
are defined with respect to SEQ ID NO:10; and (b) at least one
additional recombinant construct comprising at least one regulatory
sequence operably linked to a polynucleotide encoding a polypeptide
selected from the group consisting of a delta-4 desaturase, a
delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a
delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase,
a delta-9 desaturase, a delta-9 elongase, a C.sub.14/18 elongase, a
C.sub.16/18 elongase, a C.sub.18/20 elongase, and a C.sub.20/22
elongase.
26. The oil of claim 25, wherein the oilseed plant is selected from
the group consisting of soybean, Brassica species, sunflower,
maize, cotton, flax, and safflower.
27. Food or feed which incorporates the oil of any of claims
23-26.
28. Food or feed comprising an ingredient derived from the
processing of a transgenic seed obtained from an oilseed plant
comprising a recombinant construct comprising at least one
regulatory sequence operably linked to a polynucleotide comprising:
(a) a nucleotide sequence encoding a polypeptide having delta-8
desaturase activity wherein the polypeptide has an amino acid
sequence as set forth in SEQ ID NO:2, wherein SEQ ID NO:2 comprises
at least one mutation selected from the group consisting of S4A,
K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F,
L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A,
L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q,
A418G, G419A, and L422Q, wherein the mutations are defined with
respect to SEQ ID NO:10; (b) a nucleotide sequence encoding a
polypeptide having delta-8 desaturase activity wherein the
polypeptide has an amino acid sequence as set forth in SEQ ID
NO:198, wherein SEQ ID NO:198 comprises at least one mutation
selected from the group consisting of S4A, K5S, L9V, T12V, T16K,
T17V, D19E, V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A,
E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S,
Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V,
T279L, L280T, L293M, F297V, V298L, I309V, V310I, I346V, I347L,
T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein
the mutations are defined with respect to SEQ ID NO:10; or (c) a
complement of the nucleotide sequence of part (a) or (b), wherein
the complement and the nucleotide sequence consist of the same
number of nucleotides and are 100% complementary.
29. The food or feed of claim 28, wherein the oilseed plant is
selected from the group consisting of soybean, Brassica species,
sunflower, maize, cotton, flax, and safflower.
30. Food or feed comprising an ingredient derived from the
processing of a transgenic seed obtained from an oilseed plant
comprising: (a) a first recombinant construct comprising at least
one regulatory sequence operably linked to a polynucleotide
comprising a nucleotide sequence encoding a polypeptide having
delta-8 desaturase activity wherein the polypeptide has an amino
acid sequence as set forth in (i) SEQ ID NO:2, wherein SEQ ID NO:2
comprises at least one mutation selected from the group consisting
of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L,
G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V,
V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S,
A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are
defined with respect to SEQ ID NO:10; or (ii) SEQ ID NO:198,
wherein SEQ ID NO:198 comprises at least one mutation selected from
the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E,
V201, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V,
A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S, Q125H, M126L,
Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T,
L293M, F297V, V298L, I309V, V310I, I346V, I347L, T348S, A407S,
V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations
are defined with respect to SEQ ID NO:10; and (b) at least one
additional recombinant construct comprising at least one regulatory
sequence operably linked to a polynucleotide encoding a polypeptide
selected from the group consisting of a delta-4 desaturase, a
delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a
delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase,
a delta-9 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a
C.sub.16/18 elongase, a C.sub.18/20 elongase, and a C.sub.20/22
elongase.
31. The food or feed of claim 30, wherein the oilseed plant is
selected from the group consisting of soybean, Brassica species,
sunflower, maize, cotton, flax, and safflower.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/868,953, filed Dec. 7, 2006, the entire contents
of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of biotechnology. More
specifically, this invention pertains to the creation of nucleic
acid fragments encoding mutant delta-8 fatty acid desaturase
enzymes and the use of these desaturases in making long-chain
polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] The importance of PUFAs is undisputed. For example, certain
PUFAs are important biological components of healthy cells and are
considered "essential" fatty acids that cannot be synthesized de
novo in mammals and instead must be obtained either in the diet or
derived by further desaturation and elongation of linoleic acid
(LA; 18:2 omega-6) or .alpha.-linolenic acid (ALA; 18:3 omega-3).
Additionally PUFAs are constituents of plasma membranes of cells,
where they may be found in such forms as phospholipids or
triacylglycerols. PUFAs are necessary for proper development
(particularly in the developing infant brain) and for tissue
formation and repair and are precursors to several biologically
active eicosanoids of importance in mammals (e.g., prostacyclins,
eicosanoids, leukotrienes, prostaglandins). Studies have shown that
a high intake of long-chain omega-3 PUFAs produces cardiovascular
protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr.
28:958-966 (1975); Dyerberg, J. et al., Lancet. 2(8081):117-119
(1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); von
Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet 88:90-99
(2001)). The literature reports wide-ranging health benefits
conferred by administration of omega-3 and/or omega-6 PUFAs against
a variety of symptoms and diseases (e.g., asthma, psoriasis,
eczema, diabetes, cancer).
[0004] A variety of different hosts including plants, algae, fungi
and yeast are being investigated as means for commercial PUFA
production. Genetic engineering has demonstrated that the natural
abilities of some hosts can be substantially altered to produce
various long-chain omega-3/omega-6 PUFAs. For example, production
of arachidonic acid (ARA; 20:4 omega-6), eicosapentaenoic acid
(EPA; 20:5 omega-3) and docosahexaenoic acid (DHA; 22:6 omega-3)
all require expression of either the delta-9 elongase/delta-8
desaturase pathway or the delta-6 desaturase/delta-6 elongase
pathway. The delta-9 elongase/delta-8 desaturase pathway is present
for example in euglenoid species and is characterized by the
production of eicosadienoic acid (EDA; 20:2 omega-6] and/or
eicosatrienoic acid (ETrA; 20:3 omega-3). (FIG. 1). The delta-6
desaturase/delta-6 elongase pathway is predominantly found in
algae, mosses, fungi, nematodes and humans and is characterized by
the production of .gamma.-linoleic acid (GLA; 18:3 omega-6) and/or
stearidonic acid (STA; 18:4 omega-3) (FIG. 1).
[0005] For some applications the delta-9 elongase/delta-8
desaturase pathway is favored. However, delta-8 desaturase enzymes
are not well known in the art leaving the construction of a
recombinant delta-9 elongase/delta-8 desaturase pathway with
limited options. The few delta-8 desaturase enzymes identified thus
far have the ability to convert both EDA to
dihomo-.gamma.-linolenic acid (DGLA; 20:3) and ETrA to
eicosatetraenoic acid (ETA; 20:4) (wherein ARA are EPA are
subsequently synthesized from DGLA and ETA, respectively, following
reaction with a delta-5 desaturase, while DHA synthesis requires
subsequent expression of an additional C.sub.20/22 elongase and a
delta-4 desaturase).
[0006] Several delta-8 desaturase enzymes are known and have been
partially characterized (see for example delta-8 desaturases from
Euglena gracilis Wallis et al., Arch. Biochem. and Biophys.
365(2):307-316 (1999); WO 2000/34439; U.S. Pat. No. 6,825,017; WO
2004/057001; WO 2006/012325; WO 2006/012326). Additionally WO
2005/103253 (published Apr. 22, 2005) discloses amino acid and
nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova
salina (see also U.S. Publication No. 2005/0273885). Sayanova et
al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and
characterization of a cDNA from the free living soil amoeba
Acanthamoeba castellanii that, when expressed in Arabidopsis,
encodes a C.sub.20 delta-8 desaturase. Furthermore, commonly owned
and co-pending U.S. Provisional Application No. 60/795,810 filed
Apr. 28, 2006, discloses amino acid and nucleic acid sequences for
a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459).
[0007] A need remains therefore for additional delta-8 desaturase
enzymes to be used in recombinant pathways for the production of
PUFAs. Applicants have solved the stated need by developing a
synthetically engineered mutant Euglena gracilis delta-8
desaturase.
SUMMARY OF THE INVENTION
[0008] The invention concerns an isolated polynucleotide
comprising: [0009] (a) a nucleotide sequence encoding a mutant
polypeptide having delta-8 desaturase activity wherein the mutant
polypeptide has an amino acid sequence as set forth in SEQ ID NO:2
and further wherein SEQ ID NO:2 is not identical to SEQ ID NO:10;
or, [0010] (b) a complement of the nucleotide sequence of part (a),
wherein the complement and the nucleotide sequence consist of the
same number of nucleotides and are 100% complementary.
[0011] In a second embodiment, the invention concerns an isolated
polynucleotide of Claim 1 wherein the nucleotide sequence comprises
SEQ ID NO:1 and wherein SEQ ID NO:1 is not identical to SEQ ID
NO:9.
[0012] In a third embodiment, the invention comprises an isolated
polynucleotide comprising: [0013] (a) a nucleotide sequence
encoding a mutant polypeptide having delta-8 desaturase activity
wherein the mutant polypeptide has an amino acid sequence as set
forth in SEQ ID NO:198 and further wherein SEQ ID NO:198 is not
identical to SEQ ID NO:10; or, [0014] (b) a complement of the
nucleotide sequence of part (a), wherein the complement and the
nucleotide sequence consist of the same number of nucleotides and
are 100% complementary.
[0015] In a fourth embodiment, the invention concerns an isolated
polynucleotide of Claim 3 wherein the nucleotide sequence comprises
SEQ ID NO:197 and wherein SEQ ID NO:197 is not identical to SEQ ID
NO:9.
[0016] In a fifth embodiment, the invention concerns a recombinant
construct comprising any of the polynucleotides of the invention
operably linked to at least one regulatory sequence.
[0017] In a sixth embodiment, the invention concerns a cell
comprising in its genome the recombinant DNA construct of the
invention.
[0018] In a seventh embodiment, the invention concerns a method for
producing a transgenic plant, comprising transforming a plant cell
with at least one polynucleotide of the invention and regenerating
a transgenic plant from the transformed plant cell. A preferred
plant is soybean.
[0019] In an eighth embodiment, the invention concerns a transgenic
seed comprising the recombinant construct of the invention.
[0020] In a ninth embodiment, a method for making a long-chain
polyunsaturated fatty acid in a plant cell comprising: [0021] (a)
transforming a cell with the recombinant construct of the
invention; and [0022] (b) selecting those transformed cells that
make long-chain polyunsaturated fatty acids. Preferably, the plant
cell is a soybean cell.
[0023] In a tenth embodiment, the invention concerns a method for
producing at least one polyunsaturated fatty acid in a soybean cell
comprising: [0024] (a) transforming a soybean cell with a first
recombinant construct comprising an isolated polynucleotide
encoding at least one mutant delta-8 desaturase polypeptide,
operably linked to at least one regulatory sequence and at least
one additional recombinant construct comprising an isolated
polynucleotide, operably linked to at least one regulatory
sequence, encoding a polypeptide selected from the group consisting
of a delta-4 desaturase, a delta-5 desaturase, a delta-6
desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15
desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9
elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and a C.sub.20/22 elongase; [0025] (b)
regenerating a soybean plant from the transformed soybean cell of
step (a); and [0026] (c) selecting those seeds obtained from the
plants of step (b) having an altered level of polyunsaturated fatty
acids when compared to the level in seeds obtained from a
nontransformed soybean plant.
[0027] In an eleventh embodiment, the invention concerns an oilseed
plant comprising: [0028] (a) a first recombinant construct
comprising an isolated polynucleotide encoding at least one mutant
delta-8 desaturase polypeptide, operably linked to at least one
regulatory sequence; and [0029] (b) at least one additional
recombinant construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of a delta-4
desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8
desaturase, a delta-12 desaturase, a delta-15 desaturase, a
delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase,
C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20
elongase and a C.sub.20/22 elongase.
[0030] Also of interest are transgenic seeds obtained from such
oilseed plants and oil obtained from these seeds.
[0031] In a twelfth embodiment, the invention concerns food or feed
which incorporates oil of the invention.
[0032] In a thirteenth embodiment, the invention concerns food or
feed comprising an ingredient derived from the processing of the
seeds of the invention.
[0033] In a fourteenth embodiment, the invention concerns progeny
plants obtained from obtained from a plant made by the method of
the invention or an oilseed plant of the invention.
Biological Deposits
[0034] The following plasmid has been deposited with the American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va. 20110-2209, and bears the following designation,
Accession Number and date of deposit (Table 1).
TABLE-US-00001 TABLE 1 ATCC Deposits Plasmid Accession Number Date
of Deposit pKR72 PTA-6019 May 28, 2004
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0035] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing, which form a part of this application.
[0036] FIG. 1 is a representative PUFA biosynthetic pathway.
[0037] FIG. 2 shows a topological model of EgD8S.
[0038] FIG. 3 shows an alignment of EgD8S (SEQ ID NO:10), a delta-6
desaturase of Amylomyces rouxii (SEQ ID NO:13), a delta-6
desaturase of Rhizopus orizae (SEQ ID NO:14), a delta-8 fatty acid
desaturase-like protein of Leishmania major (GenBank Accession No.
CAJ09677; SEQ ID NO:15), and a delta-6 desaturase of Mortierella
isabellina (GenBank Accession No. AAG38104; SEQ ID NO:16). The
method of alignment used corresponds to the "Clustal W method of
alignment".
[0039] FIG. 4 shows an alignment of EgD8S (SEQ ID NO:10), the
cytochrome b.sub.5 of Saccharomyces cerevisiae (GenBank Accession
No. P40312; SEQ ID NO:178) and a probable cytochrome b.sub.5 1 of
Schizosaccharomyces pombe (GenBank Accession No. 094391; SEQ ID
NO:179). The method of alignment used corresponds to the "Clustal W
method of alignment".
[0040] FIG. 5 provides plasmid maps for the following: (A)
pZKLeuN-29E3; and, (B) pY116.
[0041] FIG. 6 provides plasmid maps for the following: (A)
pKUNFmkF2; (B) pDMW287F; (C) pDMW214; and, (D) pFmD8S.
[0042] FIG. 7 diagrams the synthesis of the Mutant EgD8S-5B, by
ligation of fragments from Mutant EgD8S-1 and Mutant EgD8S-2B.
[0043] FIG. 8A diagrams the synthesis of Mutant EgD8S-008, by
ligation of fragments from Mutant EgD8S-001 and Mutant EgD8S-003.
Similarly, FIG. 8B diagrams the synthesis of Mutant EgD8S-009, by
ligation of fragments from Mutant EgD8S-001 and Mutant
EgD8S-004.
[0044] FIG. 9A diagrams the synthesis of Mutant EgD8S-013, by
ligation of fragments from Mutant EgD8S-009 and Mutant EgD8S-23.
Similarly, FIG. 9B diagrams the synthesis of Mutant EgD8S-015, by
ligation of fragments from Mutant EgD8S-008 and Mutant
EgD8S-28.
[0045] FIG. 10 shows an alignment of EgD8S (SEQ ID NO:10), Mutant
EgD8S-23 (SEQ ID NO:4), Mutant EgD8S-013 (SEQ ID NO:6) and Mutant
EgD8S-015 (SEQ ID NO:8). The method of alignment used corresponds
to the "Clustal W method of alignment".
[0046] FIG. 11 provides plasmid maps for the following: (A)
pKo2UFm8; and, (B) pKO2UF8289.
[0047] FIG. 12 provides a plasmid map for pKR1060.
[0048] FIG. 13 provides a plasmid map for pKR1059.
[0049] FIG. 14 shows the lipid profiles of somatic soybean embryos
expressing EgD8S-23 and the Euglena gracilis delta-9 elongase for
the top 5 events (see example 17).
[0050] The sequence descriptions summarize the Sequences Listing
provided herewith. The Sequence Listing contains one letter codes
for nucleotide sequence characters and the single and three letter
codes for amino acids as defined in the IUPAC-IUB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219(2):345-373 (1984). The following sequences
comply with 37 C.F.R. .sctn.1.821-1.825 ("Requirements for Patent
Applications Containing Nucleotide Sequences and/or Amino Acid
Sequence Disclosures--the Sequence Rules") and are consistent with
World Intellectual Property Organization (WIPO) Standard ST.25
(1998) and the sequence listing requirements of the EPO and PCT
(Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the
Administrative Instructions). The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
[0051] SEQ ID NOs:1-17, 19-23, 165 and 172-177 are ORFs encoding
genes or proteins (or portions thereof) or plasmids, as identified
in Table 2.
TABLE-US-00002 TABLE 2 Summary Of Nucleic Acid And Protein SEQ ID
Numbers Nucleic acid Protein SEQ SEQ Description and Abbreviation
ID NO. ID NO. Synthetic mutant delta-8 desaturase, 1 2 derived from
Euglena gracilis ("EgD8S- (1272 bp) (422 AA) consensus") optionally
comprising M1, M2, M3, M8, M12, M15, M16, M18, M21, M26, M38, M45,
M46, M51, M63, M68, M69 and M70 mutation sites Synthetic mutant
delta-8 desaturase, 197 198 derived from Euglena gracilis ("EgD8S-
(1272 bp) (422 AA) consensus") optionally comprising M1, M2, M3,
M6, M8, M12, M14, M15, M16, M18, M19, M21, M22, M26, M38, M39, M40,
M41, M45, M46, M49, M50, M51, M53, M54, M58, M63, M68, M69 and M70
mutation sites Synthetic mutant delta-8 desaturase, 3 4 derived
from Euglena gracilis ("Mutant (1272 bp) (422 AA) EgD8S-23")
Synthetic mutant delta-8 desaturase, 5 6 derived from Euglena
gracilis ("Mutant (1272 bp) (422 AA) EgD8S-013") Synthetic mutant
delta-8 desaturase, 7 8 derived from Euglena gracilis ("Mutant
(1272 bp) (422 AA) EgD8S-015") Synthetic delta-8 desaturase,
derived from 9 10 Euglena gracilis, codon-optimized for (1272 bp)
(422 AA) expression in Yarrowia lipolytica ("EgD8S") Euglena
gracilis delta-8 desaturase (full- 11 12 length gene is nucleotides
4-1269 (Stop)) (1271 bp) (421 AA) ("EgD8") Amylomyces rouxii
delta-6 desaturase -- 13 (GenBank Accession No. AAR27297) (467 AA)
Rhizopus orizae delta-6 desaturase -- 14 (GenBank Accession No.
AAS93682) (445 AA) Leishmania major delta-8 fatty acid -- 15
desaturase-like protein (GenBank (382 AA) Accession No. CAJ09677)
Mortierella isabellina delta-6 desaturase -- 16 (GenBank Accession
No. AAG38104) (439 AA) Saccharomyces cerevisiae cytochrome b.sub.5
-- 178 (GenBank Accession No. P40312) (120 AA) Schizosaccharomyces
pombe probable -- 179 cytochrome b.sub.5 1 (GenBank Accession No.
(124 AA) O94391) Plasmid pZKLeuN-29E3 17 -- (14,655 bp) Synthetic
C.sub.16/18 elongase gene derived 19 -- from Mortierella alpina
ELO3, codon- (828 bp) optimized for expression in Yarrowia
lipolytica Plasmid pFmD8S 20 -- (8,910 bp) Plasmid pKUNFmkF2 21 --
(7,145 bp) Plasmid pDMW287F 22 -- (5,473 bp) Plasmid pDMW214 23 --
(9,513 bp) Plasmid pKO2UFkF2 165 -- (8,560 bp) Isochrysis galbana
delta-9 elongase 172 173 (GenBank Accession No. AF390174) (1064 bp)
(263 AA) Synthetic delta-9 elongase gene derived 174 173 from
Isochrysis galbana, codon-optimized (792 bp) (263 AA) for
expression in Yarrowia lipolytica Euglena gracilis delta-9 elongase
175 176 (777 bp) (258 AA) Synthetic delta-9 elongase gene derived
177 176 from Euglena gracilis, codon-optimized for (777 bp) (258
AA) expression in Yarrowia lipolytica Plasmid pY116 180 -- (8739
bp) Plasmid pKO2UF8289 181 -- (15,304 bp) Synthetic mutant delta-8
desaturase, 182 -- derived from Euglena gracilis ("modified (1288
bp) Mutant EgD8S-23"), comprising a 5' Not1 site Plasmid pKR457 183
-- (5252 bp) Plasmid pKR1058 184 -- (6532 bp) Plasmid pKR607 185 --
(7887 bp) Plasmid pKR1060 186 -- (11,766 bp) Plasmid pKR906 189 --
(4311 bp) Plasmid pKR72 190 -- (7085 bp) Plasmid pKR1010 191 --
(7873 bp) Plasmid pKR1059 192 -- (11752 bp) Euglena gracilis
delta-9 elongase -- 5' 194 -- sequence of the cDNA insert from
clone (757 bp) eeg1c.pk001.n5.f. Euglena gracilis delta-9 elongase
-- 3' 195 -- sequence of the cDNA insert from clone (774 bp)
eeg1c.pk001.n5.f. Euglena gracilis delta-9 elongase -- 196 --
sequence aligned from SEQ ID NO: 1 and (1201 bp) SEQ ID NO: 2 (full
cDNA sequence excluding polyA tail)
[0052] SEQ ID NO:18 corresponds to a LoxP recombination site that
is recognized by a Cre recombinase enzyme.
[0053] SEQ ID NOs:24-164 correspond to 70 pairs of nucleotide
primers (i.e., 1A, 1B, 2A, 2B, 3A, 3B, etc. up to 69A, 69B, 70A and
70B, respectively), used to create specific targeted mutations at
mutation sites M1, M2, M3, etc. up to M70.
[0054] SEQ ID NOs:166-171 correspond to His-rich motifs that are
featured in membrane-bound fatty acid desaturases belonging to a
super-family of membrane di-iron proteins.
[0055] SEQ ID NOs:187 and 188 correspond to primers oEugEL1-1 and
oEugEL1-2, respectively, used to amplify a Euglena gracilis delta-9
elongase.
[0056] SEQ ID NO:193 is the M13F universal primer.
DETAILED DESCRIPTION OF THE INVENTION
[0057] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
"a plant" includes a plurality of such plants, reference to "a
cell" includes one or more cells and equivalents thereof known to
those skilled in the art, and so forth.
[0058] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety. This
specifically includes the following commonly owned and copending
applications: U.S. patent application Ser. No. 10/840,478, Ser. No.
10/840,579 and Ser. No. 10/840,325 (filed May 6, 2004), U.S. patent
application Ser. No. 10/869,630 (filed Jun. 16, 2004), U.S. patent
application Ser. No. 10/882,760 (filed Jul. 1, 2004), U.S. patent
application Ser. No. 10/985,254 and Ser. No. 10/985,691 (filed Nov.
10, 2004), U.S. patent application Ser. No. 10/987,548 (filed Nov.
12, 2004), U.S. patent application Ser. No. 11/024,545 and Ser. No.
11/024,544 (filed Dec. 29, 2004), U.S. patent application Ser. No.
11/166,993 (filed Jun. 24, 2005), U.S. patent application Ser. No.
11/183,664 (filed Jul. 18, 2005), U.S. patent application Ser. No.
11/185,301 (filed Jul. 20, 2005), U.S. patent application Ser. No.
11/190,750 (filed Jul. 27, 2005), U.S. patent application Ser. No.
11/198,975 (filed Aug. 8, 2005), U.S. patent application Ser. No.
11/225,354 (filed Sep. 13, 2005), U.S. patent application Ser. No.
11/251,466 (filed Oct. 14, 2005), U.S. patent application Ser. No.
11/254,173 and Ser. No. 11/253,882 (filed Oct. 19, 2005), U.S.
patent application Ser. No. 11/264,784 and Ser. No. 11/264,737
(filed Nov. 1, 2005), U.S. patent application Ser. No. 11/265,761
(filed Nov. 2, 2005), U.S. Patent Application No. 60/739,989 (filed
Nov. 23, 2005), U.S. Patent Application No. 60/793,575 (filed Apr.
20, 2006), U.S. Patent Application No. 60/795,810 (filed Apr. 28,
2006), U.S. Patent Application No. 60/796,637 (filed May 1, 2006)
and U.S. Patent Applications No. 60/801,172 and No. 60/801,119
(filed May 17, 2006).
[0059] Additionally, commonly owned U.S. patent application Ser.
No. 10/776,311, (published Aug. 26, 2004) relating to the
production of PUFAs in plants, and U.S. patent application Ser. No.
10/776,889 (published Aug. 26, 2004) relating to annexin promoters
and their use in expression of transgenes in plants, are
incorporated by reference in their entirety.
[0060] The present invention provides mutant delta-8 desaturase
enzymes and genes encoding the same, that may be used for the
manipulation of biochemical pathways for the production of
healthful PUFAs.
[0061] PUFAs, or derivatives thereof, made by the methodology
disclosed herein can be used as dietary substitutes, or
supplements, particularly infant formulas, for patients undergoing
intravenous feeding or for preventing or treating malnutrition.
Alternatively, the purified PUFAs (or derivatives thereof) may be
incorporated into cooking oils, fats or margarines formulated so
that in normal use the recipient would receive the desired amount
for dietary supplementation. PUFAs may also be used as
anti-inflammatory or cholesterol lowering agents as components of
pharmaceutical or veterinary compositions.
[0062] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0063] "Open reading frame" is abbreviated ORF.
[0064] "Polymerase chain reaction" is abbreviated PCR.
[0065] "American Type Culture Collection" is abbreviated ATCC.
[0066] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0067] "Triacylglycerols" are abbreviated TAGs.
[0068] The term "fatty acids" refers to long-chain aliphatic acids
(alkanoic acids) of varying chain lengths, from about C.sub.12 to
C.sub.22 (although both longer and shorter chain-length acids are
known). The predominant chain lengths are between C.sub.16 and
C.sub.22. Additional details concerning the differentiation between
"saturated fatty acids" versus "unsaturated fatty acids",
"monounsaturated fatty acids" versus "polyunsaturated fatty acids"
(or "PUFAs"), and "omega-6 fatty acids" (omega-6 or n-6) versus
"omega-3 fatty acids" (omega-3 or n-3) are provided in
WO2004/101757.
[0069] Fatty acids are described herein by a simple notation system
of "X:Y", wherein X is the number of carbon (C) atoms in the
particular fatty acid and Y is the number of double bonds. The
number following the fatty acid designation indicates the position
of the double bond from the carboxyl end of the fatty acid with the
"c" affix for the cis-configuration of the double bond (e.g.,
palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, 9c),
petroselinic acid (18:1, 6c), LA (18:2, 9c, 12c), GLA (18:3, 6c,
9c, 12c) and ALA (18:3, 9c, 12c, 15c). Unless otherwise specified,
18:1, 18:2 and 18:3 refer to oleic, LA and ALA fatty acids. If not
specifically written as otherwise, double bonds are assumed to be
of the cis configuration. For instance, the double bonds in 18:2
(9, 12) would be assumed to be in the cis configuration.
[0070] Nomenclature used to describe PUFAs in the present
disclosure is shown below in Table 3. In the column titled
"Shorthand Notation", the omega-reference system is used to
indicate the number of carbons, the number of double bonds and the
position of the double bond closest to the omega carbon, counting
from the omega carbon (which is numbered 1 for this purpose). The
remainder of the Table summarizes the common names of omega-3 and
omega-6 fatty acids and their precursors, the abbreviations that
will be used throughout the specification, and each compounds'
chemical name.
TABLE-US-00003 TABLE 3 Nomenclature Of Polyunsaturated Fatty Acids
And Precursors Common Shorthand Name Abbreviation Chemical Name
Notation myristic -- tetradecanoic 14:0 palmitic PA hexadecanoic
16:0 palmitoleic -- 9-hexadecenoic 16:1 stearic -- octadecanoic
18:0 oleic -- cis-9-octadecenoic 18:1 linoleic LA
cis-9,12-octadecadienoic 18:2 omega-6 gamma- GLA cis-6,9,12- 18:3
linolenic octadecatrienoic omega-6 eicosadienoic EDA
cis-11,14-eicosadienoic 20:2 omega-6 dihomo- DGLA
cis-8,11,14-eicosatrienoic 20:3 gamma- omega-6 linolenic sciadonic
SCI cis-5,11,14-eicosatrienoic 20:3b omega-6 arachidonic ARA
cis-5,8,11,14- 20:4 eicosatetraenoic omega-6 alpha-linolenic ALA
cis-9,12,15- 18:3 octadecatrienoic omega-3 stearidonic STA
cis-6,9,12,15- 18:4 octadecatetraenoic omega-3 eicosatrienoic ETrA
or ERA cis-11,14,17- 20:3 eicosatrienoic omega-3 eicosa- ETA
cis-8,11,14,17- 20:4 tetraenoic eicosatetraenoic omega-3
juniperonic JUP cis-5,11,14,17- 20:4b eicosatrienoic omega-3
eicosa- EPA cis-5,8,11,14,17- 20:5 pentaenoic eicosapentaenoic
omega-3 docosa- DPA cis-7,10,13,16,19- 22:5 pentaenoic
docosapentaenoic omega-3 docosa- DHA cis-4,7,10,13,16,19- 22:6
hexaenoic docosahexaenoic omega-3
[0071] The terms "triacylglycerol", "oil" and "TAGs" refer to
neutral lipids composed of three fatty acyl residues esterified to
a glycerol molecule (and such terms will be used interchangeably
throughout the present disclosure herein). Such oils can contain
long-chain PUFAs, as well as shorter saturated and unsaturated
fatty acids and longer chain saturated fatty acids. Thus, "oil
biosynthesis" generically refers to the synthesis of TAGs in the
cell.
[0072] "Percent (%) PUFAs in the total lipid and oil fractions"
refers to the percent of PUFAs relative to the total fatty acids in
those fractions. The term "total lipid fraction" or "lipid
fraction" both refer to the sum of all lipids (i.e., neutral and
polar) within an oleaginous organism, thus including those lipids
that are located in the phosphatidylcholine (PC) fraction,
phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or
oil) fraction. However, the terms "lipid" and "oil" will be used
interchangeably throughout the specification.
[0073] The term "PUFA biosynthetic pathway" refers to a metabolic
process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA,
STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in
the literature (e.g., see WO 2006/052870). Briefly, this process
involves elongation of the carbon chain through the addition of
carbon atoms and desaturation of the molecule through the addition
of double bonds, via a series of special desaturation and
elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes")
present in the endoplasmic reticulim membrane. More specifically,
"PUFA biosynthetic pathway enzyme" refers to any of the following
enzymes (and genes which encode said enzymes) associated with the
biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5
desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15
desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8
desaturase, a C.sub.14/16 elongase, a delta-9 elongase, a
C.sub.16/18 elongase, a C.sub.18/20 elongase and/or a C.sub.20/22
elongase.
[0074] The term "omega-3/omega-6 fatty acid biosynthetic pathway"
refers to a set of genes which, when expressed under the
appropriate conditions encode enzymes that catalyze the production
of either or both omega-3 and omega-6 fatty acids. Typically the
genes involved in the omega-3/omega-6 fatty acid biosynthetic
pathway encode some or all of the following enzymes: delta-12
desaturase, delta-6 desaturase, C.sub.18/20 elongase, C.sub.20/22
elongase, delta-5 desaturase, delta-17 desaturase, delta-15
desaturase, delta-9 desaturase, delta-8 desaturase, a delta-9
elongase and delta-4 desaturase. A representative pathway is
illustrated in FIG. 1, providing for the conversion of oleic acid
through various intermediates to DHA, which demonstrates how both
omega-3 and omega-6 fatty acids may be produced from a common
source. The pathway is naturally divided into two portions where
one portion will generate omega-3 fatty acids and the other
portion, only omega-6 fatty acids. That portion that only generates
omega-3 fatty acids will be referred to herein as the omega-3 fatty
acid biosynthetic pathway, whereas that portion that generates only
omega-6 fatty acids will be referred to herein as the omega-6 fatty
acid biosynthetic pathway.
[0075] The term "functional" as used herein in context with the
omega-3/omega-6 fatty acid biosynthetic pathway means that some (or
all of) the genes in the pathway express active enzymes, resulting
in in vivo catalysis or substrate conversion. It should be
understood that "omega-3/omega-6 fatty acid biosynthetic pathway"
or "functional omega-3/omega-6 fatty acid biosynthetic pathway"
does not imply that all the genes listed in the above paragraph are
required, as a number of fatty acid products will only require the
expression of a subset of the genes of this pathway.
[0076] The term "desaturase" refers to a polypeptide that can
desaturate, i.e., introduce a double bond, in one or more fatty
acids to produce a fatty acid or precursor of interest. Despite use
of the omega-reference system throughout the specification to refer
to specific fatty acids, it is more convenient to indicate the
activity of a desaturase by counting from the carboxyl end of the
substrate using the delta-system. Of particular interest herein are
delta-8 desaturases that will desaturate a fatty acid between the
8.sup.th and 9.sup.th carbon atom numbered from the
carboxyl-terminal end of the molecule and that can, for example,
catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other
desaturases include: (1) delta-5 desaturases that catalyze the
conversion of DGLA to ARA and/or ETA to EPA; (2) delta-6
desaturases that catalyze the conversion of LA to GLA and/or ALA to
STA; (3) delta-4 desaturases that catalyze the conversion of DPA to
DHA; (4) delta-12 desaturases that catalyze the conversion of oleic
acid to LA; (5) delta-15 desaturases that catalyze the conversion
of LA to ALA and/or GLA to STA; (6) delta-17 desaturases that
catalyze the conversion of ARA to EPA and/or DGLA to ETA; and (7)
delta-9 desaturases that catalyze the conversion of palmitate to
palmitoleic acid (16:1) and/or stearate to oleic acid (18:1). In
the art, delta-15 and delta-17 desaturases are also occasionally
referred to as "omega-3 desaturases", "w-3 desaturases", and/or
"omega-3 desaturases", based on their ability to convert omega-6
fatty acids into their omega-3 counterparts (e.g., conversion of LA
into ALA and ARA into EPA, respectively). In some embodiments, it
is most desirable to empirically determine the specificity of a
particular fatty acid desaturase by transforming a suitable host
with the gene for the fatty acid desaturase and determining its
effect on the fatty acid profile of the host.
[0077] For the purposes herein, the term "EgD8" refers to a delta-8
desaturase enzyme (SEQ ID NO:12) isolated from Euglena gracilis,
encoded by SEQ ID NO:11 herein. EgD8 is 100% identical and
functionally equivalent to "Eg5", as described in WO 2006/012325
and WO 2006/012326 (US2005-0287652-A1).
[0078] Similarly, the term "EgD8S" refers to a synthetic delta-8
desaturase derived from Euglena gracilis that is codon-optimized
for expression in Yarrowia lipolytica herein (i.e., SEQ ID NOs:9
and 10). EgD8S is 100% identical and functionally equivalent to
"D8SF", as described in WO 2006/012325 and WO 2006/012326.
[0079] The term "mutant EgD8S" refers to a delta-8 desaturase of
the present invention that has at least one mutation with respect
to the synthetic delta-8 desaturase derived from Euglena gracilis
that is codon-optimized for expression in Yarrowia lipolytica
(i.e., EgD8S). Although "mutations" may include any deletions,
insertions and point mutations (or combinations thereof), preferred
embodiments of the mutant EgD8S are set forth in SEQ ID NO:2,
wherein: (i) SEQ ID NO:2 comprises at least one mutation selected
from the group consisting of: 4S to A, 5K to S, 12T to V, 16T to K,
17T to V, 54A to G, 55F to Y, 66P to Q, 67S to A, 108S to L, 117G
to A, 118Y to F, 120L to M, 121M to L, 125Q to H, 126M to L, 132V
to L, 133L to V, 162L to V, 163V to L, 170G to A, 171L to V, 279T
to L, 280L to T, 293L to M, 346I to V, 347I to L, 348T to S, 407A
to S, 408V to Q, 418A to G, 419G to A and 422L to Q, wherein the
mutations are defined with respect to the synthetic codon-optimized
sequence of EgD8S (i.e., SEQ ID NO:10); and (ii) SEQ ID NO:2 is not
100% identical to SEQ ID NO:10. In more preferred embodiments, the
mutant EgD8S has at least about 10-18 mutations with respect to the
synthetic codon-optimized sequence of EgD8S, more preferably at
least about 19-25 mutations, and most preferably at least about
26-33 mutations with respect to synthetic codon-optimized sequence
of EgD8S (i.e., SEQ ID NO:10). In still another embodiment, the
delta-8 desaturase activity of the mutant EgD8S is at least about
functionally equivalent to the delta-8 desaturase activity of the
synthetic codon-optimized EgD8S (SEQ ID NO:10).
[0080] A mutant EgD8S is "at least about functionally equivalent"
to EgD8S when enzymatic activity and specific selectivity of the
mutant EgD8S sequence are comparable to that of EgD8S, despite
differing polypeptide sequences. Thus, a functionally equivalent
mutant EgD8S sequence will possess delta-8 desaturase activity that
is not substantially reduced with respect to that of EgD8S when the
"conversion efficiency" of each enzyme is compared (i.e., a mutant
EgD8S will have at least about 50%, preferably at least about 75%,
more preferably at least about 85%, and most preferably at least
about 95% of the enzymatic activity of EgD8S). In more preferred
embodiments, the mutant EgD8S will have increased enzymatic
activity and specific selectivity when compared to that of EgD8S
(i.e., at least about 105%, more preferably at least about 115% and
most preferably at least about 125% of the enzymatic activity of
EgD8S).
[0081] The terms "conversion efficiency" and "percent substrate
conversion" refer to the efficiency by which a particular enzyme
(e.g., a desaturase) can convert substrate to product. The
conversion efficiency is measured according to the following
formula: ([product]/[substrate+product])*100, where `product`
includes the immediate product and all products in the pathway
derived from it.
[0082] The term "elongase system" refers to a suite of four enzymes
that are responsible for elongation of a fatty acid carbon chain to
produce a fatty acid that is 2 carbons longer than the fatty acid
substrate that the elongase system acts upon. More specifically,
the process of elongation occurs in association with fatty acid
synthase, whereby CoA is the acyl carrier (Lassner et al., The
Plant Cell, 8:281-292 (1996)). In the first step, which has been
found to be both substrate-specific and also rate-limiting,
malonyl-CoA is condensed with a long-chain acyl-CoA to yield
CO.sub.2 and a .beta.-ketoacyl-CoA (where the acyl moiety has been
elongated by two carbon atoms). Subsequent reactions include
reduction to .beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA
and a second reduction to yield the elongated acyl-CoA. Examples of
reactions catalyzed by elongase systems are the conversion of GLA
to DGLA, STA to ETA and EPA to DPA.
[0083] For the purposes herein, an enzyme catalyzing the first
condensation reaction (i.e., conversion of malonyl-CoA to
.beta.-ketoacyl-CoA) will be referred to generically as an
"elongase". In general, the substrate selectivity of elongases is
somewhat broad but segregated by both chain length and the degree
of unsaturation. Accordingly, elongases can have different
specificities. For example, a C.sub.14/16 elongase will utilize a
C.sub.14 substrate (e.g., myristic acid), a C.sub.16/18 elongase
will utilize a C.sub.16 substrate (e.g., palmitate), a C.sub.18/20
elongase will utilize a C.sub.18 substrate (e.g., GLA, STA) and a
C.sub.20/22 elongase will utilize a C.sub.20 substrate (e.g., EPA).
In like manner, a delta-9 elongase is able to catalyze the
conversion of LA and ALA to EDA and ETrA, respectively (e.g., WO
2002/077213). It is important to note that some elongases have
broad specificity and thus a single enzyme may be capable of
catalyzing several elongase reactions (e.g., thereby acting as both
a C.sub.16/18 elongase and a C.sub.18/20 elongase).
[0084] The term "delta-9 elongase/delta-8 desaturase pathway"
refers to a biosynthetic pathway for production of long-chain
PUFAs, said pathway minimally comprising a delta-9 elongase and a
delta-8 desaturase and thereby enabling biosynthesis of DGLA and/or
ETA from LA and ALA, respectively. This pathway may be advantageous
in some embodiments, as the biosynthesis of GLA and/or STA is
excluded.
[0085] The term "amino acid" will refer to the basic chemical
structural unit of a protein or polypeptide. The amino acids are
identified by either the one-letter code or the three-letter codes
for amino acids, in conformity with the IUPAC-IYUB standards
described in Nucleic Acids Research, 13:3021-3030 (1985) and in the
Biochemical Journal, 219 (2):345-373 (1984), which are herein
incorporated by reference.
[0086] The term "conservative amino acid substitution" refers to a
substitution of an amino acid residue in a given protein with
another amino acid, without altering the chemical or functional
nature of that protein. For example, it is well known in the art
that alterations in a gene that result in the production of a
chemically equivalent amino acid at a given site (but do not affect
the structural and functional properties of the encoded, folded
protein) are common. For the purposes of the present invention,
"conservative amino acid substitutions" are defined as exchanges
within one of the following five groups: [0087] 1. Small aliphatic,
nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro
[P], Gly [G]); [0088] 2. Polar, negatively charged residues and
their amides: Asp [D], Asn [N], Glu [E], Gln [Q]; [0089] 3. Polar,
positively charged residues: His [H], Arg [R], Lys [K]; [0090] 4.
Large aliphatic, nonpolar residues: Met [M], Leu [L], Ile [I], Val
[V] (Cys [C]); and [0091] 5. Large aromatic residues: Phe [F], Tyr
[Y], Trp [W]. Thus, Ala, a slightly hydrophobic amino acid, may be
substituted by another less hydrophobic residue (e.g., Gly).
Similarly, changes which result in substitution of one negatively
charged residue for another (e.g., Asp for Glu) or one positively
charged residue for another (e.g., Lys for Arg) can also be
expected to produce a functionally equivalent product. As such,
conservative amino acid substitutions generally maintain: 1.) the
structure of the polypeptide backbone in the area of the
substitution; 2.) the charge or hydrophobicity of the molecule at
the target site; or 3.) the bulk of the side chain. Additionally,
in many cases, alterations of the N-terminal and C-terminal
portions of the protein molecule would also not be expected to
alter the activity of the protein.
[0092] The term "non-conservative amino acid substitution" refers
to an amino acid substitution that is generally expected to produce
the greatest change in protein properties. Thus, for example, a
non-conservative amino acid substitution would be one whereby: 1.)
a hydrophilic residue is substituted for/by a hydrophobic residue
(e.g., Ser or Thr for/by Leu, Ile, Val); 2.) a Cys or Pro is
substituted for/by any other residue; 3.) a residue having an
electropositive side chain is substituted for/by an electronegative
residue (e.g., Lys, Arg or His for/by Asp or Glu); or, 4.) a
residue having a bulky side chain is substituted for/by one not
having a side chain (e.g., Phe for/by Gly). Sometimes,
non-conservative amino acid substitutions between two of the five
groups will not affect the activity of the encoded protein.
[0093] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", "nucleic acid fragment" and "isolated
nucleic acid fragment" are used interchangeably herein. A
polynucleotide may be a polymer of RNA or DNA that is single- or
double-stranded, that optionally contains synthetic, non-natural or
altered nucleotide bases. A polynucleotide in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA, synthetic DNA, or mixtures thereof.
[0094] A nucleic acid fragment is "hybridizable" to another nucleic
acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a
single-stranded form of the nucleic acid fragment can anneal to the
other nucleic acid fragment under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory Cold Spring Harbor,
N.Y. (1989), particularly Chapter 11 and Table 11.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. Stringency conditions can be adjusted to screen for
moderately similar fragments (such as homologous sequences from
distantly related organisms), to highly similar fragments (such as
genes that duplicate functional enzymes from closely related
organisms). Post-hybridization washes determine stringency
conditions. One set of preferred conditions uses a series of washes
starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min,
then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30
min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at
50.degree. C. for 30 min. A more preferred set of stringent
conditions uses higher temperatures in which the washes are
identical to those above except for the temperature of the final
two 30 min washes in 0.2.times.SSC, 0.5% SDS was increased to
60.degree. C. Another preferred set of highly stringent conditions
uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
An additional set of stringent conditions include hybridization at
0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC,
0.1% SDS followed by 0.1.times.SSC, 0.1% SDS, for example.
[0095] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferably a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least about 30 nucleotides.
Furthermore, the skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as length of the probe.
[0096] A "substantial portion" of an amino acid or nucleotide
sequence is that portion comprising enough of the amino acid
sequence of a polypeptide or the nucleotide sequence of a gene to
putatively identify that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)). In
general, a sequence of ten or more contiguous amino acids or thirty
or more nucleotides is necessary in order to putatively identify a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene specific oligonucleotide probes comprising 20-30 contiguous
nucleotides may be used in sequence-dependent methods of gene
identification (e.g., Southern hybridization) and isolation (e.g.,
in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12-15 bases may be
used as amplification primers in PCR in order to obtain a
particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence
comprises enough of the sequence to specifically identify and/or
isolate a nucleic acid fragment comprising the sequence. The
instant specification teaches the complete amino acid and
nucleotide sequence encoding one or more particular delta-8
desaturase proteins. The skilled artisan, having the benefit of the
sequences as reported herein, may now use all or a substantial
portion of the disclosed sequences for purposes known to those
skilled in this art. Accordingly, the instant invention comprises
the complete sequences as reported in the accompanying Sequence
Listing, as well as substantial portions of those sequences as
defined above.
[0097] The term "complementary" is used to describe the
relationship between nucleotide bases that are capable of
hybridizing to one another. For example, with respect to DNA,
adenosine is complementary to thymine and cytosine is complementary
to guanine. Accordingly, the invention herein also includes
isolated nucleic acid fragments that are complementary to the
complete sequences as reported in the accompanying Sequence
Listing, as well as those substantially similar nucleic acid
sequences.
[0098] The terms "homology", "homologous", "substantially similar"
and "corresponding substantially" are used interchangeably herein.
They refer to nucleic acid fragments wherein changes in one or more
nucleotide bases do not affect the ability of the nucleic acid
fragment to mediate gene expression or produce a certain phenotype.
These terms also refer to modifications of the nucleic acid
fragments of the instant invention such as deletion or insertion of
one or more nucleotides that do not substantially alter the
functional properties of the resulting nucleic acid fragment
relative to the initial, unmodified fragment. It is therefore
understood, as those skilled in the art will appreciate, that the
invention encompasses more than the specific exemplary
sequences.
[0099] Moreover, the skilled artisan recognizes that substantially
similar nucleic acid sequences encompassed by this invention are
also defined by their ability to hybridize, under moderately
stringent conditions (e.g., 0.5.times.SSC, 0.1% SDS, 60.degree. C.)
with the sequences exemplified herein, or to any portion of the
nucleotide sequences disclosed herein and which are functionally
equivalent to any of the nucleic acid sequences disclosed
herein.
[0100] "Codon degeneracy" refers to the nature in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment that encodes
all or a substantial portion of the amino acid sequence encoding
the instant delta-8 desaturase polypeptides as set forth in SEQ ID
NOs:2, and 12. The skilled artisan is well aware of the
"codon-bias" exhibited by a specific host cell in usage of
nucleotide codons to specify a given amino acid. Therefore, when
synthesizing a gene for improved expression in a host cell, it is
desirable to design the gene such that its frequency of codon usage
approaches the frequency of preferred codon usage of the host
cell.
[0101] "Chemically synthesized", as related to a sequence of DNA,
means that the component nucleotides were assembled in vitro.
Manual chemical synthesis of DNA may be accomplished using
well-established procedures or, automated chemical synthesis can be
performed using one of a number of commercially available machines.
"Synthetic genes" can be assembled from oligonucleotide building
blocks that are chemically synthesized using procedures known to
those skilled in the art. These building blocks are ligated and
annealed to form gene segments that are then enzymatically
assembled to construct the entire gene. Accordingly, the genes can
be tailored for optimal gene expression based on optimization of
nucleotide sequence to reflect the codon bias of the host cell. The
skilled artisan appreciates the likelihood of successful gene
expression if codon usage is biased towards those codons favored by
the host. Determination of preferred codons can be based on a
survey of genes derived from the host cell, where sequence
information is available.
[0102] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, and that may refer to the coding region alone or
may include regulatory sequences preceding (5' non-coding
sequences) and following (3' non-coding sequences) the coding
sequence. "Native gene" refers to a gene as found in nature with
its own regulatory sequences. "Chimeric gene" refers any gene that
is not a native gene, comprising regulatory and coding sequences
that are not found together in nature. Accordingly, a chimeric gene
may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of an organism. A
"foreign" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, native genes introduced into a new location
within the native host, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure. A "codon-optimized gene" is a gene having its frequency
of codon usage designed to mimic the frequency of preferred codon
usage of the host cell.
[0103] An "allele" is one of several alternative forms of a gene
occupying a given locus on a chromosome. When all the alleles
present at a given locus on a chromosome are the same that plant is
homozygous at that locus. If the alleles present at a given locus
on a chromosome differ, that plant is heterozygous at that
locus.
[0104] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Suitable regulatory sequences" refer
to nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include, but are not limited to:
promoters, translation leader sequences, introns, polyadenylation
recognition sequences, RNA processing sites, effector binding sites
and stem-loop structures.
[0105] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. The
promoter sequence may consist of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a DNA sequence that can stimulate
promoter activity, and may be an innate element of the promoter or
a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic DNA segments. It is understood by those skilled in the
art that different promoters may direct the expression of a gene in
different tissues or cell types, or at different stages of
development, or in response to different environmental or
physiological conditions. It is further recognized that since in
most cases the exact boundaries of regulatory sequences have not
been completely defined, DNA fragments of some variation may have
identical promoter activity. Promoters that cause a gene to be
expressed in most cell types at most times are commonly referred to
as "constitutive promoters". New promoters of various types useful
in plant cells are constantly being discovered; numerous examples
may be found in the compilation by Okamuro, J. K., and Goldberg, R.
B. Biochemistry of Plants, 15:1-82 (1989).
[0106] "Translation leader sequence" refers to a polynucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner, R. and Foster, G. D., Mol. Biotechnol.,
3:225-236 (1995)).
[0107] The terms "3' non-coding sequences", "transcription
terminator" and "termination sequences" refer to DNA sequences
located downstream of a coding sequence. This includes
polyadenylation recognition sequences and other sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression. The polyadenylation signal is usually characterized by
affecting the addition of polyadenylic acid tracts to the 3' end of
the mRNA precursor. The 3' region can influence the transcription,
RNA processing or stability, or translation of the associated
coding sequence. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht, I. L., et al. (Plant Cell, 1:671-680
(1989)).
[0108] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript. A RNA transcript is
referred to as the mature RNA when it is a RNA sequence derived
from post-transcriptional processing of the primary transcript.
"Messenger RNA" or "mRNA" refers to the RNA that is without introns
and that can be translated into protein by the cell. "cDNA" refers
to a DNA that is complementary to, and synthesized from, a mRNA
template using the enzyme reverse transcriptase. The cDNA can be
single-stranded or converted into double-stranded form using the
Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA
transcript that includes the mRNA and can be translated into
protein within a cell or in vitro. "Antisense RNA" refers to a RNA
transcript that is complementary to all or part of a target primary
transcript or mRNA, and that blocks the expression of a target gene
(U.S. Pat. No. 5,107,065; WO 99/28508). The complementarity of an
antisense RNA may be with any part of the specific gene transcript,
i.e., at the 5' non-coding sequence, 3' non-coding sequence,
introns, or the coding sequence. "Functional RNA" refers to
antisense RNA, ribozyme RNA, or other RNA that may not be
translated but yet has an effect on cellular processes. The terms
"complement" and "reverse complement" are used interchangeably
herein with respect to mRNA transcripts, and are meant to define
the antisense RNA of the message.
[0109] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is regulated by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in a sense or antisense orientation.
[0110] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes that are not part of
the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Expression
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that allow for
enhanced expression of that gene in a foreign host.
[0111] The terms "recombinant construct", "expression construct",
"chimeric construct", "construct", and "recombinant DNA construct"
are used interchangeably herein. A recombinant construct comprises
an artificial combination of nucleic acid fragments, e.g.,
regulatory and coding sequences that are not found together in
nature. For example, a chimeric construct may comprise regulatory
sequences and coding sequences that are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source, but arranged in a manner different than that found
in nature. Such a construct may be used by itself or may be used in
conjunction with a vector. If a vector is used, then the choice of
vector is dependent upon the method that will be used to transform
host cells as is well known to those skilled in the art. For
example, a plasmid vector can be used. The skilled artisan is well
aware of the genetic elements that must be present on the vector in
order to successfully transform, select and propagate host cells
comprising any of the isolated nucleic acid fragments of the
invention. The skilled artisan will also recognize that different
independent transformation events will result in different levels
and patterns of expression (Jones et al., EMBO J. 4:2411-2418
(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)),
and thus that multiple events must be screened in order to obtain
strains or lines displaying the desired expression level and
pattern. Such screening may be accomplished by Southern analysis of
DNA blots (Southern, J. Mol. Biol., 98:503 (1975)), Northern
analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl.,
618(1-2):133-145 (1993)), Western and/or Elisa analyses of protein
expression, phenotypic analysis or GC analysis of the PUFA
products, among others.
[0112] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragments of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0113] "Mature" protein refers to a post-translationally processed
polypeptide (i.e., one from which any pre- or propeptides present
in the primary translation product have been removed). "Precursor"
protein refers to the primary product of translation of mRNA (i.e.,
with pre- and propeptides still present). Pre- and propeptides may
be, but are not limited to, intracellular localization signals.
[0114] "Transformation" refers to the transfer of a nucleic acid
fragment into a genome of a host organism, including both nuclear
and organellar genomes, resulting in genetically stable
inheritance. In contrast, "transient transformation" refers to the
transfer of a nucleic acid fragment into the nucleus, or
DNA-containing organelle, of a host organism resulting in gene
expression without integration or stable inheritance. Thus, the
nucleic acid molecule used for transformation may be a plasmid that
replicates autonomously, for example, or, it may integrate into the
genome of the host organism. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0115] As used herein, "transgenic" refers to a plant or a cell
which comprises within its genome a heterologous polynucleotide.
Preferably, the heterologous polynucleotide is stably integrated
within the genome such that the polynucleotide is passed on to
successive generations. The heterologous polynucleotide may be
integrated into the genome alone or as part of an expression
construct. Transgenic is used herein to include any cell, cell
line, callus, tissue, plant part or plant, the genotype of which
has been altered by the presence of heterologous nucleic acid
including those transgenics initially so altered as well as those
created by sexual crosses or asexual propagation from the initial
transgenic. The term "transgenic" as used herein does not encompass
the alteration of the genome (chromosomal or extra-chromosomal) by
conventional plant breeding methods or by naturally occurring
events such as random cross-fertilization, non-recombinant viral
infection, non-recombinant bacterial transformation,
non-recombinant transposition, or spontaneous mutation.
[0116] The term "plant" refers to whole plants, plant organs, plant
tissues, seeds, plant cells, seeds and progeny of the same. Plant
cells include, without limitation, cells from seeds, suspension
cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen and
microspores.
[0117] "Progeny" comprises any subsequent generation of a
plant.
[0118] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of the target
protein. "Co-suppression" refers to the production of sense RNA
transcripts capable of suppressing the expression of identical or
substantially similar foreign or endogenous genes (U.S. Pat. No.
5,231,020). Co-suppression constructs in plants previously have
been designed by focusing on overexpression of a nucleic acid
sequence having homology to an endogenous mRNA, in the sense
orientation, which results in the reduction of all RNA having
homology to the overexpressed sequence (Vaucheret et al., Plant J.,
16:651-659 (1998); Gura, Nature, 404:804-808 (2000)). The overall
efficiency of this phenomenon is low, and the extent of the RNA
reduction is widely variable. Subsequent work has described the use
of "hairpin" structures that incorporate all, or part, of a mRNA
encoding sequence in a complementary orientation that results in a
potential "stem-loop" structure for the expressed RNA (WO 99/53050;
WO 02/00904). This increases the frequency of co-suppression in the
recovered transgenic plants. Another variation describes the use of
plant viral sequences to direct the suppression, or "silencing", of
proximal mRNA encoding sequences (WO 98/36083). Both of these
co-suppressing phenomena have not been elucidated mechanistically,
although genetic evidence has begun to unravel this complex
situation (Elmayan et al., Plant Cell, 10:1747-1757 (1998)).
[0119] The term "oleaginous" refers to those organisms that tend to
store their energy source in the form of lipid (Weete, In: Fungal
Lipid Biochemistry, 2nd Ed., Plenum, 1980). Generally, the cellular
oil or TAG content of these microorganisms follows a sigmoid curve,
wherein the concentration of lipid increases until it reaches a
maximum at the late logarithmic or early stationary growth phase
and then gradually decreases during the late stationary and death
phases (Yongmanitchai and Ward, Appl. Environ. Microbiol.,
57:419-25 (1991)).
[0120] The term "oleaginous yeast" refers to those microorganisms
classified as yeasts that can make oil. It is not uncommon for
oleaginous microorganisms to accumulate in excess of about 25% of
their dry cell weight as oil. Examples of oleaginous yeast include,
but are no means limited to, the following genera: Yarrowia,
Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon
and Lipomyces.
[0121] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in: 1.)
Computational Molecular Biology (Lesk, A. M., Ed.) Oxford
University: NY (1988); 2.) Biocomputing: Informatics and Genome
Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular
Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence
Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY
(1991). Preferred methods to determine identity are designed to
give the best match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs.
[0122] The "Clustal V method of alignment" corresponds to the
alignment method labeled Clustal V (described by Higgins and Sharp,
CABIOS. 5:151-153 (1989); Higgins, D. G. et al. (1992) Comput.
Appl. Biosci. 8:189-191) and found in the MegAlign.TM. program of
the LASERGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.). Sequence alignments and percent identity
calculations may be performed using the MegAlign.TM. program.
Multiple alignment of the sequences is performed using the Clustal
method of alignment (Higgins and Sharp, CABIOS, 5:151153 (1989);
Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992))
with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10),
unless otherwise specified. Default parameters for pairwise
alignments and calculation of percent identity of protein sequences
using the Clustal V method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5
and DIAGONALS SAVED=5. After alignment of the sequences using the
Clustal program, it is possible to obtain a "percent identity" by
viewing the "sequence distances" table in the same program.
[0123] The "Clustal W method of alignment" corresponds to the
alignment method labeled Clustal W (described by Thompson et al.,
Nucleic Acids Res. 22:4673-4680 (1994)) and found in the
MegAlign.TM. v5.07 program of the LASERGENE bioinformatics
computing suite (DNASTAR Inc., Madison, Wis.). Default parameters
for multiple alignments and calculation of percent identity of
protein sequences are GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DELAY
DIVERGENCE SEQS(%)=30, DNA TRANSITION WEIGHT=0.50, protein weight
matrix=Gonnet series and DNA weight matrix=IUB, unless otherwise
specified. Default parameters for pairwise alignments and
calculation of percent identity of protein sequences are GAP
PENALTY=10, GAP LENGTH PENALTY=0.1, protein weight matrix=Gonnet
250 and DNA weight matrix=IUB, unless otherwise specified.
[0124] "BLASTN method of alignment" is an algorithm provided by the
National Center for Biotechnology Information (NCBI) to compare
nucleotide sequences using default parameters.
[0125] It is well understood by one skilled in the art that many
levels of sequence identity are useful in identifying polypeptides,
from other species, wherein such polypeptides have the same or
similar function or activity. Useful examples of percent identities
include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95%, or any integer percentage from 50% to 100%.
Indeed, any integer amino acid identity from 50% to 100% may be
useful in describing the present invention, such as 51%, 52%, 53%,
54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, of interest is any
full-length or partial complement of this isolated nucleotide
fragment.
[0126] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include, but is not limited to: (1)
the GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.); (2) BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol., 215:403-410 (1990)); (3) DNASTAR
(DNASTAR Inc., Madison, Wis.); (4) Sequencher (Gene Codes
Corporation, Ann Arbor, Mich.); and (5) the FASTA program
incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.
Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,
111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within
the context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters that
originally load with the software when first initialized.
[0127] Suitable nucleic acid fragments (isolated polynucleotides of
the present invention) encode polypeptides that are at least about
85% identical to the amino acid sequences reported herein. More
preferred nucleic acid fragments encode amino acid sequences that
are at least about 90% identical to the amino acid sequences
reported herein while most preferred are nucleic acid fragments
that encode amino acid sequences that are at least about 95%
identical. It is well understood by one skilled in the art that
many levels of sequence identity are useful in identifying
polypeptides from other species, wherein such polypeptides have the
same or similar function or activity; although preferred ranges are
described above, any integer percentage from 85% to 100% is useful
for the purposes herein.
[0128] Suitable nucleic acid fragments not only have the above
homologies but typically encode a polypeptide having at least 50
amino acids, preferably at least 100 amino acids, more preferably
at least 150 amino acids, still more preferably at least 200 amino
acids, and most preferably at least 250 amino acids.
[0129] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter
"Maniatis"); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold
Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current
Protocols in Molecular Biology, published by Greene Publishing
Assoc. and Wiley-Interscience (1987).
An Overview: Biosynthesis of Omega Fatty Acids and
Triacylglycerols
[0130] The metabolic process wherein oleic acid is converted to
omega-3/omega-6 fatty acids involves elongation of the carbon chain
through the addition of carbon atoms and desaturation of the
molecule through the addition of double bonds. This requires a
series of special desaturation and elongation enzymes present in
the endoplasmic reticulim membrane. However, as seen in FIG. 1 and
as described below, there are often multiple alternate pathways for
production of a specific omega-3/omega-6 fatty acid.
[0131] Specifically, all pathways require the initial conversion of
oleic acid to LA, the first of the omega-6 fatty acids, by a
delta-12 desaturase. Then, using the "delta-9 elongase/delta-8
desaturase pathway", omega-6 fatty acids are formed as follows: (1)
LA is converted to EDA by a delta-9 elongase; (2) EDA is converted
to DGLA by a delta-8 desaturase; and (3) DGLA is converted to ARA
by a delta-5 desaturase. Alternatively, the "delta-9
elongase/delta-8 desaturase pathway" can be utilized for formation
of omega-3 fatty acids as follows: (1) LA is converted to ALA, the
first of the omega-3 fatty acids, by a delta-15 desaturase; (2) ALA
is converted to ETrA by a delta-9 elongase; (3) ETrA is converted
to ETA by a delta-8 desaturase; (4) ETA is converted to EPA by a
delta-5 desaturase; (5) EPA is converted to DPA by a C.sub.20/22
elongase; and (6) DPA is converted to DHA by a delta-4 desaturase.
Optionally, omega-6 fatty acids may be converted to omega-3 fatty
acids; for example, ETA and EPA are produced from DGLA and ARA,
respectively, by delta-17 desaturase activity.
[0132] Alternate pathways for the biosynthesis of omega-3/omega-6
fatty acids utilize a delta-6 desaturase and C.sub.18/20 elongase
(i.e., the "delta-6 desaturase/delta-6 elongase pathway"). More
specifically, LA and ALA may be converted to GLA and STA,
respectively, by a delta-6 desaturase; then, a C.sub.18/20 elongase
converts GLA to DGLA and/or STA to ETA.
[0133] It is contemplated that the particular functionalities
required to be expressed in a specific host organism for production
of omega-3/omega-6 fatty acids will depend on the host cell (and
its native PUFA profile and/or desaturase/elongase profile), the
availability of substrate, and the desired end product(s). For
example, expression of the delta-9 elongase/delta-8 desaturase
pathway may be preferred in some embodiments, as opposed to
expression of the delta-6 desaturase/delta-6 elongase pathway,
since PUFAs produced via the former pathway are devoid of GLA.
[0134] One skilled in the art will be able to identify various
candidate genes encoding each of the enzymes desired for
omega-3/omega-6 fatty acid biosynthesis. Useful desaturase and
elongase sequences may be derived from any source, e.g., isolated
from a natural source (from bacteria, algae, fungi, plants,
animals, etc.), produced via a semi-synthetic route or synthesized
de novo.
[0135] Although the particular source of the desaturase and
elongase genes introduced into the host is not critical,
considerations for choosing a specific polypeptide having
desaturase or elongase activity include: 1.) the substrate
specificity of the polypeptide; 2.) whether the polypeptide or a
component thereof is a rate-limiting enzyme; 3.) whether the
desaturase or elongase is essential for synthesis of a desired
PUFA; and/or 4.) co-factors required by the polypeptide. The
expressed polypeptide preferably has parameters compatible with the
biochemical environment of its location in the host cell (see WO
2004/101757).
[0136] In additional embodiments, it will also be useful to
consider the conversion efficiency of each particular desaturase
and/or elongase. More specifically, since each enzyme rarely
functions with 100% efficiency to convert substrate to product, the
final lipid profile of un-purified oils produced in a host cell
will typically be a mixture of various PUFAs consisting of the
desired omega-3/omega-6 fatty acid, as well as various upstream
intermediary PUFAs. Thus, consideration of each enzyme's conversion
efficiency is also an important variable when optimizing
biosynthesis of a desired fatty acid that must be considered in
light of the final desired lipid profile of the product.
[0137] With each of the considerations above in mind, candidate
genes having the appropriate desaturase and elongase activities
(e.g., delta-6 desaturases, C.sub.18/20 elongases, delta-5
desaturases, delta-17 desaturases, delta-15 desaturases, delta-9
desaturases, delta-12 desaturases, C.sub.14/16 elongases,
C.sub.16/18 elongases, delta-9 elongases, delta-8 desaturases,
delta-4 desaturases and C.sub.20/22 elongases) can be identified
according to publicly available literature (e.g., GenBank), the
patent literature, and experimental analysis of organisms having
the ability to produce PUFAs. These genes will be suitable for
introduction into a specific host organism, to enable or enhance
the organism's synthesis of PUFAs.
[0138] Once fatty acids are synthesized within an organism
(including saturated and unsaturated fatty acids and short-chain
and long-chain fatty acids), they may be incorporated into
triacylglycerides (TAGs). TAGs (the primary storage unit for fatty
acids, including PUFAs) are formed by a series of reactions that
involve: 1.) the esterification of one molecule of acyl-CoA to
glycerol-3-phosphate via an acyltransferase to produce
lysophosphatidic acid; 2.) the esterification of a second molecule
of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol
phosphate (commonly identified as phosphatidic acid); 3.) removal
of a phosphate by phosphatidic acid phosphatase to yield
1,2-diacylglycerol (DAG); and 4.) the addition of a third fatty
acid by the action of an acyltransferase to form TAG.
Sequence Identification of a Euglena gracilis Delta-8
Desaturase
[0139] Commonly owned WO 2006/012325 and WO 2006/012326 disclose a
Euglena gracilis delta-8 desaturase able ability to desaturate EDA
and EtrA (identified therein as "Eg5 and assigned SEQ ID NO:2). In
the present application, the Euglena gracilis delta-8 desaturase
described as "EgD8" (SEQ ID NOs:11 and 12 herein) is 100% identical
and equivalent to the nucleotide and amino acid sequences of
Eg5.
[0140] As is well known in the art, codon-optimization can be a
useful means to further optimize the expression of an enzyme in an
alternate host, since use of host-preferred codons can
substantially enhance the expression of the foreign gene encoding
the polypeptide. As such, a synthetic delta-8 desaturase derived
from Euglena gracilis and codon-optimized for expression in
Yarrowia lipolytica was also disclosed in WO 2006/012325 and WO
2006/012326 as SEQ ID NOs:112 and 113 (designated therein as
"D8SF"). Specifically, 207 bp (16.4%) of the 1263 bp coding region
were modified, corresponding to codon-optimization of 192 codons.
Additionally, "D8SF" had one additional valine amino acid inserted
between amino acid residues 1 and 2 of the wildtype Eg5; thus, the
total length of the codon-optimized desaturase is 422 amino acids.
Expression of the codon-optimized gene (i.e., "D8SF") in Y.
lipolytica demonstrated more efficient desaturation of EDA to DGLA
and/or ETrA to ETA than the wildtype gene (i.e., Eg5). In the
present application, the synthetic delta-8 desaturase derived from
Euglena gracilis and codon-optimized for expression in Y.
lipolytica described as "EgD8S" (SEQ ID NOs:9 and 10 herein) is
100% identical and equivalent to the nucleotide and amino acid
sequences of D8SF.
Engineering Targeted Mutations within the Synthetic Delta-8
Desaturase, Derived from Euglena gracilis and Codon-Optimized for
Expression in Yarrowia lipolytica
[0141] Methods for synthesizing sequences and bringing sequences
together are well established in the literature. Many techniques
are commonly employed in the literature to obtain mutations of
naturally occurring desaturase genes (wherein such mutations may
include deletions, insertions and point mutations, or combinations
thereof). This would permit production of a polypeptide having
desaturase activity, respectively, in vivo with more desirable
physical and kinetic parameters for function in the host cell such
as a longer half-life or a higher rate of production of a desired
PUFA. Or, if desired, the regions of a polypeptide of interest
(i.e., a desaturase) important for enzymatic activity can be
determined through routine mutagenesis, expression of the resulting
mutant polypeptides and determination of their activities. All such
mutant proteins and nucleotide sequences encoding them that are
derived from the wildtype (i.e., SEQ ID NOs:11 and 12) and
synthetic codon-optimized (SEQ ID NOs:9 and 10) delta-8 desaturase
described supra are within the scope of the present invention.
[0142] More specifically in the invention herein, mutant sequences
encoding delta-8 desaturases were synthetically engineered, by
making targeted mutations within the known, functional Euglena
gracilis delta-8 desaturase that was codon-optimized for expression
in Yarrowia lipolytica (i.e., "EgD8S", as set forth in SEQ ID NOs:9
and 10). The effect of each mutation on the delta-8 desaturase
activity of the resulting mutant EgD8S was screened. Although not
to be construed as limiting to the invention herein, a mutant EgD8S
enzyme (SEQ ID NO:2) was ultimately created comprising at least one
amino acid mutation (and up to about 33 amino acid mutations) with
respect to the synthetic codon-optimized EgD8S and having
functionally equivalent delta-8 desaturase activity, using the
methodology described below.
Creation of a Topological Model and Identification of Suitable
Amino Acid Sites for Mutation
[0143] General characteristics of delta-8 desaturases, based on
desaturase evolution, are well-described by P. Sperling et al.
(Prostaglandins Leukot. Essent. Fatty Acids, 68:73-95 (2003)).
Along with delta-6, delta-5 and delta-4 desaturases, delta-8
desaturases are known as long-chain PUFA "front-end" desaturases
(wherein desaturation occurs between a pre-existing double bond and
the carboxyl terminus of the fatty acid's acyl group, as opposed to
methyl-directed desaturation). These desaturases are characterized
by three histidine boxes [H(X).sub.3-4H (SEQ ID NOs:166 and 167),
H(X).sub.2-3HH (SEQ ID NOs:168 and 169) and H/Q(X).sub.2-3HH (SEQ
ID NOs:170 and 171)] and are members of the cytochrome b.sub.5
fusion superfamily, since they possess a fused cytochrome b.sub.5
domain at their N-terminus which serves as an electron donor. The
cytochrome b.sub.5 domain also contains an absolutely conserved
heme-binding motif (i.e., "HPGG") which has been shown to be
necessary for enzyme activity (J. A. Napier, et al., Prostaglandins
Leukot. Essent. Fatty Acids, 68:135-143 (2003); P. Sperling, et
al., supra). Finally, although the crystal structure of a
"front-end" desaturase is not yet available, hydropathy plots
reveal 4-6 membrane spanning helices that account for nearly 30% of
the amino acid sequence of these proteins.
[0144] Based on the generalizations above, the protein sequence of
EgD8S (SEQ ID NO:10) was specifically analyzed to enable creation
of a topological model (FIG. 2). As expected, EgD8S contained two
domains: an N-terminal cytochrome b.sub.5 domain (located between
amino acid residues 5 to 71 of SEQ ID NO:10) and a C-terminal
desaturase domain (located between amino acid residues 79 to 406 of
SEQ ID NO:10). Four membrane-spanning helices were identified at
amino acid residues 88-109, 113-132, 266-283 and 287-309 of SEQ ID
NO:10 (labeled as regions I, II, III and IV on FIG. 2), with both
the N- and C-termini located on the cytoplasmic side of the
membrane. Two additional hydrophobic regions were located at amino
acid residues 157-172 and 223-245. Finally, the three histidine
boxes were located between amino acid residues 146-150, 183-187 and
358-362, and the conserved heme-binding motif ("HPGG") was located
at amino acid residues 27-30.
[0145] Using the topological model, alignment of EgD8S with other
front-end desaturases and alignment of EgD8S's cytochrome b.sub.5
domain with other cytochrome b.sub.5 genes, 70 sites within EgD8S
were subsequently selected as possibly suitable for mutagenesis
(criteria for selection are described in detail in Example 2).
These sites corresponded to 126 individual amino acid mutations
(i.e., 57.9% conserved amino acid substitutions and 42.1%
non-conserved amino acid substitutions), as detailed in Table 7 of
Example 2.
Site-Directed Mutagenesis for Creation of EgD8S Mutants
[0146] Although a variety of approaches may be used for mutagenesis
of a delta-8 desaturase enzyme, based on the strategies herein it
was desirable to create specific point mutations within EgD8S using
oligonucleotide-mediated site-directed mutagenesis. Furthermore,
although numerous site-directed mutagenesis protocols exist (e.g.,
Ishii, T. M., et al., Methods Enzymol., 293:53-71 (1998); Ling M.
M. and B. H. Robinson, Anal. Biochem., 254:157-178 (1997); Braman
J. (ed.) In Vitro Mutagenesis Protocols. 2.sup.nd Ed., Humania:
Totowa, N.J. (2002); Kunkel T. A., et al., Methods Enzymol.,
154:367-382 (1987); Sawano A. and Miyawaki, A. Nucleic Acids Res.,
28:e78 (2000)), the QuikChange.RTM. site-directed mutagenesis kit
(Stratagene, La Jolla, Calif.) was selected for use based on its
facile implementation and high efficiency. Specifically, the kit
requires no specialized vectors, unique restriction sites, or
multiple transformations and allows site-specific mutation in
virtually any double-stranded plasmid. The basic procedure utilizes
a supercoiled double-stranded DNA vector with an insert of interest
and two synthetic oligonucleotide primers containing the desired
mutation. The oligonucleotide primers, each complementary to
opposite strands of the vector, are extended during temperature
cycling by a DNA polymerase. Incorporation of the oligonucleotide
primers generates a mutated plasmid containing staggered nicks.
Following temperature cycling, the product is treated with Dpn I
endonuclease (specific for methylated and hemi-methylated DNA) as a
means to digest the parental DNA template and to select for newly
synthesized mutant DNA. The nicked vector DNA containing the
desired mutations is then transformed and propagated in an
Escherichia coli host.
[0147] Using the techniques described above, the feasibility of
engineering a synthetic EgD8S (having multiple point mutations with
respect to EgD8S but maintaining functional equivalence with
respect to the enzyme's delta-8 desaturase activity) was then
tested. Specifically, selected individual point mutations were
introduced by site-directed mutagenesis into EgD8S (within a
plasmid construct comprising a chimeric FBAINm::EgD8S::XPR gene),
transformed into E. coli, and then screened for delta-8 desaturase
activity based on GC analyses.
[0148] The skilled person will be able to envision additional
screens for the selection of genes encoding proteins having delta-8
desaturase activity. For example, desaturase activity may be
demonstrated by assays in which a preparation containing an enzyme
is incubated with a suitable form of substrate fatty acid and
analyzed for conversion of this substrate to the predicted fatty
acid product. Alternatively, a DNA sequence proposed to encode a
desaturase protein may be incorporated into a suitable vector
construct and thereby expressed in cells of a type that do not
normally have an ability to desaturate a particular fatty acid
substrate. Activity of the desaturase enzyme encoded by the DNA
sequence can then be demonstrated by supplying a suitable form of
substrate fatty acid to cells transformed with a vector containing
the desaturase-encoding DNA sequence and to suitable control cells
(e.g., transformed with the empty vector alone). In such an
experiment, detection of the predicted fatty acid product in cells
containing the desaturase-encoding DNA sequence and not in control
cells establishes the desaturase activity.
[0149] Results from the experiment described above resulted in the
identification of some mutations that resulted in completely
non-functional mutant delta-8 desaturases having 0% delta-8
desaturase activity (e.g., simultaneous mutation of 48V to F and
49M to L or simultaneous mutation of 304G to F and 305F to G).
Despite this, .about.75% of the individual mutations tested did not
significantly diminish the mutant enzyme's delta-8 desaturase
activity as compared to the delta-8 desaturase activity of EgD8S.
More specifically, the following mutations were identified as
preferred mutations, wherein delta-8 desaturase activity was
functionally equivalent (i.e., 100%) or greater than that of EgD8S
(i.e., SEQ ID NO:10):
TABLE-US-00004 TABLE 4 Initial Preferred Mutations Within EgD8S
Sequence Mutations With Mutation Respect to EgD8S % Site (SEQ ID
NO: 10) Activity* M3 16T to K, 17T to V 100% M8 66P to Q, 67S to A
100% M12 407A to S, 408V to Q 100% M14 416Q to V, 417P to Y 100%
M16 108S to L 100% M19 122V to S 100% M38 54A to G, 55F to Y 100%
M39 64I to L, 65N to D 100% M40 69E to D, 70L to V 100% M41 75A to
G, 76V to L 100% M45 117G to A, 118Y to F 100% M46 132V to L, 133L
to V 100% M49 297F to V, 298V to L 100% M50 309I to V, 310V to I
100% M51 347I to L, 348T to S 100% M51B 346I to V, 347I to L, 348T
to S 100% M53 9L to V 100% M54 19D to E, 20V to I 100% M58 65N to Q
100% M63 279T to L, 280L to T 100% M68 162L to V, 163V to L 100%
M69 170G to A, 171L to V 100% M70 418A to G, 419G to A 100% M2 12T
to V 110% M22 127Y to Q 110% M26 293L to M 110% M6 59K to L 110% M1
4S to A, 5K to S 115% M21 125Q to H, 126M to L 120% M15 422L to Q
125% *"% Activity" refers to the delta-8 desaturase activity of
each mutant EgD8S with respect to the delta-8 desaturase activity
of EgD8S, as set forth as SEQ ID N0: 10.
[0150] It will be appreciated by one of skill in the art that the
useful mutant delta-8 desaturases of the present invention are not
limited to the 30 mutation combinations described above. For
example, although the mutation site described as M3 includes two
specific amino acid mutations (i.e., 16T to K and 17T to V), one
skilled in the art will expect that a single mutation of either 16T
to K or 17T to V will have utility in the design of a mutant
delta-8 desaturase whose delta-8 desaturase activity is at least
about functionally equivalent to the delta-8 desaturase activity of
the synthetic codon-optimized EgD8S. Thus, in actuality, Table 4
presents 52 single amino acid mutations that are useful for the
purposes herein, in the design of a mutant delta-8 desaturase
having delta-8 desaturase activity that is at least about
functionally equivalent to the delta-8 desaturase activity of SEQ
ID NO:10.
[0151] Based on the results above, experimental work was continued
in an effort to "stack" appropriate individual amino acid mutations
within the synthetic codon-optimized EgD8S sequence. This resulted
in creation of a mutant delta-8 desaturase as set forth in SEQ ID
NO:2 having "n" amino acid mutations, wherein "n" is any integer
from 1 to 33 inclusive (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33), and having delta-8 desaturase activity
comparable to that of EgD8S. Specifically, SEQ ID NO:2 comprises at
least one mutation selected from the group consisting of: 4S to A,
5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y, 66P to
Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M to L,
125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L,
170G to A, 171L to V, 279T to L, 280L to T, 293L to M, 346I to V,
347I to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A
and 422L to Q, wherein the mutations are defined with respect to
the synthetic codon-optimized sequence of EgD8S (i.e., SEQ ID
NO:10); wherein SEQ ID NO:2 is not 100% identical to SEQ ID NO:10;
and wherein the mutant EgD8S is at least about functionally
equivalent to EgD8S (SEQ ID NO:10). It will be appreciated by the
skilled person that each of the above mutations can be used in any
combination with one another. And, all such mutant proteins and
nucleotide sequences encoding them that are derived from EgD8
and/or EgD8S as described herein are within the scope of the
present invention.
[0152] In more preferred embodiments, the mutant EgD8S has at least
about 10-18 conservative and non-conservative amino acid
substitutions (i.e., mutations) with respect to the synthetic
codon-optimized sequence of EgD8S, more preferably at least about
19-25 conservative and non-conservative amino acid substitutions,
and most preferably at least about 26-33 conservative and
non-conservative amino acid substitutions with respect to the
synthetic codon-optimized sequence of EgD8S (i.e., SEQ ID NO:10).
Thus, for example, in one preferred embodiment mutant EgD8S-23 (SEQ
ID NO:4) comprises the following 24 amino acid mutations with
respect to the synthetic codon-optimized EgD8S sequence set forth
as SEQ ID NO:10: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V,
66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M,
121M to L, 125Q to H, 126M to L, 132V to L, 133 L to V, 162L to V,
163V to L, 293L to M, 407A to S, 408V to Q, 418A to G, 419G to A
and 422L to Q. Pairwise alignment of the mutant EgD8S-23 amino acid
sequence to the synthetic codon-optimized sequence of SEQ ID NO:10
using default parameters of Vector NTI.RTM.'s AlignX program
(Invitrogen Corporation, Carlsbad, Calif.) revealed 94.3% sequence
identity and 97.9% consensus between the two proteins over a length
of 422 amino acids.
[0153] In another preferred embodiment, mutant EgD8S-013 (SEQ ID
NO:6) comprises the following 28 amino acid mutations with respect
to the synthetic codon-optimized EgD8S sequence set forth as SEQ ID
NO:10: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G,
55F to Y, 66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L
to M, 121M to L, 125Q to H, 126M to L, 132V to L, 133L to V, 162L
to V, 163V to L, 170G to A, 171L to V, 293L to M, 407A to S, 408V
to Q, 418A to G, 419G to A and 422L to Q. Pairwise alignment of the
mutant EgD8S-013 amino acid sequence to the synthetic
codon-optimized sequence of SEQ ID NO:10 using default parameters
of Vector NTI.RTM.'s AlignX program revealed 93.4% sequence
identity and 97.9% consensus between the two proteins over a length
of 422 amino acids.
[0154] In another preferred embodiment, mutant EgD8S-015 (SEQ ID
NO:8) comprises the following 31 amino acid mutations with respect
to the synthetic codon-optimized EgD8S sequence set forth as SEQ ID
NO:10: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G,
55F to Y, 66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L
to M, 121M to L, 125Q to H, 126M to L, 162L to V, 163V to L, 170G
to A, 171L to V, 293L to M, 279T to L, 280L to T, 346I to V, 347I
to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A and
422L to Q. Pairwise alignment of the mutant EgD8S-015 amino acid
sequence to the synthetic codon-optimized sequence of SEQ ID NO:10
using default parameters of Vector NTI.RTM.'s AlignX program
revealed 92.7% sequence identity and 97.4% consensus between the
two proteins over a length of 422 amino acids.
[0155] Thus, in one embodiment, the present invention concerns an
isolated polynucleotide comprising: [0156] (a) a nucleotide
sequence encoding a mutant polypeptide having delta-8 desaturase
activity, wherein the mutant polypeptide has an amino acid sequence
as set forth in SEQ ID NO:2, wherein: [0157] (i) SEQ ID NO:2
comprises at least one mutation selected from the group consisting
of: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F
to Y, 66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to
M, 121M to L, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to
V, 163V to L, 170G to A, 171L to V, 279T to L, 280L to T, 293L to
M, 346I to V, 347I to L, 348T to S, 407A to S, 408V to Q, 418A to
G, 419G to A and 422L to Q, wherein the mutations are defined with
respect to the synthetic codon-optimized sequence of EgD8S (i.e.,
SEQ ID NO:10); and, [0158] (ii) SEQ ID NO:2 is not identical to SEQ
ID NO:10; or, [0159] (b) a complement of the nucleotide sequence,
wherein the complement and the nucleotide sequence consist of the
same number of nucleotides and are 100% complementary. In further
preferred embodiments, the delta-8 desaturase activity of SEQ ID
NO:2, as described above, is at least about functionally equivalent
to the delta-8 desaturase activity of SEQ ID NO:10.
Alternate Means of Mutagenesis for Creation of EgD8S Mutants
[0160] As is well known to one of skill in the art, in vitro
mutagenesis and selection or error prone PCR (Leung et al.,
Techniques, 1:11-15 (1989); Zhou et al., Nucleic Acids Res.,
19:6052-6052 (1991); Spee et al., Nucleic Acids Res., 21:777-778
(1993); Melnikov et al., Nucleic Acids Res., 27(4):1056-1062 (Feb.
15, 1999)) could also be employed as a means to obtain mutations of
naturally occurring desaturase genes, such as EgD8 or EgD8S,
wherein the mutations may include deletions, insertions and point
mutations, or combinations thereof. The principal advantage of
error-prone PCR is that all mutations introduced by this method
will be within the desired desaturase gene, and any change may be
easily controlled by changing the PCR conditions. Alternatively, in
vivo mutagenesis may be employed using commercially available
materials such as the E. coli XL1-Red strain and Epicurian coli
XL1-Red mutator strain from Stratagene (La Jolla, Calif.; Greener
and Callahan, Strategies, 7:32-34 (1994)). This strain is deficient
in three of the primary DNA repair pathways (mutS, mutD and mutT),
resulting in a mutation rate 5000-fold higher than that of
wild-type. In vivo mutagenesis does not depend on ligation
efficiency (as with error-prone PCR); however, a mutation may occur
at any region of the vector and the mutation rates are generally
much lower.
[0161] In other embodiments, it is contemplated that a mutant
delta-8 desaturase enzyme with altered or enhanced delta-8
desaturase activity may be constructed using the method of "gene
shuffling" (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S.
Pat. No. 5,830,721; and U.S. Pat. No. 5,837,458). The method of
gene shuffling is particularly attractive due to its facile
implementation and high rate of mutagenesis. The process of gene
shuffling involves the restriction of a gene of interest into
fragments of specific size in the presence of additional
populations of DNA regions of both similarity to (or difference to)
the gene of interest. This pool of fragments will then denature and
reanneal to create a mutated gene. The mutated gene is then
screened for altered activity.
[0162] Delta-8 desaturase sequences (i.e., wildtype, synthetic,
codon-optimized or mutant) may be mutated and screened for altered
or enhanced delta-8 desaturase activity by this method. The
sequences should be double-stranded and can be of various lengths
ranging from 50 bp to 10 kB. The sequences may be randomly digested
into fragments ranging from about 10 bp to 1000 bp, using
restriction endonucleases well known in the art (Maniatis, supra).
In addition to the full-length sequences, populations of fragments
that are hybridizable to all or portions of the sequence may be
added. Similarly, a population of fragments that are not
hybridizable to the wildtype sequence may also be added. Typically,
these additional fragment populations are added in about a 10- to
20-fold excess by weight as compared to the total nucleic acid.
Generally this process will allow generation of about 100 to 1000
different specific nucleic acid fragments in the mixture. The mixed
population of random nucleic acid fragments are denatured to form
single-stranded nucleic acid fragments and then reannealed. Only
those single-stranded nucleic acid fragments having regions of
homology with other single-stranded nucleic acid fragments will
reanneal. The random nucleic acid fragments may be denatured by
heating. One skilled in the art could determine the conditions
necessary to completely denature the double-stranded nucleic acid,
wherein the temperature is preferably from about 80.degree. C. to
about 100.degree. C. The nucleic acid fragments may be reannealed
by cooling, wherein the temperature is preferably from about
20.degree. C. to about 75.degree. C. Renaturation can be
accelerated by the addition of polyethylene glycol ("PEG") or salt,
wherein the salt concentration is preferably from about 0 mM to
about 200 mM. The annealed nucleic acid fragments are next
incubated in the presence of a nucleic acid polymerase and dNTP's
(i.e., dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may
be the Klenow fragment, the Taq polymerase or any other DNA
polymerase known in the art. The polymerase may be added to the
random nucleic acid fragments prior to annealing, simultaneously
with annealing or after annealing. The cycle of denaturation,
renaturation and incubation in the presence of polymerase is
repeated for a desired number of times. Preferably the cycle is
repeated from 2 to 50 times, and more preferably the sequence is
repeated from 10 to 40 times. The resulting nucleic acid is a
larger double-stranded polynucleotide of from about 50 bp to about
100 kB and may be screened for expression and altered delta-8
desaturase activity by standard cloning and expression protocols
(Maniatis, supra).
[0163] Irrespective of the method of mutagenesis, it is
contemplated that a mutant delta-8 desaturase having delta-8
desaturase activity at least about functionally equivalent to that
of EgD8 (SEQ ID NO:12) or EgD8S (SEQ ID NO:10) may be evolved as
set forth in SEQ ID NO:2, wherein: (i) SEQ ID NO:2 comprises at
least one mutation selected from the group consisting of: 4S to A,
5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y, 66P to
Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M to L,
125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L,
170G to A, 171L to V, 279T to L, 280L to T, 293L to M, 346I to V,
347I to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A
and 422L to Q, wherein the mutations are defined with respect to
the synthetic codon-optimized sequence of EgD8S (i.e., SEQ ID
NO:10); and, (ii) SEQ ID NO:2 is not 100% identical to SEQ ID
NO:10. Furthermore, it will be appreciated that the invention
encompasses not only the specific mutations described above, but
also those that allow for the substitution of chemically equivalent
amino acids. So, for example, where a substitution of an amino acid
with the aliphatic, nonpolar amino acid alanine is made, it will be
expected that the same site may be substituted with the chemically
equivalent amino acid serine.
[0164] In other embodiments, any of the delta-8 desaturase nucleic
acid fragments described herein may be used for creation of new and
improved fatty acid desaturases by domain swapping, wherein a
functional domain from any of the delta-8 desaturase nucleic acid
fragments is exchanged with a functional domain in an alternate
desaturase gene to thereby result in a novel protein.
Identification and Isolation of Homologs
[0165] Any of the instant desaturase sequences (i.e., those mutants
derived from EgD8 or EgD8S) or portions thereof may be used to
search for delta-8 desaturase homologs in the same or other
bacterial, algal, fungal, Oomycete or plant species using sequence
analysis software. In general, such computer software matches
similar sequences by assigning degrees of homology to various
substitutions, deletions and other modifications.
[0166] Alternatively, any of the instant desaturase sequences or
portions thereof may also be employed as hybridization reagents for
the identification of delta-8 homologs. The basic components of a
nucleic acid hybridization test include a probe, a sample suspected
of containing the gene or gene fragment of interest and a specific
hybridization method. Probes of the present invention are typically
single-stranded nucleic acid sequences that are complementary to
the nucleic acid sequences to be detected. Probes are
"hybridizable" to the nucleic acid sequence to be detected.
Although the probe length can vary from 5 bases to tens of
thousands of bases, typically a probe length of about 15 bases to
about 30 bases is suitable. Only part of the probe molecule need be
complementary to the nucleic acid sequence to be detected. In
addition, the complementarity between the probe and the target
sequence need not be perfect. Hybridization does occur between
imperfectly complementary molecules with the result that a certain
fraction of the bases in the hybridized region are not paired with
the proper complementary base.
[0167] Hybridization methods are well defined. Typically the probe
and sample must be mixed under conditions that will permit nucleic
acid hybridization. This involves contacting the probe and sample
in the presence of an inorganic or organic salt under the proper
concentration and temperature conditions. The probe and sample
nucleic acids must be in contact for a long enough time that any
possible hybridization between the probe and sample nucleic acid
may occur. The concentration of probe or target in the mixture will
determine the time necessary for hybridization to occur. The higher
the probe or target concentration, the shorter the hybridization
incubation time needed. Optionally, a chaotropic agent may be added
(e.g., guanidinium chloride, guanidinium thiocyanate, sodium
thiocyanate, lithium tetrachloroacetate, sodium perchlorate,
rubidium tetrachloroacetate, potassium iodide, cesium
trifluoroacetate). If desired, one can add formamide to the
hybridization mixture, typically 30-50% (v/v).
[0168] Various hybridization solutions can be employed. Typically,
these comprise from about 20 to 60% volume, preferably 30%, of a
polar organic solvent. A common hybridization solution employs
about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride,
about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES
or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g.,
sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL
(Pharmacia, Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about
250-500 kdal), and serum albumin. Also included in the typical
hybridization solution will be unlabeled carrier nucleic acids from
about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or
salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to
2% wt/vol glycine. Other additives may also be included, such as
volume exclusion agents that include a variety of polar
water-soluble or swellable agents (e.g., polyethylene glycol),
anionic polymers (e.g., polyacrylate or polymethylacrylate) and
anionic saccharidic polymers (e.g., dextran sulfate).
[0169] Nucleic acid hybridization is adaptable to a variety of
assay formats. One of the most suitable is the sandwich assay
format. The sandwich assay is particularly adaptable to
hybridization under non-denaturing conditions. A primary component
of a sandwich-type assay is a solid support. The solid support has
adsorbed to it or covalently coupled to it immobilized nucleic acid
probe that is unlabeled and complementary to one portion of the
sequence.
[0170] In additional embodiments, any of the delta-8 desaturase
nucleic acid fragments described herein (or any homologs identified
thereof) may be used to isolate genes encoding homologous proteins
from the same or other bacterial, algal, fungal, Oomycete or plant
species. Isolation of homologous genes using sequence-dependent
protocols is well known in the art. Examples of sequence-dependent
protocols include, but are not limited to: 1.) methods of nucleic
acid hybridization; 2.) methods of DNA and RNA amplification, as
exemplified by various uses of nucleic acid amplification
technologies [e.g., polymerase chain reaction (PCR), Mullis et al.,
U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et
al., Proc. Acad. Sci. USA, 82:1074 (1985); or strand displacement
amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. USA,
89:392 (1992)]; and 3.) methods of library construction and
screening by complementation.
[0171] For example, genes encoding similar proteins or polypeptides
to the delta-8 desaturases described herein could be isolated
directly by using all or a portion of the instant nucleic acid
fragments as DNA hybridization probes to screen libraries from
e.g., any desired marine euglenoid, yeast or fungus using
methodology well known to those skilled in the art (wherein those
organisms producing EDA or ETrA [or derivatives thereof] would be
preferred). Specific oligonucleotide probes based upon the instant
nucleic acid sequences can be designed and synthesized by methods
known in the art (Maniatis, supra). Moreover, the entire sequences
can be used directly to synthesize DNA probes by methods known to
the skilled artisan (e.g., random primers DNA labeling, nick
translation or end-labeling techniques), or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part of (or
full-length of) the instant sequences. The resulting amplification
products can be labeled directly during amplification reactions or
labeled after amplification reactions, and used as probes to
isolate full-length DNA fragments under conditions of appropriate
stringency.
[0172] Typically, in PCR-type amplification techniques, the primers
have different sequences and are not complementary to each other.
Depending on the desired test conditions, the sequences of the
primers should be designed to provide for both efficient and
faithful replication of the target nucleic acid. Methods of PCR
primer design are common and well known in the art (Thein and
Wallace, "The use of oligonucleotide as specific hybridization
probes in the Diagnosis of Genetic Disorders", in Human Genetic
Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50,
IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular
Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:
Current Methods and Applications. Humania: Totowa, N.J.).
[0173] Generally two short segments of the instant sequences may be
used in PCR protocols to amplify longer nucleic acid fragments
encoding homologous genes from DNA or RNA. PCR may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding eukaryotic genes.
[0174] Alternatively, the second primer sequence may be based upon
sequences derived from the cloning vector. For example, the skilled
artisan can follow the RACE protocol (Frohman et al., PNAS USA,
85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (Gibco/BRL, Gaithersburg, Md.), specific
3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA,
86:5673 (1989); Loh et al., Science, 243:217 (1989)).
Methods for Production of Various Omega-3 and/or Omega-6 Fatty
Acids
[0175] It is expected that introduction of chimeric genes encoding
the delta-8 desaturases described herein, under the control of the
appropriate promoters will result in increased production of DGLA
and/or ETA in the transformed host organism, respectively. As such,
the present invention encompasses a method for the direct
production of PUFAs comprising exposing a fatty acid substrate
(i.e., EDA or ETrA) to the desaturase enzymes described herein
(i.e., those mutants derived from EgD8 or EgD8S, or homologs
thereof), such that the substrate is converted to the desired fatty
acid product (i.e., DGLA and/or ETA).
[0176] More specifically, it is an object of the present invention
to provide a method for the production of DGLA in a host cell
(e.g., oleaginous yeast, soybean), wherein the host cell comprises:
[0177] a.) a recombinant construct encoding a delta-8 desaturase
polypeptide as set forth in SEQ ID NO:2 wherein SEQ ID NO:2 is not
100% identical to SEQ ID NO:10; and, [0178] b.) a source of EDA;
wherein the host cell is grown under conditions such that the
delta-8 desaturase is expressed and the EDA is converted to DGLA,
and wherein the DGLA is optionally recovered.
[0179] The person of skill in the art will recognize that the broad
substrate range of the delta-8 desaturase may additionally allow
for the use of the enzyme for the conversion of ETrA to ETA.
Accordingly the invention provides a method for the production of
ETA, wherein the host cell comprises: [0180] a.) a recombinant
construct encoding a delta-8 desaturase polypeptide as set forth in
SEQ ID NO:2 wherein SEQ ID NO:2 is not 100% identical to SEQ ID
NO:10; and, [0181] b.) a source of ETrA; wherein the host cell is
grown under conditions such that the delta-8 desaturase is
expressed and the ETrA is converted to ETA, and wherein the ETA is
optionally recovered.
[0182] Alternatively, each delta-8 desaturase gene and its
corresponding enzyme product described herein can be used
indirectly for the production of omega-3 fatty acids (see WO
2004/101757). Indirect production of omega-3/omega-6 PUFAs occurs
wherein the fatty acid substrate is converted indirectly into the
desired fatty acid product, via means of an intermediate step(s) or
pathway intermediate(s). Thus, it is contemplated that the delta-8
desaturases described herein (i.e., those mutants derived from EgD8
or EgD8S, or homologs thereof) may be expressed in conjunction with
additional genes encoding enzymes of the PUFA biosynthetic pathway
(e.g., delta-6 desaturases, C.sub.18/20 elongases, delta-17
desaturases, delta-15 desaturases, delta-9 desaturases, delta-12
desaturases, C.sub.14/16 elongases, C.sub.16/18 elongases, delta-9
elongases, delta-5 desaturases, delta-4 desaturases, C.sub.20/22
elongases) to result in higher levels of production of longer-chain
omega-3/omega-6 fatty acids (e.g., ARA, EPA, DPA and DHA). In
preferred embodiments, the delta-8 desaturases of the present
invention will minimally be expressed in conjunction with a delta-9
elongase (e.g., a delta-9 elongase as set forth in SEQ ID NO:173 or
SEQ ID NO:176). However, the particular genes included within a
particular expression cassette will depend on the host cell (and
its PUFA profile and/or desaturase/elongase profile), the
availability of substrate and the desired end product(s).
Plant Expression Systems, Cassettes & Vectors, and
Transformation
[0183] In one embodiment, this invention concerns a recombinant
construct comprising any one of the delta-8 desaturase
polynucleotides of the invention operably linked to at least one
regulatory sequence suitable for expression in a plant. A promoter
is a DNA sequence that directs the cellular machinery of a plant to
produce RNA from the contiguous coding sequence downstream (3') of
the promoter. The promoter region influences the rate,
developmental stage, and cell type in which the RNA transcript of
the gene is made. The RNA transcript is processed to produce mRNA
which serves as a template for translation of the RNA sequence into
the amino acid sequence of the encoded polypeptide. The 5'
non-translated leader sequence is a region of the mRNA upstream of
the protein coding region that may play a role in initiation and
translation of the mRNA. The 3' transcription
termination/polyadenylation signal is a non-translated region
downstream of the protein coding region that functions in the plant
cell to cause termination of the RNA transcript and the addition of
polyadenylate nucleotides to the 3' end of the RNA.
[0184] The origin of the promoter chosen to drive expression of the
delta-8 desaturase coding sequence is not important as long as it
has sufficient transcriptional activity to accomplish the invention
by expressing translatable mRNA for the desired nucleic acid
fragments in the desired host tissue at the right time. Either
heterologous or non-heterologous (i.e., endogenous) promoters can
be used to practice the invention. For example, suitable promoters
include, but are not limited to: the .alpha.-prime subunit of
.beta.-conglycinin promoter, the Kunitz trypsin inhibitor 3
promoter, the annexin promoter, the Gly1 promoter, the .beta.
subunit of .beta.-conglycinin promoter, the P34/Gly Bd m 30K
promoter, the albumin promoter, the Leg A1 promoter and the Leg A2
promoter.
[0185] The annexin, or P34, promoter is described in WO 2004/071178
(published Aug. 26, 2004). The level of activity of the annexin
promoter is comparable to that of many known strong promoters, such
as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.,
37:275-285 (1998); Battraw and Hall, Plant Mol. Biol., 15:527-538
(1990); Holtorf et al., Plant Mol. Biol., 29:637-646 (1995);
Jefferson et al., EMBO J., 6:3901-3907 (1987); Wilmink et al.,
Plant Mol. Biol., 28:949-955 (1995)); (2) the Arabidopsis oleosin
promoters (Plant et al., Plant Mol. Biol., 25:193-205 (1994); Li,
Texas A&M University Ph.D. dissertation, pp. 107-128 (1997));
(3) the Arabidopsis ubiquitin extension protein promoters (Callis
et al., J. Biol. Chem., 265(21):12486-93 (1990)); (4) a tomato
ubiquitin gene promoter (Rollfinke et al., Gene, 211(2):267-76
(1998)); (5) a soybean heat shock protein promoter (Schoffl et al.,
Mol. Gen. Genet., 217(2-3):246-53 (1989)); and, (6) a maize H3
histone gene promoter (Atanassova et al., Plant Mol. Biol.,
37(2):275-85 (1989)).
[0186] Another useful feature of the annexin promoter is its
expression profile in developing seeds. The annexin promoter is
most active in developing seeds at early stages (before 10 days
after pollination) and is largely quiescent in later stages. The
expression profile of the annexin promoter is different from that
of many seed-specific promoters, e.g., seed storage protein
promoters, which often provide highest activity in later stages of
development (Chen et al., Dev. Genet., 10:112-122 (1989);
Ellerstrom et al., Plant Mol. Biol., 32:1019-1027 (1996); Keddie et
al., Plant Mol. Biol., 24:327-340 (1994); Plant et al., (supra);
Li, (supra)). The annexin promoter has a more conventional
expression profile but remains distinct from other known seed
specific promoters. Thus, the annexin promoter will be a very
attractive candidate when overexpression, or suppression, of a gene
in embryos is desired at an early developing stage. For example, it
may be desirable to overexpress a gene regulating early embryo
development or a gene involved in the metabolism prior to seed
maturation.
[0187] Following identification of an appropriate promoter suitable
for expression of a specific delta-8 desaturase coding sequence,
the promoter is then operably linked in a sense orientation using
conventional means well known to those skilled in the art.
[0188] Once the recombinant construct has been made, it may then be
introduced into a plant cell of choice by methods well known to
those of ordinary skill in the art (e.g., transfection,
transformation and electroporation). Oilseed plant cells are the
preferred plant cells. The transformed plant cell is then cultured
and regenerated under suitable conditions permitting expression of
the long-chain PUFA which is then optionally recovered and
purified.
[0189] The recombinant constructs of the invention may be
introduced into one plant cell; or, alternatively, each construct
may be introduced into separate plant cells.
[0190] Expression in a plant cell may be accomplished in a
transient or stable fashion as is described above.
[0191] The desired long-chain PUFAs can be expressed in seed. Also
within the scope of this invention are seeds or plant parts
obtained from such transformed plants.
[0192] Plant parts include differentiated and undifferentiated
tissues including, but not limited to: roots, stems, shoots,
leaves, pollen, seeds, tumor tissue and various forms of cells and
culture (e.g., single cells, protoplasts, embryos and callus
tissue). The plant tissue may be in plant or in a plant organ,
tissue or cell culture.
[0193] The term "plant organ" refers to plant tissue or a group of
tissues that constitute a morphologically and functionally distinct
part of a plant. The term "genome" refers to the following: (1) the
entire complement of genetic material (genes and non-coding
sequences) that is present in each cell of an organism, or virus or
organelle; and/or (2) a complete set of chromosomes inherited as a
(haploid) unit from one parent.
[0194] Thus, this invention also concerns a method for transforming
a cell, comprising transforming a cell with a recombinant construct
of the invention and selecting those cells transformed with the
recombinant construct, wherein said recombinant construct comprises
a nucleotide sequence encoding a mutant polypeptide having delta-8
desaturase activity, wherein the amino acid sequence of the mutant
polypeptide is set forth in SEQ ID NO:2, and wherein SEQ ID NO:2 is
not identical to SEQ ID NO:10.
[0195] Also of interest is a method for producing a transformed
plant comprising transforming a plant cell with the mutant delta-8
desaturase polynucleotides of the instant invention and
regenerating a plant from the transformed plant cell.
[0196] Methods for transforming dicots (primarily by use of
Agrobacterium tumefaciens) and obtaining transgenic plants have
been published, among others, for: cotton (U.S. Pat. No. 5,004,863;
U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S.
Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut
(Cheng et al., Plant Cell Rep., 15:653-657 (1996); McKently et al.,
Plant Cell Rep., 14:699-703 (1995)); papaya (Ling, K. et al.,
Bio/technology, 9:752-758 (1991)); and pea (Grant et al., Plant
Cell Rep., 15:254-258 (1995)). For a review of other commonly used
methods of plant transformation see Newell, C. A. (Mol.
Biotechnol., 16:53-65 (2000)). One of these methods of
transformation uses Agrobacterium rhizogenes (Tepfler, M. and
Casse-Delbart, F., Microbiol. Sci., 4:24-28 (1987)). Transformation
of soybeans using direct delivery of DNA has been published using
PEG fusion (WO 92/17598), electroporation (Chowrira, G. M. et al.,
Mol. Biotechnol., 3:17-23 (1995); Christou, P. et al., Proc. Natl.
Acad. Sci. Sci. USA, 84:3962-3966 (1987)), microinjection, or
particle bombardment (McCabe, D. E. et al., Bio/Technology, 6:923
(1988); Christou et al., Plant Physiol., 87:671-674 (1988)).
[0197] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated. The regeneration, development and
cultivation of plants from single plant protoplast transformants or
from various transformed explants is well known in the art
(Weissbach and Weissbach, In: Methods for Plant Molecular Biology,
(Eds.), Academic: San Diego, Calif. (1988)). This regeneration and
growth process typically includes the steps of selection of
transformed cells and culturing those individualized cells through
the usual stages of embryonic development through the rooted
plantlet stage. Transgenic embryos and seeds are similarly
regenerated. The resulting transgenic rooted shoots are thereafter
planted in an appropriate plant growth medium such as soil.
Preferably, the regenerated plants are self-pollinated to provide
homozygous transgenic plants. Otherwise, pollen obtained from the
regenerated plants is crossed to seed-grown plants of agronomically
important lines. Conversely, pollen from plants of these important
lines is used to pollinate regenerated plants. A transgenic plant
of the present invention containing a desired polypeptide is
cultivated using methods well known to one skilled in the art.
[0198] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for: the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.); generation of recombinant DNA fragments and
recombinant expression constructs; and, the screening and isolating
of clones. See, for example: Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor: NY (1989); Maliga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995);
Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold
Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing
DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular
Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).
[0199] Examples of oilseed plants include, but are not limited to:
soybean, Brassica species, sunflower, maize, cotton, flax and
safflower.
[0200] Examples of PUFAs having at least twenty carbon atoms and
five or more carbon-carbon double bonds include, but are not
limited to, omega-3 fatty acids such as EPA, DPA and DHA. Seeds
obtained from such plants are also within the scope of this
invention as well as oil obtained from such seeds.
[0201] In one embodiment this invention concerns an oilseed plant
comprising: [0202] a) a first recombinant DNA construct comprising
an isolated polynucleotide encoding a mutant polypeptide having
delta-8 desaturase activity, operably linked to at least one
regulatory sequence; and, [0203] b) at least one additional
recombinant DNA construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of: a delta-4
desaturase, a delta-5 desaturase, delta-6 desaturase, a delta-9
desaturase, a delta-12 desaturase, a delta-15 desaturase, a
delta-17 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a
C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22
elongase. Such additional desaturases are discussed in U.S. Pat.
No. 6,075,183, No. 5,968,809, No. 6,136,574, No. 5,972,664, No.
6,051,754, No. 6,410,288 and WO 98/46763, WO 98/46764, WO 00/12720
and WO 00/40705.
[0204] The choice of combination of cassettes used depends in part
on the PUFA profile and/or desaturase/alongase profile of the
oilseed plant cells to be transformed and the long-chain PUFA(s)
which is to be expressed.
[0205] In another aspect, this invention concerns a method for
making long-chain PUFAs in a plant cell comprising: [0206] (a)
transforming a cell with a recombinant construct of the invention;
and, [0207] (b) selecting those transformed cells that make
long-chain PUFAs.
[0208] In still another aspect, this invention concerns a method
for producing at least one PUFA in a soybean cell comprising:
[0209] (a) transforming a soybean cell with a first recombinant DNA
construct comprising: [0210] (i) an isolated polynucleotide
encoding a mutant polypeptide having delta-8 desaturase activity,
operably linked to at least one regulatory sequence; and, [0211]
(ii) at least one additional recombinant DNA construct comprising
an isolated polynucleotide, operably linked to at least one
regulatory sequence, encoding a polypeptide selected from the group
consisting of: a delta-4 desaturase, a delta-5 desaturase, delta-6
desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15
desaturase, a delta-17 desaturase, a delta-9 elongase, a
C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20
elongase and a C.sub.20/22 elongase; [0212] (b) regenerating a
soybean plant from the transformed cell of step (a); and, [0213]
(c) selecting those seeds obtained from the plants of step (b)
having an altered level of PUFAs when compared to the level in
seeds obtained from a nontransformed soybean plant. In particularly
preferred embodiments, the at least one additional recombinant DNA
construct encodes a polypeptide having delta-9 elongase activity,
e.g., the delta-9 elongase isolated and/or derived from Isochrysis
galbana (GenBank Accession No. AF390174) and set forth in SEQ ID
NOs:172-174 or the delta-9 elongase isolated and/or derived from
Euglena gracilis and set forth in SEQ ID NOs:175-177.
Microbial Expression Systems, Cassettes & Vectors, and
Transformation
[0214] The delta-8 desaturase genes and gene products described
herein (i.e., those mutants derived from EgD8 or EgD8S, or homologs
thereof) may also be produced in heterologous microbial host cells,
particularly in the cells of oleaginous yeasts (e.g., Yarrowia
lipolytica).
[0215] Microbial expression systems and expression vectors
containing regulatory sequences that direct high level expression
of foreign proteins are well known to those skilled in the art. Any
of these could be used to construct chimeric genes for production
of any of the gene products of the instant sequences. These
chimeric genes could then be introduced into appropriate
microorganisms via transformation to provide high-level expression
of the encoded enzymes.
[0216] Vectors or DNA cassettes useful for the transformation of
suitable microbial host cells are well known in the art. The
specific choice of sequences present in the construct is dependent
upon the desired expression products (supra), the nature of the
host cell and the proposed means of separating transformed cells
versus non-transformed cells. Typically, however, the vector or
cassette contains sequences directing transcription and translation
of the relevant gene(s), a selectable marker and sequences allowing
autonomous replication or chromosomal integration. Suitable vectors
comprise a region 5' of the gene that controls transcriptional
initiation (e.g., a promoter) and a region 3' of the DNA fragment
that controls transcriptional termination (i.e., a terminator). It
is most preferred when both control regions are derived from genes
from the transformed microbial host cell, although it is to be
understood that such control regions need not be derived from the
genes native to the specific species chosen as a production
host.
[0217] Initiation control regions or promoters which are useful to
drive expression of the instant delta-8 desaturase ORFs in the
desired microbial host cell are numerous and familiar to those
skilled in the art. Virtually any promoter capable of directing
expression of these genes in the selected host cell is suitable for
the present invention. Expression in a microbial host cell can be
accomplished in a transient or stable fashion. Transient expression
can be accomplished by inducing the activity of a regulatable
promoter operably linked to the gene of interest. Stable expression
can be achieved by the use of a constitutive promoter operably
linked to the gene of interest. As an example, when the host cell
is yeast, transcriptional and translational regions functional in
yeast cells are provided, particularly from the host species (e.g.,
see WO 2004/101757 and WO 2006/052870 for preferred transcriptional
initiation regulatory regions for use in Yarrowia lipolytica). Any
one of a number of regulatory sequences can be used, depending upon
whether constitutive or induced transcription is desired, the
efficiency of the promoter in expressing the ORF of interest, the
ease of construction and the like.
[0218] Nucleotide sequences surrounding the translational
initiation codon `ATG` have been found to affect expression in
yeast cells. If the desired polypeptide is poorly expressed in
yeast, the nucleotide sequences of exogenous genes can be modified
to include an efficient yeast translation initiation sequence to
obtain optimal gene expression. For expression in yeast, this can
be done by site-directed mutagenesis of an inefficiently expressed
gene by fusing it in-frame to an endogenous yeast gene, preferably
a highly expressed gene. Alternatively, one can determine the
consensus translation initiation sequence in the host and engineer
this sequence into heterologous genes for their optimal expression
in the host of interest.
[0219] The termination region can be derived from the 3' region of
the gene from which the initiation region was obtained or from a
different gene. A large number of termination regions are known and
function satisfactorily in a variety of hosts (when utilized both
in the same and different genera and species from where they were
derived). The termination region usually is selected more as a
matter of convenience rather than because of any particular
property. Preferably, when the microbial host is a yeast cell, the
termination region is derived from a yeast gene (particularly
Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or
Kluyveromyces). The 3'-regions of mammalian genes encoding
.gamma.-interferon and .alpha.-2 interferon are also known to
function in yeast. Termination control regions may also be derived
from various genes native to the preferred hosts. Optionally, a
termination site may be unnecessary; however, it is most preferred
if included. Although not intended to be limiting, termination
regions useful in the disclosure herein include: .about.100 bp of
the 3' region of the Yarrowia lipolytica extracellular protease
(XPR; GenBank Accession No. M17741); the acyl-coA oxidase (Aco3:
GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank
Accession No. XP.sub.--503244) terminators; the Pex20 (GenBank
Accession No. AF054613) terminator; the Pex16 (GenBank Accession
No. U75433) terminator; the Lip1 (GenBank Accession No. Z50020)
terminator; the Lip2 (GenBank Accession No. AJ012632) terminator;
and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988)
terminator.
[0220] As one of skill in the art is aware, merely inserting a gene
into a cloning vector does not ensure that it will be successfully
expressed at the level needed. In response to the need for a high
expression rate, many specialized expression vectors have been
created by manipulating a number of different genetic elements that
control aspects of transcription, translation, protein stability,
oxygen limitation and secretion from the microbial host cell. More
specifically, some of the molecular features that have been
manipulated to control gene expression include: 1.) the nature of
the relevant transcriptional promoter and terminator sequences; 2.)
the number of copies of the cloned gene and whether the gene is
plasmid-borne or integrated into the genome of the host cell; 3.)
the final cellular location of the synthesized foreign protein; 4.)
the efficiency of translation and correct folding of the protein in
the host organism; 5.) the intrinsic stability of the mRNA and
protein of the cloned gene within the host cell; and 6.) the codon
usage within the cloned gene, such that its frequency approaches
the frequency of preferred codon usage of the host cell. Each of
these types of modifications are encompassed in the present
invention, as means to further optimize expression of the mutant
delta-8 desaturases described herein.
[0221] Once the DNA encoding a polypeptide suitable for expression
in an appropriate microbial host cell (e.g., oleaginous yeast) has
been obtained (e.g., a chimeric gene comprising a promoter, ORF and
terminator), it is placed in a plasmid vector capable of autonomous
replication in a host cell, or it is directly integrated into the
genome of the host cell. Integration of expression cassettes can
occur randomly within the host genome or can be targeted through
the use of constructs containing regions of homology with the host
genome sufficient to target recombination within the host locus.
Where constructs are targeted to an endogenous locus, all or some
of the transcriptional and translational regulatory regions can be
provided by the endogenous locus.
[0222] In the present invention, the preferred method of expressing
genes in Yarrowia lipolytica is by integration of linear DNA into
the genome of the host; and, integration into multiple locations
within the genome can be particularly useful when high level
expression of genes are desired [e.g., in the Ura3 locus (GenBank
Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No.
AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2
gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus
(Pox3: GenBank Accession No. XP.sub.--503244; or, Aco3: GenBank
Accession No. AJ001301), the delta-12 desaturase gene locus
(WO2004/104167), the Lip1 gene locus (GenBank Accession No. Z50020)
and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].
[0223] Advantageously, the Ura3 gene can be used repeatedly in
combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic
acid monohydrate; "5-FOA") selection (infra), to readily permit
genetic modifications to be integrated into the Yarrowia genome in
a facile manner.
[0224] Where two or more genes are expressed from separate
replicating vectors, it is desirable that each vector has a
different means of selection and should lack homology to the other
construct(s) to maintain stable expression and prevent reassortment
of elements among constructs. Judicious choice of regulatory
regions, selection means and method of propagation of the
introduced construct(s) can be experimentally determined so that
all introduced genes are expressed at the necessary levels to
provide for synthesis of the desired products.
[0225] Constructs comprising the gene of interest may be introduced
into a microbial host cell by any standard technique. These
techniques include transformation (e.g., lithium acetate
transformation [Methods in Enzymology, 194:186-187 (1991)]),
protoplast fusion, bolistic impact, electroporation,
microinjection, or any other method that introduces the gene of
interest into the host cell. More specific teachings applicable for
oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No.
4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl.
Microbiol. Biotechnol., 48(2):232-235 (1997)).
[0226] For convenience, a host cell that has been manipulated by
any method to take up a DNA sequence (e.g., an expression cassette)
will be referred to as "transformed" or "recombinant" herein. The
transformed host will have at least one copy of the expression
construct and may have two or more, depending upon whether the gene
is integrated into the genome, amplified or is present on an
extrachromosomal element having multiple copy numbers.
[0227] The transformed host cell can be identified by various
selection techniques, as described in WO2004/101757 and WO
2006/052870. Preferred selection methods for use herein are
resistance to kanamycin, hygromycin and the amino glycoside G418,
as well as ability to grow on media lacking uracil, leucine,
lysine, tryptophan or histidine. In alternate embodiments, 5-FOA is
used for selection of yeast Ura-mutants. The compound is toxic to
yeast cells that possess a functioning URA3 gene encoding orotidine
5'-monophosphate decarboxylase (OMP decarboxylase); thus, based on
this toxicity, 5-FOA is especially useful for the selection and
identification of Ura.sup.- mutant yeast strains (Bartel, P. L. and
Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v.
7, pp 109-147, 1997). More specifically, one can first knockout the
native Ura3 gene to produce a strain having a Ura-phenotype,
wherein selection occurs based on 5-FOA resistance. Then, a cluster
of multiple chimeric genes and a new Ura3 gene can be integrated
into a different locus of the Yarrowia genome to thereby produce a
new strain having a Ura+ phenotype. Subsequent integration produces
a new Ura3-strain (again identified using 5-FOA selection), when
the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in
combination with 5-FOA selection) can be used as a selection marker
in multiple rounds of transformation.
[0228] Following transformation, substrates suitable for the
instant delta-8 desaturases (and, optionally other PUFA enzymes
that are co-expressed within the host cell) may be produced by the
host either naturally or transgenically, or they may be provided
exogenously.
[0229] Microbial host cells for expression of the instant genes and
nucleic acid fragments may include hosts that grow on a variety of
feedstocks, including simple or complex carbohydrates, fatty acids,
organic acids, oils and alcohols, and/or hydrocarbons over a wide
range of temperature and pH values. The genes of the present
invention have been isolated for expression in an oleaginous yeast
(and in particular Yarrowia lipolytica). It is contemplated that
because transcription, translation and the protein biosynthetic
apparatus is highly conserved, any bacteria, yeast, algae, oomycete
and/or fungus will be a suitable microbial host for expression of
the present nucleic acid fragments.
[0230] Preferred microbial hosts are oleaginous yeasts. These
organisms are naturally capable of oil synthesis and accumulation,
wherein the oil can comprise greater than about 25% of the cellular
dry weight, more preferably greater than about 30% of the cellular
dry weight, and most preferably greater than about 40% of the
cellular dry weight. Genera typically identified as oleaginous
yeast include, but are not limited to: Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and
Lipomyces. More specifically, illustrative oil-synthesizing yeasts
include: Rhodosporidium toruloides, Lipomyces starkeyii, L.
lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C.
utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R.
graminis, and Yarrowia lipolytica (formerly classified as Candida
lipolytica).
[0231] Most preferred is the oleaginous yeast Yarrowia lipolytica;
and, in a further embodiment, most preferred are the Y. lipolytica
strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC
#76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G.,
Bioresour. Technol. 82(1):43-9 (2002)).
[0232] Historically, various strains of Y. lipolytica have been
used for the manufacture and production of: isocitrate lyase;
lipases; polyhydroxyalkanoates; citric acid; erythritol;
2-oxoglutaric acid; .gamma.-decalactone; .gamma.-dodecalatone; and
pyruvic acid. Specific teachings applicable for engineering ARA,
EPA and DHA production in Y. lipolytica are provided in U.S. patent
application Ser. No. 11/264,784 (WO 2006/055322), No. 11/265,761
(WO 2006/052870) and Ser. No. 11/264,737 (WO 2006/052871),
respectively.
[0233] Other preferred microbial hosts include oleaginous bacteria,
algae, Oomycetes and other fungi; and, within this broad group of
microbial hosts, of particular interest are microorganisms that
synthesize omega-3/omega-6 fatty acids (or those that can be
genetically engineered for this purpose [e.g., other yeast such as
Saccharomyces cerevisiae]). Thus, for example, transformation of
Mortierella alpina (which is commercially used for production of
ARA) with any of the present delta-8 desaturase genes under the
control of inducible or regulated promoters could yield a
transformant organism capable of synthesizing increased quantities
of EDA. The method of transformation of M. alpina is described by
Mackenzie et al. (Appl. Environ. Microbiol., 66:4655 (2000)).
Similarly, methods for transformation of Thraustochytriales
microorganisms are disclosed in U.S. Pat. No. 7,001,772.
[0234] Based on the teachings described above, in one embodiment
this invention is drawn to a method of producing either DGLA or
ETA, respectively, comprising: [0235] a) providing an oleaginous
yeast comprising: [0236] (i) a nucleotide sequence encoding a
mutant polypeptide having delta-8 desaturase activity, wherein the
mutant polypeptide has an amino acid sequence as set forth in SEQ
ID NO:2 and wherein SEQ ID NO:2 is not identical to SEQ ID NO:10;
and, [0237] (ii) a source of desaturase substrate consisting of
either EDA or ETrA, respectively; and, [0238] b) growing the yeast
of step (a) in the presence of a suitable fermentable carbon source
wherein the gene encoding the delta-8 desaturase polypeptide is
expressed and EDA is converted to DGLA or ETrA is converted to ETA,
respectively; and, [0239] c) optionally recovering the DGLA or ETA,
respectively, of step (b). Substrate feeding may be required.
[0240] Of course, since naturally produced PUFAs in oleaginous
yeast are limited to 18:2 fatty acids (i.e., LA), and less
commonly, 18:3 fatty acids (i.e., ALA), in more preferred
embodiments of the present invention the oleaginous yeast will be
genetically engineered to express multiple enzymes necessary for
long-chain PUFA biosynthesis (thereby enabling production of e.g.,
ARA, EPA, DPA and DHA), in addition to the delta-8 desaturase
described herein.
[0241] Specifically, in one embodiment this invention concerns an
oleaginous yeast comprising: [0242] a) a first recombinant DNA
construct comprising an isolated polynucleotide encoding a mutant
delta-8 desaturase polypeptide, operably linked to at least one
regulatory sequence; and, [0243] b) at least one additional
recombinant DNA construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of: a delta-4
desaturase, a delta-5 desaturase, delta-6 desaturase, a delta-9
desaturase, a delta-12 desaturase, a delta-15 desaturase, a
delta-17 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a
C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22
elongase. In particularly preferred embodiments, the at least one
additional recombinant DNA construct encodes a polypeptide having
delta-9 elongase activity, e.g., the delta-9 elongase isolated
and/or derived from Isochrysis galbana (GenBank Accession No.
AF390174) and set forth in SEQ ID NOs:172-174 or the delta-9
elongase isolated and/or derived from Euglena gracilis and set
forth in SEQ ID NOs:175-177. Metabolic Engineering of Omega-3
and/or Omega-6 Fatty Acid Biosynthesis in Microbes
[0244] Methods for manipulating biochemical pathways are well known
to those skilled in the art; and, it is expected that numerous
manipulations will be possible to maximize omega-3 and/or omega-6
fatty acid biosynthesis in oleaginous yeasts, and particularly, in
Yarrowia lipolytica. This may require metabolic engineering
directly within the PUFA biosynthetic pathway or additional
coordinated manipulation of various other metabolic pathways.
[0245] In the case of manipulations within the PUFA biosynthetic
pathway, it may be desirable to increase the production of LA to
enable increased production of omega-6 and/or omega-3 fatty acids.
Introducing and/or amplifying genes encoding delta-9 and/or
delta-12 desaturases may accomplish this. To maximize production of
omega-6 unsaturated fatty acids, it is well known to one skilled in
the art that production is favored in a host microorganism that is
substantially free of ALA; thus, preferably, the host is selected
or obtained by removing or inhibiting delta-15 or omega-3 type
desaturase activity that permits conversion of LA to ALA.
Alternatively, it may be desirable to maximize production of
omega-3 fatty acids (and minimize synthesis of omega-6 fatty
acids). In this example, one could utilize a host microorganism
wherein the delta-12 desaturase activity that permits conversion of
oleic acid to LA is removed or inhibited; subsequently, appropriate
expression cassettes would be introduced into the host, along with
appropriate substrates (e.g., ALA) for conversion to omega-3 fatty
acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA, DHA).
[0246] In alternate embodiments, biochemical pathways competing
with the omega-3 and/or omega-6 fatty acid biosynthetic pathways
for energy or carbon, or native PUFA biosynthetic pathway enzymes
that interfere with production of a particular PUFA end-product,
may be eliminated by gene disruption or down-regulated by other
means (e.g., antisense mRNA).
[0247] Detailed discussion of manipulations within the PUFA
biosynthetic pathway as a means to increase ARA, EPA or DHA (and
associated techniques thereof) are presented in WO 2006/055322, WO
2006/052870 and WO 2006/052871, respectively, as are desirable
manipulations in the TAG biosynthetic pathway and the TAG
degradation pathway (and associated techniques thereof).
[0248] Within the context of the present invention, it may be
useful to modulate the expression of the fatty acid biosynthetic
pathway by any one of the strategies described above. For example,
the present invention provides methods whereby genes encoding key
enzymes in the delta-9 elongase/delta-8 desaturase biosynthetic
pathway are introduced into oleaginous yeasts for the production of
omega-3 and/or omega-6 fatty acids. It will be particularly useful
to express the present the delta-8 desaturase genes in oleaginous
yeasts that do not naturally possess omega-3 and/or omega-6 fatty
acid biosynthetic pathways and coordinate the expression of these
genes, to maximize production of preferred PUFA products using
various means for metabolic engineering of the host organism.
Microbial Fermentation Processes for PUFA Production
[0249] The transformed microbial host cell is grown under
conditions that optimize expression of chimeric desaturase and
elongase genes and produce the greatest and the most economical
yield of the preferred PUFAs. In general, media conditions that may
be optimized include the type and amount of carbon source, the type
and amount of nitrogen source, the carbon-to-nitrogen ratio, the
oxygen level, growth temperature, pH, length of the biomass
production phase, length of the oil accumulation phase and the time
and method of cell harvest. Microorganisms of interest (i.e.,
Yarrowia lipolytica) are generally grown in complex media (e.g.,
yeast extract-peptone-dextrose broth (YPD)) or a defined minimal
media that lacks a component necessary for growth and thereby
forces selection of the desired expression cassettes (e.g., Yeast
Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
[0250] Fermentation media in the present invention must contain a
suitable carbon source. Suitable carbon sources are taught in WO
2004/101757. Although it is contemplated that the source of carbon
utilized in the present invention may encompass a wide variety of
carbon-containing sources, preferred carbon sources are sugars,
glycerol, and/or fatty acids. Most preferred is glucose and/or
fatty acids containing between 10-22 carbons.
[0251] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic (e.g., urea or glutamate)
source. In addition to appropriate carbon and nitrogen sources, the
fermentation media must also contain suitable minerals, salts,
cofactors, buffers, vitamins and other components known to those
skilled in the art suitable for the growth of the microorganism and
promotion of the enzymatic pathways necessary for PUFA production.
Particular attention is given to several metal ions (e.g.,
Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) that promote synthesis
of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell
Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
[0252] Preferred growth media in the present invention are common
commercially prepared media, such as Yeast Nitrogen Base (DIFCO
Laboratories, Detroit, Mich.). Other defined or synthetic growth
media may also be used and the appropriate medium for growth of the
transformant host cells will be known by one skilled in the art of
microbiology or fermentation science. A suitable pH range for the
fermentation is typically between about pH 4.0 to pH 8.0, wherein
pH 5.5 to pH 7.5 is preferred as the range for the initial growth
conditions. The fermentation may be conducted under aerobic or
anaerobic conditions, wherein microaerobic conditions are
preferred.
[0253] Typically, accumulation of high levels of PUFAs in
oleaginous yeast cells requires a two-stage process, since the
metabolic state must be "balanced" between growth and
synthesis/storage of fats. Thus, most preferably, a two-stage
fermentation process is necessary for the production of PUFAs in
Yarrowia lipolytica. This approach is described in WO 2004/101757,
as are various suitable fermentation process designs (i.e., batch,
fed-batch and continuous) and considerations during growth.
Purification and Processing of PUFA Oils
[0254] PUFAs may be found in the host microorganisms and plants as
free fatty acids or in esterified forms such as acylglycerols,
phospholipids, sulfolipids or glycolipids, and may be extracted
from the host cells through a variety of means well-known in the
art. One review of extraction techniques, quality analysis and
acceptability standards for yeast lipids is that of Z. Jacobs
(Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). A
brief review of downstream processing is also available by A. Singh
and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).
[0255] In general, means for the purification of PUFAs may include
extraction with organic solvents, sonication, supercritical fluid
extraction (e.g., using carbon dioxide), saponification and
physical means such as presses, or combinations thereof. One is
referred to the teachings of WO 2004/101757 for additional
details.
[0256] Methods of isolating seed oils are well known in the art
(Young et al., Processing of Fats and Oils, In The Lipid Handbook,
Gunstone et al., eds., Chapter 5, pp 253-257; Chapman & Hall:
London (1994)). For example, soybean oil is produced using a series
of steps involving the extraction and purification of an edible oil
product from the oil-bearing seed. Soybean oils and soybean
byproducts are produced using the generalized steps shown in the
Table below.
TABLE-US-00005 TABLE 5 Generalized Steps For Soybean Oil And
Byproduct Production Process Impurities Removed and/or Step Process
By-Products Obtained #1 soybean seed #2 oil extraction meal #3
degumming lecithin #4 alkali or physical refining gums, free fatty
acids, pigments #5 water washing soap #6 bleaching color, soap,
metal #7 (hydrogenation) #8 (winterization) stearine #9
deodorization free fatty acids, tocopherols, sterols, volatiles #10
oil products
[0257] More specifically, soybean seeds are cleaned, tempered,
dehulled and flaked, thereby increasing the efficiency of oil
extraction. Oil extraction is usually accomplished by solvent
(e.g., hexane) extraction but can also be achieved by a combination
of physical pressure and/or solvent extraction. The resulting oil
is called crude oil. The crude oil may be degummed by hydrating
phospholipids and other polar and neutral lipid complexes that
facilitate their separation from the nonhydrating, triglyceride
fraction (soybean oil). The resulting lecithin gums may be further
processed to make commercially important lecithin products used in
a variety of food and industrial products as emulsification and
release (i.e., anti-sticking) agents. Degummed oil may be further
refined for the removal of impurities (primarily free fatty acids,
pigments and residual gums). Refining is accomplished by the
addition of a caustic agent that reacts with free fatty acid to
form soap and hydrates phosphatides and proteins in the crude oil.
Water is used to wash out traces of soap formed during refining.
The soapstock byproduct may be used directly in animal feeds or
acidulated to recover the free fatty acids. Color is removed
through adsorption with a bleaching earth that removes most of the
chlorophyll and carotenoid compounds. The refined oil can be
hydrogenated, thereby resulting in fats with various melting
properties and textures. Winterization (fractionation) may be used
to remove stearine from the hydrogenated oil through
crystallization under carefully controlled cooling conditions.
Deodorization (principally via steam distillation under vacuum) is
the last step and is designed to remove compounds which impart odor
or flavor to the oil. Other valuable byproducts such as tocopherols
and sterols may be removed during the deodorization process.
Deodorized distillate containing these byproducts may be sold for
production of natural vitamin E and other high-value pharmaceutical
products. Refined, bleached, (hydrogenated, fractionated) and
deodorized oils and fats may be packaged and sold directly or
further processed into more specialized products. A more detailed
reference to soybean seed processing, soybean oil production and
byproduct utilization can be found in Erickson, Practical Handbook
of Soybean Processing and Utilization, The American Oil Chemists'
Society and United Soybean Board (1995). Soybean oil is liquid at
room temperature because it is relatively low in saturated fatty
acids when compared with oils such as coconut, palm, palm kernel
and cocoa butter.
[0258] Plant and microbial oils containing PUFAs that have been
refined and/or purified can be hydrogenated, to thereby result in
fats with various melting properties and textures. Many processed
fats (including spreads, confectionary fats, hard butters,
margarines, baking shortenings, etc.) require varying degrees of
solidity at room temperature and can only be produced through
alteration of the source oil's physical properties. This is most
commonly achieved through catalytic hydrogenation.
[0259] Hydrogenation is a chemical reaction in which hydrogen is
added to the unsaturated fatty acid double bonds with the aid of a
catalyst such as nickel. For example, high oleic soybean oil
contains unsaturated oleic, LA and linolenic fatty acids and each
of these can be hydrogenated. Hydrogenation has two primary
effects. First, the oxidative stability of the oil is increased as
a result of the reduction of the unsaturated fatty acid content.
Second, the physical properties of the oil are changed because the
fatty acid modifications increase the melting point resulting in a
semi-liquid or solid fat at room temperature.
[0260] There are many variables which affect the hydrogenation
reaction, which, in turn, alter the composition of the final
product. Operating conditions including pressure, temperature,
catalyst type and concentration, agitation and reactor design are
among the more important parameters that can be controlled.
Selective hydrogenation conditions can be used to hydrogenate the
more unsaturated fatty acids in preference to the less unsaturated
ones. Very light or brush hydrogenation is often employed to
increase stability of liquid oils. Further hydrogenation converts a
liquid oil to a physically solid fat. The degree of hydrogenation
depends on the desired performance and melting characteristics
designed for the particular end product. Liquid shortenings (used
in the manufacture of baking products, solid fats and shortenings
used for commercial frying and roasting operations) and base stocks
for margarine manufacture are among the myriad of possible oil and
fat products achieved through hydrogenation. A more detailed
description of hydrogenation and hydrogenated products can be found
in Patterson, H. B. W.,
[0261] Hydrogenation of Fats and Oils: Theory and Practice. The
American Oil Chemists'Society (1994). Hydrogenated oils have become
somewhat controversial due to the presence of trans-fatty acid
isomers that result from the hydrogenation process. Ingestion of
large amounts of trans-isomers has been linked with detrimental
health effects including increased ratios of low density to high
density lipoproteins in the blood plasma and increased risk of
coronary heart disease.
PUFA-Containing Oils for Use in Foodstuffs
[0262] The market place currently supports a large variety of food
and feed products, incorporating omega-3 and/or omega-6 fatty acids
(particularly ARA, EPA and DHA). It is contemplated that the
plant/seed oils, altered seeds and microbial oils of the invention
comprising PUFAs will function in food and feed products to impart
the health benefits of current formulations. Compared to other
vegetable oils, the oils of the invention are believed to function
similarly to other oils in food applications from a physical
standpoint (for example, partially hydrogenated oils such as
soybean oil are widely used as ingredients for soft spreads,
margarine and shortenings for baking and frying).
[0263] Plant/seed oils, altered seeds and microbial oils containing
omega-3 and/or omega-6 fatty acids described herein will be
suitable for use in a variety of food and feed products including,
but not limited to: food analogs, meat products, cereal products,
snack foods, baked foods and dairy products. Additionally, the
present plant/seed oils, altered seeds and microbial oils may be
used in formulations to impart health benefit in medical foods
including medical nutritionals, dietary supplements, infant formula
as well as pharmaceutical products. One of skill in the art of food
processing and food formulation will understand how the amount and
composition of the plant and microbial oil may be added to the food
or feed product. Such an amount will be referred to herein as an
"effective" amount and will depend on the food or feed product, the
diet that the product is intended to supplement or the medical
condition that the medical food or medical nutritional is intended
to correct or treat.
[0264] Food analogs can be made using processes well known to those
skilled in the art. There can be mentioned meat analogs, cheese
analogs, milk analogs and the like. Meat analogs made from soybeans
contain soy protein or tofu and other ingredients mixed together to
simulate various kinds of meats. These meat alternatives are sold
as frozen, canned or dried foods. Usually, they can be used the
same way as the foods they replace. Meat alternatives made from
soybeans are excellent sources of protein, iron and B vitamins.
Examples of meat analogs include, but are not limited to: ham
analogs, sausage analogs, bacon analogs, and the like.
[0265] Food analogs can be classified as imitation or substitutes
depending on their functional and compositional characteristics.
For example, an imitation cheese need only resemble the cheese it
is designed to replace. However, a product can generally be called
a substitute cheese only if it is nutritionally equivalent to the
cheese it is replacing and meets the minimum compositional
requirements for that cheese. Thus, substitute cheese will often
have higher protein levels than imitation cheeses and be fortified
with vitamins and minerals.
[0266] Milk analogs or nondairy food products include, but are not
limited to: imitation milk and nondairy frozen desserts (e.g.,
those made from soybeans and/or soy protein products).
[0267] Meat products encompass a broad variety of products. In the
United States "meat" includes "red meats" produced from cattle,
hogs and sheep. In addition to the red meats there are poultry
items which include chickens, turkeys, geese, guineas, ducks and
the fish and shellfish. There is a wide assortment of seasoned and
processes meat products: fresh, cured and fried, and cured and
cooked. Sausages and hot dogs are examples of processed meat
products. Thus, the term "meat products" as used herein includes,
but is not limited to, processed meat products.
[0268] A cereal food product is a food product derived from the
processing of a cereal grain. A cereal grain includes any plant
from the grass family that yields an edible grain (seed). The most
popular grains are barley, corn, millet, oats, quinoa, rice, rye,
sorghum, triticale, wheat and wild rice. Examples of a cereal food
product include, but are not limited to: whole grain, crushed
grain, grits, flour, bran, germ, breakfast cereals, extruded foods,
pastas, and the like.
[0269] A baked goods product comprises any of the cereal food
products mentioned above and has been baked or processed in a
manner comparable to baking, i.e., to dry or harden by subjecting
to heat. Examples of a baked good product include, but are not
limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs,
baked snacks, mini-biscuits, mini-crackers, mini-cookies and
mini-pretzels. As was mentioned above, oils of the invention can be
used as an ingredient.
[0270] A snack food product comprises any of the above or below
described food products.
[0271] A fried food product comprises any of the above or below
described food products that has been fried.
[0272] A health food product is any food product that imparts a
health benefit. Many oilseed-derived food products may be
considered as health foods.
[0273] A beverage can be in a liquid or in a dry powdered form.
[0274] For example, there can be mentioned non-carbonated drinks;
fruit juices, fresh, frozen, canned or concentrate; flavored or
plain milk drinks, etc. Adult and infant nutritional formulas are
well known in the art and commercially available (e.g.,
Similac.RTM., Ensure.RTM., Jevity.RTM., and Alimentum.RTM. from
Ross Products Division, Abbott Laboratories).
[0275] Infant formulas are liquids or reconstituted powders fed to
infants and young children. "Infant formula" is defined herein as
an enteral nutritional product which can be substituted for human
breast milk in feeding infants and typically is composed of a
desired percentage of fat mixed with desired percentages of
carbohydrates and proteins in an aqueous solution (e.g., see U.S.
Pat. No. 4,670,285). Based on the worldwide composition studies, as
well as levels specified by expert groups, average human breast
milk typically contains about 0.20% to 0.40% of total fatty acids
(assuming about 50% of calories from fat); and, generally the ratio
of DHA to ARA would range from about 1:1 to 1:2 (see, e.g.,
formulations of Enfamil LIPIL.TM. [Mead Johnson & Company] and
Similac Advance.TM. [Ross Products Division, Abbott Laboratories]).
Infant formulas have a special role to play in the diets of infants
because they are often the only source of nutrients for infants;
and, although breast-feeding is still the best nourishment for
infants, infant formula is a close enough second that babies not
only survive but thrive.
[0276] A dairy product is a product derived from milk. A milk
analog or nondairy product is derived from a source other than
milk, for example, soymilk as was discussed above. These products
include, but are not limited to: whole milk, skim milk, fermented
milk products such as yogurt or sour milk, cream, butter, condensed
milk, dehydrated milk, coffee whitener, coffee creamer, ice cream,
cheese, etc.
[0277] Additional food products into which the PUFA-containing oils
of the invention could be included are, for example: chewing gums,
confections and frostings, gelatins and puddings, hard and soft
candies, jams and jellies, white granulated sugar, sugar
substitutes, sweet sauces, toppings and syrups, and dry-blended
powder mixes.
Health Food Products and Pharmaceuticals
[0278] A health food product is any food product that imparts a
health benefit and includes functional foods, medical foods,
medical nutritionals and dietary supplements. Additionally,
plant/seed oils, altered seeds and microbial oils of the invention
may be used in standard pharmaceutical compositions. For example,
the oils of the invention could readily be incorporated into the
any of the above mentioned food products, to thereby produce, e.g.,
a functional or medical food. More concentrated formulations
comprising PUFAs include capsules, powders, tablets, softgels,
gelcaps, liquid concentrates and emulsions which can be used as a
dietary supplement in humans or animals other than humans.
Use in Animal Feeds
[0279] Animal feeds are generically defined herein as products
intended for use as feed or for mixing in feed for animals other
than humans. The plant/seed oils, altered seeds and microbial oils
of the invention can be used as an ingredient in various animal
feeds.
[0280] More specifically, although not limited herein, it is
expected that the oils of the invention can be used within pet food
products, ruminant and poultry food products and aquacultural food
products. Pet food products are those products intended to be fed
to a pet [e.g., a dog, cat, bird, reptile, rodent]; these products
can include the cereal and health food products above, as well as
meat and meat byproducts, soy protein products and grass and hay
products (e.g., alfalfa, timothy, oat or brome grass, vegetables).
Ruminant and poultry food products are those wherein the product is
intended to be fed to e.g., turkeys, chickens, cattle and swine. As
with the pet foods above, these products can include cereal and
health food products, soy protein products, meat and meat
byproducts, and grass and hay products as listed above.
Aquacultural food products (or "aquafeeds") are those products
intended to be used in aquafarming, i.e., which concerns the
propagation, cultivation or farming of aquatic organisms, animals
and/or plants in fresh or marine waters.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0281] One object of the present invention is the synthesis of
suitable delta-8 desaturases that will enable expression of the
delta-9 elongase/delta-8 desaturase pathway in plants and
oleaginous yeast.
[0282] In commonly owned WO 2006/012325 and WO 2006/012326
(US2005-0287652) applicants describe the isolation of a delta-8
desaturase from Euglena gracilis ("Eg5"), and the enzyme's
functional characterization upon expression in Saccharomyces
cerevisiae. The wildtype Eg5 sequence was additionally
codon-optimized for expression in Yarrowia lipolytica, resulting in
the synthesis of a synthetic, functional codon-optimized delta-8
desaturase designated as "D8SF". Upon co-expression of D8SF with a
codon-optimized delta-9 elongase (derived from Isochrysis galbana
(GenBank Accession No. 390174) in Yarrowia lipolytica, 6.4% DGLA
(with no co-synthesis of GLA) was demonstrated (Example 16 in WO
2006/012325 and WO 2006/012326 (US2005-0287652-A1)).
[0283] In the present Application, the synthetic codon-optimized
delta-8 desaturase designated as "D8SF" (and designated herein as
EgD8S'') was subjected to targeted mutations. Ultimately, a mutant
EgD8S enzyme (SEQ ID NO:2) was created comprising at least one
amino acid mutation (and up to about 33 amino acid mutations) with
respect to the synthetic codon-optimized EgD8S, wherein: (i) the at
least one mutation is selected from the group consisting of: 4S to
A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y, 66P
to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M to
L, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to
L, 170G to A, 171 L to V, 279T to L, 280L to T, 293L to M, 346I to
V, 347I to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A
and 422L to Q, wherein the mutations are defined with respect to
the synthetic codon-optimized sequence set forth in SEQ ID NO:10;
(ii) SEQ ID NO:2 is not identical to SEQ ID NO:10; and, (iii) SEQ
ID NO:2 is at least about functionally equivalent to SEQ ID
NO:10.
[0284] Given the teaching of the present application the skilled
person will recognize the commercial utility of the recombinant
genes of the present invention encoding delta-8 desaturases, to
enable production of a variety of omega-3 and/or omega-6 PUFAs via
expression of a heterologous delta-9 elongase/delta-8 desaturase
pathway.
EXAMPLES
[0285] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions. Thus,
various modifications of the invention in addition to those shown
and described herein will be apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
General Methods
[0286] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described
by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular
Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold
Spring Harbor, N.Y. (1989) (Maniatis); 2.) T. J. Silhavy, M. L.
Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold
Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3.)
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
published by Greene Publishing Assoc. and Wiley-Interscience
(1987).
[0287] Materials and methods suitable for the maintenance and
growth of microbial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society
for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock
in Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd
ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents,
restriction enzymes and materials used for the growth and
maintenance of microbial cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.),
unless otherwise specified.
[0288] General molecular cloning was performed according to
standard methods (Sambrook et al., supra). Oligonucleotides were
synthesized by Sigma-Genosys (Spring, Tex.). DNA sequence was
generated on an ABI Automatic sequencer using dye terminator
technology (U.S. Pat. No. 5,366,860; EP 272,007) using a
combination of vector and insert-specific primers. Sequence editing
was performed in Sequencher (Gene Codes Corporation, Ann Arbor,
Mich.). All sequences represent coverage at least two times in both
directions. Comparisons of genetic sequences were accomplished
using DNASTAR software (DNAStar Inc., Madison, Wis.).
Alternatively, manipulations of genetic sequences were accomplished
using the suite of programs available from the Genetics Computer
Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group
(GCG), Madison, Wis.). The GCG program "Pileup" was used with the
gap creation default value of 12, and the gap extension default
value of 4. The GCG "Gap" or "Bestfit" programs were used with the
default gap creation penalty of 50 and the default gap extension
penalty of 3. Unless otherwise stated, in all other cases GCG
program default parameters were used.
[0289] The meaning of abbreviations is as follows: "sec" means
second(s), "min" means minute(s), "hr" means hour(s), "d" means
day(s), ".mu.l" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), ".mu.mole" mean
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng"
means nanogram(s), "U" means unit(s), "bp" means base pair(s) and
"kB" means kilobase(s). Parts and percentages are by weight and
degrees are Celsius.
Transformation and Cultivation of Yarrowia lipolytica
[0290] Yarrowia lipolytica strains ATCC #20362, #76982 and #90812
were purchased from the American Type Culture Collection
(Rockville, Md.). Y. lipolytica strains were usually grown at
28.degree. C. on YPD agar (1% yeast extract, 2% bactopeptone, 2%
glucose, 2% agar).
[0291] Transformation of Yarrowia lipolytica was performed
according to the method of Chen, D. C. et al. (Appl. Microbiol.
Biotechnol., 48(2):232-235 (1997)), unless otherwise noted.
Briefly, Yarrowia was streaked onto a YPD plate and grown at
30.degree. C. for approximately 18 hr. Several large loopfuls of
cells were scraped from the plate and resuspended in 1 mL of
transformation buffer containing: 2.25 mL of 50% PEG, average MW
3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and
50 .mu.g sheared salmon sperm DNA. Then, approximately 500 ng of
linearized plasmid DNA was incubated in 100 .mu.l of resuspended
cells, and maintained at 39.degree. C. for 1 hr with vortex mixing
at 15 min intervals. The cells were plated onto selection media
plates and maintained at 30.degree. C. for 2 to 3 days.
[0292] For selection of transformants, minimal medium ("MM") was
generally used; the composition of MM is as follows: 0.17% yeast
nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium
sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1).
Supplements of uracil or leucine were added as appropriate to a
final concentration of 0.01% (thereby producing "MMU" or "MMLeu"
selection media, respectively, each prepared with 20 g/L agar).
[0293] Alternatively, transformants were selected on 5-fluoroorotic
acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate)
selection media, comprising: 0.17% yeast nitrogen base (DIFCO
Laboratories, Detroit, Mich.) without ammonium sulfate or amino
acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine,
900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L
agar.
Fatty Acid Analysis of Yarrowia lipolytica
[0294] For fatty acid analysis, cells were collected by
centrifugation and lipids were extracted as described in Bligh, E.
G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917
(1959)). Fatty acid methyl esters were prepared by
transesterification of the lipid extract with sodium methoxide
(Roughan, G., and Nishida I., Arch. Biochem. Biophys., 276(1):38-46
(1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC
fitted with a 30-m.times.0.25 mm (i.d.) HP-INNOWAX
(Hewlett-Packard) column. The oven temperature was from 170.degree.
C. (25 min hold) to 185.degree. C. at 3.5.degree. C./min.
[0295] For direct base transesterification, Yarrowia culture (3 mL)
was harvested, washed once in distilled water, and dried under
vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 .mu.l of
1%) was added to the sample, and then the sample was vortexed and
rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 .mu.l
hexane, the sample was vortexed and spun. The upper layer was
removed and analyzed by GC as described above.
Example 1
Development of a Topological Model for EgD8S
[0296] BLASTP analysis showed that EgD8S contained two domains: an
N-terminal cytochrome b.sub.5 domain (located between amino acid
residues 5 to 71 of SEQ ID NO:10) and a C-terminal desaturase
domain (located between amino acid residues 79 to 406 of SEQ ID
NO:10). In order to mutate the amino acid sequence of EgD8S without
negatively affecting the delta-8 desaturase activity, a topological
model (FIG. 2) was developed based on the logic and analyses
below.
[0297] First, the TMHMM program ("Prediction of transmembrane
helices in proteins"; TMHMM Server v. 2.0, Center for Biological
Sequence Analysis, BioCentrum-DTU, Technical University of Denmark,
DK-2800 Lyngby, Denmark) predicted that EgD8S had four
membrane-spanning helices (amino acid residues 113-132, 223-245,
266-283 and 287-309), with both the N- and C-termini located on the
cytoplasmic side of the membrane.
[0298] The membrane-bound fatty acid desaturases belong to a
super-family of membrane di-iron proteins that feature three
His-rich motifs: HX.sub.(3-4)H (SEQ ID NOs:166 and 167),
HX.sub.(2-3)HH (SEQ ID NOs:168 and 169) and (H/Q)X.sub.(2-3)HH (SEQ
ID NOs:170 and 171). These His-rich residues have been predicted to
be located in the cytoplasmic face of the membrane and have been
shown to be important for enzyme activity (Shanklin, J. et al.,
Biochemistry, 33:12787-12794 (1994); Shanklin, J., and Cahoon, E.
B., Annu. Rev. Plant Physiol. Plant Mol. Biol., 49:611-641 (1998)).
Within SEQ ID NO:10, these three histidine boxes were located
between amino acid residues 146-150, 183-187 and 358-362; two
additional His residues are located at amino acid residues 27 and
50. Each of these His residues are depicted on FIG. 2 with a small
round circle.
[0299] If the model predicted by TMHMM (supra) were accepted
without alteration, the first two His-rich regions (i.e., the
regions spanning between amino acid residues 146-150 and 183-187)
would be located in the periplasmic space, thus preventing their
participation in the iron-active site.
[0300] The conflict noted above was resolved when hydropathy plot
analysis (Kyte, J., and Doolittle, R., J. Mol. Biol., 157:105-132
(1982)) predicted one more hydrophobic region located between amino
acid residues 88-109 that immediately preceded the first predicted
transmembrane segment (i.e., residues 113-132). Since the
N-terminal cytochrome-b.sub.5 domain is located in the cytoplasmic
space, it was predicted that the hydrophobic region (i.e., residues
88-109) should be the first membrane-spanning segment (i.e., region
I, as shown in FIG. 2), while the predicted transmembrane segment
corresponding to residues 113-132 was designated as the second
membrane-spanning segment (i.e., region II, as shown in FIG. 2). As
a result, the transmembrane segment found between residues 223-245
that was originally predicted by TMHMM to span through the membrane
was instead predicted to lie in the cytoplasmic face, such that the
first two His-rich motifs (i.e., the regions spanning between amino
acid residues 146-150 and 183-187) could be adjusted to be within
the cytoplasmic side.
[0301] Finally, the hydropathy plot analysis also predicted another
hydrophobic region (i.e., residues 157-172) between the first two
His-rich motifs. Because the substrate for the desaturase is highly
hydrophobic, it was expected to most likely partition into the
lipid bilayer of the cytoplasmic membrane. This suggested that the
desaturase active site assembled from the His-rich motifs might be
at (or very near) the membrane surface. Thus, it was hypothesized
that both hydrophobic regions (i.e., residues 157-172 and residues
223-245) lie near the membrane surface to ensure that the active
site sits close the membrane.
[0302] The last two membrane-spanning helices predicted by TMHMM
(i.e., residues 266-283 and 287-309) are included within the final
topological model shown in FIG. 2 as region III and region IV.
[0303] Thus, the topological model depicted in FIG. 2 includes four
transmembrane regions labeled as regions I, II, III and IV, which
correspond to amino acid residues 88-109, 113-132, 266-283 and
287-309, respectively. Two additional hydrophobic regions are
located at amino acid residues 157-172 and 223-245. Finally, "IN"
corresponds with the cytoplasmic space, while "OUT" corresponds
with the periplasmic space.
Example 2
Strategies to Select Amino Acid Residues for Mutation
[0304] Close homologs to the EgD8S sequence were determined by
conducting BLAST (Basic Local Alignment Search Tool; Altschul, S.
F., et al., J. Mol. Biol., 215:403-410 (1993)) searches for
similarity to sequences contained in the BLAST "nr" database
(comprising all non-redundant GenBank CDS translations, sequences
derived from the 3-dimensional structure Brookhaven Protein Data
Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ
databases). Specifically, EgD8S (SEQ ID NO:10) was compared for
similarity to all publicly available protein sequences contained in
the "nr" database, using the BLASTP algorithm (Altschul et al., J.
Mol. Biol. 215:403-410 (1990)) provided by the NCBI.
[0305] Ignoring all hits to any delta-8 desaturase isolated from
Euglena gracilis, the BLASTP searches showed that EgD8S was most
homologous to the following proteins:
TABLE-US-00006 TABLE 6 Homologous Proteins To EgD8S, Based On
BLASTP Analysis % % GenBank Iden- Simi- Accession No. Protein
Organism tity larity E-Value CAE65324 hypothetical Caenorhabditis
38 56 1E-65 protein briggsae CBG10258 AAR27297 delta-6 Amylomyces
35 52 3E-65 desaturase rouxii AAS93682 delta-6 Rhizopus 32 53 4E-64
desaturase orizae *"% Identity" is defined as the percentage of
amino acids that are identical between the two proteins. **"%
Similarity" is defined as the percentage of amino acids that are
identical or conserved between the two proteins. ***"E-Value"
(Expectation Value) estimates the statistical significance of the
match, specifying the number of matches, with a given score, that
are expected in a search of a database of this size absolutely by
chance.
[0306] In order to select the amino acid residues that could be
mutated within EgD8S without affecting the delta-8 desaturase
activity, a set of criteria were developed to identify preferred
targets for mutation, as outlined below.
[0307] 1. Preferred amino acid residue targets of the EgD8S
desaturase domain (located between amino acid residues 79 to 406 of
SEQ ID NO:10) are not conserved, when compared to the delta-6
desaturase of A. rouxii (supra; "ArD6"; SEQ ID NO:13), the delta-6
desaturase of R. orizae (supra; "RoD6"; SEQ ID NO:14) and
representatives of other desaturases such as the delta-8 fatty acid
desaturase-like protein of Leishmania major (GenBank Accession No.
CAJ09677; "LmD8L"; SEQ ID NO:15), and the delta-6 desaturase of
Mortierella isabellina (GenBank Accession No. AAG38104; "MiD6"; SEQ
ID NO:16). An alignment of these proteins is shown in FIG. 3, using
the method of Clustal W (slow, accurate, Gonnet option; Thompson et
al., Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign.TM.
program of DNASTAR software. It was hypothesized that changes in
the non-conserved regions among these 5 different desaturases
should not affect the delta-8 desaturase activity of EgD8S.
[0308] 2. Preferred amino acid residue targets of the cytochrome
b.sub.5 domain of EgD8S (located between amino acids 5 to 71 of SEQ
ID NO:10) are not conserved, when compared to the cytochrome
b.sub.5 genes of Saccharomyces cerevisiae (GenBank Accession No.
P40312; "SCb5"; SEQ ID NO:178) and Schizosaccharomyces pombe
(GenBank Accession No. 094391; SPb5; SEQ ID NO:179). An alignment
of the N-terminal portion of EgD8S (i.e., amino acid residues 1-136
of SEQ ID NO:10) with SCb5 and SPb5 is shown in FIG. 4, using the
method of Clustal W (supra) of the MegAlign.TM. program of DNASTAR
software. It was hypothesized that changes in the non-conserved
region among these 3 different proteins should not affect the
electron transport function of the cytochrome b.sub.5 domain of
EgD8S and thus not affect the delta-8 desaturase activity.
[0309] 3. Preferred amino acid residue targets are on the
transmembrane helices close to the endoplasmic reticulum (ER) side
of the membrane or exposed to the ER lumen.
[0310] 4. Preferred amino acid residue targets are close to the
N-terminal or C-terminal ends of the EgD8S enzyme, since
non-conserved residues in these regions may tolerate more
mutations.
[0311] Based on the above criteria, a set of 70 target mutation
sites (comprising one, two or three amino acid residues) within
EgD8S (i.e., SEQ ID NO:10) were selected for mutation as described
below in Table 7. Of the individual 126 amino acid residue
mutations, 53 (i.e., 42.1%) were identified as "non-conservative
amino acid substitutions" while 73 (i.e., 57.9%) were identified as
"conservative amino acid substitutions".
TABLE-US-00007 TABLE 7 Selected Amino Acid Residues Suitable For
Targeted Mutation Mutation Sequence Mutations Site Within SEQ ID
NO: 10 M1 4S to A, 5K to S M2 12T to V M3 16T to K, 17T to V M4 25N
to D, 26F to E M5 31A to D, 32E to S M6 59K to L M7 61M to A, 62P
to V M8 66P to Q, 67S to A M9 72P to Q, 73Q to P M10 79A to Q, 80Q
to A M11 87R to A, 88E to I M12 407A to S, 408V to Q M13 412M to S,
413A to Q M14 416Q to V, 417P to Y M15 422L to Q M16 108S to L M17
110T to A M18 120L to M, 121M to L M19 122V to S M20 123Q to Y,
124Y to Q M21 125Q to H, 126M to L M22 127Y to Q M23 288S to N M24
289I to P, 290L to M M25 291T to V, 292S to V M26 293L to M M27
296F to T M28 298V to S M29 392N to T, 393P to T M30 394L to G,
395P to M M31 7Q to L, 8A to S M32 10P to W, 11L to Q M33 21S to F,
22A to S M34 46F to S, 47M to L M35 48V to F, 49M to L M36 37Y to
F, 38Q to N M37 51S to T, 52Q to N M38 54A to G, 55F to Y M39 64I
to L, 65N to D M40 69E to D, 70L to V M41 75A to G, 76V to L M42
89E to D, 90L to I M43 97D to E, 98A to V M44 110T to S, 111L to V
M45 117G to A, 118Y to F M46 132V to L, 133L to V M47 198D to E,
199I to L M48 231L to V, 232V to L M49 297F to V, 298V to L M50
309I to V, 310V to I M51A 347I to L, 348T to S M51B 346I to V, 347I
to L, 348T to S M52 400V to I, 401I to V M53 9L to V M54 19D to E,
20V to I M55 33I to L M56 45A to G, 46F to Y M57 57K to R, 58L to I
M58 65N to Q M59 73Q to N, 74A to G M60 96F to Y M61 239F to I,
240I to F M62 271L to M, 272A to S M63 279T to L, 280L to T M64
130G to A, 131A to G M65 304G to F, 305F to G M66 229F to Y, 230Y
to F M67 291T to S, 292S to L M68 162L to V, 163V to L M69 170G to
A, 171L to V M70 418A to G, 419G to A
Example 3
Generation of Yarrowia lipolytica Strains Y4001 And Y4001U to
Produce about 17% EDA of Total Lipids
[0312] The present Example describes the construction of strains
Y4001 and Y4001U, derived from Yarrowia lipolytica ATCC #20362, and
each capable of producing 17% EDA (C20:2) relative to the total
lipids. Both strains were engineered to test functional expression
of EgD8S and mutations thereof. Thus, it was necessary to construct
host strains capable of producing the delta-8 desaturase substrate,
EDA.
[0313] The development of strain Y4001U, having a Leu- and
Ura-phenotype, required the construction of strain Y2224 (a FOA
resistant mutant from an autonomous mutation of the Ura3 gene of
wildtype Yarrowia strain ATCC #20362) and strain Y4001.
Generation of Strain Y2224
[0314] Strain Y2224 was isolated in the following manner: Yarrowia
lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast
extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a
MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with
ammonia sulfate, without amino acid, and 20 g/L glucose) containing
250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28.degree.
C. and four of the resulting colonies were patched separately onto
MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil
and uridine to confirm uracil Ura3 auxotrophy.
Generation of Strain Y4001 to Produce 17% EDA of Total Lipids
[0315] Strain Y4001 was created via integration of construct
pZKLeuN-29E3 (FIG. 5A; comprising four chimeric genes [a delta-12
desaturase, a C.sub.16/18 elongase and two delta-9 elongases]) into
the Leu2 loci of Y2224 strain to thereby enable production of
EDA.
[0316] Construct pZKLeuN-29E3 contained the following
components:
TABLE-US-00008 TABLE 8 Description of Plasmid pZKLeuN-29E3 (SEQ ID
NO: 17) RE Sites And Nucleotides Within SEQ ID Description Of NO:
17 Fragment And Chimeric Gene Components BsiW I/Asc I 795 bp 3'
part of Yarrowia Leu2 gene (7797-7002) (GenBank Accession No.
AF260230) Sph I/Pac I 703 bp 5' part of Yarrowia Leu2 gene
(4302-3591) (GenBank Accession No. AF260230) Swa I/BsiW I
GPD::F.D12::Pex20, comprising: (10500-7797) GPD: Yarrowia
lipolytica GPD promoter (WO 2005/003310) F.D12: Fusarium
moniliforme delta-12 desaturase gene (WO 2005/047485) Pex20: Pex20
terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.
AF054613) Bgl II/Swa I Exp pro::EgD9E::Lip1, comprising:
(12526-10500) Exp pro: Yarrowia lipolytica export protein (EXP1)
promoter (WO 2006/052870 and U.S. Patent Application No.
11/265,761) EgD9E: codon-optimized delta-9 elongase gene (SEQ ID
NO: 177), derived from Euglena gracilis (SEQ ID NOs: 175 and 176;
U.S. Patent Application No. 60/739,989; see also Example 16 herein)
Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank
Accession No. Z50020) Pme I/Cla I FBAINm::EgD9S::Lip2, comprising:
(12544-1) FBAINm: Yarrowia lipolytica FBAINm promoter (WO
2005/049805) EgD9S: codon-optimized delta-9 elongase gene (SEQ ID
NO: 177; supra) Lip2: Lip2 terminator sequence from Yarrowia Lip2
gene (GenBank Accession No. AJ012632) Cla I/EcoR I
LoxP::Ura3::LoxP, comprising: (1-1736) LoxP sequence (SEQ ID NO:
18) Yarrowia Ura3 gene (GenBank Accession No. AJ306421) LoxP
sequence (SEQ ID NO: 18) EcoR I/Pac I NT::ME3S::Pex16, comprising:
(1736-3591) NT: Yarrowia lipolytica YAT1 promoter (Patent
Publication US 2006/0094102-A1) ME3S: codon-optimized C.sub.16/18
elongase gene (SEQ ID NO: 19), derived from M. alpina (see U.S.
Patent Application No. 11/253,882 and also WO 2006/052870) Pex16:
Pex16 terminator sequence of Yarrowia Pex 16 gene (GenBank
Accession No. U75433)
[0317] Plasmid pZKLeuN-29E3 was digested with Asc I/Sph I, and then
used for transformation of Y. lipolytica strain Y2224 (i.e., ATCC
#20362 Ura3-) according to the General Methods. The transformant
cells were plated onto MMLeu media plates and maintained at
30.degree. C. for 2 to 3 days. The colonies were picked and
streaked onto MM and MMLeu selection plates. The colonies that
could grow on MMLeu plates but not on MM plates were selected as
Leu-strains. Single colonies of Leu-strains were then inoculated
into liquid MMLeu at 30.degree. C. and shaken at 250 rpm/min for 2
days. The cells were collected by centrifugation, lipids were
extracted, and fatty acid methyl esters were prepared by
trans-esterification, and subsequently analyzed with a
Hewlett-Packard 6890 GC.
[0318] GC analyses showed the presence of EDA in the transformants
containing the 4 chimeric genes of pZKLeuN-29E3, but not in the
Yarrowia Y2224 control strain. Most of the selected 36 Leu-strains
produced about 12 to 16.9% EDA of total lipids. There were 3
strains (i.e., strains #11, #30 and #34) that produced about 17.4%,
17% and 17.5% EDA of total lipids; they were designated as strains
Y4001, Y4002 and Y4003, respectively.
Generation of Strain Y4001U (Leu-, Ura-) To Produce 17% EDA of
Total Lipids
[0319] Strain Y4001U was created via temporary expression of the
Cre recombinase enzyme in plasmid pY116 (FIG. 5B) within strain
Y4001 to produce a Leu- and Ura-phenotype. Construct pY116
contained the following components:
TABLE-US-00009 TABLE 9 Description of Plasmid pY116 (SEQ ID NO:
180) RE Sites And Nucleotides Within SEQ ID Description Of Fragment
And Chimeric Gene NO: 180 Components 1328-448 ColE1 plasmid origin
of replication 2258-1398 Ampicillin-resistance gene (Amp.sup.R)
3157-4461 Yarrowia autonomous replication sequence (ARS18; GenBank
Accession No. A17608) PacI/SawI Yarrowia Leu2 gene (GenBank
Accession No. 6667-4504 AF260230) 6667-180 GPAT::Cre::XPR2,
comprising: GPAT: Yarrowia lipolytica GPAT promoter (WO
2006/031937) Cre: Enterobacteria phage P1 Cre gene for recombinase
protein (GenBank Accession No. X03453) XPR2: ~100 bp of the 3'
region of the Yarrowia Xpr gene (GenBank Accession No. M17741)
[0320] Plasmid pY116 was used for transformation of freshly grown
Y4001 cells according to the General Methods. The transformant
cells were plated onto MMU plates containing 280 .mu.g/mL
sulfonylurea and maintained at 30.degree. C. for 3 to 4 days. Four
colonies were picked, inoculated into 3 mL liquid YPD media at
30.degree. C. and shaken at 250 rpm/min for 1 day. The cultures
were diluted to 1:50,000 with liquid MMU media, and 100 .mu.L was
plated onto new YPD plates and maintained at 30.degree. C. for 2
days. Colonies were picked and streaked onto MMLeu and MMLeu+Ura
selection plates. The colonies that could grow on MMLeu+Ura plates
but not on MMLeu plates were selected and analyzed by GC to confirm
the presence of C20:2 (EDA). One strain, having a Leu- and
Ura-phenotype, produced about 17% EDA of total lipids and was
designated as Y4001U.
Example 4
Generation of Auto-Replicating Plasmid pFmD8S
[0321] The present Example describes the construction of plasmid
pFmD8S comprising a chimeric FBAINm::EgD8S::XPR gene. Plasmid
pFmD8S (SEQ ID NO:20; FIG. 6D) was constructed by three-way
ligation using fragments from plasmids pKUNFmkF2, pDMW287F and
pDMW214. Plasmid pFmD8S, an auto-replicating plasmid that will
reside in Yarrowia in 1-3 copies, was utilized to test functional
expression of EgD8S (and mutations thereof), as described in
Examples 5-10, infra.
Plasmid pKUNFmkF2
[0322] pKUNFmkF2 (SEQ ID NO:21; FIG. 6A; WO 2006/012326) is a
construct comprising a chimeric FBAINm::F.D12::Lip2 gene (wherein
"FBAINmK" is the Yarrowia lipolytica FBAINm promoter [WO
2005/049805], "F.D12" is the Fusarium moniliforme delta-12
desaturase [WO 2005/047485], and "Lip2" is the Yarrowia lipolytica
Lip2 terminator sequence (GenBank Accession No. AJ012632)).
Plasmid pDMW287F
[0323] pDMW287F (SEQ ID NO:22; FIG. 6B; WO 2006/012326) is a
construct comprising the synthetic delta-8 desaturase, derived from
wildtype Euglena gracilis, and codon-optimized for expression in
Yarrowia lipolytica (wherein EgD8S is identified as "D8SF" in the
figure). The desaturase gene is flanked by a Yarrowia lipolytica
FBAIN promoter (WO 2005/049805; identified as "FBA1+intron" in the
figure) and a Pex16 terminator sequence of the Yarrowia Pex16 gene
(GenBank Accession No. U75433).
Plasmid pDMW214
[0324] pDMW214 (SEQ ID NO:23; FIG. 6C; WO 2005/049805) is a shuttle
plasmid that could replicate both in E. coli and Yarrowia
lipolytica. It contained the following components:
TABLE-US-00010 TABLE 10 Description Of Plasmid pDMW214 (SEQ ID NO:
23) RE Sites And Nucleotides Within SEQ ID Description Of NO: 23
Fragment And Chimeric Gene Components 1150-270 ColE1 plasmid origin
of replication 2080-1220 Ampicillin-resistance gene (Amp.sup.R)
2979-4256 Yarrowia autonomous replication sequence (ARS18; GenBank
Accession No. A17608) PmeI/SphI Yarrowia Leu2 gene 6501-4256
(GenBank Accession No. AF260230) 6501-1 FBA1 + intron::GUS::XPR2,
comprising: FBA1 + intron: Yarrowia lipolytica FBAIN promoter (WO
2005/049805) GUS: E. coli gene encoding .beta.-glucuronidase
(Jefferson, R. A. Nature. 342: 837-838 (1989) XPR2: ~100 bp of the
3' region of the Yarrowia Xpr gene (GenBank Accession No.
M17741)
[0325] Final Construction of Plasmid pFmD8S
[0326] The PmeI/NcoI fragment of plasmid pKUNFmkF2 (FIG. 6A;
comprising the FBAINm promoter) and the NcoI/NotI fragment of
plasmid pDMW287F (FIG. 6B; comprising the synthetic delta-8
desaturase gene EgD8S) were used directionally to replace the
PmeI/Not I fragment of pDMW214 (FIG. 6C). This resulted in
generation of pFmD8S (SEQ ID NO:20; FIG. 6D), comprising a chimeric
FBAINm::EgD8S::XPR2 gene. Thus, the components of pFmD8S are as
described in Table 11 below.
TABLE-US-00011 TABLE 11 Components Of Plasmid pFmD8S (SEQ ID NO:
20) RE Sites And Nucleotides Within SEQ ID NO: 20 Description Of
Fragment And Chimeric Gene Components Swa I/Sac II
FBAINm::EgD8S::XPR2, comprising: (7988-1461) FBAINm: Yarrowia
lipolytica FBAINm promoter (WO 2005/049805) EgD8S: codon-optimized
delta-8 desaturase gene (SEQ ID NO: 9, identified as "D8-corrected"
in FIG. 6D), derived from Euglena gracilis (SEQ ID NO: 11) XPR2:
~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank
Accession No. M17741) 2601-1721 ColE1 plasmid origin of replication
3531-2671 Ampicillin-resistance gene (Amp.sup.R) for selection in
E. coli 4430-5734 Yarrowia autonomous replication sequence (ARS18;
GenBank Accession No. A17608) 7942-5741 Yarrowia Leu2 gene (GenBank
Accession No. AF260230)
Example 5
Development of a Quick Screen to Functionally Analyze Delta-8
Desaturase Activity in Yarrowia lipolytica
[0327] A set of 40 mutations was created using pFmD8S (Example 4)
as template and 40 pairs of oligonucleotide primers to individually
mutate targeted amino acid residues within EgD8S (SEQ ID NO:10) by
site-directed mutagenesis (QuikChange.RTM. Kit, Stratagene, La
Jolla, Calif.). Specific mutations were selected from those set
forth in Table 7 of Example 2 and primer pairs were selected from
the oligonucleotides set forth in SEQ ID NOs:24-164, such that
creation of the M1 mutation (i.e., 4S to A, 5K to S within SEQ ID
NO:10) required primers 1A and 1B (SEQ ID NOs:24 and 25,
respectively), etc. Plasmids from each mutation were transformed
into E. coli XL2Blue cells (Stratagene). Four colonies from each of
the 40 transformations were picked and grown individually in liquid
media at 37.degree. C. overnight. Plasmids (i.e., 160 total) were
isolated from these cultures and sequenced individually to confirm
the mutations.
[0328] Plasmid pFmD8S and the isolated mutant plasmids were
transformed into strain Y4001 (Example 3) individually, as
described in the General Methods. The transformants were selected
on MM plates. After 2 days growth at 30.degree. C., transformants
were scraped from each plate, lipids were extracted, and fatty acid
methyl esters were prepared by trans-esterification, and
subsequently analyzed with a Hewlett-Packard 6890 GC.
[0329] GC analyses showed that there were about 7% DGLA and 12% EDA
of total lipids produced by the transformants with plasmid pFmD8S;
the average conversion efficiency whereby EgD8S converted EDA to
DGLA in the transformants was 36.8%. The conversion efficiency for
delta-8 desaturase (also referred to as the delta-8 desaturase
activity) was measured according to the following formula:
([product]/[substrate+product])*100, where `product` includes the
immediate product and all products in the pathway derived from
it.
[0330] GC analyses of transformants carrying mutations within EgD8S
showed that 30 of the 40 mutations did not affect the delta-8
desaturase activity (when compared to the synthetic codon-optimized
EgD8S delta-8 desaturase activity in transformants carrying plasmid
pFmD8S). These results suggested that the screening procedure
described herein (i.e., with pFmD8S as parent plasmid and strain
Y4001 as a host) could be used to quickly screen the
EgD8S.quadrature. mutants and identify which mutations negatively
affected delta-8 desaturase activity.
[0331] Based on these results, the remaining 30 mutations set forth
in Table 7 of Example 2 were synthesized (although some mutations
were introduced in combination for efficiency), using the
methodology described above. Table 12 describes the delta-8
desaturase relative activity attributed to each mutation site
(i.e., M1 to M70), as a percent of the delta-8 desaturase activity
resulting in each mutant EgD8S with respect to the delta-8
desaturase activity of the synthetic codon-optimized EgD8S (SEQ ID
NO:10). As seen in Table 12, delta-8 desaturase relative activity
ranged from 0% up to 125%.
TABLE-US-00012 TABLE 12 Mutant.quadrature. Delta-8 Desaturase
Activities Relative Mutations Primers Used Activity % M1 1A, 1B
115% (SEQ ID NOs: 24 and 25) M2 2A, 2B 110% (SEQ ID NOs: 26 and 27)
M3 3A, 3B 100% (SEQ ID NOs: 28 and 29) M4 4A, 4B N/A (SEQ ID NOs:
30 and 31) M5 5A, 5B N/A (SEQ ID NOs: 32 and 33) M6 6A, 6B 110%
(SEQ ID NOs: 34 and 35) M7 7A, 7B 30% (SEQ ID NOs: 36 and 37) M8
8A, 8B 100% (SEQ ID NOs: 38 and 39) M9 9A, 9B N/A (SEQ ID NOs: 40
and 41) M10 10A, 10B N/A (SEQ ID NOs: 42 and 43) M11 11A, 11B 20%
(SEQ ID NOs: 44 and 45) M12 12A, 12B 100% (SEQ ID NOs: 46 and 47)
M13 13A, 13B N/A (SEQ ID NOs: 48 and 49) M14 14A, 14B 100% (SEQ ID
NOs: 50 and 51) M15 15A, 15B 125% (SEQ ID NOs: 52 and 53) M16 16A,
16B 100% (SEQ ID NOs: 54 and 55) M17 17A, 17B 50% (SEQ ID NOs: 56
and 57) M18 18A, 18B N/A (SEQ ID NOs: 58 and 59) M19 19A, 19B 100%
(SEQ ID NOs: 60 and 61) M20 20A, 20B 80% (SEQ ID NOs: 62 and 63)
M21 21A, 21B 120% (SEQ ID NOs: 64 and 65) M22 22A, 22B 110% (SEQ ID
NOs: 66 and 67) M23 23A, 23B 80% (SEQ ID NOs: 68 and 69) M24 24A,
24B N/A (SEQ ID NOs: 70 and 71) M25 25A, 25B N/A (SEQ ID NOs: 72
and 73) M26 26A, 26B 110% (SEQ ID NOs: 74 and 75) M27 27A, 27B 80%
(SEQ ID NOs: 76 and 77) M28 28A, 28B 90% (SEQ ID NOs: 78 and 79)
M29 29A, 29B N/A (SEQ ID NOs: 80 and 81) M30 30A, 30B 85% (SEQ ID
NOs: 82 and 83) M31 31A, 31B 85% (SEQ ID NOs: 84 and 85) M32 32A,
32B N/A (SEQ ID NOs: 86 and 87) M33 33A, 33B N/A (SEQ ID NOs: 88
and 89) M34 34A, 34B N/A (SEQ ID NOs: 90 and 91) M35 35A, 35B 0%
(SEQ ID NOs: 92 and 93) M36 36A, 36B 80% (SEQ ID NOs: 94 and 95)
M37 37A, 37B 90% (SEQ ID NOs: 96 and 97) M38 38A, 38B 100% (SEQ ID
NOs: 98 and 99) M39 39A, 39B 100% (SEQ ID NOs: 100 and 101) M40
40A, 40B 100% (SEQ ID NOs: 102 and 103) M41 41A, 41B 100% (SEQ ID
NOs: 104 and 105) M42 42A, 42B 0% (SEQ ID NOs: 106 and 107) M43
43A, 43B 90% (SEQ ID NOs: 108 and 109) M44 44A, 44B N/A (SEQ ID
NOs: 110 and 111) M45 45A, 45B 100% (SEQ ID NOs: 112 and 113) M46
46A, 46B 100% (SEQ ID NOs: 114 and 115) M47 47A, 47B 0% (SEQ ID
NOs: 116 and 117) M48 48A, 48B N/A (SEQ ID NOs: 118 and 119) M49
49A, 49B 100% (SEQ ID NOs: 120 and 121) M50 50A, 50B 100% (SEQ ID
NOs: 122 and 123) M51 51A, 51B 100% (SEQ ID NOs: 124 and 125) M51B
51A, 51B 100% (SEQ ID NOs: 126 and 125) M52 52A, 52B 80% (SEQ ID
NOs: 127 and 128) M53 53A, 53B 100% (SEQ ID NOs: 129 and 130) M54
54A, 54B 100% (SEQ ID NOs: 131 and 132) M55 55A, 55B 40% (SEQ ID
NOs: 133 and 134) M56 56A, 56B 0% (SEQ ID NOs: 135 and 136) M57
57A, 57B N/A (SEQ ID NOs: 137 and 138) M58 58A, 58B 100% (SEQ ID
NOs: 139 and 140) M59 59A, 59B 90% (SEQ ID NOs: 141 and 142) M60
60A, 60B 50% (SEQ ID NOs: 143 and 144) M61 61A, 61B 50% (SEQ ID
NOs: 145 and 146) M62 62A, 62B 50% (SEQ ID NOs: 147 and 148) M63
63A, 63B 100% (SEQ ID NOs: 149 and 150) M64 64A, 64B 60% (SEQ ID
NOs: 151 and 152) M65 65A, 65B 0% (SEQ ID NOs: 153 and 154) M66
66A, 66B N/A (SEQ ID NOs: 155 and 156) M67 67A, 67B N/A (SEQ ID
NOs: 157 and 158) M68 68A, 68B 100% (SEQ ID NOs: 159 and 160) M69
69A, 69B 100% (SEQ ID NOs: 161 and 162) M70 70A, 70B 100% (SEQ ID
NOs: 163 and 164) *N/A is reported when the desired mutation was
not successfully produced of when GC data was lacking.
Example 6
Generation of pFmD8S-1, pFmD8S-001, pFmD8S-2A, pFmD8S-2B,
pFmD8S-3A, pFmD8S-3B, pFmD8S-003, pFmD8S-4, pFmD8S-004, pFmD8S-005
and pFmD8S-006 Constructs by Site-Directed Mutagenesis of EgD8S
within Construct pFmD8S
[0332] A series of plasmids were generated by consecutive rounds of
continued site-directed mutagenesis to introduce multiple select
mutations into EgD8S (SEQ ID NOs:9 and 10). pFmD8S (Example 4),
comprising the synthetic codon-optimized EgD8S, was used as the
starting template in Tables 13, 14, 16 and 17, while a mutant
created thereof (i.e., pFmD8S-M45, comprising 117G to A and 118Y to
F mutations with respect to SEQ ID NO:10) was used as the starting
template in Table 15. The resulting plasmids comprising mutant
EgD8S sequences, as well as details concerning the primers used to
produce these mutations, are described in Tables 13, 14, 15, 16 and
17.
[0333] The column titled "Mutation Site Introduced" refers to the
specific amino acid sites selected for mutation, as listed in Table
7 of Example 2. In the column titled "Total Mutations In Resultant
Plasmid With Respect to EgD8S (SEQ ID NO:10)", those amino acid
mutations that are highlighted in bold-face text correspond to
newly introduced mutations that were not present in the template in
the indicated round of site-directed mutagenesis. The number shown
in parentheses corresponds with the number of total mutations in
the resultant plasmid with respect to EgD8S (SEQ ID NO:10).
TABLE-US-00013 TABLE 13 Generation Of pFmD8S-1 And pFmD8S-001
Constructs Round - Total Mutations In Mutation Resultant Plasmid
Site Resultant With Respect to Introduced Template Primers Plasmid
EgD8S (SEQ ID NO: 10) 1 - pFmD8S 3A, 3B pFmD8S-M3 16T to K, 17T to
V M3 (SEQ ID NOs: 28 (2) and 29) 2 - pFmD8S- 1A, 1B pFmD8S-M3, 1 16
T to K, 17T to V, M1 M3 (SEQ ID NOs: 24 4S to A, 5K to S and 25)
(4) 3 - pFmD8S- 2A, 2B pFmD8S- 16 T to K, 17T to V, M2 M3, 1 (SEQ
ID NOs: 26 M3, 1, 2 4S to A, 5K to S, and 27) 12T to V (5) 4 -
pFmD8S- 8A, 8B pFmD8S-1 16 T to K, 17T to V, M8 M3, 1, 2 (SEQ ID
NOs: 38 4S to A, 5K to S, and 39) 12T to V, 66P to Q, 67S to A (7)
5 - pFmD8S-1 38A, 38B pFmD8S-001 16 T to K, 17T to V, M38 (SEQ ID
NOs: 98 4S to A, 5K to S, and 99) 12T to V, 65P to Q, 66S to A, 54A
to G, 55F to Y (9)
TABLE-US-00014 TABLE 14 Generation Of pFmD8S-2A And pFmD8S-003
Constructs Round - Total Mutations In Mutation Resultant Plasmid
Site Resultant With Respect to Introduced Template Primers Plasmid
EgD8S (SEQ ID NO: 10) 1 - pFmD8S 45A, 45B pFmD8S-M45 117G to A,
118Y to M45 (SEQ ID NOs: 112 F (2) and 113) 2 - pFmD8S- 21A, 21B
pFmD8S- 117G to A, 118Y to M21 M45 (SEQ ID NOs: 64 M45, 21 F, 125Q
to H, and 65) 126M to L (4) 3 - pFmD8S- 16A, 16B pFmD8S- 117G to A,
118Y to M16 M45, 21 (SEQ ID NOs: 54 M45, 21, 16 F, 125Q to H, and
55) 126M to L, 108S to L (5) 4 - pFmD8S- 18A, 18B pFmD8S-2A 117G to
A, 118Y to M18 M45, 21, 16 (SEQ ID NOs: 58 F, 125Q to H, and 59)
126M to L, 108S to L, 120L to M, 121M to L (7) 5 - pFmD8S- 68A, 68B
pFmD8S-003 117G to A, 118Y to M68, M69 2A (SEQ ID NOs: 159 F, 125Q
to H, and 160) 126M to L, 108S to 69A, 69B L, 120L to M, (SEQ ID
NOs: 161 121M to L, 162L to and 162) V, 163V to L, 170G to A, 171L
to V (11)
TABLE-US-00015 TABLE 15 Generation Of pFmD8S-2B And pFmD8S-004
Constructs Total Mutations In Round - Resultant Plasmid Mutation
With Respect to Site Resultant EgD8S (SEQ ID Introduced Template
Primers Plasmid NO: 10) 1 - pFmD8S- 46A, 46B pFmD8S- 117G to A,
118Y to M46 M45 (SEQ ID NOs: 114 M45, 46 F, 132V to L, and 115) 133
L to V (4) 2 - pFmD8S- 16A, 16B pFmD8S- 117G to A, 118Y to M16, M21
M45, 46 (SEQ ID NOs: 54 M45, 46, 16, 21 F, 132V to L, and 55) 133 L
to V, 108S to 21A, 21B L, 125Q to H, (SEQ ID NOs: 64 126M to L (7)
and 65) 3 - pFmD8S- 18A, 18B pFmD8S-2B 117G to A, 118Y to M18 M45,
46, 16, (SEQ ID NOs: 58 F, 132V to L, 21 and 59) 133 L to V, 108S
to L, 125Q to H, 126M to L, 120L to M, 121M to L (9) 4 - pFmD8S-
68A, 68B pFmD8S-004 117G to A, 118Y to M68, M69 2B (SEQ ID NOs: 159
F, 132V to L, and 160) 133 L to V, 108S to 69A, 69B L, 125Q to H,
(SEQ ID NOs: 161 126M to L, 120L to and 162) M, 121M to L, 162L to
V, 163V to L, 170G to A, 171L to V (13)
TABLE-US-00016 TABLE 16 Generation Of pFmD8S-3A, pFmD8S-3B And
pFmD8S-005 Constructs Round - Total Mutations In Mutation Resultant
Plasmid Site Resultant With Respect to Introduced Template Primers
Plasmid EgD8S (SEQ ID NO: 10) 1 - pFmD8S 49A, 49B pFmD8S-M49 297F
to V, 298V to M49 (SEQ ID NOs: 120 L (2) and 121) 2 - pFmD8S- 26A,
26B pFmD8S- 297F to V, 298V to M26 M49 (SEQ ID NOs: 74 M49, 26 L,
293L to M (3) and 75) 3A - pFmD8S- 61A, 61B pFmD8S-3A 297F to V,
298V to M61 M49, 26 (SEQ ID NOs: 145 L, 293L to M, and 146) 239F to
I, 240I to F (5) 3B - pFmD8S- 62A, 62B pFmD8S-3B 297F to V, 298V to
M62, M63 M49, 26 (SEQ ID NOs: 147 L, 293L to M, and 148) 271L to M,
272A to 63A, 63B S, 279T to L, (SEQ ID NOs: 149 280L to T (7) and
150) 4 - pFmD8S- 63A, 63B pFmD8S-005 297F to V, 298V to M63 3A (SEQ
ID NOs: 149 L, 293L to M, and 150) 239F to I, 240I to F, 279T to L,
280L to T (7)
TABLE-US-00017 TABLE 17 Generation Of pFmD8S-4 And pFmD8S-006
Constructs Total Mutations In Round - Resultant Plasmid Mutation
With Respect to Site Resultant EgD8S (SEQ ID Introduced Template
Primers Plasmid NO: 10) 1 - pFmD8S 51A, 51B pFmD8S-M51 346I to V,
347I to M51B (SEQ ID NOs: 126 L, 348T to S (3) and 125) 2 - pFmD8S-
15A, 15B pFmD8S- 346I to V, 347I to L, M15 M51 (SEQ ID NOs: 52 M51,
15 348T to S, and 53) 422L to Q (4) 3 - pFmD8S- 14A, 14B pFmD8S-
346I to V, 347I to L, M14 M51, 15 (SEQ ID NOs: 50 M51, 15, 14 348T
to S, and 51) 422L to Q, 416Q to V, 417P to Y (6) 4 - pFmD8S- 12A,
12B pFmD8S-4 346I to V, 347I to L, M12 M51, 15, 14 (SEQ ID NOs: 46
348T to S, and 47) 422L to Q, 416Q to V, 417P to Y, 407A to S, 408V
to Q (8) 5 - pFmD8S-4 70, 70B pFmD8S-006 346I to V, 347I to L, M70
(SEQ ID NOs: 163 348T to S, and 164) 422L to Q, 416Q to V, 417P to
Y, 407A to S, 408V to Q, 418A to G, 419G to A (10)
[0334] After each round of mutagenesis, the mutations in each
resulting plasmid was confirmed by DNA sequencing. Additionally,
the delta-8 desaturase activity of each mutant EgD8S within each
mutant plasmid was compared with the delta-8 desaturase activity of
the synthetic codon-optimized EgD8S within pFmD8S by transforming
the plasmids into strain Y4001 (Example 3) and assaying activity
based on the methodology described in Example 5. Based on these
functional analyses, it was demonstrated that the mutations in all
24 of the mutant EgD8S genes within the resultant plasmids
generated in Tables 11, 12, 13, 14 and 15 did not affect delta-8
desaturase activity (i.e., pFmD8S-M3; pFmD8S-M3,1; pFmD8S-M3,1,2;
pFmD8S1; pFmD8S-001; pFmD8S-M45; pFmD8S-M45,21; pFmD8S-M45,21,16;
pFmD8S-2A; pFmD8S-003; pFmD8S-M45,46; pFmD8S-M45,46,16,21;
pFmD8S-2B; pFmD8S-004; pFmD8S-M49; pFmD8S-M49,26; pFmD8S-3A;
pFmD8S-3B; pFmD8S-005; pFmD8S-M51; pFmD8S-M51,15; pFmD8S-M51,15,14;
pFmD8S-4; and pFmD8S-006).
Example 7
Generation of Complex Construct pFmD8S-5B by Digestion and Ligation
of Multiple Parent Plasmids
[0335] Plasmid pFmD8S-5B contained 16 mutant amino acids within the
first half of EgD8S. This plasmid was generated by 3-way ligation,
wherein the 318 bp Nco I/Bgl II fragment from pFmD8S-1 (containing
7 amino acid mutations, corresponding to M1, M2, M3 and M8) and the
954 bp Bgl II/Not I fragment from pFmD8S-2B (containing 9 amino
acid mutations, corresponding to M16, M18, M21, M45 and M46) were
used to replace the Nco I/Not I fragment of pFmD8S (Example 4; FIG.
6D). DNA sequence confirmed that pFmD8S-5B contained the expected
16 amino acid mutations within EgD8S.
[0336] The synthesis of plasmid pFmD8S-5B is schematically
diagrammed in FIG. 7 (and a similar format is used in FIG. 8 and
FIG. 9). For clarity, the pFmD8S vector backbone in which each
mutant EgD8S is contained is not included within the figure;
instead, only the 1272 bases corresponding to the mutant EgD8S are
shown (wherein the coding sequence for the delta-8 desaturase
corresponds to nucleotide bases 2-1270). Thus, the mutant EgD8S
fragment labeled as "Mutant EgD8S-1" in FIG. 7 corresponds to the
mutant EgD8S found within plasmid pFmD8S-1 and the mutant EgD8S
fragment labeled as "Mutant EgD8S-2B" in FIG. 7 corresponds to the
mutant EgD8S found within plasmid pFmD8S-2B.
[0337] Similarly, the Nco I and Not I restriction enzyme sites that
flank each mutant EgD8S gene are not included in the figure. The
Nco I nucleotide recognition sequence ("CCATGG") corresponds to the
-2 to +4 region of the mutant EgD8S, wherein the `A` position of
the `ATG` translation initiation codon is designated as +1; the
first nucleotide recognized as part of the Not I nucleotide
recognition sequence is nucleotide +1271 of mutant EgD8S, wherein
the `TAA` STOP codon of mutant EgD8S is located at +1269 to
+1270.
[0338] Mutation sites are labeled on each mutant EgD8S. Those
mutation sites shown with an asterisk correspond to a single amino
acid mutation (i.e., M2* corresponds to a mutation of 12T to V),
while those lacking an asterisk correspond to two individual amino
acid mutations (i.e., M1 corresponds to mutations 4S to A and 5K to
S); those mutation sites shown with 2 asterisks correspond to a
triple amino acid mutation (i.e., M51** corresponds to mutations
346I to V, 347I to L and 348T to S).
[0339] The delta-8 desaturase activity of mutant EgD8S-5B within
pFmD8S-5B was compared with the delta-8 desaturase activity of the
synthetic codon-optimized EgD8S within pFmD8S by transforming each
plasmid into strain Y4001 (Example 3) and assaying the activity
based on the methodology described in Example 5. Based on this
analysis, it was determined that the 16 amino acid mutations within
mutant EgD8S-5B (i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to
V, 66P to Q, 67S to A, 108S to L, 120L to M, 121M to L, 125Q to H,
126M to L, 117G to A, 118Y to F, 132V to L and 133 L to V,
corresponding to mutation sites M1, M2, M3, M8, M16, M18, M21, M45
and M46) in pFmD8S-5B did not affect the delta-8 desaturase
activity.
Example 8
Generation of pFmD8S-12, pFmD8S-13, pFmD8S-23 and pFmD8S-28
Constructs by Additional Site-Directed Mutagenesis of Mutant
EgD8S-5B within Construct pFmD8S-5B
[0340] An additional series of plasmids were generated by
consecutive rounds of continued site-directed mutagenesis to
introduce multiple select mutations into mutant EgD8S-5B, using
pFmD8S-5B (Example 7) as the starting template. The resulting
plasmids comprising mutant EgD8S sequences, as well as details
concerning the primers used to produce these mutations, are
described below in Table 18. Format and column titles of Table 18
are the same as defined above in Example 6.
TABLE-US-00018 TABLE 18 Generation Of pFmD8S-12, pFmD8S-13,
pFmD8S-23 And pFmD8S-28 Constructs Total Mutations In Round -
Resultant Mutation Plasmid With Site Resultant Respect to
Introduced Template Primers Plasmid EgD8S (SEQ ID NO: 10) 1A -
pFmD8S- 12A, 12B (SEQ ID pFmD8S-12 4S to A, 5K to S, M12, M15, 5B
NOs: 46 and 47) 12T to V, 16T to M26 15A, 15B (SEQ ID K, 17T to V,
66P NOs: 52 and 53) to Q, 67S to A, 26A, 26B (SEQ ID 108S to L,
120L NOs: 74 and 75) to M, 121M to L, 125Q to H, 126M to L, 117G to
A, 118Y to F, 132V to L, 133 L to V, 407A to S, 408V to Q, 422L to
Q, 293L to M (20) 1B - pFmD8S- 12A, 12B (SEQ ID pFmD8S-13 4S to A,
5K to S, M12, M15, 5B NOs: 46 and 47) 12T to V, 16T to M26, 15A,
15B (SEQ ID K, 17T to V, 66P M51B NOs: 52 and 53) to Q, 67S to A,
26A, 26B (SEQ ID 108S to L, 120L NOs: 74 and 75) to M, 121M to L,
51A, 51B (SEQ ID 125Q to H, 126M NOs: 126 and 125) to L, 117G to A,
118Y to F, 132V to L, 133 L to V, 407A to S, 408V to Q, 422L to Q,
293L to M, 346I to V, 347I to L, 348T to S (23) 2A - pFmD8S- 68A,
68B (SEQ ID pFmD8S-23 4S to A, 5K to S, M68, M70 12 NOs: 159 and
160) 12T to V, 16T to 70A, 70B (SEQ ID K, 17T to V, 66P NOs: 163
and 164) to Q, 67S to A, 108S to L, 120L to M, 121M to L, 125Q to
H, 126M to L, 117G to A, 118Y to F, 132V to L, 133 L to V, 407A to
S, 408V to Q, 422L to Q, 293L to M, 162L to V, 163V to L, 418A to
G, 419G to A (24) 2B - pFmD8S- 38A, 38B (SEQ ID pFmD8S-28 4S to A,
5K to S, M38, M63, 13 NOs: 98 and 99) 12T to V, 16T to M68, M69,
63A, 63B (SEQ ID K, 17T to V, 66P M70 NOs: 149 and 150) to Q, 67S
to A, 68A, 68B (SEQ ID 108S to L, 120L NOs: 159 and 160) to M, 121M
to L, 69A, 69B (SEQ ID 125Q to H, 126M NOs: 161 and 162) to L, 117G
to A, 70A, 70B (SEQ ID 118Y to F, 132V NOs: 163 and 164) to L, 133
L to V, 407A to S, 408V to Q, 422L to Q, 293L to M, 346I to V, 347I
to L, 348T to S, 54A to G, 55F to Y, 279T to L, 280L to T, 162L to
V, 163V to L, 170G to A, 171L to V, 418A to G, 419G to A (33)
[0341] After each round of mutagenesis, the mutations in the
resulting plasmid were confirmed by DNA sequencing. Additionally,
the delta-8 desaturase activity of each mutant EgD8S within each
mutated plasmid was compared with the delta-8 desaturase activity
of the synthetic codon-optimized EgD8S within pFmD8S by
transforming the plasmids into strain Y4001 (Example 3) and
assaying activity based on the methodology described in Example 5.
Based on these functional analyses, it was demonstrated that the 20
mutations in mutant EgD8S-12 within pFmD8S-12, the 23 mutations in
mutant EgD8S-13 within pFmD8S-13, the 24 mutations in mutant
EgD8S-23 within pFmD8S-23 and the 33 mutations in mutant EgD8S-28
within pFmD8S-28 did not affect the delta-8 desaturase
activity.
Example 9
Generation of Complex Constructs pFmD8S-008, pFmD8S-009, pFmD8S-013
and pFmD8S-015 by Digestion and Ligation of Multiple Parent
Plasmids
[0342] Plasmids pFmD8S-008 and pFmD8S-009 contained 20 and 22
mutant amino acids within the first half of EgD8S, respectively.
These plasmids were generated by 3-way ligation, as shown in FIGS.
8A and 8B, respectively (figure format is identical to that
described for FIG. 7 in Example 7). Specifically, the 318 bp Nco
I/Bgl II fragment from pFmD8S-001 (containing 9 amino acid
mutations in mutant EgD8S-001 corresponding to M1, M2, M3, M8 and
M38) and the 954 bp Bgl II/Not I fragment from either pFmD8S-003
(containing 11 amino acid mutations in mutant EgD8S-003
corresponding to M16, M18, M21, M45, M68 and M69) or pFmD8S-004
(containing 13 amino acid mutations in mutant EgD8S-004,
corresponding to M16, M18, M21, M45, M46, M68 and M69) were used to
replace the Nco I/Not I fragment of pFmD8S (Example 4; FIG. 6D) to
generate mutant EgD8S-008 within pFmD8S-008 and mutant EgD8S-009
within pFmD8S-009, respectively. DNA sequence confirmed that mutant
EgD8S-008 contained 20 amino acid mutations and mutant EgD8S-009
contained 22 amino acid mutations, as expected.
[0343] Plasmids pFmD8S-013 and pFmD8S-015, containing 28 and 31
amino acid mutations within mutant EgD8S-013 and mutant EgD8S-015,
respectively, were created using a similar 3-way ligation strategy
as shown in FIG. 9A and FIG. 9B (figure format is identical to that
described for FIG. 7 in Example 7). The 639 bp Nco I/Xho I fragment
from either pFmD8S-009 (containing 22 amino acid mutations within
mutant EgD8S-009) or pFmD8S-008 (containing 20 amino acid mutations
within mutant EgD8S-008) and the 633 bp Xho I/Not I fragment from
either pFmD8S-23 (Example 8, containing 6 amino acid mutations
within mutant EgD8S-23, corresponding to M12, M15, M26 and M70) or
pFmD8S-28 (Example 8, containing 11 amino acid mutations within
mutant EgD8S-28, corresponding to M12, M15, M26, M51B, M63 and M70)
were used to replace the Nco I/Not I fragment of pFmD8S (Example 4;
FIG. 6D) to generate pFmD8S-013 and pFmD8S-015, respectively. DNA
sequence confirmed that mutant EgD8S-013 and mutant EgD8S-015
contained 28 amino acid mutations and 31 amino acid mutations,
respectively.
[0344] The delta-8 desaturase activity of mutant EgD8S-008 within
pFmD8S-008, mutant EgD8S-009 within pFmD8S-009, mutant EgD8S-013
within pFmD8S-013 and mutant EgD8S-015 within pFmD8S-015 were
compared with the delta-8 desaturase activity of the synthetic
codon-optimized EgD8S within pFmD8S by transforming these plasmids
into strain Y4001 (Example 3) and assaying activity based on the
methodology described in Example 5. Based on these functional
analyses, it was demonstrated the delta-8 desaturase activity was
not affected by the 20 mutations in mutant EgD8S-008 within
pFmD8S-008 (i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to V,
66P to Q, 67S to A, 108S to L, 120L to M, 121M to L, 125Q to H,
126M to L, 54A to G, 55F to Y, 117G to A, 118Y to F, 162L to V,
163V to L, 170G to A and 171 L to V, corresponding to mutation
sites M1, M2, M3, M8, M16, M18, M21, M38, M45, M68 and M69), the 22
mutations in mutant EgD8S-009 within pFmD8S-009 (i.e., 4S to A, 5K
to S, 12T to V, 16T to K, 17T to V, 66P to Q, 67S to A, 108S to L,
120L to M, 121M to L, 125Q to H, 126M to L, 54A to G, 55F to Y,
117G to A, 118Y to F, 132V to L, 133L to V, 162L to V, 163V to L,
170G to A and 171L to V, corresponding to mutation sites M1, M2,
M3, M8, M16, M18, M21, M38, M45, M46, M68 and M69), the 28
mutations in mutant EgD8S-013 within pFmD8S-013 (i.e., 4S to A, 5K
to S, 12T to V, 16T to K, 17T to V, 66P to Q, 67S to A, 407A to S,
408V to Q, 422L to Q, 108S to L, 120L to M, 121M to L, 125Q to H,
126M to L, 293L to M, 54A to G, 55F to Y, 117G to A, 118Y to F,
132V to L, 133L to V, 162L to V, 163V to L, 170G to A, 171 L to V,
418A to G and 419G to A, corresponding to mutation sites M1, M2,
M3, M8, M12, M15, M16, M18, M21, M26, M38, M45, M46, M68, M69, M70)
or the 31 mutations in mutant EgD8S-015 within pFmD8S-015 (i.e., 4S
to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P to Q, 67S to A,
407A to S, 408V to Q, 422L to Q, 108S to L, 120L to M, 121M to L,
125Q to H, 126M to L, 293L to M, 54A to G, 55F to Y, 117G to A,
118Y to F, 346I to V, 347I to L, 348T to S, 279T to L, 280L to T,
162L to V, 163V to L, 170G to A, 171L to V, 418A to G and 419G to
A, corresponding to mutation sites M1, M2, M3, M8, M12, M15, M16,
M18, M21, M26, M38, M45, M51B, M63, M68, M69, M70).
[0345] FIG. 10 shows an alignment of EgD8S (SEQ ID NO:10), Mutant
EgD8S-23 (SEQ ID NO:4; Example 8), Mutant EgD8S-013 (SEQ ID NO:6;
Example 9) and Mutant EgD8S-015 (SEQ ID NO:8; Example 9). The
method of alignment used corresponds to the "Clustal W method of
alignment".
Example 10
Comparison of Delta-8 Desaturase Activities Among the Synthetic
Codon-Optimized EgD8S and its Mutants Upon Integration into the
Yarrowia lipolytica Genome
[0346] This Example describes quantitative analyses of the delta-8
desaturase activities of EgD8S and mutants thereof included within
pFmD8S-23, pFmD8S-013 and pFmD8S-015. This comparison required each
of the chimeric genes comprising EgD8S (or a mutant thereof) to be
inserted into the pKO2UFkF2 vector backbone. Specifically,
pKO2UFkF2 comprised a 5' and 3' portion of the Yarrowia lipolytica
delta-12 desaturase gene that was designed to target integration to
this locus (although plasmid integration could also occur via
random integration into other sites of the genome). Thus, the
activities of the chimeric genes containing the synthetic
codon-optimized EgD8S, mutant EgD8S-023, mutant EgD8S-013 and
mutant EgD8S-015 in the Yarrowia genome (i.e., 1 copy) could be
more fairly compared upon integration into the Yarrowia genome, as
opposed to the delta-8 desaturase activity levels that were
obtained upon plasmid expression (i.e., via expression in pFmD8S as
1-3 copies) and reported in previous examples.
[0347] The components of pKO2UFkF2 are as described in Table 19
below.
TABLE-US-00019 TABLE 19 Components Of Plasmid pKO2UFkF2 (SEQ ID NO:
165) RE Sites And Nucleotides Within SEQ ID Description Of NO: 165
Fragment And Chimeric Gene Components SwaI/BsiWI
FBAINm::F.D12::Pex20, comprising: 7638-1722 FBAINm: Yarrowia
lipolytica FBAINm promoter (WO 2005/049805) F.D12: Fusarium
moniliforme delta-12 desaturase gene (WO 2005/047485) Pex20: Pex20
terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.
AF054613) AscI/BsiWI 5' portion of Yarrowia lipolytica delta-12
desaturase gene 2459-1722 (WO 2004/104167) EcoRI/SphI 3' portion of
Yarrowia lipolytica delta-12 desaturase gene 5723-5167 (WO
2004/104167) EcoRI/PacI Yarrowia Ura3 gene 5723-7240 (GenBank
Accession No. AJ306421)
[0348] First, the Swa I/Not I fragment from pFmD8S (comprising the
chimeric FBAINm::EgD8S gene, wherein EgD8S is identified as
"D8-corrected" in FIG. 6D) was used to replace the SwaI/NotI
fragment of pKO2UFkF2 (comprising the chimeric FBAINm::F.D12 gene)
to generate construct pKO2UFm8 (FIG. 11A). The same methodology was
used to replace the SwaI/NotI fragment of pKO2UFkF2 with the Swa
I/Not I fragments of pFmD8S-23, pFmD8S-013 and pFmD8S-015,
respectively, thereby creating constructs pKO2UFm8-23, pKO2UFm8-013
and pKO2UFm8-015, respectively. As such, the synthetic
codon-optimized EgD8S, mutant EgD8S-023, mutant EgD8S-013 and
mutant EgD8S-015 were each under the control of the FBAINm promoter
and the Pex20 terminator.
[0349] Plasmids pKO2UFm8, pKO2UFm8-23, pKO2UFm8-013 and
pKO2UFm8-015 were digested with AscI/SphI, and then used for
transformation of strain Y4001 individually according to the
General Methods. Following transformation, cells were plated onto
MM plates and maintained at 30.degree. C. for 2 to 3 days.
[0350] A total of 6 transformants from each transformation were
picked and re-streaked onto fresh MM plates. Once grown, these
strains were individually inoculated into liquid MM at 30.degree.
C. and grown with shaking at 250 rpm/min for 1 day. The cells were
collected by centrifugation, lipids were extracted, and fatty acid
methyl esters were prepared by trans-esterification, and
subsequently analyzed with a Hewlett-Packard 6890 GC. Delta-8
desaturase activity of each delta-8 desaturase are shown below in
Table 20; conversion efficiency was calculated as described in
Example 5.
TABLE-US-00020 TABLE 20 Delta-8 Desaturase Activity In EgD8S And
Its Mutants Mutations With Respect To EgD8S Conversion Plasmid (SEQ
ID NO: 10) Efficiency pKO2UFm8 none 37.9% (comprising (average)
wildtype EgD8S [SEQ ID NO: 10]) pKO2UFm8S- 4S to A, 5K to S, 12T to
V, 16T to K, 35%, 23 17T to V, 66P to Q, 67S to A, 108S to L, 35%,
(comprising 120L to M, 121M to L, 125Q to H, 36.1%, mutant 126M to
L, 117G to A, 118Y to F, 36.2%, EgD8S-23 132V to L, 133 L to V,
407A to S, 39.8%, [SEQ ID 408V to Q, 422L to Q, 293L to M, 40% NO:
4]) 162L to V, 163V to L, 418A to G, 419G to A (24) pKO2UFm8S- 4S
to A, 5K to S, 12T to V, 16T to K, 17.8% 013 17T to V, 66P to Q,
67S to A, 407A to S, 18.4%, (comprising 408V to Q, 422L to Q, 108S
to L, 18.6%, mutant 120L to M, 121M to L, 125Q to H, 24.4%,
EgD8S-013 126M to L, 293L to M, 54A to G, 55F to 34.4% [SEQ ID Y,
117G to A, 118Y to F, 132V to L, 39.1% NO: 6]) 133L to V, 162L to
V, 163V to L, 70.8% 170G to A, 171L to V, 418A to G and 419G to A
(28) pKO2UFm8S- 4S to A, 5K to S, 12T to V, 16T to K, 17.3%, 015
17T to V, 66P to Q, 67S to A, 407A to S, 19.8%, (comprising 408V to
Q, 422L to Q, 108S to L, 20%, mutant 120L to M, 121M to L, 125Q to
H, 126M 20.1% EgD8S-015 to L, 293L to M, 54A to G, 55F to Y, 29.2%,
[SEQ ID 117G to A, 118Y to F, 346I to V, 33.5% NO: 8]) 347I to L,
348T to S, 279T to L, 280L to 38.5% T, 162L to V, 163V to L, 170G
to A, 171L to V, 418A to G and 419G to A (31)
[0351] The different conversion efficiencies observed for each
specific mutant EgD8S may be attributed to a "position effect"
based on the respective locations of each gene's integration within
the Yarrowia genome. In any case, the results demonstrate that
several of the transformants comprising mutant EgD8S-23 (SEQ ID
NO:4), mutant EgD8S-013 (SEQ ID NO:6) and mutant EgD8S-015 (SEQ ID
NO:8) possessed delta-8 desaturase activity that was at least
functionally equivalent (or increased) with respect to that of the
synthetic codon-optimized EgD8S (SEQ ID NO:10).
Example 11
Generation of Yarrowia lipolytica Strains Y4031, Y4032 and Y4033 to
Produce about 10-13.6% DGLA of Total Lipids
[0352] The present Example describes the construction of strains
Y4031, Y4032 and Y4033, derived from Yarrowia lipolytica Y4001U
(Example 3), capable of producing 10-13.6% DGLA (C20:3) relative to
the total lipids. These strains were engineered to express the
delta-9 elongase/delta-8 desaturase pathway, via expression of a
mutant delta-8 desaturase of the present invention and a delta-9
elongase.
[0353] More specifically, construct pKO2UF8289 (FIG. 11B; SEQ ID
NO:181) was created to integrate a cluster of four chimeric genes
(comprising a delta-12 desaturase, two copies of the mutant EgD8-23
and one delta-9 elongase) into the delta-12 gene locus of Yarrowia
genome in strain Y4001U. Construct pKO2UF8289 contained the
following components:
TABLE-US-00021 TABLE 21 Description Of Plasmid pKO2UF8289 (SEQ ID
NO: 181) RE Sites And Nucleotides Within SEQ ID Description Of
Fragment And Chimeric Gene NO: 181 Components AscI/BsiW I 5'
portion of Yarrowia lipolytica delta-12 (10304-9567) desaturase
gene (WO 2004/104167) EcoRI/Sph I 3' portion of Yarrowia lipolytica
delta-12 (13568-13012) desaturase gene (WO 2004/104167) Cla I/EcoR
I LoxP::Ura3::LoxP, comprising: (1-13568) LoxP sequence (SEQ ID NO:
18) Yarrowia Ura3 gene (GenBank Accession No. AJ306421) LoxP
sequence (SEQ ID NO: 18) PmeI/ClaI GPAT::EgD9E::Lip2, comprising:
(2038-1) GPAT: Yarrowia lipolytica GPAT promoter (WO 2006/031937)
EgD9E: codon-optimized delta-9 elongase gene (SEQ ID NO: 177),
derived from Euglena gracilis (SEQ ID NOs: 175 and 176; U.S. patent
application No. 60/739989; see also Example 16 herein) Lip2: Lip2
terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.
AJ012632) PmeI/PacI Exp::D8-23::Pex16, comprising: (4581-2124) Exp:
Yarrowia lipolytica export protein (EXP1) promoter (WO 2006/052870
and U.S. patent application No. 11/265761) D8-23: mutant EgD8S-23
(Example 8; SEQ ID NO: 4) Pex16: Pex16 terminator sequence of
Yarrowia Pex 16 gene (GenBank Accession No. U75433) Swa I/Pme I
YAT::F. D12::Oct, comprising: (7055-4581) YAT: Yarrowia lipolytica
YAT1 promoter (Patent Publication US 2006/0094102-A1) F.D12:
Fusarium moniliforme delta-12 desaturase gene (WO 2005/047485) OCT:
OCT terminator sequence of Yarrowia OCT gene (GenBank Accession No.
X69988) Swa I/BsiW I FBAINm::D8S-23::Pex20, comprising: (7055-9567)
FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805)
D8S-23: mutant EgD8S-23 (Example 8; SEQ ID NO: 4) Pex20: Pex20
terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.
AF054613)
[0354] Plasmid pKO2UF8289 was digested with Asc I/Sph I, and then
used for transformation of Y. lipolytica strain Y4001U (Example 3)
according to the General Methods. The transformant cells were
plated onto MMLeu media plates and maintained at 30.degree. C. for
2 to 3 days. The colonies were picked and streaked onto MMLeu
selection plates at 30.degree. C. for 2 days. These cells were then
inoculated into liquid MMLeu at 30.degree. C. and shaken at 250
rpm/min for 2 days. The cells were collected by centrifugation,
lipids were extracted, and fatty acid methyl esters were prepared
by trans-esterification, and subsequently analyzed with a
Hewlett-Packard 6890 GC.
[0355] GC analyses showed the presence of DGLA in the transformants
containing the 4 chimeric genes of pKO2UF8289, but not in the
Yarrowia Y4001U control strain. Most of the selected 12 strains
produced about 4 to 8% DGLA of total lipids. There were 3 strains
(i.e., strains #7, #8 and #12) that produced about 11.3%, 10% and
13.6% DGLA of total lipids; they were designated as strains Y4031,
Y4031 and Y4033, respectively.
Example 12
Cloning the Euglena gracilis Delta-8 Desaturase Mutant (EgD8S-23)
into a Soybean Expression Vector and Co-Expression with the
Isochrysis galbana Delta-9 Elongase
[0356] The present Example describes the construction of soybean
expression vector pKR1060, suitable for use in the production of
DGLA (C20:3). This vector was engineered to enable expression of
the delta-9 elongase/delta-8 desaturase pathway, via expression of
a mutant delta-8 desaturase of the present invention and a delta-9
elongase.
[0357] Through a number of cloning steps, a NotI site was added to
the 5' end of the EgD8S-23 gene from pKO2UFm8S-23 (Table 20,
Example 10) to produce the sequence set forth in SEQ ID NO:182.
[0358] Vector pKR457 (SEQ ID NO:183), which was previously
described in PCT Publication No. WO 2005/047479 (the contents of
which are hereby incorporated by reference), contains a NotI site
flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter
(Jofuku et al., Plant Cell 1:1079-1093 (1989)) and the KTi 3'
termination region, the isolation of which is described in U.S.
Pat. No. 6,372,965, followed by the soy albumin transcription
terminator, which was previously described in PCT Publication No.
WO 2004/071467 (Kti/NotI/Kti3'Salb3' cassette).
[0359] The NotI fragment containing EgD8S-23 described above, was
cloned into the NotI site of pKR457 to produce pKR1058 (SEQ ID
NO:184).
[0360] Plasmid pKR1058 was digested with PstI and the fragment
containing EgD8S-23 was cloned into the SbfI site of pKR607 (SEQ ID
NO:185), previously described in PCT Publication No. WO 2006/012325
(the contents of which are hereby incorporated by reference) to
produce pKR1060 (SEQ ID NO:186). In this way, EgD8S-23 is
co-expressed with the Isochrysis galbana delta-9 elongase behind
strong, seed-specific promoters. A schematic depiction of pKR1060
is shown in FIG. 12.
Example 13
Cloning the Euglena gracilis Delta-8 Desaturase Mutant (EgD8S-23)
into a Soybean Expression Vector and Co-expression with the Euglena
gracilis Delta-9 Elongase
[0361] The present Example describes the construction of soybean
expression vector pKR1059, suitable for use in the production of
DGLA (C20:3). This vector was engineered to enable expression of
the delta-9 elongase/delta-8 desaturase pathway, via expression of
a mutant delta-8 desaturase of the present invention and a delta-9
elongase.
[0362] The Euglena gracilis delta-9 elongase (SEQ ID NO:175; U.S.
Patent Application No. 60/739,989; see also Example 16 herein) was
amplified with oligonucleotide primers oEugEL1-1 (SEQ ID NO:187)
and oEugEL1-2 (SEQ ID NO:188) using the VentR.RTM. DNA Polymerase
(Cat. No. M0254S, New England Biolabs Inc., Beverly, Mass.)
following the manufacturer's protocol. The resulting DNA fragment
was cloned into the pCR-Blunt.RTM. cloning vector using the Zero
Blunt.RTM. PCR Cloning Kit (Invitrogen Corporation), following the
manufacturer's protocol, to produce pKR906 (SEQ ID NO:189).
[0363] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ
ID NO:190, 7085 bp sequence), a derivative of pKS123 which was
previously described in PCT Publication No. WO 02/008269 (the
contents of which are hereby incorporated by reference), contains
the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and
Davies, J., Gene, 25:179-188 (1983)), flanked by the T7 promoter
and transcription terminator (T7prom/hpt/T7term cassette), and a
bacterial origin of replication (ori) for selection and replication
in bacteria (e.g., E. coli). In addition, pKR72 also contains the
hygromycin B phosphotransferase gene, flanked by the 35S promoter
(Odell et al., Nature, 313:810-812 (1985)) and NOS 3' transcription
terminator (Depicker et al., J. Mol. Appl. Genet., 1:561-570
(1982)) (35S/hpt/NOS3' cassette) for selection in plants such as
soybean. pKR72 also contains a NotI restriction site, flanked by
the promoter for the .alpha.' subunit of .beta.-conglycinin (Beachy
et al., EMBO J., 4:3047-3053 (1985)) and the 3' transcription
termination region of the phaseolin gene (Doyle et al., J. Biol.
Chem. 261:9228-9238 (1986)), thus allowing for strong
tissue-specific expression in the seeds of soybean of genes cloned
into the NotI site.
[0364] The gene for the Euglena gracilis delta-9 elongase was
released from pKR906 by digestion with NotI and cloned into the
NotI site of pKR72 to produce pKR1010 (SEQ ID NO:191). In some
instances, pKR1010 is referred to as pKR912 but the two vectors are
identical.
[0365] Plasmid pKR1058 (from Example 12, SEQ ID NO:184) was
digested with PstI and the fragment containing EgD8S-23 was cloned
into the SbfI site of pKR1010 (SEQ ID NO:191), to produce pKR1059
(SEQ ID NO:192). In this way, EgD8S-23 is co-expressed with the
Euglena gracilis delta-9 elongase behind strong, seed-specific
promoters. A schematic depiction of pKR1059 is shown in FIG.
13.
Example 14
Co-Expressing Other Promoter/Gene/Terminator Cassette
Combinations
[0366] In addition to the genes, promoters, terminators and gene
cassettes described herein, one skilled in the art can appreciate
that other promoter/gene/terminator cassette combinations can be
synthesized in a way similar to, but not limited to, that described
herein for expression in plants (e.g., soybean). For instance, PCT
Publication Nos. WO 2004/071467 and WO 2004/071178 describe the
isolation of a number of promoter and transcription terminator
sequences for use in embryo-specific expression in soybean.
Furthermore, PCT Publication Nos. WO 2004/071467, WO 2005/047479
and WO 2006/012325 describe the synthesis of multiple
promoter/gene/terminator cassette combinations by ligating
individual promoters, genes and transcription terminators together
in unique combinations. Generally, a NotI site flanked by the
suitable promoter (such as those listed in, but not limited to,
Table 22) and a transcription terminator (such as those listed in,
but not limited to, Table 23) is used to clone the desired gene.
NotI sites can be added to a gene of interest such as those listed
in, but not limited to, Table 24 using PCR amplification with
oligonucleotides designed to introduce NotI sites at the 5' and 3'
ends of the gene. The resulting PCR product is then digested with
NotI and cloned into a suitable promoter/NotI/terminator
cassette.
[0367] In addition, PCT Publication Nos. WO 2004/071467, WO
2005/047479 and WO 2006/012325 describe the further linking
together of individual gene cassettes in unique combinations, along
with suitable selectable marker cassettes, in order to obtain the
desired phenotypic expression. Although this is done mainly using
different restriction enzymes sites, one skilled in the art can
appreciate that a number of techniques can be utilized to achieve
the desired promoter/gene/transcription terminator combination. In
so doing, any combination of embryo-specific
promoter/gene/transcription terminator cassettes can be achieved.
One skilled in the art can also appreciate that these cassettes can
be located on individual DNA fragments or on multiple fragments
where co-expression of genes is the outcome of co-transformation of
multiple DNA fragments.
TABLE-US-00022 TABLE 22 Seed-Specific Promoters Promoter Organism
Promoter Reference .beta.-conglycinin .alpha.'-subunit soybean
Beachy et al., EMBO J. 4: 3047-3053 (1985) kunitz trypsin inhibitor
soybean Jofuku et al., Plant Cell 1: 1079-1093 (1989) annexin
soybean WO 2004/071467 glycinin Gy1 soybean WO 2004/071467 albumin
2S soybean U.S. Pat. No. 6,177,613 legumin A1 pea Rerie et al.,
Mol. Gen. Genet. 225: 148-157 (1991) .beta.-conglycinin
.beta.-subunit soybean WO 2004/071467 BD30 (also called P34)
soybean WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen.
Genet. 225: 148-157 (1991)
TABLE-US-00023 TABLE 23 Transcription Terminators Transcription
Terminator Organism Reference phaseolin 3' bean WO 2004/071467
kunitz trypsin inhibitor 3' soybean WO 2004/071467 BD30 (also
called P34) 3' soybean WO 2004/071467 legumin A2 3' pea WO
2004/071467 albumin 2S 3' soybean WO 2004/071467
TABLE-US-00024 TABLE 24 EPA Biosynthetic Pathway Genes Gene
Organism Reference delta-6 desaturase Saprolegnia diclina WO
2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No.
5,968,809 elongase Mortierella alpina WO 2000/12720 U.S. Pat. No.
6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No.
6,075,183 delta-5 desaturase Saprolegnia diclina WO 2002/081668
delta-15 desaturase Fusarium WO 2005/047479 moniliforme delta-17
desaturase Saprolegnia diclina WO 2002/081668 elongase
Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145
elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366
(2004) delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum
delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase
Euglena gracilis U.S. Provisional Application No. 60/739,989
delta-8 desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No.
6,825,017 WO 2004/057001 WO 2006/012325 delta-8 desaturase
Acanthamoeba Sayanova et al., FEBS castellanii Lett. 580: 1946-1952
(2006) delta-8 desaturase Pavlova salina WO 2005/103253 delta-8
desaturase Pavlova lutheri U.S. Provisional Application No.
60/795,810
Example 15
Transformation of Somatic Soybean Embryo Cultures with Soybean
Expression Vectors
Culture Conditions:
[0368] Soybean embryogenic suspension cultures (cv. Jack) were
maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker,
150 rpm, 26.degree. C. with cool white fluorescent lights on 16:8
hr day/night photoperiod at light intensity of 60-85 .mu.E/m2/s.
Cultures were subcultured every 7 days to two weeks by inoculating
approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the
preferred subculture interval is every 7 days).
[0369] Soybean embryogenic suspension cultures were transformed
with the soybean expression plasmids by the method of particle gun
bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont
Biolistic PDS1000/HE instrument (helium retrofit) for all
transformations.
Soybean Embryogenic Suspension Culture Initiation:
[0370] Soybean cultures were initiated twice each month with 5-7
days between each initiation. Pods with immature seeds from
available soybean plants were picked 45-55 days after planting.
Seeds were removed from the pods and placed into a sterilized
magenta box. The soybean seeds were sterilized by shaking them for
15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95
mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of
soap, mixed well). Seeds were rinsed using 2 1-liter bottles of
sterile distilled water and those less than 4 mm were placed on
individual microscope slides. The small end of the seed was cut and
the cotyledons pressed out of the seed coat. Cotyledons were
transferred to plates containing SB199 medium (25-30 cotyledons per
plate) for 2 weeks, and then transferred to SB1 for 2-4 weeks.
Plates were wrapped with fiber tape and were maintained at
26.degree. C. with cool white fluorescent lights on 16:8 h
day/night photoperiod at light intensity of 60-80 .mu.E/m2/s. After
incubation on SB1 medium, secondary embryos were cut and placed
into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment:
[0371] A DNA fragment from soybean expression plasmid pKR1059
containing the Euglena gracilis delta-9 elongase and EgD8S-23, the
construction of which is described herein, was obtained by gel
isolation of digested plasmids. For this, 100 .mu.g of plasmid DNA
was used in 0.5 mL of the specific enzyme mix described below.
Plasmid was digested with AscI (100 units) in NEBuffer 4 (20 mM
Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1
mM dithiothreitol, pH 7.9), 100 .mu.g/mL BSA, and 5 mM
beta-mercaptoethanol at 37.degree. C. for 1.5 hr. The resulting DNA
fragments were separated by gel electrophoresis on 1% SeaPlaque GTG
agarose (BioWhitaker Molecular Applications) and the DNA fragments
containing gene cassettes were cut from the agarose gel. DNA was
purified from the agarose using the GELase digesting enzyme
following the manufacturer's protocol.
[0372] A 50 .mu.L aliquot of sterile distilled water containing 1
mg of gold particles was added to 5 .mu.L of a 1 ug/.mu.L DNA
solution (DNA fragment prepared as described herein), 50 .mu.L 2.5M
CaCl.sub.2 and 20 .mu.L of 0.1 M spermidine. The mixture was shaken
3 min on level 3 of a vortex shaker and spun for 10 sec in a bench
microfuge. The supernatant was removed, followed by a wash with 400
.mu.L 100% ethanol and another brief centrifugation. The 400 ul
ethanol was removed and the pellet was resuspended in 85 .mu.L of
100% ethanol. Five .mu.L of DNA suspension was dispensed to each
flying disk of the Biolistic PDS1000/HE instrument disk. Each 5
.mu.L aliquot contained approximately 0.058 mg gold per bombardment
(e.g., per disk).
Tissue Preparation and Bombardment with DNA:
[0373] Approximately 100-150 mg of seven day old embryogenic
suspension cultures was placed in an empty, sterile 60.times.15 mm
petri dish and the dish was covered with plastic mesh. The chamber
was evacuated to a vacuum of 27-28 inches of mercury, and tissue
was bombarded one or two shots per plate with membrane rupture
pressure set at 650 PSI. Tissue was placed approximately 2.5 inches
from the retaining/stopping screen.
Selection of Transformed Embryos and Embryo Maturation:
[0374] Transformed embryos were selected using hygromycin
(hygromycin B phosphotransferase (HPT) gene was used as the
selectable marker).
[0375] Following bombardment, the tissue was placed into fresh
SB196 media and cultured as described above. Six to eight days
post-bombardment, the SB196 was exchanged with fresh SB196
containing either 30 mg/L hygromycin. The selection media was
refreshed weekly. Four to six weeks post-selection, green,
transformed tissue was observed growing from untransformed,
necrotic embryogenic clusters.
[0376] Transformed embryogenic clusters were removed from SB196
media to 35 mL of SB228 (SHaM liquid media; Schmidt et al., Cell
Biology and Morphogenesis 24:393 (2005)) in a 250 mL Erlenmeyer
flask for 2-3 weeks. Tissue cultured in SB228 was maintained on a
rotary shaker at 130 rpm and 26.degree. C. with cool white
fluorescent lights on a 16:8 hr day/night photoperiod at a light
intensity of 60-85 .mu.E/m2/s.
After maturation in flasks on SB228 media, individual embryos were
removed from the clusters, dried and screened for alterations in
their fatty acid compositions as described supra.
Media Recipes:
SB 196
FN Lite Liquid Proliferation Medium (Per Liter)
TABLE-US-00025 [0377] MS FeEDTA - 100x Stock 1 10 mL MS Sulfate -
100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P,
B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10
mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm
(NH.sub.4).sub.2SO.sub.4 0.463 gm asparagine 1.0 gm sucrose (1%) 10
gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00026 [0378] Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x
Stock Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g
1.392 g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g
MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g
0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides
100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x
Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve
in dark bottle while stirring
SB1 Solid Medium
Per Liter
[0379] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0380] 1 mL B5 vitamins 1000.times. stock
[0381] 31.5 g glucose
[0382] 2 mL 2,4-D (20 mg/L final concentration)
[0383] pH 5.7
[0384] 8 g TC agar
SB199 Solid Medium
Per Liter
[0385] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0386] 1 mL B5 vitamins 1000.times. stock
[0387] 30 g Sucrose
[0388] 4 ml 2,4-D (40 mg/L final concentration)
[0389] pH 7.0
[0390] 2 gm Gelrite
SB 166 Solid Medium
Per Liter
[0391] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0392] 1 mL B5 vitamins 1000.times. stock
[0393] 60 g maltose
[0394] 750 mg MgCl.sub.2 hexahydrate
[0395] 5 g activated charcoal
[0396] pH 5.7
[0397] 2 g gelrite
SB 103 Solid Medium
Per Liter
[0398] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0399] 1 mL B5 vitamins 1000.times. stock
[0400] 60 g maltose
[0401] 750 mg MgCl2 hexahydrate
[0402] pH 5.7
[0403] 2 g gelrite
SB 71-4 Solid Medium (Per Liter)
[0404] 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL--Cat. No.
21153-036)
[0405] pH 5.7
[0406] 5 g TC agar
2,4-D Stock
[0407] Obtain premade from Phytotech Cat. No. D 295--concentration
1 mg/mL
B5 Vitamins Stock
Per 100 mL
[0408] Store aliquots at -20.degree. C.
[0409] 10 g myo-inositol
[0410] 100 mg nicotinic acid
[0411] 100 mg pyridoxine HCl
[0412] 1 g thiamine
If the solution does not dissolve quickly enough, apply a low level
of heat via the hot stir plate.
SB 228
Soybean Histodifferentiation & Maturation (SHaM) (Per
Liter)
TABLE-US-00027 [0413] DDI H.sub.2O 600 mL FN-Lite Macro Salts for
SHaM 10X 100 mL MS Micro Salts 1000x 1 mL MS FeEDTA 100x 10 mL CaCl
100x 6.82 mL B5 Vitamins 1000x 1 mL L-Methionine 0.149 g Sucrose 30
g Sorbitol 30 g Adjust volume to 900 mL pH 5.8 Autoclave Add to
cooled media (.ltoreq.30.degree. C.): *Glutamine (final
concentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL
after glutamine addition. Since glutamine degrades relatively
rapidly, it may be preferable to add immediately prior to using
media. Expiration 2 weeks after glutamine is added; base media can
be kept longer w/o glutamine.
FN-Lite Macro for SHAM 10.times.
Stock #1 (Per Liter)
TABLE-US-00028 [0414] (NH.sub.4)2SO.sub.4 (ammonium sulfate) 4.63 g
KNO.sub.3 (potassium nitrate) 28.3 g MgSO.sub.4*7H.sub.20
(magnesium sulfate heptahydrate) 3.7 g KH.sub.2PO.sub.4 (potassium
phosphate, monobasic) 1.85 g Bring to volume Autoclave
MS Micro 1000.times.
Stock #2 (Per 1 Liter)
TABLE-US-00029 [0415] H.sub.3BO.sub.3 (boric acid) 6.2 g
MnSO.sub.4*H.sub.2O (manganese sulfate monohydrate) 16.9 g
ZnSO4*7H20 (zinc sulfate heptahydrate) 8.6 g Na.sub.2MoO.sub.4*2H20
(sodium molybdate dihydrate) 0.25 g CuSO.sub.4*5H.sub.20 (copper
sulfate pentahydrate) 0.025 g CoCl.sub.2*6H.sub.20 (cobalt chloride
hexahydrate) 0.025 g KI (potassium iodide) 0.8300 g Bring to volume
Autoclave
FeEDTA 100.times.
Stock #3 (Per Liter)
TABLE-US-00030 [0416] Na.sub.2EDTA* (sodium EDTA) 3.73 g
FeSO.sub.4*7H.sub.20 (iron sulfate heptahydrate) 2.78 g *EDTA must
be completely dissolved before adding iron. Bring to Volume
Solution is photosensitive. Bottle(s) should be wrapped in foil to
omit light. Autoclave
Ca 100.times.
Stock #4 (Per Liter)
TABLE-US-00031 [0417] CaCl.sub.2*2H.sub.20 (calcium chloride
dihydrate) 44 g Bring to Volume Autoclave
B5 Vitamin 1000.times.
Stock #5 (Per Liter)
TABLE-US-00032 [0418] Thiamine*HCl 10 g Nicotinic Acid 1 g
Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store
frozen
4% Glutamine
Stock #6 (Per Liter)
TABLE-US-00033 [0419] DDI water heated to 30.degree. C. 900 mL
L-Glutamine 40 g Gradually add while stirring and applying low
heat. Do not exceed 35.degree. C. Bring to Volume Filter Sterilize
Store frozen* *Note: Warm thawed stock in 31.degree. C. bath to
fully dissolve crystals.
Example 16
Identification of a Delta-9 Elongase from Euglena gracilis
[0420] The present Example, disclosed in U.S. Patent Application
No. 60/739,989, describes the isolation of a delta-9 elongase from
Euglena gracilis (SEQ ID NOs:175 and 176).
Euglena gracilis Growth Conditions, Lipid Profile and mRNA
Isolation
[0421] Euglena gracilis was obtained from Dr. Richard Triemer's lab
at Michigan State University (East Lansing, Mich.). From 10 mL of
actively growing culture, a 1 mL aliquot was transferred into 250
mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg
medium was made by combining 1 g of sodium acetate, 1 g of beef
extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of
Bacto.RTM. tryptone (0123-17-3, Difco Laboratories), 2 g of
Bacto.RTM. yeast extract (0127-17-9, Difco Laboratories) in 970 mL
of water. After filter sterilizing, 30 mL of soil-water supernatant
(15-3790, Carolina Biological Supply Company, Burlington, N.C.) was
aseptically added to give the final Eg medium. Euglena gracilis
cultures were grown at 23.degree. C. with a 16 h light, 8 h dark
cycle for 2 weeks with no agitation.
[0422] After 2 weeks, 10 mL of culture was removed for lipid
analysis and centrifuged at 1,800.times.g for 5 min. The pellet was
washed once with water and re-centrifuged. The resulting pellet was
dried for 5 min under vacuum, resuspended in 100 .mu.L of
trimethylsulfonium hydroxide (TMSH) and incubated at room
temperature for 15 min with shaking. After this, 0.5 mL of hexane
was added and the vials were incubated for 15 min at room
temperature with shaking. Fatty acid methyl esters (5 .mu.L
injected from hexane layer) were separated and quantified using a
Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320
fused silica capillary column (Supelco Inc., Cat. No. 24152). The
oven temperature was programmed to hold at 220.degree. C. for 2.7
min, increase to 240.degree. C. at 20.degree. C./min and then hold
for an additional 2.3 min. Carrier gas was supplied by a Whatman
hydrogen generator. Retention times were compared to those for
methyl esters of standards commercially available (Nu-Chek Prep,
Inc. Cat. No. U-99-A).
[0423] The remaining 2 week culture (240 mL) was pelleted by
centrifugation at 1,800.times.g for 10 min, washed once with water
and re-centrifuged. Total RNA was extracted from the resulting
pellet using the RNA STAT-60.TM. reagent (TEL-TEST, Inc.,
Friendswood, Tex.) and following the manufacturer's protocol
provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water).
In this way, 1 mg of total RNA (2 mg/mL) was obtained from the
pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA
Purification Kit (Amersham Biosciences, Piscataway, N.J.) following
the manufacturer's protocol provided. In this way, 85 .mu.g of mRNA
was obtained.
Euglena gracilis cDNA Synthesis, Library Construction and
Sequencing
[0424] A cDNA library was generated using the Cloneminer.TM. cDNA
Library Construction Kit (Cat. No. 18249-029, Invitrogen
Corporation, Carlsbad, Calif.) and following the manufacturer's
protocol provided (Version B, 25-0608). Using the non-radiolabeling
method, cDNA was synthesized from 3.2 .mu.g of mRNA (described
above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of
the first and second strand, the attB1 adapter was added, ligated
and the cDNA was size fractionated using column chromatography. DNA
from fractions 7 and 8 (size ranging from .about.800-1500 bp) were
concentrated, recombined into pDONR.TM.222 and transformed into E.
coli ElectroMAX.TM. DH10B.TM. T1 Phage-Resistant cells (Invitrogen
Corporation). The Euglena gracilis library was named eeg1c.
[0425] For sequencing, clones first were recovered from archived
glycerol cultures grown/frozen in 384-well freezing media plates,
and replicated with a sterile 384 pin replicator (Genetix, Boston,
Mass.) in 384-well microtiter plates containing LB+75 .mu.g/mL
Kanamycin (replicated plates). Plasmids then were isolated, using
the Templiphi DNA sequencing template amplification kit method
(Amersham Biosciences) following the manufacturer's protocol.
Briefly, the Templiphi method uses bacteriophage .phi.29 DNA
polymerase to amplify circular single-stranded or double-stranded
DNA by isothermal rolling circle amplification (Dean et al., Genome
Res., 11:1095-1099 (2001); Nelson et al., Biotechniques, 32:S44-S47
(2002)). After growing 20 h at 37.degree. C., cells from the
replicated plate were added to 5 .mu.L of dilution buffer and
denatured at 95.degree. C. for 3 min to partially lyse cells and
release the denatured template. Templiphi premix (5 .mu.L) was then
added to each sample and the resulting reaction mixture was
incubated at 30.degree. C. for 16 h, then at 65.degree. C. for 10
min to inactivate the .phi.29 DNA polymerase activity. DNA
quantification with the PicoGreen.RTM. dsDNA Quantitation Reagent
(Molecular Probes) was performed after diluting the amplified
samples 1:3 in distilled water.
[0426] The amplified products then were denatured at 95.degree. C.
for 10 min and end-sequenced in 384-well plates, using the M13F
universal primer (SEQ ID NO:193), and the ABI Big Dye version 3.1
Prism Sequencing Kit. For the sequencing reaction, 100-200 ng of
templates and 6.4 pmol of primers were used, and the following
reaction conditions were repeated 25 times: 96.degree. C. for 10
sec, 50.degree. C. for 5 sec and 60.degree. C. for 4 min. After
ethanol-based cleanup, cycle sequencing reaction products were
resolved and detected on Perkin-Elmer ABI 3730xl automated
sequencers.
Identification of Long-Chain Polyunsaturated Fatty Acid Elongation
Enzyme Homologs from Euglena gracilis cDNA Library eeg1c
[0427] cDNA clones encoding long-chain polyunsaturated fatty acid
elongation enzyme homologs (LC-PUFA ELO homologs or delta-9
elongases) were identified by conducting BLAST (Basic Local
Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410
(1993)) searches for similarity to sequences contained in the BLAST
"nr" database (comprising all non-redundant GenBank CDS
translations, sequences derived from the 3-dimensional structure
Brookhaven Protein Data Bank, the last major release of the
SWISS-PROT protein sequence database, EMBL and DDBJ databases). The
Euglena gracilis cDNA sequences obtained above were analyzed for
similarity to all publicly available DNA sequences contained in the
"nr" database using the BLASTN algorithm provided by the National
Center for Biotechnology Information (NCBI). The DNA sequences were
translated in all reading frames and compared for similarity to all
publicly available protein sequences contained in the "nr" database
using the BLASTX algorithm (Gish and States, Nat. Genet., 3:266-272
(1993)) provided by the NCBI. For convenience, the P-value
(probability) of observing a match of a cDNA sequence to a sequence
contained in the searched databases merely by chance as calculated
by BLAST are reported herein as "pLog" values, which represent the
negative of the logarithm of the reported P-value. Accordingly, the
greater the pLog value, the greater the likelihood that the cDNA
sequence and the BLAST "hit" represent homologous proteins.
[0428] The BLASTX search using the nucleotide sequences from clone
eeg1c.pk001.n5.f revealed similarity of the protein encoded by the
cDNA to the long-chain PUFA elongation enzyme from Isochrysis
galbana (SEQ ID NO:173) (GenBank Accession No. AAL37626 (GI
17226123), locus AAL37626, CDS AF390174; Qi et al., FEBS Lett.
510(3):159-165 (2002)). The sequence of a portion of the cDNA
insert from clone eeg1c.pk001.n5.f is shown in SEQ ID NO:194 (5'
end of cDNA insert). Additional sequence was obtained from the 3'
end of the cDNA insert of eeg1c.pk001.n5.1 as described above, but
using the poly(A) tail-primed WobbleT oligonucleotides. Briefly,
the WobbleT primer is an equimolar mix of 21 mer poly(T)A,
poly(T)C, and poly(T)G, used to sequence the 3' end of cDNA
clones.
[0429] The 3' end sequence is shown in SEQ ID NO:195. Both the 5'
and 3' sequences were aligned using Sequencher.TM. (Version 4.2,
Gene Codes Corporation, Ann Arbor, Mich.) and the resulting
sequence for the cDNA is shown in SEQ ID NO:196 (1201 bp). Sequence
for the coding sequence from the cDNA in eeg1c.pk001.n5.f and the
corresponding deduced amino acid sequence is shown in SEQ ID NO:175
(777 bp) and SEQ ID NO:176 (258 amino acids), respectively.
[0430] The amino acid sequence set forth in SEQ ID NO:176 was
evaluated by BLASTP, yielding a pLog value of 38.70 (E value of
2e-39) versus the Isochrysis galbana sequence (SEQ ID NO:173). The
Euglena gracilis delta-9 elongase is 39.4% identical to the
Isochrysis galbana delta-9 elongase sequence using the Jotun Hein
method. Sequence percent identity calculations performed by the
Jotun Hein method (Hein, J. J., Meth. Enz., 183:626-645 (1990))
were done using the MegAlign.TM. v6.1 program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with
the default parameters for pairwise alignment (KTUPLE=2). The
Euglena gracilis delta-9 elongase is 31.8% identical to the
Isochrysis galbana delta-9 elongase sequence using the Clustal V
method. Sequence percent identity calculations performed by the
Clustal V method (Higgins, D. G. and Sharp, P. M., Comput. Appl.
Biosci., 5:151-153 (1989); Higgins et al., Comput. Appl. Biosci.,
8:189-191 (1992)) were done using the MegAlign.TM. v6.1 program of
the LASERGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.) with the default parameters for pairwise alignment
(KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP
LENGTH PENALTY=10). BLAST scores and probabilities indicate that
the nucleic acid fragment described herein as SEQ ID NO:175 encodes
an entire Euglena gracilis delta-9 elongase.
Example 17
Functional Analysis of EgD8S-23 and the Euglena gracilis Delta-9
Elongase in Somatic Soybean Embryos Transformed with Soybean
Expression Vector pKR1059
[0431] Mature somatic soybean embryos are a good model for zygotic
embryos. While in the globular embryo state in liquid culture,
somatic soybean embryos contain very low amounts of triacylglycerol
or storage proteins typical of maturing, zygotic soybean embryos.
At this developmental stage, the ratio of total triacylglyceride to
total polar lipid (phospholipids and glycolipid) is about 1:4, as
is typical of zygotic soybean embryos at the developmental stage
from which the somatic embryo culture was initiated. At the
globular stage as well, the mRNAs for the prominent seed proteins,
a'-subunit of .beta.-conglycinin, kunitz trypsin inhibitor 3, and
seed lectin are essentially absent. Upon transfer to hormone-free
media to allow differentiation to the maturing somatic embryo
state, triacylglycerol becomes the most abundant lipid class. As
well, mRNAs for .alpha.'-subunit of .beta.-conglycinin, kunitz
trypsin inhibitor 3 and seed lectin become very abundant messages
in the total mRNA population. On this basis, the somatic soybean
embryo system behaves very similarly to maturing zygotic soybean
embryos in vivo, and is thus a good and rapid model system for
analyzing the phenotypic effects of modifying the expression of
genes in the fatty acid biosynthesis pathway (see PCT Publication
No. WO 2002/00904, Example 3). Most importantly, the model system
is also predictive of the fatty acid composition of seeds from
plants derived from transgenic embryos.
Fatty Acid Analysis of Transgenic Somatic Soybean Embryos
Expressing pKR1059
[0432] Individual single, matured, somatic soybean embryos that had
been transformed with pKR1059 (as described in Example 15
transformants were matured on SHaM liquid media) were picked into
glass GC vials and fatty acid methyl esters were prepared by
transesterification. For transesterification, 50 .mu.L of
trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane were added
to the embryos in glass vials and incubated for 30 min at room
temperature while shaking. Fatty acid methyl esters (5 .mu.L
injected from hexane layer) were separated and quantified using a
Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320
fused silica capillary column (Catalog #24152, Supelco Inc.). The
oven temperature was programmed to hold at 220.degree. C. for 2.6
min, increase to 240.degree. C. at 20.degree. C./min and then hold
for an additional 2.4 min. Carrier gas was supplied by a Whatman
hydrogen generator. Retention times were compared to those for
methyl esters of standards commercially available (Nu-Chek Prep,
Inc.). Routinely, 6 embryos per event were analyzed by GC, using
the methodology described above.
[0433] Embryo fatty acid profiles for 31 individual events (6
embryos each) containing pKR1059 were obtained and of these, 23
events contained at least 1 embryo with greater than 1% EDA. The
lipid profiles of somatic soybean embryos expressing EgD8S-23 and
the Euglena gracilis delta-9 elongase for the top 5 events are
shown in FIG. 14. Fatty acids are identified as 16:0 (palmitate),
18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA,
ERA and ETA; and, fatty acid compositions listed in FIG. 4 are
expressed as a weight percent (wt. %) of total fatty acids. The
delta-8 desaturase activity of EgD8S-23 is expressed as percent
desaturation (% desat), calculated according to the following
formula: ([product]/[substrate+product])*100.
[0434] More specifically, the combined percent desaturation for EDA
and ERA is shown as "C20% delta-8 desat", determined as:
([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as
the overall % desaturation. The individual omega-6 delta-8
desaturation ("EDA % delta-8 desat.") was calculated as:
([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8
desaturation ("ERA % delta-8 desat.") was calculated as:
([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for
omega-6 versus omega-3 substrates ("ratio [EDA/ERA] desat.") was
obtained by dividing the EDA % delta-8 desaturation by the ERA
delta-8 desaturation.
[0435] In summary of FIG. 14, EgD8S-23 worked in soybean to convert
both EDA and ERA to DGLA and ETA, respectively. The line with the
highest average DGLA content (i.e., 2063-1-4) had embryos with an
average DGLA content of 13.2% and an average ETA content of 4.2%.
The highest DGLA and ETA content for an individual embryo from this
line was 13.5% and 4.3%, respectively. The highest average overall
% desaturation was 68.6% with the highest overall % desaturation
for an individual embryo being 69.7%. When broken down into %
desaturation for the omega-6 and omega-3 substrates, the highest
average % desaturation was 65.4% and 81.3% for EDA and ERA,
respectively. The highest % desaturation for an individual embryo
was 66.6% and 84.1% for EDA and ERA, respectively. In this example,
the D8S-23 had a preference for ERA over EDA, with the average
desaturation ratio ranging from 0.7 to 0.8. No significant levels
of GLA was found to accumulate in the embryos.
Sequence CWU 1
1
19811272DNAArtificial Sequencemutant EgD8S consensus, optionally
comprising M1, M2, M3, M8, M12, M15, M16, M18, M21, M26, M38, M45,
M46, M51, M63, M68, M69 and M70 mutation sites 1catggtgaag
nnnnnncgac aggctctgcc cctcnnnatc gacggannnn nntacgacgt 60ctccgcttgg
gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa nnnnnngaca
agctcaagcg 180aatgcccaag atcaacnnnn nntccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tcnnnaccac cctgggtctt ggcgtgcttn nnnnnttcnn 360nnnngtccag
tacnnnnnnt acttcattgg tgctnnnnnn ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacnnnnnn ggtctggtct ttggcaacnn nnnncagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagnnnnn 840nttccacctc ttctttatgc cctccatcct
gacctcgnnn ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcnnnn nnnnngactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgn
nnnnnttcgc tcgaatggcc gagaagcagc ccnnnnnnaa 1260ggctnnntaa gc
12722422PRTArtificial Sequencemutant EgD8S consensus, optionally
comprising M1, M2, M3, M8, M12, M15, M16, M18, M21, M26, M38, M45,
M46, M51, M63, M68, M69 and M70 mutation sites 2Met Val Lys Xaa Xaa
Arg Gln Ala Leu Pro Leu Xaa Ile Asp Gly Xaa1 5 10 15Xaa Tyr Asp Val
Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu
Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val 35 40 45Met His
Ser Gln Glu Xaa Xaa Asp Lys Leu Lys Arg Met Pro Lys Ile 50 55 60Asn
Xaa Xaa Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln65 70 75
80Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe
85 90 95Asp Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Xaa Thr Thr Leu
Gly 100 105 110Leu Gly Val Leu Xaa Xaa Phe Xaa Xaa Val Gln Tyr Xaa
Xaa Tyr Phe 115 120 125Ile Gly Ala Xaa Xaa Leu Gly Met His Tyr Gln
Gln Met Gly Trp Leu 130 135 140Ser His Asp Ile Cys His His Gln Thr
Phe Lys Asn Arg Asn Trp Asn145 150 155 160Asn Xaa Xaa Gly Leu Val
Phe Gly Asn Xaa Xaa Gln Gly Phe Ser Val 165 170 175Thr Trp Trp Lys
Asp Arg His Asn Ala His His Ser Ala Thr Asn Val 180 185 190Gln Gly
His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200
205Glu Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln
210 215 220Phe Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg
Phe Ile225 230 235 240Trp Cys Phe Gln Ser Val Leu Thr Val Arg Ser
Leu Lys Asp Arg Asp 245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys
Lys Glu Ala Ile Gly Leu Ala 260 265 270Leu His Trp Thr Leu Lys Xaa
Xaa Phe His Leu Phe Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Xaa
Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile
Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys305 310 315
320Ile Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile
325 330 335His Glu Thr Met Asn Ile Arg Arg Gly Xaa Xaa Xaa Asp Trp
Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp
Pro Thr Leu Pro 355 360 365Arg His Asn Leu Thr Ala Val Ser Tyr Gln
Val Glu Gln Leu Cys Gln 370 375 380Lys His Asn Leu Pro Tyr Arg Asn
Pro Leu Pro His Glu Gly Leu Val385 390 395 400Ile Leu Leu Arg Tyr
Leu Xaa Xaa Phe Ala Arg Met Ala Glu Lys Gln 405 410 415Pro Xaa Xaa
Lys Ala Xaa 42031272DNAArtificial Sequencemutant EgD8S-23
3catggtgaag gcttctcgac aggctctgcc cctcgtcatc gacggaaagg tgtacgacgt
60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca
agctcaagcg 180aatgcccaag atcaaccagg cttccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tcttgaccac cctgggtctt ggcgtgcttg ccttcttcat 360gctggtccag
taccacctgt acttcattgg tgctctcgtg ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacgtcctg ggtctggtct ttggcaacgg actccagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagaccct 840gttccacctc ttctttatgc cctccatcct
gacctcgatg ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgt
cccagttcgc tcgaatggcc gagaagcagc ccggtgccaa 1260ggctcagtaa gc
12724422PRTArtificial Sequencemutant EgD8S-23, comprising M1, M2,
M3, M8, M12, M15, M16, M18, M21, M26, M45, M46, M68 and M70
mutation sites 4Met Val Lys Ala Ser Arg Gln Ala Leu Pro Leu Val Ile
Asp Gly Lys1 5 10 15Val Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro
Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr
Asp Ala Phe Met Val 35 40 45Met His Ser Gln Glu Ala Phe Asp Lys Leu
Lys Arg Met Pro Lys Ile 50 55 60Asn Gln Ala Ser Glu Leu Pro Pro Gln
Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu Arg
Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu Trp
Tyr Ser Tyr Lys Ile Leu Thr Thr Leu Gly 100 105 110Leu Gly Val Leu
Ala Phe Phe Met Leu Val Gln Tyr His Leu Tyr Phe 115 120 125Ile Gly
Ala Leu Val Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135
140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp
Asn145 150 155 160Asn Val Leu Gly Leu Val Phe Gly Asn Gly Leu Gln
Gly Phe Ser Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala His
His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp
Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg
Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr
Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp
Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250
255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala
260 265 270Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met
Pro Ser 275 280 285Ile Leu Thr Ser Met Leu Val Phe Phe Val Ser Glu
Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn
His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val Trp Asp
Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn
Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu
Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg
His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375
380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu
Val385 390 395 400Ile Leu Leu Arg Tyr Leu Ser Gln Phe Ala Arg Met
Ala Glu Lys Gln 405 410 415Pro Gly Ala Lys Ala Gln
42051272DNAArtificial Sequencemutant EgD8S-013 5catggtgaag
gcttctcgac aggctctgcc cctcgtcatc gacggaaagg tgtacgacgt 60ctccgcttgg
gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa ggctacgaca
agctcaagcg 180aatgcccaag atcaaccagg cttccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tcttgaccac cctgggtctt ggcgtgcttg ccttcttcat 360gctggtccag
taccacctgt acttcattgg tgctctcgtg ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacgtcctg ggtctggtct ttggcaacgc tgtccagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagaccct 840gttccacctc ttctttatgc cctccatcct
gacctcgatg ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgt
cccagttcgc tcgaatggcc gagaagcagc ccggtgccaa 1260ggctcagtaa gc
12726422PRTArtificial SequenceMutant EgD8S-013, comprising M1, M2,
M3, M8, M12, M15, M16, M18, M21, M26, M38, M45, M46, M68, M69 and
M70 mutation sites 6Met Val Lys Ala Ser Arg Gln Ala Leu Pro Leu Val
Ile Asp Gly Lys1 5 10 15Val Tyr Asp Val Ser Ala Trp Val Asn Phe His
Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala
Thr Asp Ala Phe Met Val 35 40 45Met His Ser Gln Glu Gly Tyr Asp Lys
Leu Lys Arg Met Pro Lys Ile 50 55 60Asn Gln Ala Ser Glu Leu Pro Pro
Gln Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu
Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu
Trp Tyr Ser Tyr Lys Ile Leu Thr Thr Leu Gly 100 105 110Leu Gly Val
Leu Ala Phe Phe Met Leu Val Gln Tyr His Leu Tyr Phe 115 120 125Ile
Gly Ala Leu Val Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135
140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp
Asn145 150 155 160Asn Val Leu Gly Leu Val Phe Gly Asn Ala Val Gln
Gly Phe Ser Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala His
His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp
Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg
Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr
Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp
Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250
255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala
260 265 270Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met
Pro Ser 275 280 285Ile Leu Thr Ser Met Leu Val Phe Phe Val Ser Glu
Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn
His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val Trp Asp
Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn
Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu
Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg
His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375
380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu
Val385 390 395 400Ile Leu Leu Arg Tyr Leu Ser Gln Phe Ala Arg Met
Ala Glu Lys Gln 405 410 415Pro Gly Ala Lys Ala Gln
42071272DNAArtificial Sequencemutant EgD8S-015 7catggtgaag
gcttctcgac aggctctgcc cctcgtcatc gacggaaagg tgtacgacgt 60ctccgcttgg
gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa ggctacgaca
agctcaagcg 180aatgcccaag atcaaccagg cttccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tcttgaccac cctgggtctt ggcgtgcttg ccttcttcat 360gctggtccag
taccacctgt acttcattgg tgctgtgctg ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacgtcctg ggtctggtct ttggcaacgc tgtccagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagctgac 840cttccacctc ttctttatgc cctccatcct
gacctcgatg ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcgtcc tctctgactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgt
cccagttcgc tcgaatggcc gagaagcagc ccggtgccaa 1260ggctcagtaa gc
12728422PRTArtificial Sequencemutant EgD8S-015 comprising M1, M2,
M3, M8, M12, M15, M16, M18, M21, M26, M38, M45, M51, M63, M68, M69
and M70 mutation sites 8Met Val Lys Ala Ser Arg Gln Ala Leu Pro Leu
Val Ile Asp Gly Lys1 5 10 15Val Tyr Asp Val Ser Ala Trp Val Asn Phe
His Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp
Ala Thr Asp Ala Phe Met Val 35 40 45Met His Ser Gln Glu Gly Tyr Asp
Lys Leu Lys Arg Met Pro Lys Ile 50 55 60Asn Gln Ala Ser Glu Leu Pro
Pro Gln Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys
Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro
Leu Trp Tyr Ser Tyr Lys Ile Leu Thr Thr Leu Gly 100 105 110Leu Gly
Val Leu Ala Phe Phe Met Leu Val Gln Tyr His Leu Tyr Phe 115 120
125Ile Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu
130 135 140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn
Trp Asn145 150 155 160Asn Val Leu Gly Leu Val Phe Gly Asn Ala Val
Gln Gly Phe Ser Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala
His His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile
Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200
205Glu Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln
210 215 220Phe Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg
Phe Ile225 230 235 240Trp Cys Phe Gln Ser Val Leu Thr Val Arg Ser
Leu Lys Asp Arg Asp 245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys
Lys Glu Ala Ile Gly Leu Ala 260 265 270Leu His Trp Thr Leu Lys Leu
Thr Phe His Leu Phe Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Met
Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile
Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys305 310 315
320Ile Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile
325 330 335His Glu Thr Met Asn Ile Arg Arg Gly Val Leu Ser Asp Trp
Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp
Pro Thr Leu Pro 355 360 365Arg His Asn Leu Thr Ala Val Ser Tyr Gln
Val Glu Gln Leu Cys Gln 370 375 380Lys His Asn Leu Pro Tyr Arg Asn
Pro Leu Pro His Glu Gly Leu Val385 390 395 400Ile Leu Leu Arg Tyr
Leu Ser Gln Phe Ala Arg Met Ala Glu Lys Gln 405 410 415Pro Gly Ala
Lys Ala Gln 42091272DNAEuglena
gracilismisc_feature(2)..(1270)synthetic delta-8 desaturase CDS,
codon- optimized for Yarrowia lipolytica ("D8SF" or "EgD8S")
9catggtgaag tccaagcgac aggctctgcc cctcaccatc gacggaacta cctacgacgt
60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca
agctcaagcg 180aatgcccaag atcaacccct cctccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360gatggtccag
taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacctcgtg ggtctggtct ttggcaacgg actccagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagaccct 840gttccacctc ttctttatgc cctccatcct
gacctcgctc ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgg
ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260ggctctctaa gc
127210422PRTEuglena gracilisMISC_FEATUREsynthetic delta-8
desaturase codon-optimized for Yarrowia lipolytica ("D8SF" or
"EgD8S") 10Met Val Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp
Gly Thr1 5 10 15Thr Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly
Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp
Ala Phe Met Val 35 40 45Met His Ser Gln Glu Ala Phe Asp Lys Leu Lys
Arg Met Pro Lys Ile 50 55 60Asn Pro Ser Ser Glu Leu Pro Pro Gln Ala
Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu Arg Glu
Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu Trp Tyr
Ser Tyr Lys Ile Ser Thr Thr Leu Gly 100 105 110Leu Gly Val Leu Gly
Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe 115 120 125Ile Gly Ala
Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135 140Ser
His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn145 150
155 160Asn Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser
Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala
Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp Asn Leu Pro
Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg Ala Ser Pro
Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr Tyr Phe Leu
Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp Cys Phe Gln
Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250 255Asn Gln
Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265
270Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser
275 280 285Ile Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val
Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn His Tyr
Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val Trp Asp Gly His
Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn Ile Arg
Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr
Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg His Asn
Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375 380Lys
His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val385 390
395 400Ile Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys
Gln 405 410 415Pro Ala Gly Lys Ala Leu 420111271DNAEuglena
gracilismisc_feature(4)..(1269)delta-8 desaturase ("Eg5" or "EgD8")
CDS 11gaaatgaagt caaagcgcca agcgcttccc cttacaattg atggaacaac
atatgatgtg 60tctgcctggg tcaatttcca ccctggtggt gcggaaatta tagagaatta
ccaaggaagg 120gatgccactg atgccttcat ggttatgcac tctcaagaag
ccttcgacaa gctcaagcgc 180atgcccaaaa tcaatcccag ttctgagttg
ccaccccagg ctgcagtgaa tgaagctcaa 240gaggatttcc ggaagctccg
agaagagttg atcgcaactg gcatgtttga tgcctccccc 300ctctggtact
catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg
360atggttcagt atcagatgta tttcattggg gcagtgttgc ttgggatgca
ctatcaacag 420atgggctggc tttctcatga catttgccac caccagactt
tcaagaaccg gaactggaac 480aacctcgtgg gactggtatt tggcaatggt
ctgcaaggtt tttccgtgac atggtggaag 540gacagacaca atgcacatca
ttcggcaacc aatgttcaag ggcacgaccc tgatattgac 600aacctccccc
tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc
660aagctcattc agttccagca gtactatttc ttggtcatct gtatcttgtt
gcggttcatt 720tggtgtttcc agagcgtgtt gaccgtgcgc agtttgaagg
acagagataa ccaattctat 780cgctctcagt ataagaagga ggccattggc
ctcgccctgc actggacctt gaagaccctg 840ttccacttat tctttatgcc
cagcatcctc acatcgctgt tggtgttttt cgtttcggag 900ctggttggcg
gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag
960atcggggact cagtctggga tggccatgga ttctcggttg gccagatcca
tgagaccatg 1020aacattcggc gagggattat cacagattgg tttttcggag
gcttgaatta ccagattgag 1080caccatttgt ggccgaccct ccctcgccac
aacctgacag cggttagcta ccaggtggaa 1140cagctgtgcc agaagcacaa
cctgccgtat cggaacccgc tgccccatga agggttggtc 1200atcctgctgc
gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag
1260gctctataag g 127112421PRTEuglena gracilisMISC_FEATUREdelta-8
desaturase ("Eg5" or "EgD8") 12Met Lys Ser Lys Arg Gln Ala Leu Pro
Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn
Phe His Pro Gly Gly Ala Glu Ile 20 25 30Ile Glu Asn Tyr Gln Gly Arg
Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser Gln Glu Ala Phe
Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60Pro Ser Ser Glu Leu
Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu65 70 75 80Asp Phe Arg
Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95Ala Ser
Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105
110Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile
115 120 125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp
Leu Ser 130 135 140His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg
Asn Trp Asn Asn145 150 155 160Leu Val Gly Leu Val Phe Gly Asn Gly
Leu Gln Gly Phe Ser Val Thr 165 170 175Trp Trp Lys Asp Arg His Asn
Ala His His Ser Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp
Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205Asp Asp Val
Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220Gln
Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp225 230
235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp
Asn 245 250 255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly
Leu Ala Leu 260 265 270His Trp Thr Leu Lys Thr Leu Phe His Leu Phe
Phe Met Pro Ser Ile 275 280 285Leu Thr Ser Leu Leu Val Phe Phe Val
Ser Glu Leu Val Gly Gly Phe 290 295 300Gly Ile Ala Ile Val Val Phe
Met Asn His Tyr Pro Leu Glu Lys Ile305 310 315 320 Gly Asp Ser Val
Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His3 25 330 335Glu Thr
Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345
350Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg
355 360 365His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys
Gln Lys 370 375 380His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu
Gly Leu Val Ile385 390 395 400Leu Leu Arg Tyr Leu Ala Val Phe Ala
Arg Met Ala Glu Lys Gln Pro 405 410 415Ala Gly Lys Ala Leu
42013467PRTAmylomyces rouxii (GenBank Accession No. AAR27297) 13Met
Ser Ser Asp Val Gly Ala Thr Val Pro His Phe Tyr Thr Arg Ala1 5 10
15Glu Leu Ala Asp Ile His Gln Asp Val Leu Asp Lys Lys Pro Glu Ala
20 25 30Arg Lys Leu Ile Val Val Glu Asn Lys Val Tyr Asp Ile Thr Asp
Phe 35 40 45Val Phe Asp His Pro Gly Gly Glu Arg Val Leu Leu Thr Gln
Glu Gly 50 55 60Arg Asp Ala Thr Asp Val Phe His Glu Met His Pro Pro
Ser Ala Tyr65 70 75 80Glu Leu Leu Ala Asn Cys Tyr Val Gly Asp Cys
Glu Pro Lys Leu Pro 85 90 95Ile Asp Ser Thr Asp Lys Lys Ala Leu Asn
Ser Ala Ala Phe Ala Gln 100 105 110Glu Ile Arg Asp Leu Arg Asp Lys
Leu Glu Lys Gln Gly Tyr Phe Asp 115 120 125Ala Ser Thr Gly Phe Tyr
Ile Tyr Lys Val Ser Thr Thr Leu Leu Val 130 135 140Cys Ile Val Gly
Leu Ala Ile Leu Lys Ala Trp Gly Arg Glu Ser Thr145 150 155 160Leu
Ala Val Phe Ile Ala Ala Ser Leu Val Gly Leu Phe Trp Gln Gln 165 170
175Cys Gly Trp Leu Ala His Asp Tyr Ala His Tyr Gln Val Ile Lys Asp
180 185 190Pro Asn Val Asn Asn Leu Phe Leu Val Thr Phe Gly Asn Leu
Val Gln 195 200 205Gly Phe Ser Leu Ser Trp Trp Lys Asn Lys His Asn
Thr His His Ala 210 215 220Ser Thr Asn Val Ser Gly Glu Asp Pro Asp
Ile Asp Thr Ala Pro Ile225 230 235 240Leu Leu Trp Asp Glu Phe Ala
Val Ala Asn Phe Tyr Gly Ser Leu Lys 245 250 255Asp Asn Ala Ser Gly
Phe Asp Arg Phe Ile Ala Glu His Ile Leu Pro 260 265 270Tyr Gln Thr
Arg Tyr Tyr Phe Phe Ile Leu Gly Phe Ala Arg Thr Ser 275 280 285Trp
Ala Ile Gln Ser Ile Ile Tyr Ser Phe Lys Asn Glu Thr Leu Asn 290 295
300Lys Ser Lys Leu Leu Ser Trp Cys Glu Arg Ile Phe Leu Ile Val
His305 310 315 320Trp Val Phe Phe Thr Tyr Cys Thr Ile Ala Trp Ile
Ser Ser Ile Arg 325 330 335Asn Ile Ala Met Phe Phe Val Val Ser Gln
Ile Thr Thr Gly Tyr Leu 340 345 350Leu Ala Ile Val Phe Ala Met Asn
His Asn Gly Met Pro Val Tyr Ser 355 360 365Pro Glu Glu Ala Asn His
Thr Glu Phe Tyr Glu Leu Gln Cys Ile Thr 370 375 380Gly Arg Asp Val
Asn Cys Thr Val Phe Gly Asp Trp Leu Met Gly Gly385 390 395 400Leu
Asn Tyr Gln Ile Glu His His Leu Phe Pro Glu Met Pro Arg His 405 410
415His Leu Ser Lys Val Lys Ser Met Val Lys Pro Ile Ala Gln Lys Tyr
420 425 430Asn Ile Pro Tyr His Asp Thr Thr Val Ile Gly Gly Thr Ile
Glu Val 435 440 445Leu Gln Thr Leu Asp Phe Val Gln Lys Ile Ser Gln
Lys Phe Ser Lys 450 455 460Lys Met Leu46514445PRTRhizopus oryzae
(GenBank Accession No. AAS93682) 14Met Ser Thr Ser Asp Arg Gln Ser
Val Phe Thr Leu Lys Glu Leu Glu1 5 10 15Leu Ile Asn Gln Lys His Arg
Asp Gly Asp Lys Ser Ala Met Lys Phe 20 25 30Ile Ile Ile Asp Arg Lys
Val Tyr Asp Val Thr Glu Phe Leu Glu Asp 35 40 45His Gly Gly Ala Gln
Val Leu Leu Thr His Val Gly Lys Asp Ala Ser 50 55 60Asp Val Phe His
Ala Met His Glu Ser Ala Tyr Glu Ile Leu Asn Asn65 70 75 80Tyr Phe
Val Gly Asp Val Lys Asp Ala His Val Lys Glu Thr Ser Ala 85 90 95Gln
Phe Ala Ser Glu Met Arg Gln Leu Arg Asp Gln Leu Lys Lys Glu 100 105
110Gly Tyr Phe His Ser Ser Lys Ala Tyr Tyr Val Tyr Lys Val Leu Ser
115 120 125Thr Leu Ala Leu Cys Ala Ala Gly Leu Thr Leu Leu Tyr Ala
Tyr Gly 130 135 140His Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile
Ile Val Gly Ile145 150 155 160Phe Trp Gln Gln Cys Gly Trp Leu Ala
His Asp Phe Gly His His Gln 165 170 175Cys Phe Glu Asp Arg Ser Trp
Asn Asp Val Leu Val Val Phe Leu Gly 180 185 190Asn Phe Cys Gln Gly
Phe Ser Leu Ser Trp Trp Lys Asn Lys His Asn 195 200 205Thr His His
Ala Ser Thr Asn Val His Gly His Asp Asp Ile Asp Thr 210 215 220Ala
Val Leu Leu Trp Asp Glu Tyr Ala Ser Ala Ala Tyr Tyr Ala Ser225 230
235 240Leu Asp Glu Glu Thr Met Ile Ser Arg Phe Leu Ala Glu Ser Val
Leu 245 250 255His Gln Thr Arg Tyr Tyr Phe Phe Val Leu Gly Phe Ala
Arg Leu Ser 260 265 270Trp Ala Ile Gln Ser Leu Leu Tyr Ser Phe Lys
Gln Gly Ala Ile Asn 275 280 285Lys Ser His Gln Leu Asn Leu Phe Glu
Arg Phe Cys Leu Val Ser His 290 295 300Trp Thr Leu Phe Thr Tyr Cys
Thr Leu Ala Trp Cys Ser Asn Val Tyr305 310 315 320His Met Ile Leu
Phe Phe Leu Val Ser Gln Ala Thr Thr Gly Tyr Thr 325 330 335Leu Ala
Leu Val Phe Ala Leu Asn His Asn Gly Met Val Ile Thr Glu 340 345
350Glu Lys Ala Glu Ser Met Glu Phe Phe Glu Ile Gln Val Ile Thr Gly
355 360 365Arg Asp Val Thr Leu Ser Leu Gly Asp Trp Phe Met Gly Gly
Leu Asn 370 375 380Tyr Gln Ile Glu His His Val Phe Asn Met Arg His
Asn Leu Lys Val385 390 395 400Lys Met Val Lys Ser Leu Cys Lys Lys
Tyr Asp Ile Asn Tyr His Asp 405 410 415Thr Gly Phe Leu Lys Gly Thr
Leu Glu Val Leu Lys Thr Leu Asp Ile 420 425 430Thr Ser Lys Leu Ser
Leu
Gln Leu Ser Lys Lys Ser Phe 435 440 44515382PRTLeishmania major
(GenBank Accession No. CAJ09677) 15Met Val Phe Glu Leu Thr Tyr Lys
Tyr His Ser Ser Leu Lys Cys Val1 5 10 15Ile Arg Ile Asp Asp Asn Trp
Tyr Asp Cys Thr Ser Trp Arg Asn Ser 20 25 30His Gly Gly Ala Gln Met
Cys Asp Glu Phe His Gly Lys Asp Ala Thr 35 40 45Asp Ala Phe Tyr Ser
Leu His Ser Lys Glu Ala Ile Gln Lys Leu Lys 50 55 60Arg Met Lys Ala
Leu Leu Lys Glu Gly Asp Glu Arg Asp Gln Val Ser65 70 75 80Leu Asn
Phe Glu Lys Leu Leu Gln Gln Leu Arg Ser Glu Gly Trp Phe 85 90 95Glu
Arg Arg Trp Ile Ile Asp Phe Ala Arg Asn Ile Met Val Ile Val 100 105
110Leu Cys Val Val Gly Thr Tyr Leu Ser Tyr Ser Arg Phe Leu Ala Thr
115 120 125Ile Leu Ile Gly Leu Gly Met Gln Gln Gly Gly Trp Leu Ala
His Asp 130 135 140Phe Thr His Ala Arg Gly Lys Phe Ala Arg Phe Leu
Ala Asn Ala Cys145 150 155 160Gly Gly Met Ile Asn Ala Phe Ser Val
Glu Trp Trp Ser Asn Lys His 165 170 175Asn Ser His His Ile Phe Val
Asn Arg Lys Gly Met Asp Ala Asp Ile 180 185 190His Asn Glu Ala Leu
Phe Leu Trp Val Asp Val Ser Glu Asp Thr Ala 195 200 205Cys Arg Arg
Tyr Gln Tyr Thr Phe Tyr Leu Ala Ala Tyr Ala Leu Leu 210 215 220Tyr
Val Ser Trp Arg Ile Gln Ser Leu Arg Phe Ala Leu Gly Ser Gly225 230
235 240Asn Arg Leu Glu Leu Ser Leu Met Ala Leu Asn Tyr Leu Trp Leu
Ala 245 250 255Leu Leu Trp Arg Val Ser Leu Gly Ser Val Leu Leu Gly
Gly Phe Phe 260 265 270Val Ala Val Val Val Thr Val Asn His Gln Thr
Glu Glu Met Ile Glu 275 280 285His Asp Glu Tyr Asn Tyr Val Val Asp
Gln His Arg Ala Thr Arg Gly 290 295 300Val His Cys Ser Asp Phe Phe
Glu Trp Phe Phe Gly Gly Met Gln Tyr305 310 315 320Gln Leu Glu His
His Leu Leu Met Val Arg Tyr Arg Tyr Glu Val Arg 325 330 335Arg Leu
Leu Arg Lys Phe Ser Glu Asp Asn Gly Leu Phe His Val Glu 340 345
350Gly Val Trp Lys Ile Thr Lys Met Asn Phe Asp Ile Leu Tyr Ser Ile
355 360 365Ser Thr Thr Ala Leu Asn Ser Ala Glu Ala Lys Ser Gly Lys
370 375 38016439PRTMortierella isabellina (GenBank Accession No.
AAG38104) 16Met Ala Ala Ala Ser Val Arg Thr Phe Thr Arg Ala Glu Ile
Leu Asn1 5 10 15Ala Glu Ala Leu Asn Glu Gly Lys Lys Asp Ala Glu Ala
Phe Leu Met 20 25 30 Ile Ile Asp Asn Lys Val Tyr Asp Val Arg Glu
Phe Val Asp His Gly 35 40 45Gly Ser Val Ile Leu Thr His Val Gly Lys
Asp Gly Thr Asp Val Phe 50 55 60Asp Thr Phe His Glu Ala Ala Trp Glu
Thr Leu Ala Asn Phe Tyr Val65 70 75 80Gly Asp Ile Asp Glu Ser Asp
Arg Ala Ile Lys Asn Asp Asp Phe Ala 85 90 95Ala Glu Val Arg Lys Leu
Arg Thr Leu Phe Gln Ser Leu Gly Tyr Tyr 100 105 110Asp Ser Ser Lys
Ala Tyr Tyr Ala Phe Lys Val Ser Phe Asn Leu Cys 115 120 125Ile Trp
Gly Leu Ser Thr Phe Ile Val Ala Lys Trp Gly Gln Thr Ser 130 135
140Thr Leu Ala Asn Val Leu Ser Ala Ala Leu Leu Gly Leu Phe Trp
Gln145 150 155 160Gln Cys Gly Trp Leu Ala His Asp Phe Leu His His
Gln Val Phe Gln 165 170 175Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala
Phe Leu Gly Gly Val Cys 180 185 190Gln Gly Phe Ser Ser Ser Trp Trp
Lys Asp Lys His Asn Thr His His 195 200 205Ala Ala Asn Val His Gly
Glu Asp Asp Ile Asp Thr His Leu Leu Thr 210 215 220Trp Ser Glu His
Ala Leu Glu Met Phe Ser Asp Val Asp Glu Glu Leu225 230 235 240Thr
Arg Met Trp Ser Arg Phe Met Val Leu Asn Gln Thr Trp Phe Tyr 245 250
255Phe Ile Leu Ser Phe Ala Arg Leu Ser Trp Cys Leu Gln Ser Ile Leu
260 265 270Leu Val Leu Asn Gly Gln Ala His Lys Ser Gly Ala Arg Val
Ser Ile 275 280 285Ser Leu Val Glu Gln Leu Ser Leu Ala Met His Trp
Thr Trp Tyr Leu 290 295 300Ala Thr Met Phe Leu Phe Ile Lys Asp Val
Asn Met Met Val Tyr Phe305 310 315 320Leu Val Ser Gln Ala Val Cys
Gly Asn Leu Leu Ala Ile Val Phe Ser 325 330 335Leu Asn His Asn Gly
Met Val Ile Ser Lys Glu Glu Ala Val Asp Met 340 345 350Asp Phe Phe
Thr Lys Gln Ile Ile Thr Gly Arg Asp Val His Gly Leu 355 360 365Phe
Ala Asn Trp Phe Thr Gly Gly Leu Asn Tyr Gln Ile Glu His His 370 375
380Leu Phe Ser Met Arg His Asn Phe Ser Lys Ile Gln Ala Val Glu
Thr385 390 395 400Leu Cys Lys Lys Tyr Gly Val Arg Tyr His Thr Thr
Gly Met Ile Glu 405 410 415Gly Thr Ala Glu Val Phe Ser Arg Leu Asn
Glu Val Ser Lys Ala Ala 420 425 430Ser Lys Met Gly Lys Ala Gln
4351714655DNAArtificial SequencePlasmid pZKLeuN-29E3 17cgattgttgt
ctactaacta tcgtacgata acttcgtata gcatacatta tacgaagtta 60tcgcgtcgac
gagtatctgt ctgactcgtc attgccgcct ttggagtacg actccaacta
120tgagtgtgct tggatcactt tgacgataca ttcttcgttg gaggctgtgg
gtctgacagc 180tgcgttttcg gcgcggttgg ccgacaacaa tatcagctgc
aacgtcattg ctggctttca 240tcatgatcac atttttgtcg gcaaaggcga
cgcccagaga gccattgacg ttctttctaa 300tttggaccga tagccgtata
gtccagtcta tctataagtt caactaactc gtaactatta 360ccataacata
tacttcactg ccccagataa ggttccgata aaaagttctg cagactaaat
420ttatttcagt ctcctcttca ccaccaaaat gccctcctac gaagctcgag
ctaacgtcca 480caagtccgcc tttgccgctc gagtgctcaa gctcgtggca
gccaagaaaa ccaacctgtg 540tgcttctctg gatgttacca ccaccaagga
gctcattgag cttgccgata aggtcggacc 600ttatgtgtgc atgatcaaaa
cccatatcga catcattgac gacttcacct acgccggcac 660tgtgctcccc
ctcaaggaac ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa
720gttcgcagat attggcaaca ctgtcaagca ccagtaccgg tgtcaccgaa
tcgccgagtg 780gtccgatatc accaacgccc acggtgtacc cggaaccgga
atcattgctg gcctgcgagc 840tggtgccgag gaaactgtct ctgaacagaa
gaaggaggac gtctctgact acgagaactc 900ccagtacaag gagttcctag
tcccctctcc caacgagaag ctggccagag gtctgctcat 960gctggccgag
ctgtcttgca agggctctct ggccactggc gagtactcca agcagaccat
1020tgagcttgcc cgatccgacc ccgagtttgt ggttggcttc attgcccaga
accgacctaa 1080gggcgactct gaggactggc ttattctgac ccccggggtg
ggtcttgacg acaagggaga 1140cgctctcgga cagcagtacc gaactgttga
ggatgtcatg tctaccggaa cggatatcat 1200aattgtcggc cgaggtctgt
acggccagaa ccgagatcct attgaggagg ccaagcgata 1260ccagaaggct
ggctgggagg cttaccagaa gattaactgt tagaggttag actatggata
1320tgtaatttaa ctgtgtatat agagagcgtg caagtatgga gcgcttgttc
agcttgtatg 1380atggtcagac gacctgtctg atcgagtatg tatgatactg
cacaacctgt gtatccgcat 1440gatctgtcca atggggcatg ttgttgtgtt
tctcgatacg gagatgctgg gtacagtgct 1500aatacgttga actacttata
cttatatgag gctcgaagaa agctgacttg tgtatgactt 1560attctcaact
acatccccag tcacaatacc accactgcac taccactaca ccaaaaccat
1620gatcaaacca cccatggact tcctggaggc agaagaactt gttatggaaa
agctcaagag 1680agagatcata acttcgtata gcatacatta tacgaagtta
tcctgcaggt aaaggaattc 1740tggagtttct gagagaaaaa ggcaagatac
gtatgtaaca aagcgacgca tggtacaata 1800ataccggagg catgtatcat
agagagttag tggttcgatg atggcactgg tgcctggtat 1860gactttatac
ggctgactac atatttgtcc tcagacatac aattacagtc aagcacttac
1920ccttggacat ctgtaggtac cccccggcca agacgatctc agcgtgtcgt
atgtcggatt 1980ggcgtagctc cctcgctcgt caattggctc ccatctactt
tcttctgctt ggctacaccc 2040agcatgtctg ctatggctcg ttttcgtgcc
ttatctatcc tcccagtatt accaactcta 2100aatgacatga tgtgattggg
tctacacttt catatcagag ataaggagta gcacagttgc 2160ataaaaagcc
caactctaat cagcttcttc ctttcttgta attagtacaa aggtgattag
2220cgaaatctgg aagcttagtt ggccctaaaa aaatcaaaaa aagcaaaaaa
cgaaaaacga 2280aaaaccacag ttttgagaac agggaggtaa cgaaggatcg
tatatatata tatatatata 2340tatacccacg gatcccgaga ccggcctttg
attcttccct acaaccaacc attctcacca 2400ccctaattca caaccatgga
gtctggaccc atgcctgctg gcattccctt ccctgagtac 2460tatgacttct
ttatggactg gaagactccc ctggccatcg ctgccaccta cactgctgcc
2520gtcggtctct tcaaccccaa ggttggcaag gtctcccgag tggttgccaa
gtcggctaac 2580gcaaagcctg ccgagcgaac ccagtccgga gctgccatga
ctgccttcgt ctttgtgcac 2640aacctcattc tgtgtgtcta ctctggcatc
accttctact acatgtttcc tgctatggtc 2700aagaacttcc gaacccacac
actgcacgaa gcctactgcg acacggatca gtccctctgg 2760aacaacgcac
ttggctactg gggttacctc ttctacctgt ccaagttcta cgaggtcatt
2820gacaccatca tcatcatcct gaagggacga cggtcctcgc tgcttcagac
ctaccaccat 2880gctggagcca tgattaccat gtggtctggc atcaactacc
aagccactcc catttggatc 2940tttgtggtct tcaactcctt cattcacacc
atcatgtact gttactatgc cttcacctct 3000atcggattcc atcctcctgg
caaaaagtac ctgacttcga tgcagattac tcagtttctg 3060gtcggtatca
ccattgccgt gtcctacctc ttcgttcctg gctgcatccg aacacccggt
3120gctcagatgg ctgtctggat caacgtcggc tacctgtttc ccttgaccta
tctgttcgtg 3180gactttgcca agcgaaccta ctccaagcga tctgccattg
ccgctcagaa aaaggctcag 3240taagcggccg cattgatgat tggaaacaca
cacatgggtt atatctaggt gagagttagt 3300tggacagtta tatattaaat
cagctatgcc aacggtaact tcattcatgt caacgaggaa 3360ccagtgactg
caagtaatat agaatttgac caccttgcca ttctcttgca ctcctttact
3420atatctcatt tatttcttat atacaaatca cttcttcttc ccagcatcga
gctcggaaac 3480ctcatgagca ataacatcgt ggatctcgtc aatagagggc
tttttggact ccttgctgtt 3540ggccaccttg tccttgctgt ctggctcatt
ctgtttcaac gccttttaat taacggagta 3600ggtctcggtg tcggaagcga
cgccagatcc gtcatcctcc tttcgctctc caaagtagat 3660acctccgacg
agctctcgga caatgatgaa gtcggtgccc tcaacgtttc ggatggggga
3720gagatcggcg agcttgggcg acagcagctg gcagggtcgc aggttggcgt
acaggttcag 3780gtcctttcgc agcttgagga gaccctgctc gggtcgcacg
tcggttcgtc cgtcgggagt 3840ggtccatacg gtgttggcag cgcctccgac
agcaccgagc ataatagagt cagcctttcg 3900gcagatgtcg agagtagcgt
cggtgatggg ctcgccctcc ttctcaatgg cagctcctcc 3960aatgagtcgg
tcctcaaaca caaactcggt gccggaggcc tcagcaacag acttgagcac
4020cttgacggcc tcggcaatca cctcggggcc acagaagtcg ccgccgagaa
gaacaatctt 4080cttggagtca gtcttggtct tcttagtttc gggttccatt
gtggatgtgt gtggttgtat 4140gtgtgatgtg gtgtgtggag tgaaaatctg
tggctggcaa acgctcttgt atatatacgc 4200acttttgccc gtgctatgtg
gaagactaaa cctccgaaga ttgtgactca ggtagtgcgg 4260tatcggctag
ggacccaaac cttgtcgatg ccgatagcat gcgacgtcgg gcccaattcg
4320ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
tgactgggaa 4380aaccctggcg ttacccaact taatcgcctt gcagcacatc
cccctttcgc cagctggcgt 4440aatagcgaag aggcccgcac cgatcgccct
tcccaacagt tgcgcagcct gaatggcgaa 4500tggacgcgcc ctgtagcggc
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4560ccgctacact
tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
4620ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
gggttccgat 4680ttagtgcttt acggcacctc gaccccaaaa aacttgatta
gggtgatggt tcacgtagtg 4740ggccatcgcc ctgatagacg gtttttcgcc
ctttgacgtt ggagtccacg ttctttaata 4800gtggactctt gttccaaact
ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4860tataagggat
tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
4920ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
attttctcct 4980tacgcatctg tgcggtattt cacaccgcat caggtggcac
ttttcgggga aatgtgcgcg 5040gaacccctat ttgtttattt ttctaaatac
attcaaatat gtatccgctc atgagacaat 5100aaccctgata aatgcttcaa
taatattgaa aaaggaagag tatgagtatt caacatttcc 5160gtgtcgccct
tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
5220cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
tacatcgaac 5280tggatctcaa cagcggtaag atccttgaga gttttcgccc
cgaagaacgt tttccaatga 5340tgagcacttt taaagttctg ctatgtggcg
cggtattatc ccgtattgac gccgggcaag 5400agcaactcgg tcgccgcata
cactattctc agaatgactt ggttgagtac tcaccagtca 5460cagaaaagca
tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
5520tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
aaggagctaa 5580ccgctttttt gcacaacatg ggggatcatg taactcgcct
tgatcgttgg gaaccggagc 5640tgaatgaagc cataccaaac gacgagcgtg
acaccacgat gcctgtagca atggcaacaa 5700cgttgcgcaa actattaact
ggcgaactac ttactctagc ttcccggcaa caattaatag 5760actggatgga
ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
5820ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
attgcagcac 5880tggggccaga tggtaagccc tcccgtatcg tagttatcta
cacgacgggg agtcaggcaa 5940ctatggatga acgaaataga cagatcgctg
agataggtgc ctcactgatt aagcattggt 6000aactgtcaga ccaagtttac
tcatatatac tttagattga tttaaaactt catttttaat 6060ttaaaaggat
ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
6120agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
tcttgagatc 6180ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
accaccgcta ccagcggtgg 6240tttgtttgcc ggatcaagag ctaccaactc
tttttccgaa ggtaactggc ttcagcagag 6300cgcagatacc aaatactgtt
cttctagtgt agccgtagtt aggccaccac ttcaagaact 6360ctgtagcacc
gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
6420gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
aaggcgcagc 6480ggtcgggctg aacggggggt tcgtgcacac agcccagctt
ggagcgaacg acctacaccg 6540aactgagata cctacagcgt gagctatgag
aaagcgccac gcttcccgaa gggagaaagg 6600cggacaggta tccggtaagc
ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 6660ggggaaacgc
ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
6720gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
aacgcggcct 6780ttttacggtt cctggccttt tgctggcctt ttgctcacat
gttctttcct gcgttatccc 6840ctgattctgt ggataaccgt attaccgcct
ttgagtgagc tgataccgct cgccgcagcc 6900gaacgaccga gcgcagcgag
tcagtgagcg aggaagcgga agagcgccca atacgcaaac 6960cgcctctccc
cgcgcgttgg ccgattcatt aatgcagctg gcgcgcccac tgagctcgtc
7020taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt
ctttgtatca 7080tttgtaacaa ttaccctgta caaactaagg tattgaaatc
ccacaatatt cccaaagtcc 7140acccctttcc aaattgtcat gcctacaact
catataccaa gcactaacct accaaacacc 7200actaaaaccc cacaaaatat
atcttaccga atatacagta acaagctacc accacactcg 7260ttgggtgcag
tcgccagctt aaagatatct atccacatca gccacaactc ccttccttta
7320ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta
gacaagacac 7380tttcaagaag actgtttcca aaacgtacca ctgtcctcca
ctacaaacac acccaatctg 7440cttcttctag tcaaggttgc tacaccggta
aattataaat catcatttca ttagcagggc 7500agggcccttt ttatagagtc
ttatacacta gcggaccctg ccggtagacc aacccgcagg 7560cgcgtcagtt
tgctccttcc atcaatgcgt cgtagaaacg acttactcct tcttgagcag
7620ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga
gcctccgata 7680tcggcggtag tgataccagc ctcgacggac tccttgacgg
cagcctcaac agcgtcaccg 7740gcgggcttca tgttaagaga gaacttgagc
atcatggcgg cagacagaat ggtggcgtac 7800gcaactaaca tgaatgaata
cgatatacat caaagactat gatacgcagt attgcacact 7860gtacgagtaa
gagcactagc cactgcactc aagtgaaacc gttgcccggg tacgagtatg
7920agtatgtaca gtatgtttag tattgtactt ggacagtgct tgtatcgtac
attctcaagt 7980gtcaaacata aatatccgtt gctatatcct cgcaccacca
cgtagctcgc tatatccctg 8040tgttgaatcc atccatcttg gattgccaat
tgtgcacaca gaaccgggca ctcacttccc 8100catccacact tgcggccgcg
cctacttaag caacgggctt gataacagcg gggggggtgc 8160ccacgttgtt
gcggttgcgg aagaacagaa cacccttacc agcaccctcg gcaccagcgc
8220tgggctcaac ccactggcac atacgcgcac tgcggtacat ggcgcggatg
aagccacgag 8280gaccatcctg gacatcagcc cggtagtgct tgcccatgat
gggcttaatg gcctcggtgg 8340cctcgtccgc gttgtagaag gggatgctgc
tgacgtagtg gtggaggaca tgagtctcga 8400tgatgccgtg gagaaggtgg
cggccgatga agcccatctc acggtcaatg gtagcagcgg 8460caccacggac
gaagttccac tcgtcgttgg tgtagtgggg aagggtaggg tcggtgtgct
8520ggaggaaggt gatggcaacg agccagtggt taacccagag gtagggaaca
aagtaccaga 8580tggccatgtt gtagaaaccg aacttctgaa cgaggaagta
cagagcagtg gccatcagac 8640cgataccaat atcgctgagg acgatgagct
tagcgtcact gttctcgtac agagggctgc 8700ggggatcgaa gtggttaaca
ccaccgccga ggccgttatg cttgcccttg ccgcgaccct 8760cacgctggcg
ctcgtggtag ttgtggccgg taacattggt gatgaggtag ttgggccagc
8820cnacgannnn ctcagtaaga tgagcgagct cgtgggtcat ctttccgaga
cgagtagcct 8880gctgctcgcg ggttcgggga acgaagacca tgtcacgctc
catgttgcca gtggccttgt 8940ggtgctttcg gtgggagatt tgccagctga
agtaggggac aaggagggaa gagtgaagaa 9000cccagccagt aatgtcgttg
atgatgcgag aatcggagaa agcaccgtga ccgcactcat 9060gggcaataac
ccagagacca gtaccgaaaa gaccctgaag aacggtgtac acggcccaca
9120gaccagcgcg ggcgggggtg gaggggatat attcgggggt cacaaagttg
taccagatgc 9180tgaaagtggt agtcaggagg acaatgtcgc ggaggatata
accgtatccc ttgagagcgg 9240agcgcttgaa gcagtgctta gggatggcat
tgtagatgtc cttgatggta aagtcgggaa 9300cctcgaactg gttgccgtag
gtgtcgagca tgacaccata ctcggacttg ggcttggcga 9360tatcaacctc
ggacatggac gagagcgatg tggaagaggc cgagtggcgg ggagagtctg
9420aaggagagac ggcggcagac tcagaatccg tcacagtagt tgaggtgacg
gtgcgtctaa 9480gcgcagggtt ctgcttgggc agagccgaag tggacgccat
ggttgatgtg tgtttaattc 9540aagaatgaat atagagaaga gaagaagaaa
aaagattcaa ttgagccggc gatgcagacc 9600cttatataaa tgttgccttg
gacagacgga gcaagcccgc ccaaacctac gttcggtata 9660atatgttaag
ctttttaaca caaaggtttg gcttggggta acctgatgtg gtgcaaaaga
9720ccgggcgttg gcgagccatt gcgcgggcga atggggccgt gactcgtctc
aaattcgagg 9780gcgtgcctca attcgtgccc ccgtggcttt ttcccgccgt
ttccgccccg tttgcaccac 9840tgcagccgct
tctttggttc ggacaccttg ctgcgagcta ggtgccttgt gctacttaaa
9900aagtggcctc ccaacaccaa catgacatga gtgcgtgggc caagacacgt
tggcggggtc 9960gcagtcggct caatggcccg gaaaaaacgc tgctggagct
ggttcggacg cagtccgccg 10020cggcgtatgg atatccgcaa ggttccatag
cgccattgcc ctccgtcggc gtctatcccg 10080caacctctaa atagagcggg
aatataaccc aagcttcttt tttttccttt aacacgcaca 10140cccccaacta
tcatgttgct gctgctgttt gactctactc tgtggagggg tgctcccacc
10200caacccaacc tacaggtgga tccggcgctg tgattggctg ataagtctcc
tatccggact 10260aattctgacc aatgggacat gcgcgcagga cccaaatgcc
gcaattacgt aaccccaacg 10320aaatgcctac ccctctttgg agcccagcgg
ccccaaatcc ccccaagcag cccggttcta 10380ccggcttcca tctccaagca
caagcagccc ggttctaccg gcttccatct ccaagcaccc 10440ctttctccac
accccacaaa aagacccgtg caggacatcc tactgcgtcg acatcattta
10500aattccttca cttcaagttc attcttcatc tgcttctgtt ttactttgac
aggcaaatga 10560agacatggta cgacttgatg gaggccaaga acgccatttc
accccgagac accgaagtgc 10620ctgaaatcct ggctgccccc attgataaca
tcggaaacta cggtattccg gaaagtgtat 10680atagaacctt tccccagctt
gtgtctgtgg atatggatgg tgtaatcccc tttgagtact 10740cgtcttggct
tctctccgag cagtatgagg ctctctaatc tagcgcattt aatatctcaa
10800tgtatttata tatttatctt ctcatgcggc cgctcactga atctttttgg
ctcccttgtg 10860cttcctgacg atatacgttt gcacatagaa attcaagaac
aaacacaaga ctgtgccaac 10920ataaaagtaa ttgaagaacc agccaaacat
cctcatccca tcttggcgat aacagggaat 10980gttcctgtac ttccagacaa
tgtagaaacc aacattgaat tgaatgatct gcattgatgt 11040aatcagggat
tttggcatgg ggaacttcag cttgatcaat ctggtccaat aataaccgta
11100catgatccag tggatgaaac cattcaacag cacaaaaatc caaacagctt
catttcggta 11160attatagaac agccacatat ccatcggtgc ccccaaatga
tggaagaatt gcaaccaggt 11220cagaggcttg cccatcagtg gcaaatagaa
ggagtcaata tactccagga acttgctcaa 11280atagaacaac tgcgtggtga
tcctgaagac gttgttgtca aaagccttct cgcagttgtc 11340agacataaca
ccgatggtgt acatggcata tgccattgag aggaatgatc ccaacgaata
11400aatggacatg agaaggttgt aattggtgaa aacaaacttc atacgagact
gaccttttgg 11460accaaggggg ccaagagtga acttcaagat gacaaatgcg
atggacaagt aaagcacctc 11520acagtgactg gcatcactcc agagttgggc
ataatcaact ggttgggtaa aacttcctgc 11580ccaattgaga ctatttcatt
caccacctcc atggccattg ctgtagatat gtcttgtgtg 11640taagggggtt
ggggtggttg tttgtgttct tgacttttgt gttagcaagg gaagacgggc
11700aaaaaagtga gtgtggttgg gagggagaga cgagccttat atataatgct
tgtttgtgtt 11760tgtgcaagtg gacgccgaaa cgggcaggag ccaaactaaa
caaggcagac aatgcgagct 11820taattggatt gcctgatggg caggggttag
ggctcgatca atgggggtgc gaagtgacaa 11880aattgggaat taggttcgca
agcaaggctg acaagacttt ggcccaaaca tttgtacgcg 11940gtggacaaca
ggagccaccc atcgtctgtc acgggctagc cggtcgtgcg tcctgtcagg
12000ctccacctag gctccatgcc actccataca atcccactag tgtaccgcta
ggccgctttt 12060agctcccatc taagaccccc ccaaaacctc cactgtacag
tgcactgtac tgtgtggcga 12120tcaagggcaa gggaaaaaag gcgcaaacat
gcacgcatgg aatgacgtag gtaaggcgtt 12180actagactga aaagtggcac
atttcggcgt gccaaagggt cctaggtgcg tttcgcgagc 12240tgggcgccag
gccaagccgc tccaaaacgc ctctccgact ccctccagcg gcctccatat
12300ccccatccct ctccacagca atgttgttaa gccttgcaaa cgaaaaaata
gaaaggctaa 12360taagcttcca atattgtggt gtacgctgca taacgcaaca
atgagcgcca aacaacacac 12420acacacagca cacagcagca ttaaccacga
tgaacagcat gacattacag gtgggtgtgt 12480aatcagggcc ctgattgctg
gtggtgggag cccccatcat gggcagatct gcgtacactg 12540tttaaacagt
gtacgcagat ctactataga ggaacattta aattgccccg gagaagacgg
12600ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca
ttgccactag 12660gggggggcct ttttatatgg ccaagccaag ctctccacgt
cggttgggct gcacccaaca 12720ataaatgggt agggttgcac caacaaaggg
atgggatggg gggtagaaga tacgaggata 12780acggggctca atggcacaaa
taagaacgaa tactgccatt aagactcgtg atccagcgac 12840tgacaccatt
gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg
12900acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg
tgcaggcaga 12960aaacgctgga acagcgtgta cagtttgtct taacaaaaag
tgagggcgct gaggtcgagc 13020agggtggtgt gacttgttat agcctttaga
gctgcgaaag cgcgtatgga tttggctcat 13080caggccagat tgagggtctg
tggacacatg tcatgttagt gtacttcaat cgccccctgg 13140atatagcccc
gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct
13200cgatacccac accttgcttc tcctgcactt gccaacctta atactggttt
acattgacca 13260acatcttaca agcggggggc ttgtctaggg tatatataaa
cagtggctct cccaatcggt 13320tgccagtctc ttttttcctt tctttcccca
cagattcgaa atctaaacta cacatcacag 13380aattccgagc cgtgagtatc
cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat 13440gacacaatcc
gaaagtcgct agcaacacac actctctaca caaactaacc cagctctggt
13500accatggagg tcgtgaacga aatcgtctcc attggccagg aggttcttcc
caaggtcgac 13560tatgctcagc tctggtctga tgcctcgcac tgcgaggtgc
tgtacctctc catcgccttc 13620gtcatcctga agttcaccct tggtcctctc
ggacccaagg gtcagtctcg aatgaagttt 13680gtgttcacca actacaacct
gctcatgtcc atctactcgc tgggctcctt cctctctatg 13740gcctacgcca
tgtacaccat tggtgtcatg tccgacaact gcgagaaggc tttcgacaac
13800aatgtcttcc gaatcaccac tcagctgttc tacctcagca agttcctcga
gtacattgac 13860tccttctatc tgcccctcat gggcaagcct ctgacctggt
tgcagttctt tcaccatctc 13920ggagctccta tggacatgtg gctgttctac
aactaccgaa acgaagccgt ttggatcttt 13980gtgctgctca acggcttcat
tcactggatc atgtacggct actattggac ccgactgatc 14040aagctcaagt
tccctatgcc caagtccctg attacttcta tgcagatcat tcagttcaac
14100gttggcttct acatcgtctg gaagtaccgg aacattccct gctaccgaca
agatggaatg 14160agaatgtttg gctggttttt caactacttc tacgttggta
ctgtcctgtg tctgttcctc 14220aacttctacg tgcagaccta catcgtccga
aagcacaagg gagccaaaaa gattcagtga 14280gcggccgcat gtacatacaa
gattatttat agaaatgaat cgcgatcgaa caaagagtac 14340gagtgtacga
gtaggggatg atgataaaag tggaagaagt tccgcatctt tggatttatc
14400aacgtgtagg acgatacttc ctgtaaaaat gcaatgtctt taccataggt
tctgctgtag 14460atgttattaa ctaccattaa catgtctact tgtacagttg
cagaccagtt ggagtataga 14520atggtacact taccaaaaag tgttgatggt
tgtaactacg atatataaaa ctgttgacgg 14580gatccccgct gatatgccta
aggaacaatc aaagaggaag atattaattc agaatgctag 14640tatacagtta gggat
146551834DNAEscherichia coli 18ataacttcgt ataatgtatg ctatacgaag
ttat 3419828DNAMortierella alpinamisc_featuresynthetic C16/18
elongase codon-optimized for Yarrowia lipolytica; U.S. Patent
Application No. 11/253882, filed October 19, 2005 19atggagtctg
gacccatgcc tgctggcatt cccttccctg agtactatga cttctttatg 60gactggaaga
ctcccctggc catcgctgcc acctacactg ctgccgtcgg tctcttcaac
120cccaaggttg gcaaggtctc ccgagtggtt gccaagtcgg ctaacgcaaa
gcctgccgag 180cgaacccagt ccggagctgc catgactgcc ttcgtctttg
tgcacaacct cattctgtgt 240gtctactctg gcatcacctt ctactacatg
tttcctgcta tggtcaagaa cttccgaacc 300cacacactgc acgaagccta
ctgcgacacg gatcagtccc tctggaacaa cgcacttggc 360tactggggtt
acctcttcta cctgtccaag ttctacgagg tcattgacac catcatcatc
420atcctgaagg gacgacggtc ctcgctgctt cagacctacc accatgctgg
agccatgatt 480accatgtggt ctggcatcaa ctaccaagcc actcccattt
ggatctttgt ggtcttcaac 540tccttcattc acaccatcat gtactgttac
tatgccttca cctctatcgg attccatcct 600cctggcaaaa agtacctgac
ttcgatgcag attactcagt ttctggtcgg tatcaccatt 660gccgtgtcct
acctcttcgt tcctggctgc atccgaacac ccggtgctca gatggctgtc
720tggatcaacg tcggctacct gtttcccttg acctatctgt tcgtggactt
tgccaagcga 780acctactcca agcgatctgc cattgccgct cagaaaaagg ctcagtaa
828208910DNAArtificial SequencePlasmid pFmD8S 20catggtgaag
tccaagcgac aggctctgcc cctcaccatc gacggaacta cctacgacgt 60ctccgcttgg
gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg
120agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca
agctcaagcg 180aatgcccaag atcaacccct cctccgagct gcctccccag
gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct
gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga
tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360gatggtccag
taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca
420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc
gaaactggaa 480taacctcgtg ggtctggtct ttggcaacgg actccagggc
ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac
caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct
ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt
cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat
720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg
cactggactc tcaagaccct 840gttccacctc ttctttatgc cctccatcct
gacctcgctc ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa
ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat
tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat
1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact
accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact
gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta
ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgg
ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260ggctctctaa
gcggccgcca ccgcggcccg agattccggc ctcttcggcc gccaagcgac
1320ccgggtggac gtctagaggt acctagcaat taacagatag tttgccggtg
ataattctct 1380taacctccca cactcctttg acataacgat ttatgtaacg
aaactgaaat ttgaccagat 1440attgtgtccg cggtggagct ccagcttttg
ttccctttag tgagggttaa tttcgagctt 1500ggcgtaatca tggtcatagc
tgtttcctgt gtgaaattgt tatccgctca caattccaca 1560caacatacga
gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
1620cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
cgtgccagct 1680gcattaatga atcggccaac gcgcggggag aggcggtttg
cgtattgggc gctcttccgc 1740ttcctcgctc actgactcgc tgcgctcggt
cgttcggctg cggcgagcgg tatcagctca 1800ctcaaaggcg gtaatacggt
tatccacaga atcaggggat aacgcaggaa agaacatgtg 1860agcaaaaggc
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
1920taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
ggtggcgaaa 1980cccgacagga ctataaagat accaggcgtt tccccctgga
agctccctcg tgcgctctcc 2040tgttccgacc ctgccgctta ccggatacct
gtccgccttt ctcccttcgg gaagcgtggc 2100gctttctcat agctcacgct
gtaggtatct cagttcggtg taggtcgttc gctccaagct 2160gggctgtgtg
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
2220tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
ctggtaacag 2280gattagcaga gcgaggtatg taggcggtgc tacagagttc
ttgaagtggt ggcctaacta 2340cggctacact agaaggacag tatttggtat
ctgcgctctg ctgaagccag ttaccttcgg 2400aaaaagagtt ggtagctctt
gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2460tgtttgcaag
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
2520ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
tggtcatgag 2580attatcaaaa aggatcttca cctagatcct tttaaattaa
aaatgaagtt ttaaatcaat 2640ctaaagtata tatgagtaaa cttggtctga
cagttaccaa tgcttaatca gtgaggcacc 2700tatctcagcg atctgtctat
ttcgttcatc catagttgcc tgactccccg tcgtgtagat 2760aactacgata
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
2820acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
ccgagcgcag 2880aagtggtcct gcaactttat ccgcctccat ccagtctatt
aattgttgcc gggaagctag 2940agtaagtagt tcgccagtta atagtttgcg
caacgttgtt gccattgcta caggcatcgt 3000ggtgtcacgc tcgtcgtttg
gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3060agttacatga
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
3120tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
tgcataattc 3180tcttactgtc atgccatccg taagatgctt ttctgtgact
ggtgagtact caaccaagtc 3240attctgagaa tagtgtatgc ggcgaccgag
ttgctcttgc ccggcgtcaa tacgggataa 3300taccgcgcca catagcagaa
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3360aaaactctca
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
3420caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
aaacaggaag 3480gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
tgttgaatac tcatactctt 3540cctttttcaa tattattgaa gcatttatca
gggttattgt ctcatgagcg gatacatatt 3600tgaatgtatt tagaaaaata
aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3660acctgacgcg
ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
3720gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
cttcctttct 3780cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
gggctccctt tagggttccg 3840atttagtgct ttacggcacc tcgaccccaa
aaaacttgat tagggtgatg gttcacgtag 3900tgggccatcg ccctgataga
cggtttttcg ccctttgacg ttggagtcca cgttctttaa 3960tagtggactc
ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
4020tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
tttaacaaaa 4080atttaacgcg aattttaaca aaatattaac gcttacaatt
tccattcgcc attcaggctg 4140cgcaactgtt gggaagggcg atcggtgcgg
gcctcttcgc tattacgcca gctggcgaaa 4200gggggatgtg ctgcaaggcg
attaagttgg gtaacgccag ggttttccca gtcacgacgt 4260tgtaaaacga
cggccagtga attgtaatac gactcactat agggcgaatt gggtaccggg
4320ccccccctcg aggtcgatgg tgtcgataag cttgatatcg aattcatgtc
acacaaaccg 4380atcttcgcct caaggaaacc taattctaca tccgagagac
tgccgagatc cagtctacac 4440tgattaattt tcgggccaat aatttaaaaa
aatcgtgtta tataatatta tatgtattat 4500atatatacat catgatgata
ctgacagtca tgtcccattg ctaaatagac agactccatc 4560tgccgcctcc
aactgatgtt ctcaatattt aaggggtcat ctcgcattgt ttaataataa
4620acagactcca tctaccgcct ccaaatgatg ttctcaaaat atattgtatg
aacttatttt 4680tattacttag tattattaga caacttactt gctttatgaa
aaacacttcc tatttaggaa 4740acaatttata atggcagttc gttcatttaa
caatttatgt agaataaatg ttataaatgc 4800gtatgggaaa tcttaaatat
ggatagcata aatgatatct gcattgccta attcgaaatc 4860aacagcaacg
aaaaaaatcc cttgtacaac ataaatagtc atcgagaaat atcaactatc
4920aaagaacagc tattcacacg ttactattga gattattatt ggacgagaat
cacacactca 4980actgtctttc tctcttctag aaatacaggt acaagtatgt
actattctca ttgttcatac 5040ttctagtcat ttcatcccac atattccttg
gatttctctc caatgaatga cattctatct 5100tgcaaattca acaattataa
taagatatac caaagtagcg gtatagtggc aatcaaaaag 5160cttctctggt
gtgcttctcg tatttatttt tattctaatg atccattaaa ggtatatatt
5220tatttcttgt tatataatcc ttttgtttat tacatgggct ggatacataa
aggtattttg 5280atttaatttt ttgcttaaat tcaatccccc ctcgttcagt
gtcaactgta atggtaggaa 5340attaccatac ttttgaagaa gcaaaaaaaa
tgaaagaaaa aaaaaatcgt atttccaggt 5400tagacgttcc gcagaatcta
gaatgcggta tgcggtacat tgttcttcga acgtaaaagt 5460tgcgctccct
gagatattgt acatttttgc ttttacaagt acaagtacat cgtacaacta
5520tgtactactg ttgatgcatc cacaacagtt tgttttgttt ttttttgttt
tttttttttc 5580taatgattca ttaccgctat gtatacctac ttgtacttgt
agtaagccgg gttattggcg 5640ttcaattaat catagactta tgaatctgca
cggtgtgcgc tgcgagttac ttttagctta 5700tgcatgctac ttgggtgtaa
tattgggatc tgttcggaaa tcaacggatg ctcaaccgat 5760ttcgacagta
ataatttgaa tcgaatcgga gcctaaaatg aacccgagta tatctcataa
5820aattctcggt gagaggtctg tgactgtcag tacaaggtgc cttcattatg
ccctcaacct 5880taccatacct cactgaatgt agtgtacctc taaaaatgaa
atacagtgcc aaaagccaag 5940gcactgagct cgtctaacgg acttgatata
caaccaatta aaacaaatga aaagaaatac 6000agttctttgt atcatttgta
acaattaccc tgtacaaact aaggtattga aatcccacaa 6060tattcccaaa
gtccacccct ttccaaattg tcatgcctac aactcatata ccaagcacta
6120acctaccaaa caccactaaa accccacaaa atatatctta ccgaatatac
agtaacaagc 6180taccaccaca ctcgttgggt gcagtcgcca gcttaaagat
atctatccac atcagccaca 6240actcccttcc tttaataaac cgactacacc
cttggctatt gaggttatga gtgaatatac 6300tgtagacaag acactttcaa
gaagactgtt tccaaaacgt accactgtcc tccactacaa 6360acacacccaa
tctgcttctt ctagtcaagg ttgctacacc ggtaaattat aaatcatcat
6420ttcattagca gggcagggcc ctttttatag agtcttatac actagcggac
cctgccggta 6480gaccaacccg caggcgcgtc agtttgctcc ttccatcaat
gcgtcgtaga aacgacttac 6540tccttcttga gcagctcctt gaccttgttg
gcaacaagtc tccgacctcg gaggtggagg 6600aagagcctcc gatatcggcg
gtagtgatac cagcctcgac ggactccttg acggcagcct 6660caacagcgtc
accggcgggc ttcatgttaa gagagaactt gagcatcatg gcggcagaca
6720gaatggtggc aatggggttg accttctgct tgccgagatc gggggcagat
ccgtgacagg 6780gctcgtacag accgaacgcc tcgttggtgt cgggcagaga
agccagagag gcggagggca 6840gcagacccag agaaccgggg atgacggagg
cctcgtcgga gatgatatcg ccaaacatgt 6900tggtggtgat gatgatacca
ttcatcttgg agggctgctt gatgaggatc atggcggccg 6960agtcgatcag
ctggtggttg agctcgagct gggggaattc gtccttgagg actcgagtga
7020cagtctttcg ccaaagtcga gaggaggcca gcacgttggc cttgtcaaga
gaccacacgg 7080gaagaggggg gttgtgctga agggccagga aggcggccat
tcgggcaatt cgctcaacct 7140caggaacgga gtaggtctcg gtgtcggaag
cgacgccaga tccgtcatcc tcctttcgct 7200ctccaaagta gatacctccg
acgagctctc ggacaatgat gaagtcggtg ccctcaacgt 7260ttcggatggg
ggagagatcg gcgagcttgg gcgacagcag ctggcagggt cgcaggttgg
7320cgtacaggtt caggtccttt cgcagcttga ggagaccctg ctcgggtcgc
acgtcggttc 7380gtccgtcggg agtggtccat acggtgttgg cagcgcctcc
gacagcaccg agcataatag 7440agtcagcctt tcggcagatg tcgagagtag
cgtcggtgat gggctcgccc tccttctcaa 7500tggcagctcc tccaatgagt
cggtcctcaa acacaaactc ggtgccggag gcctcagcaa 7560cagacttgag
caccttgacg gcctcggcaa tcacctcggg gccacagaag tcgccgccga
7620gaagaacaat cttcttggag tcagtcttgg tcttcttagt ttcgggttcc
attgtggatg 7680tgtgtggttg tatgtgtgat gtggtgtgtg gagtgaaaat
ctgtggctgg caaacgctct 7740tgtatatata cgcacttttg cccgtgctat
gtggaagact aaacctccga agattgtgac 7800tcaggtagtg cggtatcggc
tagggaccca aaccttgtcg atgccgatag cgctatcgaa 7860cgtaccccag
ccggccggga gtatgtcgga ggggacatac gagatcgtca agggtttgtg
7920gccaactggt aaataaatga tgtcgacgtt taaacagtgt acgcagatct
actatagagg 7980aacatttaaa ttgccccgga gaagacggcc aggccgccta
gatgacaaat tcaacaactc 8040acagctgact ttctgccatt gccactaggg
gggggccttt ttatatggcc aagccaagct 8100ctccacgtcg gttgggctgc
acccaacaat aaatgggtag ggttgcacca acaaagggat 8160gggatggggg
gtagaagata cgaggataac ggggctcaat ggcacaaata agaacgaata
8220ctgccattaa gactcgtgat ccagcgactg acaccattgc atcatctaag
ggcctcaaaa 8280ctacctcgga actgctgcgc tgatctggac accacagagg
ttccgagcac tttaggttgc 8340accaaatgtc ccaccaggtg caggcagaaa
acgctggaac agcgtgtaca gtttgtctta 8400acaaaaagtg agggcgctga
ggtcgagcag ggtggtgtga cttgttatag cctttagagc 8460tgcgaaagcg
cgtatggatt tggctcatca ggccagattg agggtctgtg gacacatgtc
8520atgttagtgt acttcaatcg ccccctggat atagccccga caataggccg
tggcctcatt 8580tttttgcctt ccgcacattt ccattgctcg gtacccacac
cttgcttctc ctgcacttgc 8640caaccttaat actggtttac attgaccaac
atcttacaag cggggggctt gtctagggta 8700tatataaaca gtggctctcc
caatcggttg ccagtctctt ttttcctttc tttccccaca 8760gattcgaaat
ctaaactaca catcacagaa ttccgagccg tgagtatcca cgacaagatc
8820agtgtcgaga cgacgcgttt tgtgtaatga cacaatccga aagtcgctag
caacacacac 8880tctctacaca aactaaccca gctctggtac
8910217145DNAArtificial SequencePlasmid pKUNFmkF2 21catggcgtcc
acttcggctc tgcccaagca gaaccctgcg cttagacgca ccgtcacctc 60aactactgtg
acggattctg
agtctgccgc cgtctctcct tcagactctc cccgccactc 120ggcctcttcc
acatcgctct cgtccatgtc cgaggttgat atcgccaagc ccaagtccga
180gtatggtgtc atgctcgaca cctacggcaa ccagttcgag gttcccgact
ttaccatcaa 240ggacatctac aatgccatcc ctaagcactg cttcaagcgc
tccgctctca agggatacgg 300ttatatcctc cgcgacattg tcctcctgac
taccactttc agcatctggt acaactttgt 360gacccccgaa tatatcccct
ccacccccgc ccgcgctggt ctgtgggccg tgtacaccgt 420tcttcagggt
cttttcggta ctggtctctg ggttattgcc catgagtgcg gtcacggtgc
480tttctccgat tctcgcatca tcaacgacat tactggctgg gttcttcact
cttccctcct 540tgtcccctac ttcagctggc aaatctccca ccgaaagcac
cacaaggcca ctggcaacat 600ggagcgtgac atggtcttcg ttccccgaac
ccgcgagcag caggctactc gtctcggaaa 660gatgacccac gagctcgctc
atcttactga gnnnntcgtn ggctggccca actacctcat 720caccaatgtt
accggccaca actaccacga gcgccagcgt gagggtcgcg gcaagggcaa
780gcataacggc ctcggcggtg gtgttaacca cttcgatccc cgcagccctc
tgtacgagaa 840cagtgacgct aagctcatcg tcctcagcga tattggtatc
ggtctgatgg ccactgctct 900gtacttcctc gttcagaagt tcggtttcta
caacatggcc atctggtact ttgttcccta 960cctctgggtt aaccactggc
tcgttgccat caccttcctc cagcacaccg accctaccct 1020tccccactac
accaacgacg agtggaactt cgtccgtggt gccgctgcta ccattgaccg
1080tgagatgggc ttcatcggcc gccaccttct ccacggcatc atcgagactc
atgtcctcca 1140ccactacgtc agcagcatcc ccttctacaa cgcggacgag
gccaccgagg ccattaagcc 1200catcatgggc aagcactacc gggctgatgt
ccaggatggt cctcgtggct tcatccgcgc 1260catgtaccgc agtgcgcgta
tgtgccagtg ggttgagccc agcgctggtg ccgagggtgc 1320tggtaagggt
gttctgttct tccgcaaccg caacaacgtg ggcacccccc ccgctgttat
1380caagcccgtt gcttaagtag gcgcggccgc tatttatcac tctttacaac
ttctacctca 1440actatctact ttaataaatg aatatcgttt attctctatg
attactgtat atgcgttcct 1500ctaagacaaa tcgaaaccag catgtgatcg
aatggcatac aaaagtttct tccgaagttg 1560atcaatgtcc tgatagtcag
gcagcttgag aagattgaca caggtggagg ccgtagggaa 1620ccgatcaacc
tgtctaccag cgttacgaat ggcaaatgac gggttcaaag ccttgaatcc
1680ttgcaatggt gccttggata ctgatgtcac aaacttaaga agcagccgct
tgtcctcttc 1740ctcgatcgat ggtcatagct gtttcctgtg tgaaattgtt
atccgctcac aattccacac 1800aacgtacgaa gtcgtcaatg atgtcgatat
gggttttgat catgcacaca taaggtccga 1860ccttatcggc aagctcaatg
agctccttgg tggtggtaac atccagagaa gcacacaggt 1920tggttttctt
ggctgccacg agcttgagca ctcgagcggc aaaggcggac ttgtggacgt
1980tagctcgagc ttcgtaggag ggcattttgg tggtgaagag gagactgaaa
taaatttagt 2040ctgcagaact ttttatcgga accttatctg gggcagtgaa
gtatatgtta tggtaatagt 2100tacgagttag ttgaacttat agatagactg
gactatacgg ctatcggtcc aaattagaaa 2160gaacgtcaat ggctctctgg
gcgtcgcctt tgccgacaaa aatgtgatca tgatgaaagc 2220cagcaatgac
gttgcagctg atattgttgt cggccaaccg cgccgaaaac gcagctgtca
2280gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat ccaagcacac
tcatagttgg 2340agtcgtactc caaaggcggc aatgacgagt cagacagata
ctcgtcgacc ttttccttgg 2400gaaccaccac cgtcagccct tctgactcac
gtattgtagc caccgacaca ggcaacagtc 2460cgtggatagc agaatatgtc
ttgtcggtcc atttctcacc aactttaggc gtcaagtgaa 2520tgttgcagaa
gaagtatgtg ccttcattga gaatcggtgt tgctgatttc aataaagtct
2580tgagatcagt ttggcgcgcc agctgcatta atgaatcggc caacgcgcgg
ggagaggcgg 2640tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct cggtcgttcg 2700gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca cagaatcagg 2760ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2820ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
2880acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 2940tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat acctgtccgc 3000ctttctccct tcgggaagcg tggcgctttc
tcatagctca cgctgtaggt atctcagttc 3060ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3120ctgcgcctta
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
3180actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 3240gttcttgaag tggtggccta actacggcta cactagaaga
acagtatttg gtatctgcgc 3300tctgctgaag ccagttacct tcggaaaaag
agttggtagc tcttgatccg gcaaacaaac 3360caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3420atctcaagaa
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
3480acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
tccttttaaa 3540ttaaaaatga agttttaaat caatctaaag tatatatgag
taaacttggt ctgacagtta 3600ccaatgctta atcagtgagg cacctatctc
agcgatctgt ctatttcgtt catccatagt 3660tgcctgactc cccgtcgtgt
agataactac gatacgggag ggcttaccat ctggccccag 3720tgctgcaatg
ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
3780gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
ccatccagtc 3840tattaattgt tgccgggaag ctagagtaag tagttcgcca
gttaatagtt tgcgcaacgt 3900tgttgccatt gctacaggca tcgtggtgtc
acgctcgtcg tttggtatgg cttcattcag 3960ctccggttcc caacgatcaa
ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4020tagctccttc
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
4080ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
gcttttctgt 4140gactggtgag tactcaacca agtcattctg agaatagtgt
atgcggcgac cgagttgctc 4200ttgcccggcg tcaatacggg ataataccgc
gccacatagc agaactttaa aagtgctcat 4260cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4320ttcgatgtaa
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
4380ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
gggcgacacg 4440gaaatgttga atactcatac tcttcctttt tcaatattat
tgaagcattt atcagggtta 4500ttgtctcatg agcggataca tatttgaatg
tatttagaaa aataaacaaa taggggttcc 4560gcgcacattt ccccgaaaag
tgccacctga tgcggtgtga aataccgcac agatgcgtaa 4620ggagaaaata
ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
4680tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
tcccttataa 4740atcaaaagaa tagaccgaga tagggttgag tgttgttcca
gtttggaaca agagtccact 4800attaaagaac gtggactcca acgtcaaagg
gcgaaaaacc gtctatcagg gcgatggccc 4860actacgtgaa ccatcaccct
aatcaagttt tttggggtcg aggtgccgta aagcactaaa 4920tcggaaccct
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
4980gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
gtgtagcggt 5040cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
ccgctacagg gcgcgtccat 5100tcgccattca ggctgcgcaa ctgttgggaa
gggcgatcgg tgcgggcctc ttcgctatta 5160cgccagctgg cgaaaggggg
atgtgctgca aggcgattaa gttgggtaac gccagggttt 5220tcccagtcac
gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
5280gaattgggcc cgacgtcgca tgcagtggtg gtattgtgac tggggatgta
gttgagaata 5340agtcatacac aagtcagctt tcttcgagcc tcatataagt
ataagtagtt caacgtatta 5400gcactgtacc cagcatctcc gtatcgagaa
acacaacaac atgccccatt ggacagatca 5460tgcggataca caggttgtgc
agtatcatac atactcgatc agacaggtcg tctgaccatc 5520atacaagctg
aacaagcgct ccatacttgc acgctctcta tatacacagt taaattacat
5580atccatagtc taacctctaa cagttaatct tctggtaagc ctcccagcca
gccttctggt 5640atcgcttggc ctcctcaata ggatctcggt tctggccgta
cagacctcgg ccgacaatta 5700tgatatccgt tccggtagac atgacatcct
caacagttcg gtactgctgt ccgagagcgt 5760ctcccttgtc gtcaagaccc
accccggggg tcagaataag ccagtcctca gagtcgccct 5820taattaattt
gaatcgaatc gatgagccta aaatgaaccc gagtatatct cataaaattc
5880tcggtgagag gtctgtgact gtcagtacaa ggtgccttca ttatgccctc
aaccttacca 5940tacctcactg aatgtagtgt acctctaaaa atgaaataca
gtgccaaaag ccaaggcact 6000gagctcgtct aacggacttg atatacaacc
aattaaaaca aatgaaaaga aatacagttc 6060tttgtatcat ttgtaacaat
taccctgtac aaactaaggt attgaaatcc cacaatattc 6120ccaaagtcca
cccctttcca aattgtcatg cctacaactc atataccaag cactaaccta
6180ccgtttaaac agtgtacgca gatctactat agaggaacat ttaaattgcc
ccggagaaga 6240cggccaggcc gcctagatga caaattcaac aactcacagc
tgactttctg ccattgccac 6300tagggggggg cctttttata tggccaagcc
aagctctcca cgtcggttgg gctgcaccca 6360acaataaatg ggtagggttg
caccaacaaa gggatgggat ggggggtaga agatacgagg 6420ataacggggc
tcaatggcac aaataagaac gaatactgcc attaagactc gtgatccagc
6480gactgacacc attgcatcat ctaagggcct caaaactacc tcggaactgc
tgcgctgatc 6540tggacaccac agaggttccg agcactttag gttgcaccaa
atgtcccacc aggtgcaggc 6600agaaaacgct ggaacagcgt gtacagtttg
tcttaacaaa aagtgagggc gctgaggtcg 6660agcagggtgg tgtgacttgt
tatagccttt agagctgcga aagcgcgtat ggatttggct 6720catcaggcca
gattgagggt ctgtggacac atgtcatgtt agtgtacttc aatcgccccc
6780tggatatagc cccgacaata ggccgtggcc tcattttttt gccttccgca
catttccatt 6840gctcgatacc cacaccttgc ttctcctgca cttgccaacc
ttaatactgg tttacattga 6900ccaacatctt acaagcgggg ggcttgtcta
gggtatatat aaacagtggc tctcccaatc 6960ggttgccagt ctcttttttc
ctttctttcc ccacagattc gaaatctaaa ctacacatca 7020cagaattccg
agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt
7080aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta
acccagctct 7140ggtac 7145225473DNAArtificial SequencePlasmid
pDMW287F 22ggccgcattg atgattggaa acacacacat gggttatatc taggtgagag
ttagttggac 60agttatatat taaatcagct atgccaacgg taacttcatt catgtcaacg
aggaaccagt 120gactgcaagt aatatagaat ttgaccacct tgccattctc
ttgcactcct ttactatatc 180tcatttattt cttatataca aatcacttct
tcttcccagc atcgagctcg gaaacctcat 240gagcaataac atcgtggatc
tcgtcaatag agggcttttt ggactccttg ctgttggcca 300ccttgtcctt
gctgtctggc tcattctgtt tcaacgcctt ttaattaatc gagcttggcg
360taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
tccacacaac 420atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
aatgagtgag ctaactcaca 480ttaattgcgt tgcgctcact gcccgctttc
cagtcgggaa acctgtcgtg ccagctgcat 540taatgaatcg gccaacgcgc
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600tcgctcactg
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
660aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
catgtgagca 720aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg 780ctccgccccc ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg 840acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900ccgaccctgc
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
960tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc 1020tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt 1080gagtccaacc cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt 1140agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 1200tacactagaa
ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
1260agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 1320tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 1380acggggtctg acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgagatta 1440tcaaaaagga tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500agtatatatg
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
1560tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact 1620acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg agacccacgc 1680tcaccggctc cagatttatc agcaataaac
cagccagccg gaagggccga gcgcagaagt 1740ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 1800agtagttcgc
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
1860tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
aaggcgagtt 1920acatgatccc ccatgttgtg caaaaaagcg gttagctcct
tcggtcctcc gatcgttgtc 1980agaagtaagt tggccgcagt gttatcactc
atggttatgg cagcactgca taattctctt 2040actgtcatgc catccgtaag
atgcttttct gtgactggtg agtactcaac caagtcattc 2100tgagaatagt
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
2160gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
ggggcgaaaa 2220ctctcaagga tcttaccgct gttgagatcc agttcgatgt
aacccactcg tgcacccaac 2280tgatcttcag catcttttac tttcaccagc
gtttctgggt gagcaaaaac aggaaggcaa 2340aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400tttcaatatt
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
2460tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
agtgccacct 2520gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
tggttacgcg cagcgtgacc 2580gctacacttg ccagcgccct agcgcccgct
cctttcgctt tcttcccttc ctttctcgcc 2640acgttcgccg gctttccccg
tcaagctcta aatcgggggc tccctttagg gttccgattt 2700agtgctttac
ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
2760ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
ctttaatagt 2820ggactcttgt tccaaactgg aacaacactc aaccctatct
cggtctattc ttttgattta 2880taagggattt tgccgatttc ggcctattgg
ttaaaaaatg agctgattta acaaaaattt 2940aacgcgaatt ttaacaaaat
attaacgctt acaatttcca ttcgccattc aggctgcgca 3000actgttggga
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
3060gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
cgacgttgta 3120aaacgacggc cagtgaattg taatacgact cactataggg
cgaattgggt accgggcccc 3180ccctcgaggt cgacgtttaa acagtgtacg
cagtactata gaggaacatc gattgccccg 3240gagaagacgg ccaggccgcc
tagatgacaa attcaacaac tcacagctga ctttctgcca 3300ttgccactag
gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct
3360gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg
gggtagaaga 3420tacgaggata acggggctca atggcacaaa taagaacgaa
tactgccatt aagactcgtg 3480atccagcgac tgacaccatt gcatcatcta
agggcctcaa aactacctcg gaactgctgc 3540gctgatctgg acaccacaga
ggttccgagc actttaggtt gcaccaaatg tcccaccagg 3600tgcaggcaga
aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct
3660gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag
cgcgtatgga 3720tttggctcat caggccagat tgagggtctg tggacacatg
tcatgttagt gtacttcaat 3780cgccccctgg atatagcccc gacaataggc
cgtggcctca tttttttgcc ttccgcacat 3840ttccattgct cggtacccac
accttgcttc tcctgcactt gccaacctta atactggttt 3900acattgacca
acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct
3960cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa
atctaaacta 4020cacatcacac aatgcctgtt actgacgtcc ttaagcgaaa
gtccggtgtc atcgtcggcg 4080acgatgtccg agccgtgagt atccacgaca
agatcagtgt cgagacgacg cgttttgtgt 4140aatgacacaa tccgaaagtc
gctagcaaca cacactctct acacaaacta acccagctct 4200ccatggtgaa
gtccaagcga caggctctgc ccctcaccat cgacggaact acctacgacg
4260tctccgcttg ggtgaacttc caccctggtg gagctgaaat cattgagaac
taccagggac 4320gagatgctac tgacgccttc atggttatgc actctcagga
agccttcgac aagctcaagc 4380gaatgcccaa gatcaacccc tcctccgagc
tgcctcccca ggctgccgtc aacgaagctc 4440aggaggattt ccgaaagctc
cgagaagagc tgatcgccac tggcatgttt gacgcctctc 4500ccctctggta
ctcgtacaag atctccacca ccctgggtct tggcgtgctt ggatacttcc
4560tgatggtcca gtaccagatg tacttcattg gtgctgtgct gctcggtatg
cactaccagc 4620aaatgggatg gctgtctcat gacatctgcc accaccagac
cttcaagaac cgaaactgga 4680ataacctcgt gggtctggtc tttggcaacg
gactccaggg cttctccgtg acctggtgga 4740aggacagaca caacgcccat
cattctgcta ccaacgttca gggtcacgat cccgacattg 4800ataacctgcc
tctgctcgcc tggtccgagg acgatgtcac tcgagcttct cccatctccc
4860gaaagctcat tcagttccaa cagtactatt tcctggtcat ctgtattctc
ctgcgattca 4920tctggtgttt ccagtctgtg ctgaccgttc gatccctcaa
ggaccgagac aaccagttct 4980accgatctca gtacaagaaa gaggccattg
gactcgctct gcactggact ctcaagaccc 5040tgttccacct cttctttatg
ccctccatcc tgacctcgct cctggtgttc tttgtttccg 5100agctcgtcgg
tggcttcgga attgccatcg tggtcttcat gaaccactac cctctggaga
5160agatcggtga ttccgtctgg gacggacatg gcttctctgt gggtcagatc
catgagacca 5220tgaacattcg acgaggcatc attactgact ggttctttgg
aggcctgaac taccagatcg 5280agcaccatct ctggcccacc ctgcctcgac
acaacctcac tgccgtttcc taccaggtgg 5340aacagctgtg ccagaagcac
aacctcccct accgaaaccc tctgccccat gaaggtctcg 5400tcatcctgct
ccgatacctg gccgtgttcg ctcgaatggc cgagaagcag cccgctggca
5460aggctctcta agc 5473239513DNAArtificial SequencePlasmid pDMW214
23ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
60ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
120ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
acattaattg 180cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa 240tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca 300ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360taatacggtt
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
420agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
aggctccgcc 480cccctgacga gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac 540tataaagata ccaggcgttt ccccctggaa
gctccctcgt gcgctctcct gttccgaccc 600tgccgcttac cggatacctg
tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660gctcacgctg
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
720acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
cttgagtcca 780acccggtaag acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag 840cgaggtatgt aggcggtgct acagagttct
tgaagtggtg gcctaactac ggctacacta 900gaaggacagt atttggtatc
tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960gtagctcttg
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
1020agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt 1080ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcaaaaa 1140ggatcttcac ctagatcctt ttaaattaaa
aatgaagttt taaatcaatc taaagtatat 1200atgagtaaac ttggtctgac
agttaccaat gcttaatcag tgaggcacct atctcagcga 1260tctgtctatt
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
1320gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
cgctcaccgg 1380ctccagattt atcagcaata aaccagccag ccggaagggc
cgagcgcaga agtggtcctg 1440caactttatc cgcctccatc cagtctatta
attgttgccg ggaagctaga gtaagtagtt 1500cgccagttaa tagtttgcgc
aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560cgtcgtttgg
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
1620cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
gtcagaagta 1680agttggccgc agtgttatca ctcatggtta tggcagcact
gcataattct cttactgtca 1740tgccatccgt aagatgcttt tctgtgactg
gtgagtactc aaccaagtca ttctgagaat 1800agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
1920ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
aactgatctt 1980cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg 2040caaaaaaggg aataagggcg acacggaaat
gttgaatact catactcttc ctttttcaat 2100attattgaag catttatcag
ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160agaaaaataa
acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
2220cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
accgctacac 2280ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
ttcctttctc gccacgttcg
2340ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
tttagtgctt 2400tacggcacct cgaccccaaa aaacttgatt agggtgatgg
ttcacgtagt gggccatcgc 2460cctgatagac ggtttttcgc cctttgacgt
tggagtccac gttctttaat agtggactct 2520tgttccaaac tggaacaaca
ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580ttttgccgat
ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
2640attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
gcaactgttg 2700ggaagggcga tcggtgcggg cctcttcgct attacgccag
ctggcgaaag ggggatgtgc 2760tgcaaggcga ttaagttggg taacgccagg
gttttcccag tcacgacgtt gtaaaacgac 2820ggccagtgaa ttgtaatacg
actcactata gggcgaattg ggtaccgggc cccccctcga 2880ggtcgatggt
gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc
2940aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact
gattaatttt 3000cgggccaata atttaaaaaa atcgtgttat ataatattat
atgtattata tatatacatc 3060atgatgatac tgacagtcat gtcccattgc
taaatagaca gactccatct gccgcctcca 3120actgatgttc tcaatattta
aggggtcatc tcgcattgtt taataataaa cagactccat 3180ctaccgcctc
caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt
3240attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa
caatttataa 3300tggcagttcg ttcatttaac aatttatgta gaataaatgt
tataaatgcg tatgggaaat 3360cttaaatatg gatagcataa atgatatctg
cattgcctaa ttcgaaatca acagcaacga 3420aaaaaatccc ttgtacaaca
taaatagtca tcgagaaata tcaactatca aagaacagct 3480attcacacgt
tactattgag attattattg gacgagaatc acacactcaa ctgtctttct
3540ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact
tctagtcatt 3600tcatcccaca tattccttgg atttctctcc aatgaatgac
attctatctt gcaaattcaa 3660caattataat aagatatacc aaagtagcgg
tatagtggca atcaaaaagc ttctctggtg 3720tgcttctcgt atttattttt
attctaatga tccattaaag gtatatattt atttcttgtt 3780atataatcct
tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt
3840tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa
ttaccatact 3900tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta
tttccaggtt agacgttccg 3960cagaatctag aatgcggtat gcggtacatt
gttcttcgaa cgtaaaagtt gcgctccctg 4020agatattgta catttttgct
tttacaagta caagtacatc gtacaactat gtactactgt 4080tgatgcatcc
acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat
4140taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt
tcaattaatc 4200atagacttat gaatctgcac ggtgtgcgct gcgagttact
tttagcttat gcatgctact 4260tgggtgtaat attgggatct gttcggaaat
caacggatgc tcaaccgatt tcgacagtaa 4320taatttgaat cgaatcggag
cctaaaatga acccgagtat atctcataaa attctcggtg 4380agaggtctgt
gactgtcagt acaaggtgcc ttcattatgc cctcaacctt accatacctc
4440actgaatgta gtgtacctct aaaaatgaaa tacagtgcca aaagccaagg
cactgagctc 4500gtctaacgga cttgatatac aaccaattaa aacaaatgaa
aagaaataca gttctttgta 4560tcatttgtaa caattaccct gtacaaacta
aggtattgaa atcccacaat attcccaaag 4620tccacccctt tccaaattgt
catgcctaca actcatatac caagcactaa cctaccaaac 4680accactaaaa
ccccacaaaa tatatcttac cgaatataca gtaacaagct accaccacac
4740tcgttgggtg cagtcgccag cttaaagata tctatccaca tcagccacaa
ctcccttcct 4800ttaataaacc gactacaccc ttggctattg aggttatgag
tgaatatact gtagacaaga 4860cactttcaag aagactgttt ccaaaacgta
ccactgtcct ccactacaaa cacacccaat 4920ctgcttcttc tagtcaaggt
tgctacaccg gtaaattata aatcatcatt tcattagcag 4980ggcagggccc
tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc
5040aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact
ccttcttgag 5100cagctccttg accttgttgg caacaagtct ccgacctcgg
aggtggagga agagcctccg 5160atatcggcgg tagtgatacc agcctcgacg
gactccttga cggcagcctc aacagcgtca 5220ccggcgggct tcatgttaag
agagaacttg agcatcatgg cggcagacag aatggtggca 5280atggggttga
ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga
5340ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag
cagacccaga 5400gaaccgggga tgacggaggc ctcgtcggag atgatatcgc
caaacatgtt ggtggtgatg 5460atgataccat tcatcttgga gggctgcttg
atgaggatca tggcggccga gtcgatcagc 5520tggtggttga gctcgagctg
ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580caaagtcgag
aggaggccag cacgttggcc ttgtcaagag accacacggg aagagggggg
5640ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc gctcaacctc
aggaacggag 5700taggtctcgg tgtcggaagc gacgccagat ccgtcatcct
cctttcgctc tccaaagtag 5760atacctccga cgagctctcg gacaatgatg
aagtcggtgc cctcaacgtt tcggatgggg 5820gagagatcgg cgagcttggg
cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880aggtcctttc
gcagcttgag gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga
5940gtggtccata cggtgttggc agcgcctccg acagcaccga gcataataga
gtcagccttt 6000cggcagatgt cgagagtagc gtcggtgatg ggctcgccct
ccttctcaat ggcagctcct 6060ccaatgagtc ggtcctcaaa cacaaactcg
gtgccggagg cctcagcaac agacttgagc 6120accttgacgg cctcggcaat
cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180ttcttggagt
cagtcttggt cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt
6240atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc aaacgctctt
gtatatatac 6300gcacttttgc ccgtgctatg tggaagacta aacctccgaa
gattgtgact caggtagtgc 6360ggtatcggct agggacccaa accttgtcga
tgccgatagc gctatcgaac gtaccccagc 6420cggccgggag tatgtcggag
gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480aataaatgat
gtcgacgttt aaacagtgta cgcagtacta tagaggaaca attgccccgg
6540agaagacggc caggccgcct agatgacaaa ttcaacaact cacagctgac
tttctgccat 6600tgccactagg ggggggcctt tttatatggc caagccaagc
tctccacgtc ggttgggctg 6660cacccaacaa taaatgggta gggttgcacc
aacaaaggga tgggatgggg ggtagaagat 6720acgaggataa cggggctcaa
tggcacaaat aagaacgaat actgccatta agactcgtga 6780tccagcgact
gacaccattg catcatctaa gggcctcaaa actacctcgg aactgctgcg
6840ctgatctgga caccacagag gttccgagca ctttaggttg caccaaatgt
cccaccaggt 6900gcaggcagaa aacgctggaa cagcgtgtac agtttgtctt
aacaaaaagt gagggcgctg 6960aggtcgagca gggtggtgtg acttgttata
gcctttagag ctgcgaaagc gcgtatggat 7020ttggctcatc aggccagatt
gagggtctgt ggacacatgt catgttagtg tacttcaatc 7080gccccctgga
tatagccccg acaataggcc gtggcctcat ttttttgcct tccgcacatt
7140tccattgctc ggtacccaca ccttgcttct cctgcacttg ccaaccttaa
tactggttta 7200cattgaccaa catcttacaa gcggggggct tgtctagggt
atatataaac agtggctctc 7260ccaatcggtt gccagtctct tttttccttt
ctttccccac agattcgaaa tctaaactac 7320acatcacaca atgcctgtta
ctgacgtcct taagcgaaag tccggtgtca tcgtcggcga 7380cgatgtccga
gccgtgagta tccacgacaa gatcagtgtc gagacgacgc gttttgtgta
7440atgacacaat ccgaaagtcg ctagcaacac acactctcta cacaaactaa
cccagctctc 7500catggcatgg atggtacgtc ctgtagaaac cccaacccgt
gaaatcaaaa aactcgacgg 7560cctgtgggca ttcagtctgg atcgcgaaaa
ctgtggaatt gatcagcgtt ggtgggaaag 7620cgcgttacaa gaaagccggg
caattgctgt gccaggcagt tttaacgatc agttcgccga 7680tgcagatatt
cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa
7740aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt
acggcaaagt 7800gtgggtcaat aatcaggaag tgatggagca tcagggcggc
tatacgccat ttgaagccga 7860tgtcacgccg tatgttattg ccgggaaaag
tgtacgtatc accgtttgtg tgaacaacga 7920actgaactgg cagactatcc
cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa 7980gcagtcttac
ttccatgatt tctttaacta tgccgggatc catcgcagcg taatgctcta
8040caccacgccg aacacctggg tggacgatat caccgtggtg acgcatgtcg
cgcaagactg 8100taaccacgcg tctgttgact ggcaggtggt ggccaatggt
gatgtcagcg ttgaactgcg 8160tgatgcggat caacaggtgg ttgcaactgg
acaaggcact agcgggactt tgcaagtggt 8220gaatccgcac ctctggcaac
cgggtgaagg ttatctctat gaactgtgcg tcacagccaa 8280aagccagaca
gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa
8340gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg
gtcgtcatga 8400agatgcggac ttacgtggca aaggattcga taacgtgctg
atggtgcacg accacgcatt 8460aatggactgg attggggcca actcctaccg
tacctcgcat tacccttacg ctgaagagat 8520gctcgactgg gcagatgaac
atggcatcgt ggtgattgat gaaactgctg ctgtcggctt 8580taacctctct
ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga
8640agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag
agctgatagc 8700gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt
gccaacgaac cggatacccg 8760tccgcaagtg cacgggaata tttcgccact
ggcggaagca acgcgtaaac tcgacccgac 8820gcgtccgatc acctgcgtca
atgtaatgtt ctgcgacgct cacaccgata ccatcagcga 8880tctctttgat
gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt
8940ggaaacggca gagaaggtac tggaaaaaga acttctggcc tggcaggaga
aactgcatca 9000gccgattatc atcaccgaat acggcgtgga tacgttagcc
gggctgcact caatgtacac 9060cgacatgtgg agtgaagagt atcagtgtgc
atggctggat atgtatcacc gcgtctttga 9120tcgcgtcagc gccgtcgtcg
gtgaacaggt atggaatttc gccgattttg cgacctcgca 9180aggcatattg
cgcgttggcg gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa
9240gtcggcggct tttctgctgc aaaaacgctg gactggcatg aacttcggtg
aaaaaccgca 9300gcagggaggc aaacaatgat taattaacta gagcggccgc
caccgcggcc cgagattccg 9360gcctcttcgg ccgccaagcg acccgggtgg
acgtctagag gtacctagca attaacagat 9420agtttgccgg tgataattct
cttaacctcc cacactcctt tgacataacg atttatgtaa 9480cgaaactgaa
atttgaccag atattgtgtc cgc 95132439DNAArtificial SequencePrimer 1A
24tggtaccatg gtgaaggctt ctcgacaggc tctgcccct 392539DNAArtificial
SequencePrimer 1B 25aggggcagag cctgtcgaga agccttcacc atggtacca
392632DNAArtificial SequencePrimer 2A 26caggctctgc ccctcgtcat
cgacggaact ac 322732DNAArtificial SequencePrimer 2B 27gtagttccgt
cgatgacgag gggcagagcc tg 322837DNAArtificial SequencePrimer 3A
28ctcaccatcg acggaaaggt gtacgacgtc tccgctt 372937DNAArtificial
SequencePrimer 3B 29aagcggagac gtcgtacacc tttccgtcga tggtgag
373039DNAArtificial SequencePrimer 4A 30acgtctccgc ttgggtggac
gagcaccctg gtggagctg 393139DNAArtificial SequencePrimer 4B
31cagctccacc agggtgctcg tccacccaag cggagacgt 393239DNAArtificial
SequencePrimer 5A 32cttccaccct ggtggagact ctatcattga gaactacca
393339DNAArtificial SequencePrimer 5B 33tggtagttct caatgataga
gtctccacca gggtggaag 393434DNAArtificial SequencePrimer 6A
34agccttcgac aagctcctgc gaatgcccaa gatc 343534DNAArtificial
SequencePrimer 6B 35gatcttgggc attcgcagga gcttgtcgaa ggct
343639DNAArtificial SequencePrimer 7A 36tcgacaagct caagcgagcc
gtcaagatca acccctcct 393739DNAArtificial SequencePrimer 7B
37aggaggggtt gatcttgacg gctcgcttga gcttgtcga 393839DNAArtificial
SequencePrimer 8A 38aatgcccaag atcaaccagg cttccgagct gcctcccca
393939DNAArtificial SequencePrimer 8B 39tggggaggca gctcggaagc
ctggttgatc ttgggcatt 394037DNAArtificial SequencePrimer 9A
40tcctccgagc tgcctcagcc tgctgccgtc aacgaag 374137DNAArtificial
SequencePrimer 9B 41cttcgttgac ggcagcaggc tgaggcagct cggagga
374240DNAArtificial SequencePrimer 10A 42aggctgccgt caacgaacag
gctgaggatt tccgaaagct 404340DNAArtificial SequencePrimer 10B
43agctttcgga aatcctcagc ctgttcgttg acggcagcct 404440DNAArtificial
SequencePrimer 11A 44aggatttccg aaagctcgcc atcgagctga tcgccactgg
404540DNAArtificial SequencePrimer 11B 45ccagtggcga tcagctcgat
ggcgagcttt cggaaatcct 404640DNAArtificial SequencePrimer 12A
46tcctgctccg atacctgtcc cagttcgctc gaatggccga 404739DNAArtificial
SequencePrimer 12B 47tcggccattc gagcgaactg ggacaggtat cggagcagg
394838DNAArtificial SequencePrimer 13A 48tggccgtgtt cgctcgactg
tccgagaagc agcccgct 384938DNAArtificial SequencePrimer 13B
49agcgggctgc ttctcggaca gtcgagcgaa cacggcca 385037DNAArtificial
SequencePrimer 14A 50tcgaatggcc gagaaggtgt acgctggcaa ggctctc
375137DNAArtificial SequencePrimer 14B 51gagagccttg ccagcgtaca
ccttctcggc cattcga 375237DNAArtificial SequencePrimer 15A
52agcccgctgg caaggctcag taagcggccg ccaccgc 375337DNAArtificial
SequencePrimer 15B 53gcggtggcgg ccgcttactg agccttgcca gcgggct
375433DNAArtificial SequencePrimer 16A 54gtactcgtac aagatcgtca
ccaccctggg tct 335533DNAArtificial SequencePrimer 16B 55agacccaggg
tggtgacgat cttgtacgag tac 335640DNAArtificial SequencePrimer 17A
56actcgtacaa gatctccttc gctctgggtc ttggcgtgct 405740DNAArtificial
SequencePrimer 17B 57agcacgccaa gacccagagc gaaggagatc ttgtacgagt
405838DNAArtificial SequencePrimer 18A 58gcgtgcttgg atacttcatg
ctggtccagt accagatg 385938DNAArtificial SequencePrimer 18B
59catctggtac tggaccagca tgaagtatcc aagcacgc 386034DNAArtificial
SequencePrimer 19A 60tggatacttc ctgatgtccc agtaccagat gtac
346134DNAArtificial SequencePrimer 19B 61gtacatctgg tactgggaca
tcaggaagta tcca 346240DNAArtificial SequencePrimer 20A 62gatacttcct
gatggtctac cagcagatgt acttcattgg 406340DNAArtificial SequencePrimer
20B 63ccaatgaagt acatctgctg gtagaccatc aggaagtatc
406434DNAArtificial SequencePrimer 21A 64tgatggtcca gtaccacctg
tacttcattg gtgc 346534DNAArtificial SequencePrimer 21B 65gcaccaatga
agtacaggtg gtactggacc atca 346637DNAArtificial SequencePrimer 22A
66tggtccagta ccagatgcag ttcattggtg ctgtgct 376737DNAArtificial
SequencePrimer 22B 67agcacagcac caatgaactg catctggtac tggacca
376834DNAArtificial SequencePrimer 23A 68cctcttcttt atgcccaaca
tcctgacctc gctc 346934DNAArtificial SequencePrimer 23B 69gagcgaggtc
aggatgttgg gcataaagaa gagg 347038DNAArtificial SequencePrimer 24A
70tcttctttat gccctcccct atgacctcgc tcctggtg 387138DNAArtificial
SequencePrimer 24B 71caccaggagc gaggtcatag gggagggcat aaagaaga
387239DNAArtificial SequencePrimer 25A 72ttatgccctc catcctggtc
gtgctcctgg tgttctttg 397339DNAArtificial SequencePrimer 25B
73caaagaacac caggagcacg accaggatgg agggcataa 397437DNAArtificial
SequencePrimer 26A 74cctccatcct gacctcgatg ctggtgttct ttgtttc
377537DNAArtificial SequencePrimer 26B 75gaaacaaaga acaccagcat
cgaggtcagg atggagg 377634DNAArtificial SequencePrimer 27A
76gacctcgctc ctggtgacct ttgtttccga gctc 347734DNAArtificial
SequencePrimer 27B 77gagctcggaa acaaaggtca ccaggagcga ggtc
347834DNAArtificial SequencePrimer 28A 78gctcctggtg ttcttttctt
ccgagctcgt cggt 347934DNAArtificial SequencePrimer 28B 79accgacgagc
tcggaagaaa agaacaccag gagc 348038DNAArtificial SequencePrimer 29A
80acaacctccc ctaccgaacc actctgcccc atgaaggt 388138DNAArtificial
SequencePrimer 29B 81accttcatgg ggcagagtgg ttcggtaggg gaggttgt
388240DNAArtificial SequencePrimer 30A 82tcccctaccg aaaccctggc
atgcatgaag gtctcgtcat 408340DNAArtificial SequencePrimer 30B
83atgacgagac cttcatgcat gccagggttt cggtagggga 408436DNAArtificial
SequencePrimer 31A 84gtgaagtcca agcgactgtc tctgcccctc accatc
368536DNAArtificial SequencePrimer 31B 85gatggtgagg ggcagagaca
gtcgcttgga cttcac 368640DNAArtificial SequencePrimer 32A
86ccaagcgaca ggctctgtgg cagaccatcg acggaactac 408740DNAArtificial
SequencePrimer 32B 87gtagttccgt cgatggtctg ccacagagcc tgtcgcttgg
408837DNAArtificial SequencePrimer 33A 88aactacctac gacgtcttct
cttgggtgaa cttccac 378937DNAArtificial SequencePrimer 33B
89gtggaagttc acccaagaga agacgtcgta ggtagtt 379037DNAArtificial
SequencePrimer 34A 90agatgctact gacgcctccc tggttatgca ctctcag
379137DNAArtificial SequencePrimer 34B 91ctgagagtgc ataaccaggg
aggcgtcagt agcatct 379238DNAArtificial SequencePrimer 35A
92ctactgacgc cttcatgttc ctgcactctc aggaagcc 389338DNAArtificial
SequencePrimer 35B 93ggcttcctga gagtgcagga acatgaaggc gtcagtag
389439DNAArtificial SequencePrimer 36A 94tgaaatcatt gagaacttca
acggacgaga tgctactga 399539DNAArtificial SequencePrimer 36B
95tcagtagcat ctcgtccgtt gaagttctca atgatttca 399640DNAArtificial
SequencePrimer 37A 96ccttcatggt tatgcacacc aacgaagcct tcgacaagct
409740DNAArtificial SequencePrimer 37B 97agcttgtcga aggcttcgtt
ggtgtgcata accatgaagg 409836DNAArtificial SequencePrimer 38A
98atgcactctc aggaaggcta cgacaagctc aagcga 369936DNAArtificial
SequencePrimer 38B 99tcgcttgagc ttgtcgtagc cttcctgaga gtgcat
3610038DNAArtificial SequencePrimer 39A 100ctcaagcgaa tgcccaagct
tgatccctcc tttgagct 3810138DNAArtificial SequencePrimer 39B
101agctcaaagg agggatcaag cttgggcatt cgcttgag 3810233DNAArtificial
SequencePrimer 40A 102tcaacccctc ctttgatgtg cctccccagg ctg
3310333DNAArtificial SequencePrimer 40B 103cagcctgggg aggcacatca
aaggaggggt tga 3310433DNAArtificial SequencePrimer 41A
104tgcctcccca ggctggactc aacgaagctc agg 3310533DNAArtificial
SequencePrimer 41B 105cctgagcttc gttgagtcca gcctggggag gca
3310636DNAArtificial SequencePrimer 42A 106gaaagctccg
agaagacatc atcgccactg gcatgt 3610736DNAArtificial SequencePrimer
42B 107acatgccagt ggcgatgatg tcttctcgga gctttc 3610835DNAArtificial
SequencePrimer 43A 108ccactggcat gtttgaggtc tctcccctct ggtac
3510935DNAArtificial SequencePrimer 43B 109gtaccagagg ggagagacct
caaacatgcc agtgg 3511036DNAArtificial SequencePrimer 44A
110gtacaagatc tccacctcgg tgggtcttgg cgtgct 3611136DNAArtificial
SequencePrimer 44B 111agcacgccaa gacccaccga ggtggagatc ttgtac
3611236DNAArtificial SequencePrimer 45A 112ggtcttggcg tgcttgcctt
cttcctgatg gtccag 3611336DNAArtificial SequencePrimer 45B
113ctggaccatc aggaagaagg caagcacgcc aagacc 3611436DNAArtificial
SequencePrimer 46A 114gtacttcatt ggtgctctcg tgctcggtat gcacta
3611536DNAArtificial SequencePrimer 46B 115tagtgcatac cgagcacgag
agcaccaatg aagtac 3611634DNAArtificial SequencePrimer 47A
116agggtcacga tcccgagctt gataacctgc ctct 3411734DNAArtificial
SequencePrimer 47B 117agaggcaggt tatcaagctc gggatcgtga ccct
3411836DNAArtificial SequencePrimer 48A 118ccaacagtac tatttcgtcc
tcatctgtat tctcct 3611936DNAArtificial SequencePrimer 48B
119aggagaatac agatgaggac gaaatagtac tgttgg 3612037DNAArtificial
SequencePrimer 49A 120ctcgctcctg gtgttcgttc tttccgagct cgtcggt
3712137DNAArtificial SequencePrimer 49B 121accgacgagc tcggaaagaa
cgaacaccag gagcgag 3712240DNAArtificial SequencePrimer 50A
122gtggcttcgg aattgccgtg atcgtcttca tgaaccacta 4012340DNAArtificial
SequencePrimer 50B 123tagtggttca tgaagacgat cacggcaatt ccgaagccac
4012436DNAArtificial SequencePrimer 51A (for 347I to L and 348T to
S mutations) 124cattcgacga ggcatcctct ctgactggtt ctttgg
3612536DNAArtificial SequencePrimer 51B 125ccaaagaacc agtcagagag
gatgcctcgt cgaatg 3612639DNAArtificial SequencePrimer 51A (for 346I
to V, 347I to L and 348T to S mutations) 126gaacattcga cgaggcgtcc
tctctgactg gttctttgg 3912737DNAArtificial SequencePrimer 52A
127tgccccatga aggtctcatc gtcctgctcc gatacct 3712837DNAArtificial
SequencePrimer 52B 128aggtatcgga gcaggacgat gagaccttca tggggca
3712935DNAArtificial SequencePrimer 53A 129gtccaagcga caggctgttc
ccctcaccat cgacg 3513035DNAArtificial SequencePrimer 53B
130cgtcgatggt gaggggaaca gcctgtcgct tggac 3513136DNAArtificial
SequencePrimer 54A 131gacggaacta cctacgagat ctccgcttgg gtgaac
3613236DNAArtificial SequencePrimer 54B 132gttcacccaa gcggagatct
cgtaggtagt tccgtc 3613336DNAArtificial SequencePrimer 55A
133accctggtgg agctgaactg attgagaact accagg 3613436DNAArtificial
SequencePrimer 55B 134cctggtagtt ctcaatcagt tcagctccac cagggt
3613538DNAArtificial SequencePrimer 56A 135acgagatgct actgacggct
acatggttat gcactctc 3813638DNAArtificial SequencePrimer 56B
136gagagtgcat aaccatgtag ccgtcagtag catctcgt 3813738DNAArtificial
SequencePrimer 57A 137ctcaggaagc cttcgaccga atcaagcgaa tgcccaag
3813838DNAArtificial SequencePrimer 57B 138cttgggcatt cgcttgattc
ggtcgaaggc ttcctgag 3813935DNAArtificial SequencePrimer 58A
139gcgaatgccc aagatccaac cctcctccga gctgc 3514035DNAArtificial
SequencePrimer 58B 140gcagctcgga ggagggttgg atcttgggca ttcgc
3514139DNAArtificial SequencePrimer 59A 141cctccgagct gcctcccaac
ggtgccgtca acgaagctc 3914239DNAArtificial SequencePrimer 59B
142gagcttcgtt gacggcaccg ttgggaggca gctcggagg 3914336DNAArtificial
SequencePrimer 60A 143gatcgccact ggcatgtacg acgcctctcc cctctg
3614436DNAArtificial SequencePrimer 60B 144cagaggggag aggcgtcgta
catgccagtg gcgatc 3614536DNAArtificial SequencePrimer 61A
145ctgtattctc ctgcgaatct tctggtgttt ccagtc 3614636DNAArtificial
SequencePrimer 61B 146gactggaaac accagaagat tcgcaggaga atacag
3614736DNAArtificial SequencePrimer 62A 147gaaagaggcc attggaatgt
ctctgcactg gactct 3614836DNAArtificial SequencePrimer 62B
148agagtccagt gcagagacat tccaatggcc tctttc 3614940DNAArtificial
SequencePrimer 63A 149tgcactggac tctcaagctg accttccacc tcttctttat
4015040DNAArtificial SequencePrimer 63B 150ataaagaaga ggtggaaggt
cagcttgaga gtccagtgca 4015136DNAArtificial SequencePrimer 64A
151cacctgtact tcattgctgg tctcgtgctc ggtatg 3615236DNAArtificial
SequencePrimer 64B 152cataccgagc acgagaccag caatgaagta caggtg
3615337DNAArtificial SequencePrimer 65A 153ttccgagctc gtcggtttcg
gcggaattgc catcgtg 3715437DNAArtificial SequencePrimer 65B
154cacgatggca attccgccga aaccgacgag ctcggaa 3715536DNAArtificial
SequencePrimer 66A 155agttccaaca gtacttctat ctggtcatct gtattc
3615636DNAArtificial SequencePrimer 66B 156gaatacagat gaccagatag
aagtactgtt ggaact 3615736DNAArtificial SequencePrimer 67A
157atgccctcca tcctgagcct gatgttcgtg ttcgtt 3615836DNAArtificial
SequencePrimer 67B 158aacgaacacg aacatcaggc tcaggatgga gggcat
3615934DNAArtificial SequencePrimer 68A 159cgaaactgga ataacgtcct
gggtctggtc tttg 3416034DNAArtificial SequencePrimer 68B
160caaagaccag acccaggacg ttattccagt ttcg 3416133DNAArtificial
SequencePrimer 69A 161tggtctttgg caacgctgtc cagggcttct ccg
3316233DNAArtificial SequencePrimer 69B 162cggagaagcc ctggacagcg
ttgccaaaga cca 3316336DNAArtificial SequencePrimer 70A
163gccgagaagg tgtacggtgc caaggctcag taagcg 3616436DNAArtificial
SequencePrimer 70B 164cgcttactga gccttggcac cgtacacctt ctcggc
361658560DNAArtificial SequencePlasmid pKO2UFkF2 165catggcgtcc
acttcggctc tgcccaagca gaaccctgcg cttagacgca ccgtcacctc 60aactactgtg
acggattctg agtctgccgc cgtctctcct tcagactctc cccgccactc
120ggcctcttcc acatcgctct cgtccatgtc cgaggttgat atcgccaagc
ccaagtccga 180gtatggtgtc atgctcgaca cctacggcaa ccagttcgag
gttcccgact ttaccatcaa 240ggacatctac aatgccatcc ctaagcactg
cttcaagcgc tccgctctca agggatacgg 300ttatatcctc cgcgacattg
tcctcctgac taccactttc agcatctggt acaactttgt 360gacccccgaa
tatatcccct ccacccccgc ccgcgctggt ctgtgggccg tgtacaccgt
420tcttcagggt cttttcggta ctggtctctg ggttattgcc catgagtgcg
gtcacggtgc 480tttctccgat tctcgcatca tcaacgacat tactggctgg
gttcttcact cttccctcct 540tgtcccctac ttcagctggc aaatctccca
ccgaaagcac cacaaggcca ctggcaacat 600ggagcgtgac atggtcttcg
ttccccgaac ccgcgagcag caggctactc gtctcggaaa 660gatgacccac
gagctcgctc atcttactga gnnnntcgtn ggctggccca actacctcat
720caccaatgtt accggccaca actaccacga gcgccagcgt gagggtcgcg
gcaagggcaa 780gcataacggc ctcggcggtg gtgttaacca cttcgatccc
cgcagccctc tgtacgagaa 840cagtgacgct aagctcatcg tcctcagcga
tattggtatc ggtctgatgg ccactgctct 900gtacttcctc gttcagaagt
tcggtttcta caacatggcc atctggtact ttgttcccta 960cctctgggtt
aaccactggc tcgttgccat caccttcctc cagcacaccg accctaccct
1020tccccactac accaacgacg agtggaactt cgtccgtggt gccgctgcta
ccattgaccg 1080tgagatgggc ttcatcggcc gccaccttct ccacggcatc
atcgagactc atgtcctcca 1140ccactacgtc agcagcatcc ccttctacaa
cgcggacgag gccaccgagg ccattaagcc 1200catcatgggc aagcactacc
gggctgatgt ccaggatggt cctcgtggct tcatccgcgc 1260catgtaccgc
agtgcgcgta tgtgccagtg ggttgagccc agcgctggtg ccgagggtgc
1320tggtaagggt gttctgttct tccgcaaccg caacaacgtg ggcacccccc
ccgctgttat 1380caagcccgtt gcttaagtag gcgcggccgc aagtgtggat
ggggaagtga gtgcccggtt 1440ctgtgtgcac aattggcaat ccaagatgga
tggattcaac acagggatat agcgagctac 1500gtggtggtgc gaggatatag
caacggatat ttatgtttga cacttgagaa tgtacgatac 1560aagcactgtc
caagtacaat actaaacata ctgtacatac tcatactcgt acccgggcaa
1620cggtttcact tgagtgcagt ggctagtgct cttactcgta cagtgtgcaa
tactgcgtat 1680catagtcttt gatgtatatc gtattcattc atgttagttg
cgtacgggtg aagcttccac 1740tggtcggcgt ggtagtgggg cagagtgggg
tcggtgtgct gcaggtaggt gatggccacg 1800agccagtggt tgacccacag
gtaggggatc aggtagtaga gggtgacgga agccaggccc 1860catcggttga
tggagtatgc gatgacggac atggtgatac caataccgac gttagagatc
1920cagatgttga accagtcctt cttctcaaac agcggggcgt tggggttgaa
gtggttgaca 1980gcccatttgt tgagcttggg gtacttctgt ccggtaacgt
aagacagcag atacagaggc 2040catccaaaca cctgctgggt gatgaggccg
tagagggtca tgaggggagc gtcctcagca 2100agctcagacc agtcatgggc
gcctcggttc tccataaact cctttcggtc cttgggcaca 2160aacaccatat
cacgggtgag gtgaccagtg gacttgtggt gcatggagtg ggtcagcttc
2220caggcgtagt aagggaccag catggaggag tgcagaaccc atccggtgac
gttgttgacg 2280gtgttagagt cggagaaagc agagtggcca cactcgtggg
caagaaccca cagaccggtg 2340ccaaacagac cctggacaat ggagtacatg
gcccaggcca cagctcggcc ggaagccgag 2400ggaataagag gcaggtacgc
gtaggccatg taggcaaaaa cggcgataaa gaagcaggcg 2460cgccagctgc
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
2520tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
gcgagcggta 2580tcagctcact caaaggcggt aatacggtta tccacagaat
caggggataa cgcaggaaag 2640aacatgtgag caaaaggcca gcaaaaggcc
aggaaccgta aaaaggccgc gttgctggcg 2700tttttccata ggctccgccc
ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2760tggcgaaacc
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
2820cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
cccttcggga 2880agcgtggcgc tttctcatag ctcacgctgt aggtatctca
gttcggtgta ggtcgttcgc 2940tccaagctgg gctgtgtgca cgaacccccc
gttcagcccg accgctgcgc cttatccggt 3000aactatcgtc ttgagtccaa
cccggtaaga cacgacttat cgccactggc agcagccact 3060ggtaacagga
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
3120cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
gaagccagtt 3180accttcggaa aaagagttgg tagctcttga tccggcaaac
aaaccaccgc tggtagcggt 3240ggtttttttg tttgcaagca gcagattacg
cgcagaaaaa aaggatctca agaagatcct 3300ttgatctttt ctacggggtc
tgacgctcag tggaacgaaa actcacgtta agggattttg 3360gtcatgagat
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
3420aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
cttaatcagt 3480gaggcaccta tctcagcgat ctgtctattt cgttcatcca
tagttgcctg actccccgtc 3540gtgtagataa ctacgatacg ggagggctta
ccatctggcc ccagtgctgc aatgataccg 3600cgagacccac gctcaccggc
tccagattta tcagcaataa accagccagc cggaagggcc 3660gagcgcagaa
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
3720gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
cattgctaca 3780ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
tcagctccgg ttcccaacga 3840tcaaggcgag ttacatgatc ccccatgttg
tgcaaaaaag cggttagctc cttcggtcct 3900ccgatcgttg tcagaagtaa
gttggccgca gtgttatcac tcatggttat ggcagcactg 3960cataattctc
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
4020accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
ggcgtcaata 4080cgggataata ccgcgccaca tagcagaact ttaaaagtgc
tcatcattgg aaaacgttct 4140tcggggcgaa aactctcaag gatcttaccg
ctgttgagat ccagttcgat gtaacccact 4200cgtgcaccca actgatcttc
agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4260acaggaaggc
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
4320atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
catgagcgga 4380tacatatttg aatgtattta gaaaaataaa caaatagggg
ttccgcgcac atttccccga 4440aaagtgccac ctgatgcggt gtgaaatacc
gcacagatgc gtaaggagaa aataccgcat 4500caggaaattg taagcgttaa
tattttgtta aaattcgcgt taaatttttg ttaaatcagc 4560tcatttttta
accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
4620gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
gaacgtggac 4680tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
gcccactacg tgaaccatca 4740ccctaatcaa gttttttggg gtcgaggtgc
cgtaaagcac taaatcggaa ccctaaaggg 4800agcccccgat ttagagcttg
acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 4860aaagcgaaag
gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
4920accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca
ttcaggctgc 4980gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
attacgccag ctggcgaaag 5040ggggatgtgc tgcaaggcga ttaagttggg
taacgccagg gttttcccag tcacgacgtt 5100gtaaaacgac ggccagtgaa
ttgtaatacg actcactata gggcgaattg ggcccgacgt 5160cgcatgcttg
aatctacaag taggagggtt ggagtgatta agtgaaactt ctttaacggc
5220tctatgccag ttctattgat atccgaaaca tcagtatgaa ggtctgataa
gggtgacttc 5280ttcccacaga ttcgtatcag tacgagtacg agaccggtac
ttgtaacagt attgatacta 5340aagggaaact acaacggttg tcagcgtaat
gtgacttcgc ccatgaacgc agacacgcag 5400tgccgagtgc ggtgatatcg
cctactcgtt acgtccatgg actacacaac ccctcggctt 5460cgcttggctt
agcctcgggc tcggtgctgt tcagttaaaa cacaatcaaa taacatttct
5520actttttaga aggcaggccg tcaggagcaa ctccgactcc attgacgttt
ctaaacatct 5580gaatgccttc cttaccttca acaaactggc aggttcgggc
gacagtgtaa agagacttga 5640tgaagttggt gtcgtcgtgt cggtagtgct
tgcccatgac cttcttgatc ttctcagtgg 5700cgattcgggc gttgtagaag
ggaattccgt cgtcgcctga gtcgacgagt atctgtctga 5760ctcgtcattg
ccgcctttgg agtacgactc caactatgag tgtgcttgga tcactttgac
5820gatacattct tcgttggagg ctgtgggtct gacagctgcg ttttcggcgc
ggttggccga 5880caacaatatc agctgcaacg tcattgctgg ctttcatcat
gatcacattt ttgtcggcaa 5940aggcgacgcc cagagagcca ttgacgttct
ttctaatttg gaccgatagc cgtatagtcc 6000agtctatcta taagttcaac
taactcgtaa ctattaccat aacatatact tcactgcccc 6060agataaggtt
ccgataaaaa gttctgcaga ctaaatttat ttcagtctcc tcttcaccac
6120caaaatgccc tcctacgaag ctcgagctaa cgtccacaag tccgcctttg
ccgctcgagt 6180gctcaagctc gtggcagcca agaaaaccaa cctgtgtgct
tctctggatg ttaccaccac 6240caaggagctc attgagcttg ccgataaggt
cggaccttat gtgtgcatga tcaaaaccca 6300tatcgacatc attgacgact
tcacctacgc cggcactgtg ctccccctca aggaacttgc 6360tcttaagcac
ggtttcttcc tgttcgagga cagaaagttc gcagatattg gcaacactgt
6420caagcaccag taccggtgtc accgaatcgc cgagtggtcc gatatcacca
acgcccacgg 6480tgtacccgga accggaatca ttgctggcct gcgagctggt
gccgaggaaa ctgtctctga 6540acagaagaag gaggacgtct ctgactacga
gaactcccag tacaaggagt tcctagtccc 6600ctctcccaac gagaagctgg
ccagaggtct gctcatgctg gccgagctgt cttgcaaggg 6660ctctctggcc
actggcgagt actccaagca gaccattgag cttgcccgat ccgaccccga
6720gtttgtggtt ggcttcattg cccagaaccg acctaagggc gactctgagg
actggcttat 6780tctgaccccc ggggtgggtc ttgacgacaa gggagacgct
ctcggacagc agtaccgaac 6840tgttgaggat gtcatgtcta ccggaacgga
tatcataatt gtcggccgag gtctgtacgg 6900ccagaaccga gatcctattg
aggaggccaa gcgataccag aaggctggct gggaggctta 6960ccagaagatt
aactgttaga ggttagacta tggatatgta atttaactgt gtatatagag
7020agcgtgcaag tatggagcgc ttgttcagct tgtatgatgg tcagacgacc
tgtctgatcg 7080agtatgtatg atactgcaca acctgtgtat ccgcatgatc
tgtccaatgg ggcatgttgt 7140tgtgtttctc gatacggaga tgctgggtac
agtgctaata cgttgaacta cttatactta 7200tatgaggctc gaagaaagct
gacttgtgta tgacttaatt aatttgaatc gaatcgatga 7260gcctaaaatg
aacccgagta tatctcataa aattctcggt gagaggtctg tgactgtcag
7320tacaaggtgc cttcattatg ccctcaacct taccatacct cactgaatgt
agtgtacctc 7380taaaaatgaa atacagtgcc aaaagccaag gcactgagct
cgtctaacgg acttgatata 7440caaccaatta aaacaaatga aaagaaatac
agttctttgt atcatttgta acaattaccc 7500tgtacaaact aaggtattga
aatcccacaa tattcccaaa gtccacccct ttccaaattg 7560tcatgcctac
aactcatata ccaagcacta acctaccgtt taaacagtgt acgcagatct
7620actatagagg aacatttaaa ttgccccgga gaagacggcc aggccgccta
gatgacaaat 7680tcaacaactc acagctgact ttctgccatt gccactaggg
gggggccttt ttatatggcc 7740aagccaagct ctccacgtcg gttgggctgc
acccaacaat aaatgggtag ggttgcacca 7800acaaagggat gggatggggg
gtagaagata cgaggataac ggggctcaat ggcacaaata 7860agaacgaata
ctgccattaa gactcgtgat ccagcgactg acaccattgc atcatctaag
7920ggcctcaaaa ctacctcgga actgctgcgc tgatctggac accacagagg
ttccgagcac 7980tttaggttgc accaaatgtc ccaccaggtg caggcagaaa
acgctggaac agcgtgtaca 8040gtttgtctta acaaaaagtg agggcgctga
ggtcgagcag ggtggtgtga cttgttatag 8100cctttagagc tgcgaaagcg
cgtatggatt tggctcatca ggccagattg agggtctgtg 8160gacacatgtc
atgttagtgt acttcaatcg ccccctggat atagccccga caataggccg
8220tggcctcatt tttttgcctt ccgcacattt ccattgctcg atacccacac
cttgcttctc 8280ctgcacttgc caaccttaat actggtttac attgaccaac
atcttacaag cggggggctt 8340gtctagggta tatataaaca gtggctctcc
caatcggttg ccagtctctt ttttcctttc 8400tttccccaca gattcgaaat
ctaaactaca catcacagaa ttccgagccg tgagtatcca 8460cgacaagatc
agtgtcgaga cgacgcgttt tgtgtaatga cacaatccga aagtcgctag
8520caacacacac tctctacaca aactaaccca gctctggtac
85601665PRTArtificial SequenceHis-rich motif 166His Xaa Xaa Xaa
His1 51676PRTArtificial SequenceHis-rich motif 167His Xaa Xaa Xaa
Xaa His1 51685PRTArtificial SequenceHis-rich motif 168His Xaa Xaa
His His1 51696PRTArtificial SequenceHis-rich motif 169His Xaa Xaa
Xaa His His1 51705PRTArtificial SequenceHis-rich motif 170Xaa Xaa
Xaa His His1 51716PRTArtificial SequenceHis-rich motif 171Xaa Xaa
Xaa Xaa His His1 51721064DNAIsochrysis galbana (GenBank Accession
No. AF390174)CDS(2)..(793)delta-9 elongase 172g atg gcc ctc gca
aac gac gcg gga gag cgc atc tgg gcg gct gtg acc 49Met Ala Leu Ala
Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr1 5 10 15gac ccg gaa
atc ctc att ggc acc ttc tcg tac ttg cta ctc aaa ccg 97Asp Pro Glu
Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro 20 25 30ctg ctc
cgc aat tcc ggg ctg gtg gat gag aag aag ggc gca tac agg 145Leu Leu
Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg 35 40 45acg
tcc atg atc tgg tac aac gtt ctg ctg gcg ctc ttc tct gcg ctg 193Thr
Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu 50 55
60agc ttc tac gtg acg gcg acc gcc ctc ggc tgg gac tat ggt acg ggc
241Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr
Gly65 70 75 80gcg tgg ctg cgc agg caa acc ggc gac aca ccg cag ccg
ctc ttc cag 289Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro
Leu Phe Gln 85 90 95tgc ccg tcc ccg gtt tgg gac tcg aag ctc ttc aca
tgg acc gcc aag 337Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr
Trp Thr Ala Lys 100 105 110gca ttc tat tac tcc aag tac gtg gag tac
ctc gac acg gcc tgg ctg 385Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr
Leu Asp Thr Ala Trp Leu 115 120 125gtg ctc aag ggc aag agg gtc tcc
ttt ctc cag gcc ttc cac cac ttt 433Val Leu Lys Gly Lys Arg Val Ser
Phe Leu Gln Ala Phe His His Phe 130 135 140ggc gcg ccg tgg gat gtg
tac ctc ggc att cgg ctg cac aac gag ggc 481Gly Ala Pro Trp Asp Val
Tyr Leu Gly Ile Arg Leu His Asn Glu Gly145 150 155 160gta tgg atc
ttc atg ttt ttc aac tcg ttc att cac acc atc atg tac 529Val Trp Ile
Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr 165 170 175acc
tac tac ggc ctc acc gcc gcc ggg tat aag ttc aag gcc aag ccg 577Thr
Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro 180 185
190ctc atc acc gcg atg cag atc tgc cag ttc gtg ggc ggc ttc ctg ttg
625Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu
195 200 205gtc tgg gac tac atc aac gtc ccc tgc ttc aac tcg gac aaa
ggg aag 673Val Trp Asp Tyr Ile Asn Val Pro Cys Phe Asn Ser Asp Lys
Gly Lys 210 215 220ttg ttc agc tgg gct ttc aac tat gca tac gtc ggc
tcg gtc ttc ttg 721Leu Phe Ser Trp Ala Phe Asn Tyr Ala Tyr Val Gly
Ser Val Phe Leu225 230 235 240ctc ttc tgc cac ttt ttc tac cag gac
aac ttg gca acg aag aaa tcg 769Leu Phe Cys His Phe Phe Tyr Gln Asp
Asn Leu Ala Thr Lys Lys Ser 245 250 255 gcc aag gcg ggc aag cag ctc
tag gcctcgagcc ggctcgcggg ttcaaggagg 823Ala Lys Ala Gly Lys Gln Leu
260gcgacacggg ggtgggacgt ttgcatggag atggattgtg gatgtcctta
cgccttactc 883atcaatgtcc tcccatctct cccctctaga ccttctacta
gccatctaga agggcagctc 943agagacggat accgttcccc ctccccttcc
ttttcgtctt tgctttgcca ttgtttgttt 1003gtctctattt tttaaactat
tgacgctaac gcgttacgct cgcaaaaaaa aaaaaaaaaa 1063a
1064173263PRTIsochrysis galbana (GenBank Accession No. AF390174)
173Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr1
5 10 15Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys
Pro 20 25 30Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala
Tyr Arg 35 40 45Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe
Ser Ala Leu 50 55 60Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp
Tyr Gly Thr Gly65 70 75 80Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr
Pro Gln Pro Leu Phe Gln 85 90 95Cys Pro Ser Pro Val Trp Asp Ser Lys
Leu Phe Thr Trp Thr Ala Lys 100 105 110Ala Phe Tyr Tyr Ser Lys Tyr
Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125Val Leu Lys Gly Lys
Arg Val Ser Phe Leu Gln Ala Phe His His Phe 130 135 140Gly Ala Pro
Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly145 150 155
160Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr
165 170 175Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys Ala
Lys Pro 180 185 190Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly
Gly Phe Leu Leu 195 200 205Val Trp Asp Tyr Ile Asn Val Pro Cys Phe
Asn Ser Asp Lys Gly Lys 210 215 220Leu Phe Ser Trp Ala Phe Asn Tyr
Ala Tyr Val Gly Ser Val Phe Leu225 230 235 240Leu Phe Cys His Phe
Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser 245 250 255Ala Lys Ala
Gly Lys Gln Leu 260174792DNAArtificial SequenceIgD9e synthetic
delta-9 elongase (codon- optimized for Yarrowia lipolytica)
174atggctctgg ccaacgacgc tggcgagcga atctgggctg ccgtcaccga
tcccgaaatc 60ctcattggca ccttctccta cctgctcctg aagcctctcc tgcgaaactc
tggtctcgtg 120gacgagaaga aaggagccta ccgaacctcc atgatctggt
acaacgtcct cctggctctc 180ttctctgccc tgtccttcta cgtgactgcc
accgctctcg gctgggacta cggtactgga 240gcctggctgc gaagacagac
cggtgatact ccccagcctc tctttcagtg tccctctcct 300gtctgggact
ccaagctgtt cacctggact gccaaggcct tctactattc taagtacgtg
360gagtacctcg acaccgcttg gctggtcctc aagggcaagc gagtgtcctt
tctgcaggcc 420ttccatcact ttggagctcc ctgggacgtc tacctcggca
ttcgactgca caacgagggt 480gtgtggatct tcatgttctt taactcgttc
attcacacca tcatgtacac ctactatgga 540ctgactgccg ctggctacaa
gttcaaggcc aagcctctga tcactgccat gcagatttgc 600cagttcgtcg
gtggctttct cctggtctgg gactacatca acgttccctg cttcaactct
660gacaagggca agctgttctc ctgggctttc aactacgcct acgtcggatc
tgtctttctc 720ctgttctgtc acttctttta ccaggacaac ctggccacca
agaaatccgc taaggctggt 780aagcagcttt ag 792175777DNAEuglena
gracilismisc_feature(1)..(777)delta-9 elongase; U.S. Patent
Application No. 60/739989, filed 11/23/2005 175atggaggtgg
tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60gcccaactct
ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc
120atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat
gaagtttgtt 180ttcaccaatt acaaccttct catgtccatt tattcgttgg
gatcattcct ctcaatggca 240tatgccatgt acaccatcgg tgttatgtct
gacaactgcg agaaggcttt tgacaacaac 300gtcttcagga tcaccacgca
gttgttctat ttgagcaagt tcctggagta tattgactcc 360ttctatttgc
cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg
420gcaccgatgg atatgtggct gttctataat taccgaaatg aagctgtttg
gatttttgtg 480ctgttgaatg gtttcatcca ctggatcatg tacggttatt
attggaccag attgatcaag 540ctgaagttcc ccatgccaaa atccctgatt
acatcaatgc agatcattca attcaatgtt 600ggtttctaca ttgtctggaa
gtacaggaac attccctgtt atcgccaaga tgggatgagg 660atgtttggct
ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat
720ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcagtga
777176258PRTEuglena gracilisMISC_FEATURE(1)..(258)delta-9 elongase;
U.S. Patent Application No. 60/739989, filed 11/23/2005 176Met Glu
Val Val Asn Glu Ile Val Ser Ile Gly Gln Glu Val Leu Pro1 5 10 15Lys
Val Asp Tyr Ala Gln Leu Trp Ser Asp Ala Ser His Cys Glu Val 20 25
30Leu Tyr Leu Ser Ile Ala Phe Val Ile Leu Lys Phe Thr Leu Gly Pro
35 40 45Leu Gly Pro Lys Gly Gln Ser Arg Met Lys Phe Val Phe Thr Asn
Tyr 50 55 60Asn Leu Leu Met Ser Ile Tyr Ser Leu Gly Ser Phe Leu Ser
Met Ala65 70 75 80Tyr Ala Met Tyr Thr Ile Gly Val Met Ser Asp Asn
Cys Glu Lys Ala 85 90 95Phe Asp Asn Asn Val Phe Arg Ile Thr Thr Gln
Leu Phe Tyr Leu Ser 100 105 110Lys Phe Leu Glu Tyr Ile Asp Ser Phe
Tyr Leu Pro Leu Met Gly Lys 115 120 125Pro Leu Thr Trp Leu Gln Phe
Phe His His Leu Gly Ala Pro Met Asp 130 135 140Met Trp Leu Phe Tyr
Asn Tyr Arg Asn Glu Ala Val Trp Ile Phe Val145 150 155 160Leu Leu
Asn Gly Phe Ile His Trp Ile Met Tyr Gly Tyr Tyr Trp Thr 165 170
175Arg Leu Ile Lys Leu Lys Phe Pro Met Pro Lys Ser Leu Ile Thr Ser
180 185 190Met Gln Ile Ile Gln Phe Asn Val Gly Phe Tyr Ile Val Trp
Lys Tyr 195 200 205Arg Asn Ile Pro Cys Tyr Arg Gln Asp Gly Met Arg
Met Phe Gly Trp 210 215 220Phe Phe Asn Tyr Phe Tyr Val Gly Thr Val
Leu Cys Leu Phe Leu Asn225 230 235 240Phe Tyr Val Gln Thr Tyr Ile
Val Arg Lys His Lys Gly Ala Lys Lys 245 250 255Ile Gln
177777DNAEuglena gracilismisc_feature(1)..(777)synthetic delta-9
elongase codon-optimized for Yarrowia lipolytica; U.S. Patent
Application No. 60/739989, filed 11/23/2005 177atggaggtcg
tgaacgaaat cgtctccatt ggccaggagg ttcttcccaa ggtcgactat 60gctcagctct
ggtctgatgc ctcgcactgc gaggtgctgt acctctccat cgccttcgtc
120atcctgaagt tcacccttgg tcctctcgga cccaagggtc agtctcgaat
gaagtttgtg 180ttcaccaact acaacctgct catgtccatc tactcgctgg
gctccttcct ctctatggcc 240tacgccatgt acaccattgg tgtcatgtcc
gacaactgcg agaaggcttt cgacaacaat 300gtcttccgaa tcaccactca
gctgttctac ctcagcaagt tcctcgagta cattgactcc 360ttctatctgc
ccctcatggg caagcctctg acctggttgc agttctttca ccatctcgga
420gctcctatgg acatgtggct gttctacaac taccgaaacg aagccgtttg
gatctttgtg 480ctgctcaacg gcttcattca ctggatcatg tacggctact
attggacccg actgatcaag 540ctcaagttcc ctatgcccaa gtccctgatt
acttctatgc agatcattca gttcaacgtt 600ggcttctaca tcgtctggaa
gtaccggaac attccctgct accgacaaga tggaatgaga 660atgtttggct
ggtttttcaa ctacttctac gttggtactg tcctgtgtct gttcctcaac
720ttctacgtgc agacctacat cgtccgaaag cacaagggag ccaaaaagat tcagtga
777178120PRTSaccharomyces cerevisiae (GenBank Accession No. P40312)
178Met Pro Lys Val Tyr Ser Tyr Gln Glu Val Ala Glu His Asn Gly Pro1
5 10 15Glu Asn Phe Trp Ile Ile Ile Asp Asp Lys Val Tyr Asp Val Ser
Gln 20 25 30Phe Lys Asp Glu His Pro Gly Gly Asp Glu Ile Ile Met Asp
Leu Gly 35 40 45Gly Gln Asp Ala Thr Glu Ser Phe Val Asp Ile Gly His
Ser Asp Glu 50 55 60Ala Leu Arg Leu Leu Lys Gly Leu Tyr Ile Gly Asp
Val Asp Lys Thr65 70 75 80Ser Glu Arg Val Ser Val Glu Lys Val Ser
Thr Ser Glu Asn Gln Ser 85 90 95Lys Gly Ser Gly Thr Leu Val Val Ile
Leu Ala Ile Leu Met Leu Gly 100 105 110Val Ala Tyr Tyr Leu Leu Asn
Glu 115 120179124PRTSchizosaccharomyces pombe (GenBank Accession
No. O94391) 179Met Ser Val Lys Tyr Phe Glu Pro Glu Glu Ile Val Glu
His Asn Asn1 5 10 15Ser Lys Asp Met Tyr Met Val Ile Asn Gly Lys Val
Tyr Asp Val Ser 20 25 30Asn Phe Ala Asp Asp His Pro Gly Gly Leu Asp
Ile Met Leu Asp Tyr 35 40 45Ala Gly Gln Asp Ala Thr Lys Ala Tyr Gln
Asp Ile Gly His Ser Ile 50 55 60Ala Ala Asp Glu Leu Leu Glu Glu Met
Tyr Ile Gly Asp Leu Lys Pro65 70 75 80Gly Thr Glu Glu Arg Leu Lys
Glu Leu Lys Lys Pro Arg Ser Phe Asp 85 90 95Asn Asp Thr Pro Pro Leu
Pro Leu Leu Ile Ala Leu Ile Val Leu Pro 100 105 110Ala Ile Ala Val
Ile Val Phe Val Lys Leu Asn Lys 115 1201808739DNAArtificial
SequencePlasmid pY116 180ggccgccacc gcggcccgag attccggcct
cttcggccgc caagcgaccc gggtggacgt 60ctagaggtac ctagcaatta acagatagtt
tgccggtgat aattctctta acctcccaca 120ctcctttgac ataacgattt
atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180gtggagctcc
agcttttgtt ccctttagtg agggtttaaa cgagcttggc gtaatcatgg
240tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
cgtacgagcc 300ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
gctaactcac attaattgcg 360ttgcgctcac tgcccgcttt ccagtcggga
aacctgtcgt gccagctgca ttaatgaatc 420ggccaacgcg cggggagagg
cggtttgcgt attgggcgct cttccgcttc ctcgctcact 480gactcgctgc
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
540atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
aaaaggccag 600caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
ttttccatag gctccgcccc 660cctgacgagc atcacaaaaa tcgacgctca
agtcagaggt ggcgaaaccc gacaggacta 720taaagatacc aggcgtttcc
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 780ccgcttaccg
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
840tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
ctgtgtgcac 900gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
actatcgtct tgagtccaac 960ccggtaagac acgacttatc gccactggca
gcagccactg gtaacaggat tagcagagcg 1020aggtatgtag gcggtgctac
agagttcttg aagtggtggc ctaactacgg ctacactaga 1080aggacagtat
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
1140agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
ttgcaagcag 1200cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
tgatcttttc tacggggtct 1260gacgctcagt ggaacgaaaa ctcacgttaa
gggattttgg tcatgagatt atcaaaaagg 1320atcttcacct agatcctttt
aaattaaaaa tgaagtttta aatcaatcta aagtatatat 1380gagtaaactt
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
1440tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
tacgatacgg 1500gagggcttac catctggccc cagtgctgca atgataccgc
gagacccacg ctcaccggct 1560ccagatttat cagcaataaa ccagccagcc
ggaagggccg agcgcagaag tggtcctgca 1620actttatccg cctccatcca
gtctattaat tgttgccggg aagctagagt aagtagttcg 1680ccagttaata
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
1740tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
tacatgatcc 1800cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
cgatcgttgt cagaagtaag 1860ttggccgcag tgttatcact catggttatg
gcagcactgc ataattctct tactgtcatg 1920ccatccgtaa gatgcttttc
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 1980tgtatgcggc
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
2040agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
actctcaagg 2100atcttaccgc tgttgagatc cagttcgatg taacccactc
gtgcacccaa ctgatcttca 2160gcatctttta ctttcaccag cgtttctggg
tgagcaaaaa caggaaggca aaatgccgca 2220aaaaagggaa taagggcgac
acggaaatgt tgaatactca tactcttcct ttttcaatat 2280tattgaagca
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
2340aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
tgacgcgccc 2400tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
gcagcgtgac cgctacactt 2460gccagcgccc tagcgcccgc tcctttcgct
ttcttccctt cctttctcgc cacgttcgcc 2520ggctttcccc gtcaagctct
aaatcggggg ctccctttag ggttccgatt tagtgcttta 2580cggcacctcg
accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
2640tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
tggactcttg 2700ttccaaactg gaacaacact caaccctatc tcggtctatt
cttttgattt ataagggatt 2760ttgccgattt cggcctattg gttaaaaaat
gagctgattt aacaaaaatt taacgcgaat 2820tttaacaaaa tattaacgct
tacaatttcc attcgccatt caggctgcgc aactgttggg 2880aagggcgatc
ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
2940caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
aaaacgacgg 3000ccagtgaatt gtaatacgac tcactatagg gcgaattggg
taccgggccc cccctcgagg 3060tcgatggtgt cgataagctt gatatcgaat
tcatgtcaca caaaccgatc ttcgcctcaa 3120ggaaacctaa ttctacatcc
gagagactgc cgagatccag tctacactga ttaattttcg 3180ggccaataat
ttaaaaaaat cgtgttatat aatattatat gtattatata tatacatcat
3240gatgatactg acagtcatgt cccattgcta aatagacaga ctccatctgc
cgcctccaac 3300tgatgttctc aatatttaag gggtcatctc gcattgttta
ataataaaca gactccatct 3360accgcctcca aatgatgttc tcaaaatata
ttgtatgaac ttatttttat tacttagtat 3420tattagacaa cttacttgct
ttatgaaaaa cacttcctat ttaggaaaca atttataatg 3480gcagttcgtt
catttaacaa tttatgtaga ataaatgtta taaatgcgta tgggaaatct
3540taaatatgga tagcataaat gatatctgca ttgcctaatt cgaaatcaac
agcaacgaaa 3600aaaatccctt gtacaacata aatagtcatc gagaaatatc
aactatcaaa gaacagctat 3660tcacacgtta ctattgagat tattattgga
cgagaatcac acactcaact gtctttctct 3720cttctagaaa tacaggtaca
agtatgtact attctcattg ttcatacttc tagtcatttc 3780atcccacata
ttccttggat ttctctccaa tgaatgacat tctatcttgc aaattcaaca
3840attataataa gatataccaa agtagcggta tagtggcaat caaaaagctt
ctctggtgtg 3900cttctcgtat ttatttttat tctaatgatc cattaaaggt
atatatttat ttcttgttat 3960ataatccttt tgtttattac atgggctgga
tacataaagg tattttgatt taattttttg 4020cttaaattca atcccccctc
gttcagtgtc aactgtaatg gtaggaaatt accatacttt 4080tgaagaagca
aaaaaaatga aagaaaaaaa aaatcgtatt tccaggttag acgttccgca
4140gaatctagaa tgcggtatgc ggtacattgt tcttcgaacg taaaagttgc
gctccctgag 4200atattgtaca tttttgcttt tacaagtaca agtacatcgt
acaactatgt actactgttg 4260atgcatccac aacagtttgt tttgtttttt
tttgtttttt ttttttctaa tgattcatta 4320ccgctatgta tacctacttg
tacttgtagt aagccgggtt attggcgttc aattaatcat 4380agacttatga
atctgcacgg tgtgcgctgc gagttacttt tagcttatgc atgctacttg
4440ggtgtaatat tgggatctgt
tcggaaatca acggatgctc aaccgatttc gacagtaatt 4500aattaatttg
aatcgaatcg gagcctaaaa tgaacccgag tatatctcat aaaattctcg
4560gtgagaggtc tgtgactgtc agtacaaggt gccttcatta tgccctcaac
cttaccatac 4620ctcactgaat gtagtgtacc tctaaaaatg aaatacagtg
ccaaaagcca aggcactgag 4680ctcgtctaac ggacttgata tacaaccaat
taaaacaaat gaaaagaaat acagttcttt 4740gtatcatttg taacaattac
cctgtacaaa ctaaggtatt gaaatcccac aatattccca 4800aagtccaccc
ctttccaaat tgtcatgcct acaactcata taccaagcac taacctacca
4860aacaccacta aaaccccaca aaatatatct taccgaatat acagtaacaa
gctaccacca 4920cactcgttgg gtgcagtcgc cagcttaaag atatctatcc
acatcagcca caactccctt 4980cctttaataa accgactaca cccttggcta
ttgaggttat gagtgaatat actgtagaca 5040agacactttc aagaagactg
tttccaaaac gtaccactgt cctccactac aaacacaccc 5100aatctgcttc
ttctagtcaa ggttgctaca ccggtaaatt ataaatcatc atttcattag
5160cagggcaggg ccctttttat agagtcttat acactagcgg accctgccgg
tagaccaacc 5220cgcaggcgcg tcagtttgct ccttccatca atgcgtcgta
gaaacgactt actccttctt 5280gagcagctcc ttgaccttgt tggcaacaag
tctccgacct cggaggtgga ggaagagcct 5340ccgatatcgg cggtagtgat
accagcctcg acggactcct tgacggcagc ctcaacagcg 5400tcaccggcgg
gcttcatgtt aagagagaac ttgagcatca tggcggcaga cagaatggtg
5460gcaatggggt tgaccttctg cttgccgaga tcgggggcag atccgtgaca
gggctcgtac 5520agaccgaacg cctcgttggt gtcgggcaga gaagccagag
aggcggaggg cagcagaccc 5580agagaaccgg ggatgacgga ggcctcgtcg
gagatgatat cgccaaacat gttggtggtg 5640atgatgatac cattcatctt
ggagggctgc ttgatgagga tcatggcggc cgagtcgatc 5700agctggtggt
tgagctcgag ctgggggaat tcgtccttga ggactcgagt gacagtcttt
5760cgccaaagtc gagaggaggc cagcacgttg gccttgtcaa gagaccacac
gggaagaggg 5820gggttgtgct gaagggccag gaaggcggcc attcgggcaa
ttcgctcaac ctcaggaacg 5880gagtaggtct cggtgtcgga agcgacgcca
gatccgtcat cctcctttcg ctctccaaag 5940tagatacctc cgacgagctc
tcggacaatg atgaagtcgg tgccctcaac gtttcggatg 6000ggggagagat
cggcgagctt gggcgacagc agctggcagg gtcgcaggtt ggcgtacagg
6060ttcaggtcct ttcgcagctt gaggagaccc tgctcgggtc gcacgtcggt
tcgtccgtcg 6120ggagtggtcc atacggtgtt ggcagcgcct ccgacagcac
cgagcataat agagtcagcc 6180tttcggcaga tgtcgagagt agcgtcggtg
atgggctcgc cctccttctc aatggcagct 6240cctccaatga gtcggtcctc
aaacacaaac tcggtgccgg aggcctcagc aacagacttg 6300agcaccttga
cggcctcggc aatcacctcg gggccacaga agtcgccgcc gagaagaaca
6360atcttcttgg agtcagtctt ggtcttctta gtttcgggtt ccattgtgga
tgtgtgtggt 6420tgtatgtgtg atgtggtgtg tggagtgaaa atctgtggct
ggcaaacgct cttgtatata 6480tacgcacttt tgcccgtgct atgtggaaga
ctaaacctcc gaagattgtg actcaggtag 6540tgcggtatcg gctagggacc
caaaccttgt cgatgccgat agcgctatcg aacgtacccc 6600agccggccgg
gagtatgtcg gaggggacat acgagatcgt caagggtttg tggccaactg
6660gtatttaaat gtagctaacg gtagcaggcg aactactggt acatacctcc
cccggaatat 6720gtacaggcat aatgcgtatc tgtgggacat gtggtcgttg
cgccattatg taagcagcgt 6780gtactcctct gactgtccat atggtttgct
ccatctcacc ctcatcgttt tcattgttca 6840caggcggcca caaaaaaact
gtcttctctc cttctctctt cgccttagtc tactcggacc 6900agttttagtt
tagcttggcg ccactggata aatgagacct caggccttgt gatgaggagg
6960tcacttatga agcatgttag gaggtgcttg tatggataga gaagcaccca
aaataataag 7020aataataata aaacaggggg cgttgtcatt tcatatcgtg
ttttcaccat caatacacct 7080ccaaacaatg cccttcatgt ggccagcccc
aatattgtcc tgtagttcaa ctctatgcag 7140ctcgtatctt attgagcaag
taaaactctg tcagccgata ttgcccgacc cgcgacaagg 7200gtcaacaagg
tggtgtaagg ccttcgcaga agtcaaaact gtgccaaaca aacatctaga
7260gtctctttgg tgtttctcgc atatatttwa tcggctgtct tacgtatttg
cgcctcggta 7320ccggactaat ttcggatcat ccccaatacg ctttttcttc
gcagctgtca acagtgtcca 7380tgatctatcc acctaaatgg gtcatatgag
gcgtataatt tcgtggtgct gataataatt 7440cccatatatt tgacacaaaa
cttccccccc tagacataca tctcacaatc tcacttcttg 7500tgcttctgtc
acacatctcc tccagctgac ttcaactcac acctctgccc cagttggtct
7560acagcggtat aaggtttctc cgcatagagg tgcaccactc ctcccgatac
ttgtttgtgt 7620gacttgtggg tcacgacata tatatctaca cacattgcgc
caccctttgg ttcttccagc 7680acaacaaaaa cacgacacgc taaccatggc
caatttactg accgtacacc aaaatttgcc 7740tgcattaccg gtcgatgcaa
cgagtgatga ggttcgcaag aacctgatgg acatgttcag 7800ggatcgccag
gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg
7860ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg
aagatgttcg 7920cgattatctt ctatatcttc aggcgcgcgg tctggcagta
aaaactatcc agcaacattt 7980gggccagcta aacatgcttc atcgtcggtc
cgggctgcca cgaccaagtg acagcaatgc 8040tgtttcactg gttatgcggc
ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa 8100acaggctcta
gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag
8160cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata
acaccctgtt 8220acgtatagcc gaaattgcca ggatcagggt taaagatatc
tcacgtactg acggtgggag 8280aatgttaatc catattggca gaacgaaaac
gctggttagc accgcaggtg tagagaaggc 8340acttagcctg ggggtaacta
aactggtcga gcgatggatt tccgtctctg gtgtagctga 8400tgatccgaat
aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc
8460caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc
atcgattgat 8520ttacggcgct aaggatgact ctggtcagag atacctggcc
tggtctggac acagtgcccg 8580tgtcggagcc gcgcgagata tggcccgcgc
tggagtttca ataccggaga tcatgcaagc 8640tggtggctgg accaatgtaa
atattgtcat gaactatatc cgtaacctgg atagtgaaac 8700aggggcaatg
gtgcgcctgc tggaagatgg cgattaagc 873918115304DNAArtificial
SequencePlasmid pKO2UF8289 181cgatcgagga agaggacaag cggctgcttc
ttaagtttgt gacatcagta tccaaggcac 60cattgcaagg attcaaggct ttgaacccgt
catttgccat tcgtaacgct ggtagacagg 120ttgatcggtt ccctacggcc
tccacctgtg tcaatcttct caagctgcct gactatcagg 180acattgatca
acttcggaag aaacttttgt atgccattcg atcacatgct ggtttcgatt
240tgtcttagag gaacgcatat acagtaatca tagagaataa acgatattca
tttattaaag 300tagatagttg aggtagaagt tgtaaagagt gataaatagc
ggccgctcac tgaatctttt 360tggctccctt gtgcttcctg acgatatacg
tttgcacata gaaattcaag aacaaacaca 420agactgtgcc aacataaaag
taattgaaga accagccaaa catcctcatc ccatcttggc 480gataacaggg
aatgttcctg tacttccaga caatgtagaa accaacattg aattgaatga
540tctgcattga tgtaatcagg gattttggca tggggaactt cagcttgatc
aatctggtcc 600aataataacc gtacatgatc cagtggatga aaccattcaa
cagcacaaaa atccaaacag 660cttcatttcg gtaattatag aacagccaca
tatccatcgg tgcccccaaa tgatggaaga 720attgcaacca ggtcagaggc
ttgcccatca gtggcaaata gaaggagtca atatactcca 780ggaacttgct
caaatagaac aactgcgtgg tgatcctgaa gacgttgttg tcaaaagcct
840tctcgcagtt gtcagacata acaccgatgg tgtacatggc atatgccatt
gagaggaatg 900atcccaacga ataaatggac atgagaaggt tgtaattggt
gaaaacaaac ttcatacgag 960actgaccttt tggaccaagg gggccaagag
tgaacttcaa gatgacaaat gcgatggaca 1020agtaaagcac ctcacagtga
ctggcatcac tccagagttg ggcataatca actggttggg 1080taaaacttcc
tgcccaattg agactatttc attcaccacc tccatggtta gcgtgtcgtg
1140tttttgttgt gctggaagaa ccaaagggtg gcgcaatgtg tgtagatata
tatgtcgtga 1200cccacaagtc acacaaacaa gtatcgggag gagtggtgca
cctctatgcg gagaaacctt 1260ataccgctgt agaccaactg gggcagaggt
gtgagttgaa gtcagctgga ggagatgtgt 1320gacagaagca caagaagtga
gattgtgaga tgtatgtcta gggggggaag ttttgtgtca 1380aatatatggg
aattattatc agcaccacga aattatacgc ctcatatgac ccatttaggt
1440ggatagatca tggacactgt tgacagctgc gaagaaaaag cgtattgggg
atgatccgaa 1500attagtccgg taccgaggcg caaatacgta agacagccga
twaaatatat gcgagaaaca 1560ccaaagagac tctagatgtt tgtttggcac
agttttgact tctgcgaagg ccttacacca 1620ccttgttgac ccttgtcgcg
ggtcgggcaa tatcggctga cagagtttta cttgctcaat 1680aagatacgag
ctgcatagag ttgaactaca ggacaatatt ggggctggcc acatgaaggg
1740cattgtttgg aggtgtattg atggtgaaaa cacgatatga aatgacaacg
ccccctgttt 1800tattattatt cttattattt tgggtgcttc tctatccata
caagcacctc ctaacatgct 1860tcataagtga cctcctcatc acaaggcctg
aggtctcatt tatccagtgg cgccaagcta 1920aactaaaact ggtccgagta
gactaaggcg aagagagaag gagagaagac agtttttttg 1980tggccgcctg
tgaacaatga aaacgatgag ggtgagatgg agcaaaccat atggtttaaa
2040cagtcagagg agtacacgct gcttacataa tggcgcaacg accacatgtc
ccacagatac 2100gcatcgattc gattcaaatt aattaaaagg cgttgaaaca
gaatgagcca gacagcaagg 2160acaaggtggc caacagcaag gagtccaaaa
agccctctat tgacgagatc cacgatgtta 2220ttgctcatga ggtttccgag
ctcgatgctg ggaagaagaa gtgatttgta tataagaaat 2280aaatgagata
tagtaaagga gtgcaagaga atggcaaggt ggtcaaattc tatattactt
2340gcagtcactg gttcctcgtt gacatgaatg aagttaccgt tggcatagct
gatttaatat 2400ataactgtcc aactaactct cacctagata taacccatgt
gtgtgtttcc aatcatcaat 2460gcggccgctt actgagcctt ggcaccgggc
tgcttctcgg ccattcgagc gaactgggac 2520aggtatcgga gcaggatgac
gagaccttca tggggcagag ggtttcggta ggggaggttg 2580tgcttctggc
acagctgttc cacctggtag gaaacggcag tgaggttgtg tcgaggcagg
2640gtgggccaga gatggtgctc gatctggtag ttcaggcctc caaagaacca
gtcagtaatg 2700atgcctcgtc gaatgttcat ggtctcatgg atctgaccca
cagagaagcc atgtccgtcc 2760cagacggaat caccgatctt ctccagaggg
tagtggttca tgaagaccac gatggcaatt 2820ccgaagccac cgacgagctc
ggaaacaaag aacaccagca tcgaggtcag gatggagggc 2880ataaagaaga
ggtggaacag ggtcttgaga gtccagtgca gagcgagtcc aatggcctct
2940ttcttgtact gagatcggta gaactggttg tctcggtcct tgagggatcg
aacggtcagc 3000acagactgga aacaccagat gaatcgcagg agaatacaga
tgaccaggaa atagtactgt 3060tggaactgaa tgagctttcg ggagatggga
gaagctcgag tgacatcgtc ctcggaccag 3120gcgagcagag gcaggttatc
aatgtcggga tcgtgaccct gaacgttggt agcagaatga 3180tgggcgttgt
gtctgtcctt ccaccaggtc acggagaagc cctggagtcc gttgccaaag
3240accagaccca ggacgttatt ccagtttcgg ttcttgaagg tctggtggtg
gcagatgtca 3300tgagacagcc atcccatttg ctggtagtgc ataccgagca
cgagagcacc aatgaagtac 3360aggtggtact ggaccagcat gaagaaggca
agcacgccaa gacccagggt ggtcaagatc 3420ttgtacgagt accagagggg
agaggcgtca aacatgccag tggcgatcag ctcttctcgg 3480agctttcgga
aatcctcctg agcttcgttg acggcagcct ggggaggcag ctcggaagcc
3540tggttgatct tgggcattcg cttgagcttg tcgaaggctt cctgagagtg
cataaccatg 3600aaggcgtcag tagcatctcg tccctggtag ttctcaatga
tttcagctcc accagggtgg 3660aagttcaccc aagcggagac gtcgtacacc
tttccgtcga tgacgagggg cagagcctgt 3720cgagaagcct tcaccatggc
cattgctgta gatatgtctt gtgtgtaagg gggttggggt 3780ggttgtttgt
gttcttgact tttgtgttag caagggaaga cgggcaaaaa agtgagtgtg
3840gttgggaggg agagacgagc cttatatata atgcttgttt gtgtttgtgc
aagtggacgc 3900cgaaacgggc aggagccaaa ctaaacaagg cagacaatgc
gagcttaatt ggattgcctg 3960atgggcaggg gttagggctc gatcaatggg
ggtgcgaagt gacaaaattg ggaattaggt 4020tcgcaagcaa ggctgacaag
actttggccc aaacatttgt acgcggtgga caacaggagc 4080cacccatcgt
ctgtcacggg ctagccggtc gtgcgtcctg tcaggctcca cctaggctcc
4140atgccactcc atacaatccc actagtgtac cgctaggccg cttttagctc
ccatctaaga 4200cccccccaaa acctccactg tacagtgcac tgtactgtgt
ggcgatcaag ggcaagggaa 4260aaaaggcgca aacatgcacg catggaatga
cgtaggtaag gcgttactag actgaaaagt 4320ggcacatttc ggcgtgccaa
agggtcctag gtgcgtttcg cgagctgggc gccaggccaa 4380gccgctccaa
aacgcctctc cgactccctc cagcggcctc catatcccca tccctctcca
4440cagcaatgtt gttaagcctt gcaaacgaaa aaatagaaag gctaataagc
ttccaatatt 4500gtggtgtacg ctgcataacg caacaatgag cgccaaacaa
cacacacaca cagcacacag 4560cagcattaac cacgatgttt aaacagtgta
cgcagatccc gtcaacagtt ttatatatcg 4620tagttacaac catcaacact
ttttggtaag tgtaccattc tatactccaa ctggtctgca 4680actgtacaag
tagacatgtt aatggtagtt aataacatct acagcagaac ctatggtaaa
4740gacattgcat ttttacagga agtatcgtcc tacacgttga taaatccaaa
gatgcggaac 4800ttcttccact tttatcatca tcccctactc gtacactcgt
actctttgtt cgatcgcgat 4860tcatttctat aaataatctt gtatgtacat
gcggccgcgc ctacttaagc aacgggcttg 4920ataacagcgg ggggggtgcc
cacgttgttg cggttgcgga agaacagaac acccttacca 4980gcaccctcgg
caccagcgct gggctcaacc cactggcaca tacgcgcact gcggtacatg
5040gcgcggatga agccacgagg accatcctgg acatcagccc ggtagtgctt
gcccatgatg 5100ggcttaatgg cctcggtggc ctcgtccgcg ttgtagaagg
ggatgctgct gacgtagtgg 5160tggaggacat gagtctcgat gatgccgtgg
agaaggtggc ggccgatgaa gcccatctca 5220cggtcaatgg tagcagcggc
accacggacg aagttccact cgtcgttggt gtagtgggga 5280agggtagggt
cggtgtgctg gaggaaggtg atggcaacga gccagtggtt aacccagagg
5340tagggaacaa agtaccagat ggccatgttg tagaaaccga acttctgaac
gaggaagtac 5400agagcagtgg ccatcagacc gataccaata tcgctgagga
cgatgagctt agcgtcactg 5460ttctcgtaca gagggctgcg gggatcgaag
tggttaacac caccgccgag gccgttatgc 5520ttgcccttgc cgcgaccctc
acgctggcgc tcgtggtagt tgtggccggt aacattggtg 5580atgaggtagt
tgggccagcc nacgannnnc tcagtaagat gagcgagctc gtgggtcatc
5640tttccgagac gagtagcctg ctgctcgcgg gttcggggaa cgaagaccat
gtcacgctcc 5700atgttgccag tggccttgtg gtgctttcgg tgggagattt
gccagctgaa gtaggggaca 5760aggagggaag agtgaagaac ccagccagta
atgtcgttga tgatgcgaga atcggagaaa 5820gcaccgtgac cgcactcatg
ggcaataacc cagagaccag taccgaaaag accctgaaga 5880acggtgtaca
cggcccacag accagcgcgg gcgggggtgg aggggatata ttcgggggtc
5940acaaagttgt accagatgct gaaagtggta gtcaggagga caatgtcgcg
gaggatataa 6000ccgtatccct tgagagcgga gcgcttgaag cagtgcttag
ggatggcatt gtagatgtcc 6060ttgatggtaa agtcgggaac ctcgaactgg
ttgccgtagg tgtcgagcat gacaccatac 6120tcggacttgg gcttggcgat
atcaacctcg gacatggacg agagcgatgt ggaagaggcc 6180gagtggcggg
gagagtctga aggagagacg gcggcagact cagaatccgt cacagtagtt
6240gaggtgacgg tgcgtctaag cgcagggttc tgcttgggca gagccgaagt
ggacgccatg 6300gttgtgaatt agggtggtga gaatggttgg ttgtagggaa
gaatcaaagg ccggtctcgg 6360gatccgtggg tatatatata tatatatata
tatacgatcc ttcgttacct ccctgttctc 6420aaaactgtgg tttttcgttt
ttcgtttttt gctttttttg atttttttag ggccaactaa 6480gcttccagat
ttcgctaatc acctttgtac taattacaag aaaggaagaa gctgattaga
6540gttgggcttt ttatgcaact gtgctactcc ttatctctga tatgaaagtg
tagacccaat 6600cacatcatgt catttagagt tggtaatact gggaggatag
ataaggcacg aaaacgagcc 6660atagcagaca tgctgggtgt agccaagcag
aagaaagtag atgggagcca attgacgagc 6720gagggagcta cgccaatccg
acatacgaca cgctgagatc gtcttggccg gggggtacct 6780acagatgtcc
aagggtaagt gcttgactgt aattgtatgt ctgaggacaa atatgtagtc
6840agccgtataa agtcatacca ggcaccagtg ccatcatcga accactaact
ctctatgata 6900catgcctccg gtattattgt accatgcgtc gctttgttac
atacgtatct tgcctttttc 6960tctcagaaac tccagacttt ggctattggt
cgagataagc ccggaccata gtgagtcttt 7020cacactctac atttctccct
tgctccaact atttaaattg ccccggagaa gacggccagg 7080ccgcctagat
gacaaattca acaactcaca gctgactttc tgccattgcc actagggggg
7140ggccttttta tatggccaag ccaagctctc cacgtcggtt gggctgcacc
caacaataaa 7200tgggtagggt tgcaccaaca aagggatggg atggggggta
gaagatacga ggataacggg 7260gctcaatggc acaaataaga acgaatactg
ccattaagac tcgtgatcca gcgactgaca 7320ccattgcatc atctaagggc
ctcaaaacta cctcggaact gctgcgctga tctggacacc 7380acagaggttc
cgagcacttt aggttgcacc aaatgtccca ccaggtgcag gcagaaaacg
7440ctggaacagc gtgtacagtt tgtcttaaca aaaagtgagg gcgctgaggt
cgagcagggt 7500ggtgtgactt gttatagcct ttagagctgc gaaagcgcgt
atggatttgg ctcatcaggc 7560cagattgagg gtctgtggac acatgtcatg
ttagtgtact tcaatcgccc cctggatata 7620gccccgacaa taggccgtgg
cctcattttt ttgccttccg cacatttcca ttgctcggta 7680cccacacctt
gcttctcctg cacttgccaa ccttaatact ggtttacatt gaccaacatc
7740ttacaagcgg ggggcttgtc tagggtatat ataaacagtg gctctcccaa
tcggttgcca 7800gtctcttttt tcctttcttt ccccacagat tcgaaatcta
aactacacat cacagaattc 7860cgagccgtga gtatccacga caagatcagt
gtcgagacga cgcgttttgt gtaatgacac 7920aatccgaaag tcgctagcaa
cacacactct ctacacaaac taacccagct ctggtaccat 7980ggtgaaggct
tctcgacagg ctctgcccct cgtcatcgac ggaaaggtgt acgacgtctc
8040cgcttgggtg aacttccacc ctggtggagc tgaaatcatt gagaactacc
agggacgaga 8100tgctactgac gccttcatgg ttatgcactc tcaggaagcc
ttcgacaagc tcaagcgaat 8160gcccaagatc aaccaggctt ccgagctgcc
tccccaggct gccgtcaacg aagctcagga 8220ggatttccga aagctccgag
aagagctgat cgccactggc atgtttgacg cctctcccct 8280ctggtactcg
tacaagatct tgaccaccct gggtcttggc gtgcttgcct tcttcatgct
8340ggtccagtac cacctgtact tcattggtgc tctcgtgctc ggtatgcact
accagcaaat 8400gggatggctg tctcatgaca tctgccacca ccagaccttc
aagaaccgaa actggaataa 8460cgtcctgggt ctggtctttg gcaacggact
ccagggcttc tccgtgacct ggtggaagga 8520cagacacaac gcccatcatt
ctgctaccaa cgttcagggt cacgatcccg acattgataa 8580cctgcctctg
ctcgcctggt ccgaggacga tgtcactcga gcttctccca tctcccgaaa
8640gctcattcag ttccaacagt actatttcct ggtcatctgt attctcctgc
gattcatctg 8700gtgtttccag tctgtgctga ccgttcgatc cctcaaggac
cgagacaacc agttctaccg 8760atctcagtac aagaaagagg ccattggact
cgctctgcac tggactctca agaccctgtt 8820ccacctcttc tttatgccct
ccatcctgac ctcgatgctg gtgttctttg tttccgagct 8880cgtcggtggc
ttcggaattg ccatcgtggt cttcatgaac cactaccctc tggagaagat
8940cggtgattcc gtctgggacg gacatggctt ctctgtgggt cagatccatg
agaccatgaa 9000cattcgacga ggcatcatta ctgactggtt ctttggaggc
ctgaactacc agatcgagca 9060ccatctctgg cccaccctgc ctcgacacaa
cctcactgcc gtttcctacc aggtggaaca 9120gctgtgccag aagcacaacc
tcccctaccg aaaccctctg ccccatgaag gtctcgtcat 9180cctgctccga
tacctgtccc agttcgctcg aatggccgag aagcagcccg gtgccaaggc
9240tcagtaagcg gccgcaagtg tggatgggga agtgagtgcc cggttctgtg
tgcacaattg 9300gcaatccaag atggatggat tcaacacagg gatatagcga
gctacgtggt ggtgcgagga 9360tatagcaacg gatatttatg tttgacactt
gagaatgtac gatacaagca ctgtccaagt 9420acaatactaa acatactgta
catactcata ctcgtacccg ggcaacggtt tcacttgagt 9480gcagtggcta
gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt
9540atatcgtatt cattcatgtt agttgcgtac gggtgaagct tccactggtc
ggcgtggtag 9600tggggcagag tggggtcggt gtgctgcagg taggtgatgg
ccacgagcca gtggttgacc 9660cacaggtagg ggatcaggta gtagagggtg
acggaagcca ggccccatcg gttgatggag 9720tatgcgatga cggacatggt
gataccaata ccgacgttag agatccagat gttgaaccag 9780tccttcttct
caaacagcgg ggcgttgggg ttgaagtggt tgacagccca tttgttgagc
9840ttggggtact tctgtccggt aacgtaagac agcagataca gaggccatcc
aaacacctgc 9900tgggtgatga ggccgtagag ggtcatgagg ggagcgtcct
cagcaagctc agaccagtca 9960tgggcgcctc ggttctccat aaactccttt
cggtccttgg gcacaaacac catatcacgg 10020gtgaggtgac cagtggactt
gtggtgcatg gagtgggtca gcttccaggc gtagtaaggg 10080accagcatgg
aggagtgcag aacccatccg gtgacgttgt tgacggtgtt agagtcggag
10140aaagcagagt ggccacactc gtgggcaaga acccacagac cggtgccaaa
cagaccctgg 10200acaatggagt acatggccca ggccacagct cggccggaag
ccgagggaat aagaggcagg 10260tacgcgtagg ccatgtaggc aaaaacggcg
ataaagaagc aggcgcgcca gctgcattaa 10320tgaatcggcc aacgcgcggg
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 10380ctcactgact
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
10440gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
gtgagcaaaa 10500ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
tggcgttttt ccataggctc 10560cgcccccctg acgagcatca caaaaatcga
cgctcaagtc agaggtggcg aaacccgaca 10620ggactataaa gataccaggc
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 10680accctgccgc
ttaccggata cctgtccgcc tttctccctt
cgggaagcgt ggcgctttct 10740catagctcac gctgtaggta tctcagttcg
gtgtaggtcg ttcgctccaa gctgggctgt 10800gtgcacgaac cccccgttca
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 10860tccaacccgg
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
10920agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
ctacggctac 10980actagaagaa cagtatttgg tatctgcgct ctgctgaagc
cagttacctt cggaaaaaga 11040gttggtagct cttgatccgg caaacaaacc
accgctggta gcggtggttt ttttgtttgc 11100aagcagcaga ttacgcgcag
aaaaaaagga tctcaagaag atcctttgat cttttctacg 11160gggtctgacg
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
11220aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
aatctaaagt 11280atatatgagt aaacttggtc tgacagttac caatgcttaa
tcagtgaggc acctatctca 11340gcgatctgtc tatttcgttc atccatagtt
gcctgactcc ccgtcgtgta gataactacg 11400atacgggagg gcttaccatc
tggccccagt gctgcaatga taccgcgaga cccacgctca 11460ccggctccag
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
11520cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
tagagtaagt 11580agttcgccag ttaatagttt gcgcaacgtt gttgccattg
ctacaggcat cgtggtgtca 11640cgctcgtcgt ttggtatggc ttcattcagc
tccggttccc aacgatcaag gcgagttaca 11700tgatccccca tgttgtgcaa
aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 11760agtaagttgg
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
11820gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
gtcattctga 11880gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
caatacggga taataccgcg 11940ccacatagca gaactttaaa agtgctcatc
attggaaaac gttcttcggg gcgaaaactc 12000tcaaggatct taccgctgtt
gagatccagt tcgatgtaac ccactcgtgc acccaactga 12060tcttcagcat
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
12120gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
cttccttttt 12180caatattatt gaagcattta tcagggttat tgtctcatga
gcggatacat atttgaatgt 12240atttagaaaa ataaacaaat aggggttccg
cgcacatttc cccgaaaagt gccacctgat 12300gcggtgtgaa ataccgcaca
gatgcgtaag gagaaaatac cgcatcagga aattgtaagc 12360gttaatattt
tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
12420taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
agggttgagt 12480gttgttccag tttggaacaa gagtccacta ttaaagaacg
tggactccaa cgtcaaaggg 12540cgaaaaaccg tctatcaggg cgatggccca
ctacgtgaac catcacccta atcaagtttt 12600ttggggtcga ggtgccgtaa
agcactaaat cggaacccta aagggagccc ccgatttaga 12660gcttgacggg
gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
12720ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
acccgccgcg 12780cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
gctgcgcaac tgttgggaag 12840ggcgatcggt gcgggcctct tcgctattac
gccagctggc gaaaggggga tgtgctgcaa 12900ggcgattaag ttgggtaacg
ccagggtttt cccagtcacg acgttgtaaa acgacggcca 12960gtgaattgta
atacgactca ctatagggcg aattgggccc gacgtcgcat gcttgaatct
13020acaagtagga gggttggagt gattaagtga aacttcttta acggctctat
gccagttcta 13080ttgatatccg aaacatcagt atgaaggtct gataagggtg
acttcttccc acagattcgt 13140atcagtacga gtacgagacc ggtacttgta
acagtattga tactaaaggg aaactacaac 13200ggttgtcagc gtaatgtgac
ttcgcccatg aacgcagaca cgcagtgccg agtgcggtga 13260tatcgcctac
tcgttacgtc catggactac acaacccctc ggcttcgctt ggcttagcct
13320cgggctcggt gctgttcagt taaaacacaa tcaaataaca tttctacttt
ttagaaggca 13380ggccgtcagg agcaactccg actccattga cgtttctaaa
catctgaatg ccttccttac 13440cttcaacaaa ctggcaggtt cgggcgacag
tgtaaagaga cttgatgaag ttggtgtcgt 13500cgtgtcggta gtgcttgccc
atgaccttct tgatcttctc agtggcgatt cgggcgttgt 13560agaagggaat
tcctttacct gcaggataac ttcgtataat gtatgctata cgaagttatg
13620atctctctct tgagcttttc cataacaagt tcttctgcct ccaggaagtc
catgggtggt 13680ttgatcatgg ttttggtgta gtggtagtgc agtggtggta
ttgtgactgg ggatgtagtt 13740gagaataagt catacacaag tcagctttct
tcgagcctca tataagtata agtagttcaa 13800cgtattagca ctgtacccag
catctccgta tcgagaaaca caacaacatg ccccattgga 13860cagatcatgc
ggatacacag gttgtgcagt atcatacata ctcgatcaga caggtcgtct
13920gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat
acacagttaa 13980attacatatc catagtctaa cctctaacag ttaatcttct
ggtaagcctc ccagccagcc 14040ttctggtatc gcttggcctc ctcaatagga
tctcggttct ggccgtacag acctcggccg 14100acaattatga tatccgttcc
ggtagacatg acatcctcaa cagttcggta ctgctgtccg 14160agagcgtctc
ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag
14220tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc
ggatcgggca 14280agctcaatgg tctgcttgga gtactcgcca gtggccagag
agcccttgca agacagctcg 14340gccagcatga gcagacctct ggccagcttc
tcgttgggag aggggactag gaactccttg 14400tactgggagt tctcgtagtc
agagacgtcc tccttcttct gttcagagac agtttcctcg 14460gcaccagctc
gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata
14520tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt
gccaatatct 14580gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa
gagcaagttc cttgaggggg 14640agcacagtgc cggcgtaggt gaagtcgtca
atgatgtcga tatgggtttt gatcatgcac 14700acataaggtc cgaccttatc
ggcaagctca atgagctcct tggtggtggt aacatccaga 14760gaagcacaca
ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg
14820gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa
gaggagactg 14880aaataaattt agtctgcaga actttttatc ggaaccttat
ctggggcagt gaagtatatg 14940ttatggtaat agttacgagt tagttgaact
tatagataga ctggactata cggctatcgg 15000tccaaattag aaagaacgtc
aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga 15060tcatgatgaa
agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa
15120aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt
gatccaagca 15180cactcatagt tggagtcgta ctccaaaggc ggcaatgacg
agtcagacag atactcgtcg 15240acgcgataac ttcgtataat gtatgctata
cgaagttatc gtacgatagt tagtagacaa 15300caat
153041821288DNAArtificial Sequencemodified mutant EgD8S-23,
comprising a 5' Not1 site 182gcggccgcac catggtgaag gcttctcgac
aggctctgcc cctcgtcatc gacggaaagg 60tgtacgacgt ctccgcttgg gtgaacttcc
accctggtgg agctgaaatc attgagaact 120accagggacg agatgctact
gacgccttca tggttatgca ctctcaggaa gccttcgaca 180agctcaagcg
aatgcccaag atcaaccagg cttccgagct gcctccccag gctgccgtca
240acgaagctca ggaggatttc cgaaagctcc gagaagagct gatcgccact
ggcatgtttg 300acgcctctcc cctctggtac tcgtacaaga tcttgaccac
cctgggtctt ggcgtgcttg 360ccttcttcat gctggtccag taccacctgt
acttcattgg tgctctcgtg ctcggtatgc 420actaccagca aatgggatgg
ctgtctcatg acatctgcca ccaccagacc ttcaagaacc 480gaaactggaa
taacgtcctg ggtctggtct ttggcaacgg actccagggc ttctccgtga
540cctggtggaa ggacagacac aacgcccatc attctgctac caacgttcag
ggtcacgatc 600ccgacattga taacctgcct ctgctcgcct ggtccgagga
cgatgtcact cgagcttctc 660ccatctcccg aaagctcatt cagttccaac
agtactattt cctggtcatc tgtattctcc 720tgcgattcat ctggtgtttc
cagtctgtgc tgaccgttcg atccctcaag gaccgagaca 780accagttcta
ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc
840tcaagaccct gttccacctc ttctttatgc cctccatcct gacctcgatg
ctggtgttct 900ttgtttccga gctcgtcggt ggcttcggaa ttgccatcgt
ggtcttcatg aaccactacc 960ctctggagaa gatcggtgat tccgtctggg
acggacatgg cttctctgtg ggtcagatcc 1020atgagaccat gaacattcga
cgaggcatca ttactgactg gttctttgga ggcctgaact 1080accagatcga
gcaccatctc tggcccaccc tgcctcgaca caacctcact gccgtttcct
1140accaggtgga acagctgtgc cagaagcaca acctccccta ccgaaaccct
ctgccccatg 1200aaggtctcgt catcctgctc cgatacctgt cccagttcgc
tcgaatggcc gagaagcagc 1260ccggtgccaa ggctcagtaa gcggccgc
12881835252DNAArtificial SequencePlasmid pKR457 183gtacgtgggc
ggatcccccg ggctgcagga attcactggc cgtcgtttta caacgtcgtg 60actgggaaaa
ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
120gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
cgcagcctga 180atggcgaatg gcgcctgatg cggtattttc tccttacgca
tctgtgcggt atttcacacc 240gcatatggtg cactctcagt acaatctgct
ctgatgccgc atagttaagc cagccccgac 300acccgccaac acccgctgac
gcgccctgac gggcttgtct gctcccggca tccgcttaca 360gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
420aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
gtcatgataa 480taatggtttc ttagacgtca ggtggcactt ttcggggaaa
tgtgcgcgga acccctattt 540gtttattttt ctaaatacat tcaaatatgt
atccgctcat gagacaataa ccctgataaa 600tgcttcaata atattgaaaa
aggaagagta tgagtattca acatttccgt gtcgccctta 660ttcccttttt
tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
720taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
gatctcaaca 780gcggtaagat ccttgagagt tttcgccccg aagaacgttt
tccaatgatg agcactttta 840aagttctgct atgtggcgcg gtattatccc
gtattgacgc cgggcaagag caactcggtc 900gccgcataca ctattctcag
aatgacttgg ttgagtactc accagtcaca gaaaagcatc 960ttacggatgg
catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
1020ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
gcttttttgc 1080acaacatggg ggatcatgta actcgccttg atcgttggga
accggagctg aatgaagcca 1140taccaaacga cgagcgtgac accacgatgc
ctgtagcaat ggcaacaacg ttgcgcaaac 1200tattaactgg cgaactactt
actctagctt cccggcaaca attaatagac tggatggagg 1260cggataaagt
tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
1320ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
gggccagatg 1380gtaagccctc ccgtatcgta gttatctaca cgacggggag
tcaggcaact atggatgaac 1440gaaatagaca gatcgctgag ataggtgcct
cactgattaa gcattggtaa ctgtcagacc 1500aagtttactc atatatactt
tagattgatt taaaacttca tttttaattt aaaaggatct 1560aggtgaagat
cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
1620actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
ttttttctgc 1680gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
agcggtggtt tgtttgccgg 1740atcaagagct accaactctt tttccgaagg
taactggctt cagcagagcg cagataccaa 1800atactgtcct tctagtgtag
ccgtagttag gccaccactt caagaactct gtagcaccgc 1860ctacatacct
cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
1920gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
tcgggctgaa 1980cggggggttc gtgcacacag cccagcttgg agcgaacgac
ctacaccgaa ctgagatacc 2040tacagcgtga gctatgagaa agcgccacgc
ttcccgaagg gagaaaggcg gacaggtatc 2100cggtaagcgg cagggtcgga
acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2160ggtatcttta
tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
2220gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
ttacggttcc 2280tggccttttg ctggcctttt gctcacatgt tctttcctgc
gttatcccct gattctgtgg 2340ataaccgtat taccgccttt gagtgagctg
ataccgctcg ccgcagccga acgaccgagc 2400gcagcgagtc agtgagcgag
gaagcggaag agcgcccaat acgcaaaccg cctctccccg 2460cgcgttggcc
gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
2520gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
gctttacact 2580ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
ataacaattt cacacaggaa 2640acagctatga ccatgattac gccaagcttg
catgcctgca ggtcgactcg acgtacgtcc 2700tcgaagagaa gggttaataa
cacatttttt aacattttta acacaaattt tagttattta 2760aaaatttatt
aaaaaattta aaataagaag aggaactctt taaataaatc taacttacaa
2820aatttatgat ttttaataag ttttcaccaa taaaaaatgt cataaaaata
tgttaaaaag 2880tatattatca atattctctt tatgataaat aaaaagaaaa
aaaaaataaa agttaagtga 2940aaatgagatt gaagtgactt taggtgtgta
taaatatatc aaccccgcca acaatttatt 3000taatccaaat atattgaagt
atattattcc atagccttta tttatttata tatttattat 3060ataaaagctt
tatttgttct aggttgttca tgaaatattt ttttggtttt atctccgttg
3120taagaaaatc atgtgctttg tgtcgccact cactattgca gctttttcat
gcattggtca 3180gattgacggt tgattgtatt tttgtttttt atggttttgt
gttatgactt aagtcttcat 3240ctctttatct cttcatcagg tttgatggtt
acctaatatg gtccatgggt acatgcatgg 3300ttaaattagg tggccaactt
tgttgtgaac gatagaattt tttttatatt aagtaaacta 3360tttttatatt
atgaaataat aataaaaaaa atattttatc attattaaca aaatcatatt
3420agttaatttg ttaactctat aataaaagaa atactgtaac attcacatta
catggtaaca 3480tctttccacc ctttcatttg ttttttgttt gatgactttt
tttcttgttt aaatttattt 3540cccttctttt aaatttggaa tacattatca
tcatatataa actaaaatac taaaaacagg 3600attacacaaa tgataaataa
taacacaaat atttataaat ctagctgcaa tatatttaaa 3660ctagctatat
cgatattgta aaataaaact agctgcattg atactgataa aaaaatatca
3720tgtgctttct ggactgatga tgcagtatac ttttgacatt gcctttattt
tatttttcag 3780aaaagctttc ttagttctgg gttcttcatt atttgtttcc
catctccatt gtgaattgaa 3840tcatttgctt cgtgtcacaa atacaattta
gntaggtaca tgcattggtc agattcacgg 3900tttattatgt catgacttaa
gttcatggta gtacattacc tgccacgcat gcattatatt 3960ggttagattt
gataggcaaa tttggttgtc aacaatataa atataaataa tgtttttata
4020ttacgaaata acagtgatca aaacaaacag ttttatcttt attaacaaga
ttttgttttt 4080gtttgatgac gttttttaat gtttacgctt tcccccttct
tttgaattta gaacacttta 4140tcatcataaa atcaaatact aaaaaaatta
catatttcat aaataataac acaaatattt 4200ttaaaaaatc tgaaataata
atgaacaata ttacatatta tcacgaaaat tcattaataa 4260aaatattata
taaataaaat gtaatagtag ttatatgtag gaaaaaagta ctgcacgcat
4320aatatataca aaaagattaa aatgaactat tataaataat aacactaaat
taatggtgaa 4380tcatatcaaa ataatgaaaa agtaaataaa atttgtaatt
aacttctata tgtattacac 4440acacaaataa taaataatag taaaaaaaat
tatgataaat atttaccatc tcataagata 4500tttaaaataa tgataaaaat
atagattatt ttttatgcaa ctagctagcc aaaaagagaa 4560cacgggtata
tataaaaaga gtacctttaa attctactgt acttccttta ttcctgacgt
4620ttttatatca agtggacata cgtgaagatt ttaattatca gtctaaatat
ttcattagca 4680cttaatactt ttctgtttta ttcctatcct ataagtagtc
ccgattctcc caacattgct 4740tattcacaca actaactaag aaagtcttcc
atagcccccc aagcggccgc gacacaagtg 4800tgagagtact aaataaatgc
tttggttgta cgaaatcatt acactaaata aaataatcaa 4860agcttatata
tgccttccgc taaggccgaa tgcaaagaaa ttggttcttt ctcgttatct
4920tttgccactt ttactagtac gtattaatta ctacttaatc atctttgttt
acggctcatt 4980atatccggtc tagaggatcc aaggccgcga agttaaaagc
aatgttgtca cttgtcgtac 5040taacacatga tgtgatagtt tatgctagct
agctataaca taagctgtct ctgagtgtgt 5100tgtatattaa taaagatcat
cactggtgaa tggtgatcgt gtacgtaccc tacttagtag 5160gcaatggaag
cacttagagt gtgctttgtg catggccttg cctctgtttt gagacttttg
5220taatgttttc gagtttaaat ctttgccttt gc 52521846532DNAArtificial
SequencePlasmid pKR1058 184ggccgcgaca caagtgtgag agtactaaat
aaatgctttg gttgtacgaa atcattacac 60taaataaaat aatcaaagct tatatatgcc
ttccgctaag gccgaatgca aagaaattgg 120ttctttctcg ttatcttttg
ccacttttac tagtacgtat taattactac ttaatcatct 180ttgtttacgg
ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg
240ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct
ataacataag 300ctgtctctga gtgtgttgta tattaataaa gatcatcact
ggtgaatggt gatcgtgtac 360gtaccctact tagtaggcaa tggaagcact
tagagtgtgc tttgtgcatg gccttgcctc 420tgttttgaga cttttgtaat
gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg 480atcccccggg
ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
540ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
tggcgtaata 600gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
cagcctgaat ggcgaatggc 660gcctgatgcg gtattttctc cttacgcatc
tgtgcggtat ttcacaccgc atatggtgca 720ctctcagtac aatctgctct
gatgccgcat agttaagcca gccccgacac ccgccaacac 780ccgctgacgc
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
840ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
cgcgcgagac 900gaaagggcct cgtgatacgc ctatttttat aggttaatgt
catgataata atggtttctt 960agacgtcagg tggcactttt cggggaaatg
tgcgcggaac ccctatttgt ttatttttct 1020aaatacattc aaatatgtat
ccgctcatga gacaataacc ctgataaatg cttcaataat 1080attgaaaaag
gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
1140cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
aaagatgctg 1200aagatcagtt gggtgcacga gtgggttaca tcgaactgga
tctcaacagc ggtaagatcc 1260ttgagagttt tcgccccgaa gaacgttttc
caatgatgag cacttttaaa gttctgctat 1320gtggcgcggt attatcccgt
attgacgccg ggcaagagca actcggtcgc cgcatacact 1380attctcagaa
tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
1440tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
gcggccaact 1500tacttctgac aacgatcgga ggaccgaagg agctaaccgc
ttttttgcac aacatggggg 1560atcatgtaac tcgccttgat cgttgggaac
cggagctgaa tgaagccata ccaaacgacg 1620agcgtgacac cacgatgcct
gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 1680aactacttac
tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
1740caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
aaatctggag 1800ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
gccagatggt aagccctccc 1860gtatcgtagt tatctacacg acggggagtc
aggcaactat ggatgaacga aatagacaga 1920tcgctgagat aggtgcctca
ctgattaagc attggtaact gtcagaccaa gtttactcat 1980atatacttta
gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
2040tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
tgagcgtcag 2100accccgtaga aaagatcaaa ggatcttctt gagatccttt
ttttctgcgc gtaatctgct 2160gcttgcaaac aaaaaaacca ccgctaccag
cggtggtttg tttgccggat caagagctac 2220caactctttt tccgaaggta
actggcttca gcagagcgca gataccaaat actgtccttc 2280tagtgtagcc
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
2340ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
cttaccgggt 2400tggactcaag acgatagtta ccggataagg cgcagcggtc
gggctgaacg gggggttcgt 2460gcacacagcc cagcttggag cgaacgacct
acaccgaact gagataccta cagcgtgagc 2520tatgagaaag cgccacgctt
cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2580gggtcggaac
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
2640gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
tcgtcagggg 2700ggcggagcct atggaaaaac gccagcaacg cggccttttt
acggttcctg gccttttgct 2760ggccttttgc tcacatgttc tttcctgcgt
tatcccctga ttctgtggat aaccgtatta 2820ccgcctttga gtgagctgat
accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2880tgagcgagga
agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
2940ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
gagcgcaacg 3000caattaatgt gagttagctc actcattagg caccccaggc
tttacacttt atgcttccgg 3060ctcgtatgtt gtgtggaatt gtgagcggat
aacaatttca cacaggaaac agctatgacc 3120atgattacgc caagcttgca
tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg 3180gttaataaca
cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa
3240aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa
tttatgattt 3300ttaataagtt ttcaccaata aaaaatgtca taaaaatatg
ttaaaaagta tattatcaat 3360attctcttta tgataaataa aaagaaaaaa
aaaataaaag ttaagtgaaa atgagattga 3420agtgacttta ggtgtgtata
aatatatcaa ccccgccaac aatttattta atccaaatat 3480attgaagtat
attattccat agcctttatt tatttatata tttattatat aaaagcttta
3540tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta
agaaaatcat 3600gtgctttgtg tcgccactca ctattgcagc tttttcatgc
attggtcaga ttgacggttg
3660attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct
ctttatctct 3720tcatcaggtt tgatggttac ctaatatggt ccatgggtac
atgcatggtt aaattaggtg 3780gccaactttg ttgtgaacga tagaattttt
tttatattaa gtaaactatt tttatattat 3840gaaataataa taaaaaaaat
attttatcat tattaacaaa atcatattag ttaatttgtt 3900aactctataa
taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct
3960ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc
cttcttttaa 4020atttggaata cattatcatc atatataaac taaaatacta
aaaacaggat tacacaaatg 4080ataaataata acacaaatat ttataaatct
agctgcaata tatttaaact agctatatcg 4140atattgtaaa ataaaactag
ctgcattgat actgataaaa aaatatcatg tgctttctgg 4200actgatgatg
cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt
4260agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc
atttgcttcg 4320tgtcacaaat acaatttagn taggtacatg cattggtcag
attcacggtt tattatgtca 4380tgacttaagt tcatggtagt acattacctg
ccacgcatgc attatattgg ttagatttga 4440taggcaaatt tggttgtcaa
caatataaat ataaataatg tttttatatt acgaaataac 4500agtgatcaaa
acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt
4560tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc
atcataaaat 4620caaatactaa aaaaattaca tatttcataa ataataacac
aaatattttt aaaaaatctg 4680aaataataat gaacaatatt acatattatc
acgaaaattc attaataaaa atattatata 4740aataaaatgt aatagtagtt
atatgtagga aaaaagtact gcacgcataa tatatacaaa 4800aagattaaaa
tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat
4860aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac
acaaataata 4920aataatagta aaaaaaatta tgataaatat ttaccatctc
ataagatatt taaaataatg 4980ataaaaatat agattatttt ttatgcaact
agctagccaa aaagagaaca cgggtatata 5040taaaaagagt acctttaaat
tctactgtac ttcctttatt cctgacgttt ttatatcaag 5100tggacatacg
tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt
5160ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta
ttcacacaac 5220taactaagaa agtcttccat agccccccaa gcggccgcac
catggtgaag gcttctcgac 5280aggctctgcc cctcgtcatc gacggaaagg
tgtacgacgt ctccgcttgg gtgaacttcc 5340accctggtgg agctgaaatc
attgagaact accagggacg agatgctact gacgccttca 5400tggttatgca
ctctcaggaa gccttcgaca agctcaagcg aatgcccaag atcaaccagg
5460cttccgagct gcctccccag gctgccgtca acgaagctca ggaggatttc
cgaaagctcc 5520gagaagagct gatcgccact ggcatgtttg acgcctctcc
cctctggtac tcgtacaaga 5580tcttgaccac cctgggtctt ggcgtgcttg
ccttcttcat gctggtccag taccacctgt 5640acttcattgg tgctctcgtg
ctcggtatgc actaccagca aatgggatgg ctgtctcatg 5700acatctgcca
ccaccagacc ttcaagaacc gaaactggaa taacgtcctg ggtctggtct
5760ttggcaacgg actccagggc ttctccgtga cctggtggaa ggacagacac
aacgcccatc 5820attctgctac caacgttcag ggtcacgatc ccgacattga
taacctgcct ctgctcgcct 5880ggtccgagga cgatgtcact cgagcttctc
ccatctcccg aaagctcatt cagttccaac 5940agtactattt cctggtcatc
tgtattctcc tgcgattcat ctggtgtttc cagtctgtgc 6000tgaccgttcg
atccctcaag gaccgagaca accagttcta ccgatctcag tacaagaaag
6060aggccattgg actcgctctg cactggactc tcaagaccct gttccacctc
ttctttatgc 6120cctccatcct gacctcgatg ctggtgttct ttgtttccga
gctcgtcggt ggcttcggaa 6180ttgccatcgt ggtcttcatg aaccactacc
ctctggagaa gatcggtgat tccgtctggg 6240acggacatgg cttctctgtg
ggtcagatcc atgagaccat gaacattcga cgaggcatca 6300ttactgactg
gttctttgga ggcctgaact accagatcga gcaccatctc tggcccaccc
6360tgcctcgaca caacctcact gccgtttcct accaggtgga acagctgtgc
cagaagcaca 6420acctccccta ccgaaaccct ctgccccatg aaggtctcgt
catcctgctc cgatacctgt 6480cccagttcgc tcgaatggcc gagaagcagc
ccggtgccaa ggctcagtaa gc 65321857887DNAArtificial SequencePlasmid
pKR607 185ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac
tggttcttga 60tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt
taacatgcat 120ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt
tgaaataaac aaaaatgagg 180tggcggaaga gaatacgagt ttgaggttgg
gttagaaaca acaaatgtga gggctcatga 240tgggttgagt tggtgaatgt
tttgggctgc tcgattgaca cctttgtgag tacgtgttgt 300tgtgcatggc
ttttggggtc cagttttttt ttcttgacgc ggcgatcctg atcagctagt
360ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg
aacttagaca 420ttgctatgca aaggatactc tcattgtgtt ttgtcttctt
ttgttccttg gctttttctt 480atgatccaag agactagtca gtgttgtggc
attcgagact accaagatta attatgatgg 540gggaaggata agtaactgat
tagtacggac tgttaccaaa ttaattaata agcggcaaat 600gaagggcatg
gatcaaaagc ttggatctcc tgcaggatct ggccggccgg atctcgtacg
660gatccgtcga cggcgcgccc gatcatccgg atatagttcc tcctttcagc
aaaaaacccc 720tcaagacccg tttagaggcc ccaaggggtt atgctagtta
ttgctcagcg gtggcagcag 780ccaactcagc ttcctttcgg gctttgttag
cagccggatc gatccaagct gtacctcact 840attcctttgc cctcggacga
gtgctggggc gtcggtttcc actatcggcg agtacttcta 900cacagccatc
ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc
960cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca
tcatcgaaat 1020tgccgtcaac caagctctga tagagttggt caagaccaat
gcggagcata tacgcccgga 1080gccgcggcga tcctgcaagc tccggatgcc
tccgctcgaa gtagcgcgtc tgctgctcca 1140tacaagccaa ccacggcctc
cagaagaaga tgttggcgac ctcgtattgg gaatccccga 1200acatcgcctc
gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt
1260tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg
gcccaaagca 1320tcagctcatc gagagcctgc gcgacggacg cactgacggt
gtcgtccatc acagtttgcc 1380agtgatacac atggggatca gcaatcgcgc
atatgaaatc acgccatgta gtgtattgac 1440cgattccttg cggtccgaat
gggccgaacc cgctcgtctg gctaagatcg gccgcagcga 1500tcgcatccat
agcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt
1560cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc
tcgctgaatt 1620ccccaatgtc aagcacttcc ggaatcggga gcgcggccga
tgcaaagtgc cgataaacat 1680aacgatcttt gtagaaacca tcggcgcagc
tatttacccg caggacatat ccacgccctc 1740ctacatcgaa gctgaaagca
cgagattctt cgccctccga gagctgcatc aggtcggaga 1800cgctgtcgaa
cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt
1860ccatgggtat atctccttct taaagttaaa caaaattatt tctagaggga
aaccgttgtg 1920gtctccctat agtgagtcgt attaatttcg cgggatcgag
atcgatccaa ttccaatccc 1980acaaaaatct gagcttaaca gcacagttgc
tcctctcaga gcagaatcgg gtattcaaca 2040ccctcatatc aactactacg
ttgtgtataa cggtccacat gccggtatat acgatgactg 2100gggttgtaca
aaggcggcaa caaacggcgt tcccggagtt gcacacaaga aatttgccac
2160tattacagag gcaagagcag cagctgacgc gtacacaaca agtcagcaaa
cagacaggtt 2220gaacttcatc cccaaaggag aagctcaact caagcccaag
agctttgcta aggccctaac 2280aagcccacca aagcaaaaag cccactggct
cacgctagga accaaaaggc ccagcagtga 2340tccagcccca aaagagatct
cctttgcccc ggagattaca atggacgatt tcctctatct 2400ttacgatcta
ggaaggaagt tcgaaggtga aggtgacgac actatgttca ccactgataa
2460tgagaaggtt agcctcttca atttcagaaa gaatgctgac ccacagatgg
ttagagaggc 2520ctacgcagca ggtctcatca agacgatcta cccgagtaac
aatctccagg agatcaaata 2580ccttcccaag aaggttaaag atgcagtcaa
aagattcagg actaattgca tcaagaacac 2640agagaaagac atatttctca
agatcagaag tactattcca gtatggacga ttcaaggctt 2700gcttcataaa
ccaaggcaag taatagagat tggagtctct aaaaaggtag ttcctactga
2760atctaaggcc atgcatggag tctaagattc aaatcgagga tctaacagaa
ctcgccgtga 2820agactggcga acagttcata cagagtcttt tacgactcaa
tgacaagaag aaaatcttcg 2880tcaacatggt ggagcacgac actctggtct
actccaaaaa tgtcaaagat acagtctcag 2940aagaccaaag ggctattgag
acttttcaac aaaggataat ttcgggaaac ctcctcggat 3000tccattgccc
agctatctgt cacttcatcg aaaggacagt agaaaaggaa ggtggctcct
3060acaaatgcca tcattgcgat aaaggaaagg ctatcattca agatgcctct
gccgacagtg 3120gtcccaaaga tggaccccca cccacgagga gcatcgtgga
aaaagaagac gttccaacca 3180cgtcttcaaa gcaagtggat tgatgtgaca
tctccactga cgtaagggat gacgcacaat 3240cccactatcc ttcgcaagac
ccttcctcta tataaggaag ttcatttcat ttggagagga 3300cacgctcgag
ctcatttctc tattacttca gccataacaa aagaactctt ttctcttctt
3360attaaaccat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt
ctgatcgaaa 3420agttcgacag cgtctccgac ctgatgcagc tctcggaggg
cgaagaatct cgtgctttca 3480gcttcgatgt aggagggcgt ggatatgtcc
tgcgggtaaa tagctgcgcc gatggtttct 3540acaaagatcg ttatgtttat
cggcactttg catcggccgc gctcccgatt ccggaagtgc 3600ttgacattgg
ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
3660tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
gtcgcggagg 3720ccatggatgc gatcgctgcg gccgatctta gccagacgag
cgggttcggc ccattcggac 3780cgcaaggaat cggtcaatac actacatggc
gtgatttcat atgcgcgatt gctgatcccc 3840atgtgtatca ctggcaaact
gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc 3900tcgatgagct
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
3960atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
attgactgga 4020gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
catcttcttc tggaggccgt 4080ggttggcttg tatggagcag cagacgcgct
acttcgagcg gaggcatccg gagcttgcag 4140gatcgccgcg gctccgggcg
tatatgctcc gcattggtct tgaccaactc tatcagagct 4200tggttgacgg
caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
4260gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
gccgtctgga 4320ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
acgccccagc actcgtccga 4380gggcaaagga atagtgaggt acctaaagaa
ggagtgcgtc gaagcagatc gttcaaacat 4440ttggcaataa agtttcttaa
gattgaatcc tgttgccggt cttgcgatga ttatcatata 4500atttctgttg
aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
4560gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga
tagaaaacaa 4620aatatagcgc gcaaactagg ataaattatc gcgcgcggtg
tcatctatgt tactagatcg 4680atgtcgaatc gatcaacctg cattaatgaa
tcggccaacg cgcggggaga ggcggtttgc 4740gtattgggcg ctcttccgct
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4800ggcgagcggt
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
4860acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
aaaaaggccg 4920cgttgctggc gtttttccat aggctccgcc cccctgacga
gcatcacaaa aatcgacgct 4980caagtcagag gtggcgaaac ccgacaggac
tataaagata ccaggcgttt ccccctggaa 5040gctccctcgt gcgctctcct
gttccgaccc tgccgcttac cggatacctg tccgcctttc 5100tcccttcggg
aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
5160aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg 5220ccttatccgg taactatcgt cttgagtcca acccggtaag
acacgactta tcgccactgg 5280cagcagccac tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct 5340tgaagtggtg gcctaactac
ggctacacta gaaggacagt atttggtatc tgcgctctgc 5400tgaagccagt
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
5460ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
aaaggatctc 5520aagaagatcc tttgatcttt tctacggggt ctgacgctca
gtggaacgaa aactcacgtt 5580aagggatttt ggtcatgaca ttaacctata
aaaataggcg tatcacgagg ccctttcgtc 5640tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 5700cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
5760ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 5820accatatgga catattgtcg ttagaacgcg gctacaatta
atacataacc ttatgtatca 5880tacacatacg atttaggtga cactatagaa
cggcgcgcca agcttgttga aacatccctg 5940aagtgtctca ttttatttta
tttattcttt gctgataaaa aaataaaata aaagaagcta 6000agcacacggt
caaccattgc tctactgcta aaagggttat gtgtagtgtt ttactgcata
6060aattatgcag caaacaagac aactcaaatt aaaaaatttc ctttgcttgt
ttttttgttg 6120tctctgactt gactttcttg tggaagttgg ttgtataagg
attgggacac cattgtcctt 6180cttaatttaa ttttattctt tgctgataaa
aaaaaaaatt tcatatagtg ttaaataata 6240atttgttaaa taaccaaaaa
gtcaaatatg tttactctcg tttaaataat tgagattcgt 6300ccagcaaggc
taaacgattg tatagattta tgacaatatt tactttttta tagataaatg
6360ttatattata ataaatttat atacatatat tatatgttat ttattattat
tttaaatcct 6420tcaatatttt atcaaaccaa ctcataattt tttttttatc
tgtaagaagc aataaaatta 6480aatagaccca ctttaaggat gatccaacct
ttatacagag taagagagtt caaatagtac 6540cctttcatat acatatcaac
taaaatatta gaaatatcat ggatcaaacc ttataaagac 6600attaaataag
tggataagta taatatataa atgggtagta tataatatat aaatggatac
6660aaacttctct ctttataatt gttatgtctc cttaacatcc taatataata
cataagtggg 6720taatatataa tatataaatg gagacaaact tcttccatta
taattgttat gtcttcttaa 6780cacttatgtc tcgttcacaa tgctaaggtt
agaattgttt agaaagtctt atagtacaca 6840tttgtttttg tactatttga
agcattccat aagccgtcac gattcagatg atttataata 6900ataagaggaa
atttatcata gaacaataag gtgcatagat agagtgttaa tatatcataa
6960catcctttgt ttattcatag aagaagtgag atggagctca gttattatac
tgttacatgg 7020tcggatacaa tattccatgc tctccatgag ctcttacacc
tacatgcatt ttagttcata 7080cttgcggccg ctaaagctgc ttaccagcct
tagcggattt cttggtggcc aggttgtcct 7140ggtaaaagaa gtgacagaac
aggagaaaga cagatccgac gtaggcgtag ttgaaagccc 7200aggagaacag
cttgcccttg tcagagttga agcagggaac gttgatgtag tcccagacca
7260ggagaaagcc accgacgaac tggcaaatct gcatggcagt gatcagaggc
ttggccttga 7320acttgtagcc agcggcagtc agtccatagt aggtgtacat
gatggtgtga atgaacgagt 7380taaagaacat gaagatccac acaccctcgt
tgtgcagtcg aatgccgagg tagacgtccc 7440agggagctcc aaagtgatgg
aaggcctgca gaaaggacac tcgcttgccc ttgaggacca 7500gccaagcggt
gtcgaggtac tccacgtact tagaatagta gaaggccttg gcagtccagg
7560tgaacagctt ggagtcccag acaggagagg gacactgaaa gagaggctgg
ggagtatcac 7620cggtctgtct tcgcagccag gctccagtac cgtagtccca
gccgagagcg gtggcagtca 7680cgtagaagga cagggcagag aagagagcca
ggaggacgtt gtaccagatc atggaggttc 7740ggtaggctcc tttcttctcg
tccacgagac cagagtttcg caggagaggc ttcaggagca 7800ggtaggagaa
ggtgccaatg aggatttcgg gatcggtgac ggcagcccag attcgctcgc
7860cagcgtcgtt ggccagagcc atggtgc 788718611766DNAArtificial
SequencePlasmid pKR1060 186ggagatccaa gcttttgatc catgcccttc
atttgccgct tattaattaa tttggtaaca 60gtccgtacta atcagttact tatccttccc
ccatcataat taatcttggt agtctcgaat 120gccacaacac tgactagtct
cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180aacacaatga
gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac
240gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc
gcgtcaagaa 300aaaaaaactg gaccccaaaa gccatgcaca acaacacgta
ctcacaaagg tgtcaatcga 360gcagcccaaa acattcacca actcaaccca
tcatgagccc tcacatttgt tgtttctaac 420ccaacctcaa actcgtattc
tcttccgcca cctcattttt gtttatttca acacccgtca 480aactgcatgc
caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa
540tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc
ctagtacacc 600gtattaaaga atttaagata tactgcggcc gcaccatggc
tctggccaac gacgctggcg 660agcgaatctg ggctgccgtc accgatcccg
aaatcctcat tggcaccttc tcctacctgc 720tcctgaagcc tctcctgcga
aactctggtc tcgtggacga gaagaaagga gcctaccgaa 780cctccatgat
ctggtacaac gtcctcctgg ctctcttctc tgccctgtcc ttctacgtga
840ctgccaccgc tctcggctgg gactacggta ctggagcctg gctgcgaaga
cagaccggtg 900atactcccca gcctctcttt cagtgtccct ctcctgtctg
ggactccaag ctgttcacct 960ggactgccaa ggccttctac tattctaagt
acgtggagta cctcgacacc gcttggctgg 1020tcctcaaggg caagcgagtg
tcctttctgc aggccttcca tcactttgga gctccctggg 1080acgtctacct
cggcattcga ctgcacaacg agggtgtgtg gatcttcatg ttctttaact
1140cgttcattca caccatcatg tacacctact atggactgac tgccgctggc
tacaagttca 1200aggccaagcc tctgatcact gccatgcaga tttgccagtt
cgtcggtggc tttctcctgg 1260tctgggacta catcaacgtt ccctgcttca
actctgacaa gggcaagctg ttctcctggg 1320ctttcaacta cgcctacgtc
ggatctgtct ttctcctgtt ctgtcacttc ttttaccagg 1380acaacctggc
caccaagaaa tccgctaagg ctggtaagca gctttagcgg ccgcaagtat
1440gaactaaaat gcatgtaggt gtaagagctc atggagagca tggaatattg
tatccgacca 1500tgtaacagta taataactga gctccatctc acttcttcta
tgaataaaca aaggatgtta 1560tgatatatta acactctatc tatgcacctt
attgttctat gataaatttc ctcttattat 1620tataaatcat ctgaatcgtg
acggcttatg gaatgcttca aatagtacaa aaacaaatgt 1680gtactataag
actttctaaa caattctaac cttagcattg tgaacgagac ataagtgtta
1740agaagacata acaattataa tggaagaagt ttgtctccat ttatatatta
tatattaccc 1800acttatgtat tatattagga tgttaaggag acataacaat
tataaagaga gaagtttgta 1860tccatttata tattatatac tacccattta
tatattatac ttatccactt atttaatgtc 1920tttataaggt ttgatccatg
atatttctaa tattttagtt gatatgtata tgaaagggta 1980ctatttgaac
tctcttactc tgtataaagg ttggatcatc cttaaagtgg gtctatttaa
2040ttttattgct tcttacagat aaaaaaaaaa ttatgagttg gtttgataaa
atattgaagg 2100atttaaaata ataataaata acatataata tatgtatata
aatttattat aatataacat 2160ttatctataa aaaagtaaat attgtcataa
atctatacaa tcgtttagcc ttgctggacg 2220aatctcaatt atttaaacga
gagtaaacat atttgacttt ttggttattt aacaaattat 2280tatttaacac
tatatgaaat tttttttttt atcagcaaag aataaaatta aattaagaag
2340gacaatggtg tcccaatcct tatacaacca acttccacaa gaaagtcaag
tcagagacaa 2400caaaaaaaca agcaaaggaa attttttaat ttgagttgtc
ttgtttgctg cataatttat 2460gcagtaaaac actacacata acccttttag
cagtagagca atggttgacc gtgtgcttag 2520cttcttttat tttatttttt
tatcagcaaa gaataaataa aataaaatga gacacttcag 2580ggatgtttca
acaagcttgg cgcgccgttc tatagtgtca cctaaatcgt atgtgtatga
2640tacataaggt tatgtattaa ttgtagccgc gttctaacga caatatgtcc
atatggtgca 2700ctctcagtac aatctgctct gatgccgcat agttaagcca
gccccgacac ccgccaacac 2760ccgctgacgc gccctgacgg gcttgtctgc
tcccggcatc cgcttacaga caagctgtga 2820ccgtctccgg gagctgcatg
tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 2880gaaagggcct
cgtgatacgc ctatttttat aggttaatgt catgaccaaa atcccttaac
2940gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag 3000atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg 3060tggtttgttt gccggatcaa gagctaccaa
ctctttttcc gaaggtaact ggcttcagca 3120gagcgcagat accaaatact
gtccttctag tgtagccgta gttaggccac cacttcaaga 3180actctgtagc
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
3240gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc 3300agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca 3360ccgaactgag atacctacag cgtgagcatt
gagaaagcgc cacgcttccc gaagggagaa 3420aggcggacag gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc 3480cagggggaaa
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
3540gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg 3600cctttttacg gttcctggcc ttttgctggc cttttgctca
catgttcttt cctgcgttat 3660cccctgattc tgtggataac cgtattaccg
cctttgagtg agctgatacc gctcgccgca 3720gccgaacgac cgagcgcagc
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 3780aaccgcctct
ccccgcgcgt tggccgattc attaatgcag gttgatcgat tcgacatcga
3840tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg
ctatattttg 3900ttttctatcg cgtattaaat gtataattgc gggactctaa
tcataaaaac ccatctcata 3960aataacgtca tgcattacat gttaattatt
acatgcttaa cgtaattcaa cagaaattat 4020atgataatca tcgcaagacc
ggcaacagga ttcaatctta agaaacttta ttgccaaatg 4080tttgaacgat
ctgcttcgac gcactccttc tttaggtacc tcactattcc tttgccctcg
4140gacgagtgct ggggcgtcgg tttccactat cggcgagtac
ttctacacag ccatcggtcc 4200agacggccgc gcttctgcgg gcgatttgtg
tacgcccgac agtcccggct ccggatcgga 4260cgattgcgtc gcatcgaccc
tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc 4320tctgatagag
ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg
4380caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa
gccaaccacg 4440gcctccagaa gaagatgttg gcgacctcgt attgggaatc
cccgaacatc gcctcgctcc 4500agtcaatgac cgctgttatg cggccattgt
ccgtcaggac attgttggag ccgaaatccg 4560cgtgcacgag gtgccggact
tcggggcagt cctcggccca aagcatcagc tcatcgagag 4620cctgcgcgac
ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg
4680gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt
ccttgcggtc 4740cgaatgggcc gaacccgctc gtctggctaa gatcggccgc
agcgatcgca tccatggcct 4800ccgcgaccgg ctgcagaaca gcgggcagtt
cggtttcagg caggtcttgc aacgtgacac 4860cctgtgcacg gcgggagatg
caataggtca ggctctcgct gaattcccca atgtcaagca 4920cttccggaat
cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga
4980aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca
tcgaagctga 5040aagcacgaga ttcttcgccc tccgagagct gcatcaggtc
ggagacgctg tcgaactttt 5100cgatcagaaa cttctcgaca gacgtcgcgg
tgagttcagg ctttttcatg gtttaataag 5160aagagaaaag agttcttttg
ttatggctga agtaatagag aaatgagctc gagcgtgtcc 5220tctccaaatg
aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt
5280gtgcgtcatc ccttacgtca gtggagatgt cacatcaatc cacttgcttt
gaagacgtgg 5340ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg
gggtccatct ttgggaccac 5400tgtcggcaga ggcatcttga atgatagcct
ttcctttatc gcaatgatgg catttgtagg 5460agccaccttc cttttctact
gtcctttcga tgaagtgaca gatagctggg caatggaatc 5520cgaggaggtt
tcccgaaatt atcctttgtt gaaaagtctc aatagccctt tggtcttctg
5580agactgtatc tttgacattt ttggagtaga ccagagtgtc gtgctccacc
atgttgacga 5640agattttctt cttgtcattg agtcgtaaaa gactctgtat
gaactgttcg ccagtcttca 5700cggcgagttc tgttagatcc tcgatttgaa
tcttagactc catgcatggc cttagattca 5760gtaggaacta cctttttaga
gactccaatc tctattactt gccttggttt atgaagcaag 5820ccttgaatcg
tccatactgg aatagtactt ctgatcttga gaaatatgtc tttctctgtg
5880ttcttgatgc aattagtcct gaatcttttg actgcatctt taaccttctt
gggaaggtat 5940ttgatctcct ggagattgtt actcgggtag atcgtcttga
tgagacctgc tgcgtaggcc 6000tctctaacca tctgtgggtc agcattcttt
ctgaaattga agaggctaac cttctcatta 6060tcagtggtga acatagtgtc
gtcaccttca ccttcgaact tccttcctag atcgtaaaga 6120tagaggaaat
cgtccattgt aatctccggg gcaaaggaga tctcttttgg ggctggatca
6180ctgctgggcc ttttggttcc tagcgtgagc cagtgggctt tttgctttgg
tgggcttgtt 6240agggccttag caaagctctt gggcttgagt tgagcttctc
ctttggggat gaagttcaac 6300ctgtctgttt gctgacttgt tgtgtacgcg
tcagctgctg ctcttgcctc tgtaatagtg 6360gcaaatttct tgtgtgcaac
tccgggaacg ccgtttgttg ccgcctttgt acaaccccag 6420tcatcgtata
taccggcatg tggaccgtta tacacaacgt agtagttgat atgagggtgt
6480tgaatacccg attctgctct gagaggagca actgtgctgt taagctcaga
tttttgtggg 6540attggaattg gatcgatctc gatcccgcga aattaatacg
actcactata gggagaccac 6600aacggtttcc ctctagaaat aattttgttt
aactttaaga aggagatata cccatggaaa 6660agcctgaact caccgcgacg
tctgtcgaga agtttctgat cgaaaagttc gacagcgtct 6720ccgacctgat
gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
6780ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
gatcgttatg 6840tttatcggca ctttgcatcg gccgcgctcc cgattccgga
agtgcttgac attggggaat 6900tcagcgagag cctgacctat tgcatctccc
gccgtgcaca gggtgtcacg ttgcaagacc 6960tgcctgaaac cgaactgccc
gctgttctgc agccggtcgc ggaggctatg gatgcgatcg 7020ctgcggccga
tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
7080aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
tatcactggc 7140aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
ggctctcgat gagctgatgc 7200tttgggccga ggactgcccc gaagtccggc
acctcgtgca cgcggatttc ggctccaaca 7260atgtcctgac ggacaatggc
cgcataacag cggtcattga ctggagcgag gcgatgttcg 7320gggattccca
atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
7380agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
ccgcggctcc 7440gggcgtatat gctccgcatt ggtcttgacc aactctatca
gagcttggtt gacggcaatt 7500tcgatgatgc agcttgggcg cagggtcgat
gcgacgcaat cgtccgatcc ggagccggga 7560ctgtcgggcg tacacaaatc
gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 7620aagtactcgc
cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagt
7680gaggtacagc ttggatcgat ccggctgcta acaaagcccg aaaggaagct
gagttggctg 7740ctgccaccgc tgagcaataa ctagcataac cccttggggc
ctctaaacgg gtcttgaggg 7800gttttttgct gaaaggagga actatatccg
gatgatcggg cgcgccgtcg acggatccgt 7860acgagatccg gccggccaga
tcctgcagcc cgggggatcc gcccacgtac gcaaaggcaa 7920agatttaaac
tcgaaaacat tacaaaagtc tcaaaacaga ggcaaggcca tgcacaaagc
7980acactctaag tgcttccatt gcctactaag tagggtacgt acacgatcac
cattcaccag 8040tgatgatctt tattaatata caacacactc agagacagct
tatgttatag ctagctagca 8100taaactatca catcatgtgt tagtacgaca
agtgacaaca ttgcttttaa cttcgcggcc 8160ttggatcctc tagaccggat
ataatgagcc gtaaacaaag atgattaagt agtaattaat 8220acgtactagt
aaaagtggca aaagataacg agaaagaacc aatttctttg cattcggcct
8280tagcggaagg catatataag ctttgattat tttatttagt gtaatgattt
cgtacaacca 8340aagcatttat ttagtactct cacacttgtg tcgcggccgc
ttactgagcc ttggcaccgg 8400gctgcttctc ggccattcga gcgaactggg
acaggtatcg gagcaggatg acgagacctt 8460catggggcag agggtttcgg
taggggaggt tgtgcttctg gcacagctgt tccacctggt 8520aggaaacggc
agtgaggttg tgtcgaggca gggtgggcca gagatggtgc tcgatctggt
8580agttcaggcc tccaaagaac cagtcagtaa tgatgcctcg tcgaatgttc
atggtctcat 8640ggatctgacc cacagagaag ccatgtccgt cccagacgga
atcaccgatc ttctccagag 8700ggtagtggtt catgaagacc acgatggcaa
ttccgaagcc accgacgagc tcggaaacaa 8760agaacaccag catcgaggtc
aggatggagg gcataaagaa gaggtggaac agggtcttga 8820gagtccagtg
cagagcgagt ccaatggcct ctttcttgta ctgagatcgg tagaactggt
8880tgtctcggtc cttgagggat cgaacggtca gcacagactg gaaacaccag
atgaatcgca 8940ggagaataca gatgaccagg aaatagtact gttggaactg
aatgagcttt cgggagatgg 9000gagaagctcg agtgacatcg tcctcggacc
aggcgagcag aggcaggtta tcaatgtcgg 9060gatcgtgacc ctgaacgttg
gtagcagaat gatgggcgtt gtgtctgtcc ttccaccagg 9120tcacggagaa
gccctggagt ccgttgccaa agaccagacc caggacgtta ttccagtttc
9180ggttcttgaa ggtctggtgg tggcagatgt catgagacag ccatcccatt
tgctggtagt 9240gcataccgag cacgagagca ccaatgaagt acaggtggta
ctggaccagc atgaagaagg 9300caagcacgcc aagacccagg gtggtcaaga
tcttgtacga gtaccagagg ggagaggcgt 9360caaacatgcc agtggcgatc
agctcttctc ggagctttcg gaaatcctcc tgagcttcgt 9420tgacggcagc
ctggggaggc agctcggaag cctggttgat cttgggcatt cgcttgagct
9480tgtcgaaggc ttcctgagag tgcataacca tgaaggcgtc agtagcatct
cgtccctggt 9540agttctcaat gatttcagct ccaccagggt ggaagttcac
ccaagcggag acgtcgtaca 9600cctttccgtc gatgacgagg ggcagagcct
gtcgagaagc cttcaccatg gtgcggccgc 9660ttggggggct atggaagact
ttcttagtta gttgtgtgaa taagcaatgt tgggagaatc 9720gggactactt
ataggatagg aataaaacag aaaagtatta agtgctaatg aaatatttag
9780actgataatt aaaatcttca cgtatgtcca cttgatataa aaacgtcagg
aataaaggaa 9840gtacagtaga atttaaaggt actcttttta tatatacccg
tgttctcttt ttggctagct 9900agttgcataa aaaataatct atatttttat
cattatttta aatatcttat gagatggtaa 9960atatttatca taattttttt
tactattatt tattatttgt gtgtgtaata catatagaag 10020ttaattacaa
attttattta ctttttcatt attttgatat gattcaccat taatttagtg
10080ttattattta taatagttca ttttaatctt tttgtatata ttatgcgtgc
agtacttttt 10140tcctacatat aactactatt acattttatt tatataatat
ttttattaat gaattttcgt 10200gataatatgt aatattgttc attattattt
cagatttttt aaaaatattt gtgttattat 10260ttatgaaata tgtaattttt
ttagtatttg attttatgat gataaagtgt tctaaattca 10320aaagaagggg
gaaagcgtaa acattaaaaa acgtcatcaa acaaaaacaa aatcttgtta
10380ataaagataa aactgtttgt tttgatcact gttatttcgt aatataaaaa
cattatttat 10440atttatattg ttgacaacca aatttgccta tcaaatctaa
ccaatataat gcatgcgtgg 10500caggtaatgt actaccatga acttaagtca
tgacataata aaccgtgaat ctgaccaatg 10560catgtaccta nctaaattgt
atttgtgaca cgaagcaaat gattcaattc acaatggaga 10620tgggaaacaa
ataatgaaga acccagaact aagaaagctt ttctgaaaaa taaaataaag
10680gcaatgtcaa aagtatactg catcatcagt ccagaaagca catgatattt
ttttatcagt 10740atcaatgcag ctagttttat tttacaatat cgatatagct
agtttaaata tattgcagct 10800agatttataa atatttgtgt tattatttat
catttgtgta atcctgtttt tagtatttta 10860gtttatatat gatgataatg
tattccaaat ttaaaagaag ggaaataaat ttaaacaaga 10920aaaaaagtca
tcaaacaaaa aacaaatgaa agggtggaaa gatgttacca tgtaatgtga
10980atgttacagt atttctttta ttatagagtt aacaaattaa ctaatatgat
tttgttaata 11040atgataaaat atttttttta ttattatttc ataatataaa
aatagtttac ttaatataaa 11100aaaaattcta tcgttcacaa caaagttggc
cacctaattt aaccatgcat gtacccatgg 11160accatattag gtaaccatca
aacctgatga agagataaag agatgaagac ttaagtcata 11220acacaaaacc
ataaaaaaca aaaatacaat caaccgtcaa tctgaccaat gcatgaaaaa
11280gctgcaatag tgagtggcga cacaaagcac atgattttct tacaacggag
ataaaaccaa 11340aaaaatattt catgaacaac ctagaacaaa taaagctttt
atataataaa tatataaata 11400aataaaggct atggaataat atacttcaat
atatttggat taaataaatt gttggcgggg 11460ttgatatatt tatacacacc
taaagtcact tcaatctcat tttcacttaa cttttatttt 11520ttttttcttt
ttatttatca taaagagaat attgataata tactttttaa catattttta
11580tgacattttt tattggtgaa aacttattaa aaatcataaa ttttgtaagt
tagatttatt 11640taaagagttc ctcttcttat tttaaatttt ttaataaatt
tttaaataac taaaatttgt 11700gttaaaaatg ttaaaaaatg tgttattaac
ccttctcttc gaggacgtac gtcgagtcga 11760cctgca
1176618730DNAArtificial SequencePrimer oEugEL1-1 187agcggccgca
ccatggaggt ggtgaatgaa 3018830DNAArtificial SequencePrimer oEugEL1-2
188tgcggccgct cactgaatct ttttggctcc 301894311DNAArtificial
SequencePlasmid pKR906 189agcggccgca ccatggaggt ggtgaatgaa
atagtctcaa ttgggcagga agttttaccc 60aaagttgatt atgcccaact ctggagtgat
gccagtcact gtgaggtgct ttacttgtcc 120atcgcatttg tcatcttgaa
gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180atgaagtttg
ttttcaccaa ttacaacctt ctcatgtcca tttattcgtt gggatcattc
240ctctcaatgg catatgccat gtacaccatc ggtgttatgt ctgacaactg
cgagaaggct 300tttgacaaca acgtcttcag gatcaccacg cagttgttct
atttgagcaa gttcctggag 360tatattgact ccttctattt gccactgatg
ggcaagcctc tgacctggtt gcaattcttc 420catcatttgg gggcaccgat
ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480tggatttttg
tgctgttgaa tggtttcatc cactggatca tgtacggtta ttattggacc
540agattgatca agctgaagtt ccccatgcca aaatccctga ttacatcaat
gcagatcatt 600caattcaatg ttggtttcta cattgtctgg aagtacagga
acattccctg ttatcgccaa 660gatgggatga ggatgtttgg ctggttcttc
aattactttt atgttggcac agtcttgtgt 720ttgttcttga atttctatgt
gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780attcagtgag
cggccgcacc tgaattccag cacactggcg gccgttacta gtggatccga
840gctcggtacc aagcttgatg catagcttga gtattctaac gcgtcaccta
aatagcttgg 900cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
tccgctcaca attccacaca 960acatacgagc cggaagcata aagtgtaaag
cctggggtgc ctaatgagtg agctaactca 1020cattaattgc gttgcgctca
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080attaatgaat
cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
1140cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
tcagctcact 1200caaaggcggt aatacggtta tccacagaat caggggataa
cgcaggaaag aacatgtgag 1260caaaaggcca gcaaaagccc aggaaccgta
aaaaggccgc gttgctggcg tttttccata 1320ggctccgccc ccctgacgag
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380cgacaggact
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
1440ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
agcgtggcgc 1500tttctcatag ctcacgctgt aggtatctca gttcggtgta
ggtcgttcgc tccaagctgg 1560gctgtgtgca cgaacccccc gttcagcccg
accgctgcgc cttatccggt aactatcgtc 1620ttgagtccaa cccggtaaga
cacgacttat cgccactggc agcagccact ggtaacagga 1680ttagcagagc
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
1740gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
accttcggaa 1800aaagagttgg tagctcttga tccggcaaac aaaccaccgc
tggtagcggt ggtttttttg 1860tttgcaagca gcagattacg cgcagaaaaa
aaggatctca agaagatcct ttgatctttt 1920ctacggggtc tgacgctcag
tggaacgaaa actcacgtta agggattttg gtcatgagat 1980tatcaaaaag
gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgtc
2040agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc
agggcgaact 2100cccgccccca cggctgctcg ccgatctcgg tcatggccgg
cccggaggcg tcccggaagt 2160tcgtggacac gacctccgac cactcggcgt
acagctcgtc caggccgcgc acccacaccc 2220aggccagggt gttgtccggc
accacctggt cctggaccgc gctgatgaac agggtcacgt 2280cgtcccggac
cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt
2340cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga
acggcactgg 2400tcaacttggc catggtggcc ctcctcacgt gctattattg
aagcatttat cagggttatt 2460gtctcatgag cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc 2520gcacatttcc ccgaaaagtg
ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580gagaaaatac
cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag
2640gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
cgaggaagcg 2700gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
gtagccaacg ctatgtcctg 2760atagcggtcc gccacaccca gccggccaca
gtcgatgaat ccagaaaagc ggccattttc 2820caccatgata ttcggcaagc
aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880catgctcgcc
ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
2940cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
cgatgcgatg 3000tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
gtatgcagcc gccgcattgc 3060atcagccatg atggatactt tctcggcagg
agcaaggtga gatgacagga gatcctgccc 3120cggcacttcg cccaatagca
gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180tgcgcaagga
acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc
3240attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
gcgctgacag 3300ccggaacacg gcggcatcag agcagccgat tgtctgttgt
gcccagtcat agccgaatag 3360cctctccacc caagcggccg gagaacctgc
gtgcaatcca tcttgttcaa tcatgcgaaa 3420cgatcctcat cctgtctctt
gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480cgagaaagcc
atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
3540tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc
gccatgtaag 3600cccactgcaa gctacctgct ttctctttgc gcttgcgttt
tcccttgtcc agatagccca 3660gtagctgaca ttcatccggg gtcagcaccg
tttctgcgga ctggctttct acgtgaaaag 3720gatctaggtg aagatccttt
ttgataatct catgcctgac atttatattc cccagaacat 3780caggttaatg
gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc
3840gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
tttcatcccc 3900gatatgcacc accgggtaaa gttcacggga gactttatct
gacagcagac gtgcactggc 3960cagggggatc accatccgtc gccccggcgt
gtcaataata tcactctgta catccacaaa 4020cagacgataa cggctctctc
ttttataggt gtaaacctta aactgccgta cgtataggct 4080gcgcaactgt
tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
4140agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
agtcacgacg 4200ttgtaaaacg acggccagtg aattgtaata cgactcacta
tagggcgaat tgggccctct 4260agatgcatgc tcgagcggcc gccagtgtga
tggatatctg cagaattcag g 43111907085DNAArtificial SequencePlasmid
pKR72 190gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt
tcagcaaaaa 60acccctcaag acccgtttag aggccccaag gggttatgct agttattgct
cagcggtggc 120agcagccaac tcagcttcct ttcgggcttt gttagcagcc
ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg gacgagtgct
ggggcgtcgg tttccactat cggcgagtac 240ttctacacag ccatcggtcc
agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300agtcccggct
ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc
360gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga
gcatatacgc 420ccggagccgc ggcgatcctg caagctccgg atgcctccgc
tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg gcctccagaa
gaagatgttg gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc
agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600attgttggag
ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca
660aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt
ccatcacagt 720ttgccagtga tacacatggg gatcagcaat cgcgcatatg
aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc cgaatgggcc
gaacccgctc gtctggctaa gatcggccgc 840agcgatcgca tccatagcct
ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900caggtcttgc
aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct
960gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa
agtgccgata 1020aacataacga tctttgtaga aaccatcggc gcagctattt
acccgcagga catatccacg 1080ccctcctaca tcgaagctga aagcacgaga
ttcttcgccc tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt
cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200cttttccatg
ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg
1260ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
tccaattcca 1320atcccacaaa aatctgagct taacagcaca gttgctcctc
tcagagcaga atcgggtatt 1380caacaccctc atatcaacta ctacgttgtg
tataacggtc cacatgccgg tatatacgat 1440gactggggtt gtacaaaggc
ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500gccactatta
cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac
1560aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt
tgctaaggcc 1620ctaacaagcc caccaaagca aaaagcccac tggctcacgc
taggaaccaa aaggcccagc 1680agtgatccag ccccaaaaga gatctccttt
gccccggaga ttacaatgga cgatttcctc 1740tatctttacg atctaggaag
gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800gataatgaga
aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga
1860gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct
ccaggagatc 1920aaataccttc ccaagaaggt taaagatgca gtcaaaagat
tcaggactaa ttgcatcaag 1980aacacagaga aagacatatt tctcaagatc
agaagtacta ttccagtatg gacgattcaa 2040ggcttgcttc ataaaccaag
gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100actgaatcta
aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc
2160cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca
agaagaaaat 2220cttcgtcaac atggtggagc acgacactct ggtctactcc
aaaaatgtca aagatacagt 2280ctcagaagac caaagggcta ttgagacttt
tcaacaaagg ataatttcgg gaaacctcct 2340cggattccat tgcccagcta
tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400ctcctacaaa
tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga
2460cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag
aagacgttcc 2520aaccacgtct tcaaagcaag tggattgatg tgacatctcc
actgacgtaa gggatgacgc 2580acaatcccac tatccttcgc aagacccttc
ctctatataa ggaagttcat ttcatttgga 2640gaggacacgc tcgagctcat
ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700ttcttattaa
accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
2760cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
aatctcgtgc
2820tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
gcgccgatgg 2880tttctacaaa gatcgttatg tttatcggca ctttgcatcg
gccgcgctcc cgattccgga 2940agtgcttgac attggggaat tcagcgagag
cctgacctat tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc
tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060ggaggccatg
gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
3120cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
cgattgctga 3180tccccatgtg tatcactggc aaactgtgat ggacgacacc
gtcagtgcgt ccgtcgcgca 3240ggctctcgat gagctgatgc tttgggccga
ggactgcccc gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca
atgtcctgac ggacaatggc cgcataacag cggtcattga 3360ctggagcgag
gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
3420gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
atccggagct 3480tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
ggtcttgacc aactctatca 3540gagcttggtt gacggcaatt tcgatgatgc
agcttgggcg cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga
ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660ctggaccgat
ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
3720tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc
agatcgttca 3780aacatttggc aataaagttt cttaagattg aatcctgttg
ccggtcttgc gatgattatc 3840atataatttc tgttgaatta cgttaagcat
gtaataatta acatgtaatg catgacgtta 3900tttatgagat gggtttttat
gattagagtc ccgcaattat acatttaata cgcgatagaa 3960aacaaaatat
agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta
4020gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg
ggagaggcgg 4080tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc
tcaatgctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg
acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag
agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
4920acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca
cgaggccctt 4980tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
cacatgcagc tcccggagac 5040ggtcacagct tgtctgtaag cggatgccgg
gagcagacaa gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg
gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160agtgcaccat
atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg
5220tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt
gttgaaacat 5280ccctgaagtg tctcatttta ttttatttat tctttgctga
taaaaaaata aaataaaaga 5340agctaagcac acggtcaacc attgctctac
tgctaaaagg gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac
aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460tgttgtctct
gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg
5520tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata
tagtgttaaa 5580taataatttg ttaaataacc aaaaagtcaa atatgtttac
tctcgtttaa ataattgaga 5640ttcgtccagc aaggctaaac gattgtatag
atttatgaca atatttactt ttttatagat 5700aaatgttata ttataataaa
tttatataca tatattatat gttatttatt attattttaa 5760atccttcaat
attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa
5820aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga
gagttcaaat 5880agtacccttt catatacata tcaactaaaa tattagaaat
atcatggatc aaaccttata 5940aagacattaa ataagtggat aagtataata
tataaatggg tagtatataa tatataaatg 6000gatacaaact tctctcttta
taattgttat gtctccttaa catcctaata taatacataa 6060gtgggtaata
tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt
6120cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa
gtcttatagt 6180acacatttgt ttttgtacta tttgaagcat tccataagcc
gtcacgattc agatgattta 6240taataataag aggaaattta tcatagaaca
ataaggtgca tagatagagt gttaatatat 6300cataacatcc tttgtttatt
catagaagaa gtgagatgga gctcagttat tatactgtta 6360catggtcgga
tacaatattc catgctctcc atgagctctt acacctacat gcattttagt
6420tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag
gatattgaac 6480tggttcttga tgatgaaaac ctgggccgag attgcagcta
tttatagtca taggtcttgt 6540taacatgcat ggacatttgg ccacggggtg
gcatgcagtt tgacgggtgt tgaaataaac 6600aaaaatgagg tggcggaaga
gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660gggctcatga
tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag
6720tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc
ggcgatcctg 6780atcagctagt ggataagtga tgtccactgt gtgtgattgc
gtttttgttt gaattttatg 6840aacttagaca ttgctatgca aaggatactc
tcattgtgtt ttgtcttctt ttgttccttg 6900gctttttctt atgatccaag
agactagtca gtgttgtggc attcgagact accaagatta 6960attatgatgg
gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata
7020agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct
ggccggccgg 7080atctc 70851917873DNAArtificial SequencePlasmid
pKR1010 191cgcgccgttc tatagtgtca cctaaatcgt atgtgtatga tacataaggt
tatgtattaa 60ttgtagccgc gttctaacga caatatgtcc atatggtgca ctctcagtac
aatctgctct 120gatgccgcat agttaagcca gccccgacac ccgccaacac
ccgctgacgc gccctgacgg 180gcttgtctgc tcccggcatc cgcttacaga
caagctgtga ccgtctccgg gagctgcatg 240tgtcagaggt tttcaccgtc
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 300ctatttttat
aggttaatgt catgaccaaa atcccttaac gtgagttttc gttccactga
360gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
tctgcgcgta 420atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
tggtttgttt gccggatcaa 480gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat accaaatact 540gtccttctag tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca 600tacctcgctc
tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
660accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
ctgaacgggg 720ggttcgtgca cacagcccag cttggagcga acgacctaca
ccgaactgag atacctacag 780cgtgagcatt gagaaagcgc cacgcttccc
gaagggagaa aggcggacag gtatccggta 840agcggcaggg tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat 900ctttatagtc
ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
960tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
gttcctggcc 1020ttttgctggc cttttgctca catgttcttt cctgcgttat
cccctgattc tgtggataac 1080cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac cgagcgcagc 1140gagtcagtga gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 1200tggccgattc
attaatgcag gttgatcgat tcgacatcga tctagtaaca tagatgacac
1260cgcgcgcgat aatttatcct agtttgcgcg ctatattttg ttttctatcg
cgtattaaat 1320gtataattgc gggactctaa tcataaaaac ccatctcata
aataacgtca tgcattacat 1380gttaattatt acatgcttaa cgtaattcaa
cagaaattat atgataatca tcgcaagacc 1440ggcaacagga ttcaatctta
agaaacttta ttgccaaatg tttgaacgat ctgcttcgac 1500gcactccttc
tttaggtacc tcactattcc tttgccctcg gacgagtgct ggggcgtcgg
1560tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc
gcttctgcgg 1620gcgatttgtg tacgcccgac agtcccggct ccggatcgga
cgattgcgtc gcatcgaccc 1680tgcgcccaag ctgcatcatc gaaattgccg
tcaaccaagc tctgatagag ttggtcaaga 1740ccaatgcgga gcatatacgc
ccggagccgc ggcgatcctg caagctccgg atgcctccgc 1800tcgaagtagc
gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg
1860gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac
cgctgttatg 1920cggccattgt ccgtcaggac attgttggag ccgaaatccg
cgtgcacgag gtgccggact 1980tcggggcagt cctcggccca aagcatcagc
tcatcgagag cctgcgcgac ggacgcactg 2040acggtgtcgt ccatcacagt
ttgccagtga tacacatggg gatcagcaat cgcgcatatg 2100aaatcacgcc
atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
2160gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg
ctgcagaaca 2220gcgggcagtt cggtttcagg caggtcttgc aacgtgacac
cctgtgcacg gcgggagatg 2280caataggtca ggctctcgct gaattcccca
atgtcaagca cttccggaat cgggagcgcg 2340gccgatgcaa agtgccgata
aacataacga tctttgtaga aaccatcggc gcagctattt 2400acccgcagga
catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
2460tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa
cttctcgaca 2520gacgtcgcgg tgagttcagg ctttttcatg gtttaataag
aagagaaaag agttcttttg 2580ttatggctga agtaatagag aaatgagctc
gagcgtgtcc tctccaaatg aaatgaactt 2640ccttatatag aggaagggtc
ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca 2700gtggagatgt
cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc
2760acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga
ggcatcttga 2820atgatagcct ttcctttatc gcaatgatgg catttgtagg
agccaccttc cttttctact 2880gtcctttcga tgaagtgaca gatagctggg
caatggaatc cgaggaggtt tcccgaaatt 2940atcctttgtt gaaaagtctc
aatagccctt tggtcttctg agactgtatc tttgacattt 3000ttggagtaga
ccagagtgtc gtgctccacc atgttgacga agattttctt cttgtcattg
3060agtcgtaaaa gactctgtat gaactgttcg ccagtcttca cggcgagttc
tgttagatcc 3120tcgatttgaa tcttagactc catgcatggc cttagattca
gtaggaacta cctttttaga 3180gactccaatc tctattactt gccttggttt
atgaagcaag ccttgaatcg tccatactgg 3240aatagtactt ctgatcttga
gaaatatgtc tttctctgtg ttcttgatgc aattagtcct 3300gaatcttttg
actgcatctt taaccttctt gggaaggtat ttgatctcct ggagattgtt
3360actcgggtag atcgtcttga tgagacctgc tgcgtaggcc tctctaacca
tctgtgggtc 3420agcattcttt ctgaaattga agaggctaac cttctcatta
tcagtggtga acatagtgtc 3480gtcaccttca ccttcgaact tccttcctag
atcgtaaaga tagaggaaat cgtccattgt 3540aatctccggg gcaaaggaga
tctcttttgg ggctggatca ctgctgggcc ttttggttcc 3600tagcgtgagc
cagtgggctt tttgctttgg tgggcttgtt agggccttag caaagctctt
3660gggcttgagt tgagcttctc ctttggggat gaagttcaac ctgtctgttt
gctgacttgt 3720tgtgtacgcg tcagctgctg ctcttgcctc tgtaatagtg
gcaaatttct tgtgtgcaac 3780tccgggaacg ccgtttgttg ccgcctttgt
acaaccccag tcatcgtata taccggcatg 3840tggaccgtta tacacaacgt
agtagttgat atgagggtgt tgaatacccg attctgctct 3900gagaggagca
actgtgctgt taagctcaga tttttgtggg attggaattg gatcgatctc
3960gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc
ctctagaaat 4020aattttgttt aactttaaga aggagatata cccatggaaa
agcctgaact caccgcgacg 4080tctgtcgaga agtttctgat cgaaaagttc
gacagcgtct ccgacctgat gcagctctcg 4140gagggcgaag aatctcgtgc
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 4200gtaaatagct
gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
4260gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
cctgacctat 4320tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
tgcctgaaac cgaactgccc 4380gctgttctgc agccggtcgc ggaggctatg
gatgcgatcg ctgcggccga tcttagccag 4440acgagcgggt tcggcccatt
cggaccgcaa ggaatcggtc aatacactac atggcgtgat 4500ttcatatgcg
cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
4560gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
ggactgcccc 4620gaagtccggc acctcgtgca cgcggatttc ggctccaaca
atgtcctgac ggacaatggc 4680cgcataacag cggtcattga ctggagcgag
gcgatgttcg gggattccca atacgaggtc 4740gccaacatct tcttctggag
gccgtggttg gcttgtatgg agcagcagac gcgctacttc 4800gagcggaggc
atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
4860ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
agcttgggcg 4920cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
ctgtcgggcg tacacaaatc 4980gcccgcagaa gcgcggccgt ctggaccgat
ggctgtgtag aagtactcgc cgatagtgga 5040aaccgacgcc ccagcactcg
tccgagggca aaggaatagt gaggtacagc ttggatcgat 5100ccggctgcta
acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
5160ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
gaaaggagga 5220actatatccg gatgatcggg cgcgccgtcg acggatccgt
acgagatccg gccggccaga 5280tcctgcagga gatccaagct tttgatccat
gcccttcatt tgccgcttat taattaattt 5340ggtaacagtc cgtactaatc
agttacttat ccttccccca tcataattaa tcttggtagt 5400ctcgaatgcc
acaacactga ctagtctctt ggatcataag aaaaagccaa ggaacaaaag
5460aagacaaaac acaatgagag tatcctttgc atagcaatgt ctaagttcat
aaaattcaaa 5520caaaaacgca atcacacaca gtggacatca cttatccact
agctgatcag gatcgccgcg 5580tcaagaaaaa aaaactggac cccaaaagcc
atgcacaaca acacgtactc acaaaggtgt 5640caatcgagca gcccaaaaca
ttcaccaact caacccatca tgagccctca catttgttgt 5700ttctaaccca
acctcaaact cgtattctct tccgccacct catttttgtt tatttcaaca
5760cccgtcaaac tgcatgccac cccgtggcca aatgtccatg catgttaaca
agacctatga 5820ctataaatag ctgcaatctc ggcccaggtt ttcatcatca
agaaccagtt caatatccta 5880gtacaccgta ttaaagaatt taagatatac
tgcggccgca ccatggaggt ggtgaatgaa 5940atagtctcaa ttgggcagga
agttttaccc aaagttgatt atgcccaact ctggagtgat 6000gccagtcact
gtgaggtgct ttacttgtcc atcgcatttg tcatcttgaa gttcactctt
6060ggcccccttg gtccaaaagg tcagtctcgt atgaagtttg ttttcaccaa
ttacaacctt 6120ctcatgtcca tttattcgtt gggatcattc ctctcaatgg
catatgccat gtacaccatc 6180ggtgttatgt ctgacaactg cgagaaggct
tttgacaaca acgtcttcag gatcaccacg 6240cagttgttct atttgagcaa
gttcctggag tatattgact ccttctattt gccactgatg 6300ggcaagcctc
tgacctggtt gcaattcttc catcatttgg gggcaccgat ggatatgtgg
6360ctgttctata attaccgaaa tgaagctgtt tggatttttg tgctgttgaa
tggtttcatc 6420cactggatca tgtacggtta ttattggacc agattgatca
agctgaagtt ccccatgcca 6480aaatccctga ttacatcaat gcagatcatt
caattcaatg ttggtttcta cattgtctgg 6540aagtacagga acattccctg
ttatcgccaa gatgggatga ggatgtttgg ctggttcttc 6600aattactttt
atgttggcac agtcttgtgt ttgttcttga atttctatgt gcaaacgtat
6660atcgtcagga agcacaaggg agccaaaaag attcagtgag cggccgcaag
tatgaactaa 6720aatgcatgta ggtgtaagag ctcatggaga gcatggaata
ttgtatccga ccatgtaaca 6780gtataataac tgagctccat ctcacttctt
ctatgaataa acaaaggatg ttatgatata 6840ttaacactct atctatgcac
cttattgttc tatgataaat ttcctcttat tattataaat 6900catctgaatc
gtgacggctt atggaatgct tcaaatagta caaaaacaaa tgtgtactat
6960aagactttct aaacaattct aaccttagca ttgtgaacga gacataagtg
ttaagaagac 7020ataacaatta taatggaaga agtttgtctc catttatata
ttatatatta cccacttatg 7080tattatatta ggatgttaag gagacataac
aattataaag agagaagttt gtatccattt 7140atatattata tactacccat
ttatatatta tacttatcca cttatttaat gtctttataa 7200ggtttgatcc
atgatatttc taatatttta gttgatatgt atatgaaagg gtactatttg
7260aactctctta ctctgtataa aggttggatc atccttaaag tgggtctatt
taattttatt 7320gcttcttaca gataaaaaaa aaattatgag ttggtttgat
aaaatattga aggatttaaa 7380ataataataa ataacatata atatatgtat
ataaatttat tataatataa catttatcta 7440taaaaaagta aatattgtca
taaatctata caatcgttta gccttgctgg acgaatctca 7500attatttaaa
cgagagtaaa catatttgac tttttggtta tttaacaaat tattatttaa
7560cactatatga aatttttttt tttatcagca aagaataaaa ttaaattaag
aaggacaatg 7620gtgtcccaat ccttatacaa ccaacttcca caagaaagtc
aagtcagaga caacaaaaaa 7680acaagcaaag gaaatttttt aatttgagtt
gtcttgtttg ctgcataatt tatgcagtaa 7740aacactacac ataacccttt
tagcagtaga gcaatggttg accgtgtgct tagcttcttt 7800tattttattt
ttttatcagc aaagaataaa taaaataaaa tgagacactt cagggatgtt
7860tcaacaagct tgg 787319211752DNAArtificial SequencePlasmid
pKR1059 192ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa
tttggtaaca 60gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt
agtctcgaat 120gccacaacac tgactagtct cttggatcat aagaaaaagc
caaggaacaa aagaagacaa 180aacacaatga gagtatcctt tgcatagcaa
tgtctaagtt cataaaattc aaacaaaaac 240gcaatcacac acagtggaca
tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300aaaaaaactg
gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga
360gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt
tgtttctaac 420ccaacctcaa actcgtattc tcttccgcca cctcattttt
gtttatttca acacccgtca 480aactgcatgc caccccgtgg ccaaatgtcc
atgcatgtta acaagaccta tgactataaa 540tagctgcaat ctcggcccag
gttttcatca tcaagaacca gttcaatatc ctagtacacc 600gtattaaaga
atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct
660caattgggca ggaagtttta cccaaagttg attatgccca actctggagt
gatgccagtc 720actgtgaggt gctttacttg tccatcgcat ttgtcatctt
gaagttcact cttggccccc 780ttggtccaaa aggtcagtct cgtatgaagt
ttgttttcac caattacaac cttctcatgt 840ccatttattc gttgggatca
ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900tgtctgacaa
ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt
960tctatttgag caagttcctg gagtatattg actccttcta tttgccactg
atgggcaagc 1020ctctgacctg gttgcaattc ttccatcatt tgggggcacc
gatggatatg tggctgttct 1080ataattaccg aaatgaagct gtttggattt
ttgtgctgtt gaatggtttc atccactgga 1140tcatgtacgg ttattattgg
accagattga tcaagctgaa gttccccatg ccaaaatccc 1200tgattacatc
aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca
1260ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc
ttcaattact 1320tttatgttgg cacagtcttg tgtttgttct tgaatttcta
tgtgcaaacg tatatcgtca 1380ggaagcacaa gggagccaaa aagattcagt
gagcggccgc aagtatgaac taaaatgcat 1440gtaggtgtaa gagctcatgg
agagcatgga atattgtatc cgaccatgta acagtataat 1500aactgagctc
catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac
1560tctatctatg caccttattg ttctatgata aatttcctct tattattata
aatcatctga 1620atcgtgacgg cttatggaat gcttcaaata gtacaaaaac
aaatgtgtac tataagactt 1680tctaaacaat tctaacctta gcattgtgaa
cgagacataa gtgttaagaa gacataacaa 1740ttataatgga agaagtttgt
ctccatttat atattatata ttacccactt atgtattata 1800ttaggatgtt
aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt
1860atatactacc catttatata ttatacttat ccacttattt aatgtcttta
taaggtttga 1920tccatgatat ttctaatatt ttagttgata tgtatatgaa
agggtactat ttgaactctc 1980ttactctgta taaaggttgg atcatcctta
aagtgggtct atttaatttt attgcttctt 2040acagataaaa aaaaaattat
gagttggttt gataaaatat tgaaggattt aaaataataa 2100taaataacat
ataatatatg tatataaatt tattataata taacatttat ctataaaaaa
2160gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc
tcaattattt 2220aaacgagagt aaacatattt gactttttgg ttatttaaca
aattattatt taacactata 2280tgaaattttt ttttttatca gcaaagaata
aaattaaatt aagaaggaca atggtgtccc 2340aatccttata caaccaactt
ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400aaggaaattt
tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta
2460cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc
ttttatttta 2520tttttttatc agcaaagaat aaataaaata aaatgagaca
cttcagggat gtttcaacaa 2580gcttggcgcg ccgttctata gtgtcaccta
aatcgtatgt gtatgataca taaggttatg 2640tattaattgt agccgcgttc
taacgacaat atgtccatat ggtgcactct cagtacaatc 2700tgctctgatg
ccgcatagtt aagccagccc cgacacccgc
caacacccgc tgacgcgccc 2760tgacgggctt gtctgctccc ggcatccgct
tacagacaag ctgtgaccgt ctccgggagc 2820tgcatgtgtc agaggttttc
accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880atacgcctat
ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc
2940cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
tttttttctg 3000cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
cagcggtggt ttgtttgccg 3060gatcaagagc taccaactct ttttccgaag
gtaactggct tcagcagagc gcagatacca 3120aatactgtcc ttctagtgta
gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180cctacatacc
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
3240tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
gtcgggctga 3300acggggggtt cgtgcacaca gcccagcttg gagcgaacga
cctacaccga actgagatac 3360ctacagcgtg agcattgaga aagcgccacg
cttcccgaag ggagaaaggc ggacaggtat 3420ccggtaagcg gcagggtcgg
aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480tggtatcttt
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
3540tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
tttacggttc 3600ctggcctttt gctggccttt tgctcacatg ttctttcctg
cgttatcccc tgattctgtg 3660gataaccgta ttaccgcctt tgagtgagct
gataccgctc gccgcagccg aacgaccgag 3720cgcagcgagt cagtgagcga
ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780gcgcgttggc
cgattcatta atgcaggttg atcgattcga catcgatcta gtaacataga
3840tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt
ctatcgcgta 3900ttaaatgtat aattgcggga ctctaatcat aaaaacccat
ctcataaata acgtcatgca 3960ttacatgtta attattacat gcttaacgta
attcaacaga aattatatga taatcatcgc 4020aagaccggca acaggattca
atcttaagaa actttattgc caaatgtttg aacgatctgc 4080ttcgacgcac
tccttcttta ggtacctcac tattcctttg ccctcggacg agtgctgggg
4140cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac
ggccgcgctt 4200ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg
atcggacgat tgcgtcgcat 4260cgaccctgcg cccaagctgc atcatcgaaa
ttgccgtcaa ccaagctctg atagagttgg 4320tcaagaccaa tgcggagcat
atacgcccgg agccgcggcg atcctgcaag ctccggatgc 4380ctccgctcga
agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
4440atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc
aatgaccgct 4500gttatgcggc cattgtccgt caggacattg ttggagccga
aatccgcgtg cacgaggtgc 4560cggacttcgg ggcagtcctc ggcccaaagc
atcagctcat cgagagcctg cgcgacggac 4620gcactgacgg tgtcgtccat
cacagtttgc cagtgataca catggggatc agcaatcgcg 4680catatgaaat
cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
4740ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc
gaccggctgc 4800agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg
tgacaccctg tgcacggcgg 4860gagatgcaat aggtcaggct ctcgctgaat
tccccaatgt caagcacttc cggaatcggg 4920agcgcggccg atgcaaagtg
ccgataaaca taacgatctt tgtagaaacc atcggcgcag 4980ctatttaccc
gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
5040tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat
cagaaacttc 5100tcgacagacg tcgcggtgag ttcaggcttt ttcatggttt
aataagaaga gaaaagagtt 5160cttttgttat ggctgaagta atagagaaat
gagctcgagc gtgtcctctc caaatgaaat 5220gaacttcctt atatagagga
agggtcttgc gaaggatagt gggattgtgc gtcatccctt 5280acgtcagtgg
agatgtcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct
5340ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc
ggcagaggca 5400tcttgaatga tagcctttcc tttatcgcaa tgatggcatt
tgtaggagcc accttccttt 5460tctactgtcc tttcgatgaa gtgacagata
gctgggcaat ggaatccgag gaggtttccc 5520gaaattatcc tttgttgaaa
agtctcaata gccctttggt cttctgagac tgtatctttg 5580acatttttgg
agtagaccag agtgtcgtgc tccaccatgt tgacgaagat tttcttcttg
5640tcattgagtc gtaaaagact ctgtatgaac tgttcgccag tcttcacggc
gagttctgtt 5700agatcctcga tttgaatctt agactccatg catggcctta
gattcagtag gaactacctt 5760tttagagact ccaatctcta ttacttgcct
tggtttatga agcaagcctt gaatcgtcca 5820tactggaata gtacttctga
tcttgagaaa tatgtctttc tctgtgttct tgatgcaatt 5880agtcctgaat
cttttgactg catctttaac cttcttggga aggtatttga tctcctggag
5940attgttactc gggtagatcg tcttgatgag acctgctgcg taggcctctc
taaccatctg 6000tgggtcagca ttctttctga aattgaagag gctaaccttc
tcattatcag tggtgaacat 6060agtgtcgtca ccttcacctt cgaacttcct
tcctagatcg taaagataga ggaaatcgtc 6120cattgtaatc tccggggcaa
aggagatctc ttttggggct ggatcactgc tgggcctttt 6180ggttcctagc
gtgagccagt gggctttttg ctttggtggg cttgttaggg ccttagcaaa
6240gctcttgggc ttgagttgag cttctccttt ggggatgaag ttcaacctgt
ctgtttgctg 6300acttgttgtg tacgcgtcag ctgctgctct tgcctctgta
atagtggcaa atttcttgtg 6360tgcaactccg ggaacgccgt ttgttgccgc
ctttgtacaa ccccagtcat cgtatatacc 6420ggcatgtgga ccgttataca
caacgtagta gttgatatga gggtgttgaa tacccgattc 6480tgctctgaga
ggagcaactg tgctgttaag ctcagatttt tgtgggattg gaattggatc
6540gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg
gtttccctct 6600agaaataatt ttgtttaact ttaagaagga gatataccca
tggaaaagcc tgaactcacc 6660gcgacgtctg tcgagaagtt tctgatcgaa
aagttcgaca gcgtctccga cctgatgcag 6720ctctcggagg gcgaagaatc
tcgtgctttc agcttcgatg taggagggcg tggatatgtc 6780ctgcgggtaa
atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
6840gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
cgagagcctg 6900acctattgca tctcccgccg tgcacagggt gtcacgttgc
aagacctgcc tgaaaccgaa 6960ctgcccgctg ttctgcagcc ggtcgcggag
gctatggatg cgatcgctgc ggccgatctt 7020agccagacga gcgggttcgg
cccattcgga ccgcaaggaa tcggtcaata cactacatgg 7080cgtgatttca
tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
7140gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
ggccgaggac 7200tgccccgaag tccggcacct cgtgcacgcg gatttcggct
ccaacaatgt cctgacggac 7260aatggccgca taacagcggt cattgactgg
agcgaggcga tgttcgggga ttcccaatac 7320gaggtcgcca acatcttctt
ctggaggccg tggttggctt gtatggagca gcagacgcgc 7380tacttcgagc
ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
7440cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
tgatgcagct 7500tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
ccgggactgt cgggcgtaca 7560caaatcgccc gcagaagcgc ggccgtctgg
accgatggct gtgtagaagt actcgccgat 7620agtggaaacc gacgccccag
cactcgtccg agggcaaagg aatagtgagg tacagcttgg 7680atcgatccgg
ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
7740caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
tttgctgaaa 7800ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg
atccgtacga gatccggccg 7860gccagatcct gcagcccggg ggatccgccc
acgtacgcaa aggcaaagat ttaaactcga 7920aaacattaca aaagtctcaa
aacagaggca aggccatgca caaagcacac tctaagtgct 7980tccattgcct
actaagtagg gtacgtacac gatcaccatt caccagtgat gatctttatt
8040aatatacaac acactcagag acagcttatg ttatagctag ctagcataaa
ctatcacatc 8100atgtgttagt acgacaagtg acaacattgc ttttaacttc
gcggccttgg atcctctaga 8160ccggatataa tgagccgtaa acaaagatga
ttaagtagta attaatacgt actagtaaaa 8220gtggcaaaag ataacgagaa
agaaccaatt tctttgcatt cggccttagc ggaaggcata 8280tataagcttt
gattatttta tttagtgtaa tgatttcgta caaccaaagc atttatttag
8340tactctcaca cttgtgtcgc ggccgcttac tgagccttgg caccgggctg
cttctcggcc 8400attcgagcga actgggacag gtatcggagc aggatgacga
gaccttcatg gggcagaggg 8460tttcggtagg ggaggttgtg cttctggcac
agctgttcca cctggtagga aacggcagtg 8520aggttgtgtc gaggcagggt
gggccagaga tggtgctcga tctggtagtt caggcctcca 8580aagaaccagt
cagtaatgat gcctcgtcga atgttcatgg tctcatggat ctgacccaca
8640gagaagccat gtccgtccca gacggaatca ccgatcttct ccagagggta
gtggttcatg 8700aagaccacga tggcaattcc gaagccaccg acgagctcgg
aaacaaagaa caccagcatc 8760gaggtcagga tggagggcat aaagaagagg
tggaacaggg tcttgagagt ccagtgcaga 8820gcgagtccaa tggcctcttt
cttgtactga gatcggtaga actggttgtc tcggtccttg 8880agggatcgaa
cggtcagcac agactggaaa caccagatga atcgcaggag aatacagatg
8940accaggaaat agtactgttg gaactgaatg agctttcggg agatgggaga
agctcgagtg 9000acatcgtcct cggaccaggc gagcagaggc aggttatcaa
tgtcgggatc gtgaccctga 9060acgttggtag cagaatgatg ggcgttgtgt
ctgtccttcc accaggtcac ggagaagccc 9120tggagtccgt tgccaaagac
cagacccagg acgttattcc agtttcggtt cttgaaggtc 9180tggtggtggc
agatgtcatg agacagccat cccatttgct ggtagtgcat accgagcacg
9240agagcaccaa tgaagtacag gtggtactgg accagcatga agaaggcaag
cacgccaaga 9300cccagggtgg tcaagatctt gtacgagtac cagaggggag
aggcgtcaaa catgccagtg 9360gcgatcagct cttctcggag ctttcggaaa
tcctcctgag cttcgttgac ggcagcctgg 9420ggaggcagct cggaagcctg
gttgatcttg ggcattcgct tgagcttgtc gaaggcttcc 9480tgagagtgca
taaccatgaa ggcgtcagta gcatctcgtc cctggtagtt ctcaatgatt
9540tcagctccac cagggtggaa gttcacccaa gcggagacgt cgtacacctt
tccgtcgatg 9600acgaggggca gagcctgtcg agaagccttc accatggtgc
ggccgcttgg ggggctatgg 9660aagactttct tagttagttg tgtgaataag
caatgttggg agaatcggga ctacttatag 9720gataggaata aaacagaaaa
gtattaagtg ctaatgaaat atttagactg ataattaaaa 9780tcttcacgta
tgtccacttg atataaaaac gtcaggaata aaggaagtac agtagaattt
9840aaaggtactc tttttatata tacccgtgtt ctctttttgg ctagctagtt
gcataaaaaa 9900taatctatat ttttatcatt attttaaata tcttatgaga
tggtaaatat ttatcataat 9960tttttttact attatttatt atttgtgtgt
gtaatacata tagaagttaa ttacaaattt 10020tatttacttt ttcattattt
tgatatgatt caccattaat ttagtgttat tatttataat 10080agttcatttt
aatctttttg tatatattat gcgtgcagta cttttttcct acatataact
10140actattacat tttatttata taatattttt attaatgaat tttcgtgata
atatgtaata 10200ttgttcatta ttatttcaga ttttttaaaa atatttgtgt
tattatttat gaaatatgta 10260atttttttag tatttgattt tatgatgata
aagtgttcta aattcaaaag aagggggaaa 10320gcgtaaacat taaaaaacgt
catcaaacaa aaacaaaatc ttgttaataa agataaaact 10380gtttgttttg
atcactgtta tttcgtaata taaaaacatt atttatattt atattgttga
10440caaccaaatt tgcctatcaa atctaaccaa tataatgcat gcgtggcagg
taatgtacta 10500ccatgaactt aagtcatgac ataataaacc gtgaatctga
ccaatgcatg tacctancta 10560aattgtattt gtgacacgaa gcaaatgatt
caattcacaa tggagatggg aaacaaataa 10620tgaagaaccc agaactaaga
aagcttttct gaaaaataaa ataaaggcaa tgtcaaaagt 10680atactgcatc
atcagtccag aaagcacatg atattttttt atcagtatca atgcagctag
10740ttttatttta caatatcgat atagctagtt taaatatatt gcagctagat
ttataaatat 10800ttgtgttatt atttatcatt tgtgtaatcc tgtttttagt
attttagttt atatatgatg 10860ataatgtatt ccaaatttaa aagaagggaa
ataaatttaa acaagaaaaa aagtcatcaa 10920acaaaaaaca aatgaaaggg
tggaaagatg ttaccatgta atgtgaatgt tacagtattt 10980cttttattat
agagttaaca aattaactaa tatgattttg ttaataatga taaaatattt
11040tttttattat tatttcataa tataaaaata gtttacttaa tataaaaaaa
attctatcgt 11100tcacaacaaa gttggccacc taatttaacc atgcatgtac
ccatggacca tattaggtaa 11160ccatcaaacc tgatgaagag ataaagagat
gaagacttaa gtcataacac aaaaccataa 11220aaaacaaaaa tacaatcaac
cgtcaatctg accaatgcat gaaaaagctg caatagtgag 11280tggcgacaca
aagcacatga ttttcttaca acggagataa aaccaaaaaa atatttcatg
11340aacaacctag aacaaataaa gcttttatat aataaatata taaataaata
aaggctatgg 11400aataatatac ttcaatatat ttggattaaa taaattgttg
gcggggttga tatatttata 11460cacacctaaa gtcacttcaa tctcattttc
acttaacttt tatttttttt ttctttttat 11520ttatcataaa gagaatattg
ataatatact ttttaacata tttttatgac attttttatt 11580ggtgaaaact
tattaaaaat cataaatttt gtaagttaga tttatttaaa gagttcctct
11640tcttatttta aattttttaa taaattttta aataactaaa atttgtgtta
aaaatgttaa 11700aaaatgtgtt attaaccctt ctcttcgagg acgtacgtcg
agtcgacctg ca 1175219318DNAArtificial SequenceM13F Universal primer
193tgtaaaacga cggccagt 18194757DNAEuglena
gracilismisc_feature(677)..(677)n is a, c, g, or t 194ttttttttcg
aacacttaat ggaggtggtg aatgaaatag tctcaattgg gcaggaagtt 60ttacccaaag
ttgattatgc ccaactctgg agtgatgcca gtcactgtga ggtgctttac
120ttgtccatcg catttgtcat cttgaagttc actcttggcc cccttggtcc
aaaaggtcag 180tctcgtatga agtttgtttt caccaattac aaccttctca
tgtccattta ttcgttggga 240tcattcctct caatggcata tgccatgtac
accatcggtg ttatgtctga caactgcgag 300aaggcttttg acaacaacgt
cttcaggatc accacgcagt tgttctattt gagcaagttc 360ctggagtata
ttgactcctt ctatttgcca ctgatgggca agcctctgac ctggttgcaa
420ttcttccatc atttgggggc accgatggat atgtggctgt tctataatta
ccgaaatgaa 480gctgtttgga tttttgtgct gttgaatggt ttcatccact
ggatcatgta cggttattat 540tggaccagat tgatcaagct gaagttcccc
atgccaaaat ccctgattac atcaatgcag 600atcattcaat tcaatgttgg
tttctacatt gtctggaagt acaggaacat tccctgttat 660cgccaagatg
ggatgangat gtttggctgg ttcttcaatt acttttatgt tggcacagtc
720ttgtgtttgt tcttgaattt ctatgtgcaa acgtata 757195774DNAEuglena
gracilismisc_feature(34)..(34)n is a, c, g, or t 195tcaggatcac
cacgcagttg ttctatttga gcangttcct ggagtatatt gactccttct 60atttgccant
gatgggcaag cntctgacct ggttgcaatt cttccatcat tngggggcac
120cgatggatat gtggctgttc tataattacc gaaatgaagc tgtttggatt
tttgtgctgt 180tgaatggttt catccactgg atcatgtacg gttattannn
gaccagattg atcaagctga 240agttccccat gccaaaatcc ctgattacat
caatgcagat cattcaattc aatgttggtt 300tctacattgt ctggaagtac
aggaacattc cctgttatcg ccaagatggg atgaggatgt 360ttggctggtt
cttcaattac ttttatgttg gcacagtctt gtgtttgttc ttgaatttct
420atgtgcaaac gtatatcgtc aggaagcaca agggagccaa aaagattcag
tgatatttcc 480tcctctgcgg tggcctcttt tgacctcccc ttgacaccta
taatgtggag gtgtcgggct 540ctctccgtct caccagcact tgactctgca
ggtgctcact tttatttttt acccatcttt 600gcttgttgac cattcacctc
tcccacttcc acatagtcca ttctaactgt tgcagactgc 660ggtccatttt
ttccagagct cccaatgacc atacgcgaca ccttgtnnnc ncccagccca
720ttgtgcacaa ttcatagtgg catcgttttg ccttgatacg tgtgcatcca gcgg
7741961201DNAEuglena gracilismisc_feature(1134)..(1136)n is a, c,
g, or t 196gacatggcaa ctatgatttt attttgactg atagtgacct gttcgttgca
acaaattgat 60gagcaatgct tttttataat gccaactttg tacaaaaaag ttggattttt
tttcgaacac 120ttaatggagg tggtgaatga aatagtctca attgggcagg
aagttttacc caaagttgat 180tatgcccaac tctggagtga tgccagtcac
tgtgaggtgc tttacttgtc catcgcattt 240gtcatcttga agttcactct
tggccccctt ggtccaaaag gtcagtctcg tatgaagttt 300gttttcacca
attacaacct tctcatgtcc atttattcgt tgggatcatt cctctcaatg
360gcatatgcca tgtacaccat cggtgttatg tctgacaact gcgagaaggc
ttttgacaac 420aacgtcttca ggatcaccac gcagttgttc tatttgagca
agttcctgga gtatattgac 480tccttctatt tgccactgat gggcaagcct
ctgacctggt tgcaattctt ccatcatttg 540ggggcaccga tggatatgtg
gctgttctat aattaccgaa atgaagctgt ttggattttt 600gtgctgttga
atggtttcat ccactggatc atgtacggtt attattggac cagattgatc
660aagctgaagt tccccatgcc aaaatccctg attacatcaa tgcagatcat
tcaattcaat 720gttggtttct acattgtctg gaagtacagg aacattccct
gttatcgcca agatgggatg 780aggatgtttg gctggttctt caattacttt
tatgttggca cagtcttgtg tttgttcttg 840aatttctatg tgcaaacgta
tatcgtcagg aagcacaagg gagccaaaaa gattcagtga 900tatttcctcc
tctgcggtgg cctcttttga cctccccttg acacctataa tgtggaggtg
960tcgggctctc tccgtctcac cagcacttga ctctgcaggt gctcactttt
attttttacc 1020catctttgct tgttgaccat tcacctctcc cacttccaca
tagtccattc taactgttgc 1080agactgcggt ccattttttc cagagctccc
aatgaccata cgcgacacct tgtnnncncc 1140cagcccattg tgcacaattc
atagtggcat cgttttgcct tgatacgtgt gcatccagcg 1200g
12011971272DNAArtificial Sequencemutant EgD8S consensus, optionally
comprising M1, M2, M3, M6, M8, M12, M14, M15, M16, M18, M19, M21,
M22, M26, M38, M39, M40, M41, M45, M46, M49, M50, M51, M53, M54,
M58, M63, M68, M69 and M70 mutation sites 197catggtgaag nnnnnncgac
aggctnnncc cctcnnnatc gacggannnn nntacnnnnn 60ntccgcttgg gtgaacttcc
accctggtgg agctgaaatc attgagaact accagggacg 120agatgctact
gacgccttca tggttatgca ctctcaggaa nnnnnngaca agctcnnncg
180aatgcccaag nnnnnnnnnn nntccnnnnn ncctccccag gctnnnnnna
acgaagctca 240ggaggatttc cgaaagctcc gagaagagct gatcgccact
ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga tcnnnaccac
cctgggtctt ggcgtgcttn nnnnnttcnn 360nnnnnnncag tacnnnnnnn
nnttcattgg tgctnnnnnn ctcggtatgc actaccagca 420aatgggatgg
ctgtctcatg acatctgcca ccaccagacc ttcaagaacc gaaactggaa
480taacnnnnnn ggtctggtct ttggcaacnn nnnncagggc ttctccgtga
cctggtggaa 540ggacagacac aacgcccatc attctgctac caacgttcag
ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct ggtccgagga
cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt cagttccaac
agtactattt cctggtcatc tgtattctcc tgcgattcat 720ctggtgtttc
cagtctgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta
780ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc
tcaagnnnnn 840nttccacctc ttctttatgc cctccatcct gacctcgnnn
ctggtgttcn nnnnntccga 900gctcgtcggt ggcttcggaa ttgccnnnnn
ngtcttcatg aaccactacc ctctggagaa 960gatcggtgat tccgtctggg
acggacatgg cttctctgtg ggtcagatcc atgagaccat 1020gaacattcga
cgaggcnnnn nnnnngactg gttctttgga ggcctgaact accagatcga
1080gcaccatctc tggcccaccc tgcctcgaca caacctcact gccgtttcct
accaggtgga 1140acagctgtgc cagaagcaca acctccccta ccgaaaccct
ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgn nnnnnttcgc
tcgaatggcc gagaagnnnn nnnnnnnnaa 1260ggctnnntaa gc
1272198422PRTArtificial Sequencemutant EgD8S consensus, optionally
comprising M1, M2, M3, M6, M8, M12, M14, M15, M16, M18, M19, M21,
M22, M26, M38, M39, M40, M41, M45, M46, M49, M50, M51, M53, M54,
M58, M63, M68, M69 and M70 mutation sites 198Met Val Lys Xaa Xaa
Arg Gln Ala Xaa Pro Leu Xaa Ile Asp Gly Xaa1 5 10 15Xaa Tyr Xaa Xaa
Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu 20 25 30 Ile Ile
Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val 35 40 45Met
His Ser Gln Glu Xaa Xaa Asp Lys Leu Xaa Arg Met Pro Lys Xaa 50 55
60Xaa Xaa Xaa Ser Xaa Xaa Pro Pro Gln Ala Xaa Xaa Asn Glu Ala Gln65
70 75 80Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met
Phe 85 90 95Asp Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Xaa Thr Thr
Leu Gly 100 105 110Leu Gly Val Leu Xaa Xaa Phe Xaa Xaa Xaa Gln Tyr
Xaa Xaa Xaa Phe 115 120 125Ile Gly Ala Xaa Xaa Leu Gly Met His Tyr
Gln Gln Met Gly Trp Leu 130 135 140Ser His Asp Ile Cys His His Gln
Thr Phe Lys Asn Arg Asn Trp Asn145 150 155 160Asn Xaa Xaa Gly Leu
Val Phe Gly Asn Xaa Xaa Gln Gly Phe Ser Val 165 170 175Thr Trp Trp
Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val 180 185 190Gln
Gly His Asp Pro Asp
Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val
Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln
Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235
240Trp Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp
245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly
Leu Ala 260 265 270Leu His Trp Thr Leu Lys Xaa Xaa Phe His Leu Phe
Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Xaa Leu Val Phe Xaa Xaa
Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Xaa Xaa Val Phe
Met Asn His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val
Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr
Met Asn Ile Arg Arg Gly Xaa Xaa Xaa Asp Trp Phe Phe 340 345 350Gly
Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360
365Arg His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln
370 375 380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly
Leu Val385 390 395 400Ile Leu Leu Arg Tyr Leu Xaa Xaa Phe Ala Arg
Met Ala Glu Lys Xaa 405 410 415Xaa Xaa Xaa Lys Ala Xaa 420
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