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| United States Patent Application |
20060260012
|
| Kind Code
|
A1
|
|
Khan; Rafiqul
|
November 16, 2006
|
Method of transforming soybean
Abstract
The present disclosure provides methods for Agrobacterium-mediated
transformation of soybean cells or tissue and regeneration of the
transformed cells or tissue into transformed plants. The methods may be
used for transforming many soybean cultivars.
| Inventors: |
Khan; Rafiqul; (RESEARCH TRIANGLE PARK, NC)
|
| Correspondence Address:
|
SYNGENTA BIOTECHNOLOGY, INC.;PATENT DEPARTMENT
3054 CORNWALLIS ROAD
P.O. BOX 12257
RESEARCH TRIANGLE PARK
NC
27709-2257
US
|
| Assignee: |
SYNGENTA PARTICIPATIONS AG
|
| Family ID:
|
30000570
|
| Appl. No.:
|
10/517039
|
| Filed:
|
June 17, 2003 |
| PCT Filed:
|
June 17, 2003 |
| PCT NO:
|
PCT/US03/19212 |
| 371 Date:
|
December 7, 2004 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 60390562 | Jun 22, 2002 | |
|
|
| Current U.S. Class: |
800/287 ; 435/415; 435/468; 800/312 |
| Current CPC Class: |
C12N 15/8205 20130101 |
| Class at Publication: |
800/287 ; 800/312; 435/468; 435/415 |
| International Class: |
A01H 5/00 20060101 A01H005/00; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
1. A method for transforming soybean cells or tissue, comprising: (a)
preparing an explant from a soybean seed by. (i) removing a hypocotyl
from said soybean seed; (ii) removing one cotyledon along with its
adjacent axillary bud, leaving primary leaves attached to a remaining
cotyledon; and (iii) removing a portion of a primary leaf from said
remaining cotyledon, thereby generating a primary leaf base; and (b)
co-cultivating said explant with Agrobacterium comprising at least one
nucleic acid of interest to be incorporated into a genome of one or more
soybean cells.
2. The method of claim 1, further comprising cultivating at least one
formed shoot in a medium containing a selection agent.
3. The method of claim 2, wherein said at least one nucleic acid of
interest comprises a selectable marker gene.
4. The method of claim 3, wherein said selectable marker gene is a
phosphomannose isomerase gene.
5. The method of claim 4, wherein said selection agent is mannose.
6. The method of claim 4, wherein co-cultivation with said Agrobacterium
is carried out in the presence of mannose.
7. The method of claim 2, further comprising inducing shoot formation
from said primary leaf base.
8. The method of claim 7, wherein shoot formation is induced by culturing
said primary leaf base in a medium comprising a shoot-inducing hormone.
9. The method of claim 8, wherein said shoot-inducing hormone comprises
at least one of an auxin, a cytokinin, and a gibberellic acid.
10. The method of claim 9, wherein said auxin is selected from the group
consisting of IAA, NAA, and IBA.
11. The method of claim 9, wherein said cytokinin is selected from the
group consisting of benzylaminopurine (BAP), thidiazuron, kinetin, and
isopentenyl adenine.
12. The method of claim 7, wherein induction of shoot formation comprises
removing one or more of a primary meristem, a secondary meristem, and an
axillary meristem attached to a cotyledon.
13. The method of claim 7, further comprising selecting a transformed
shoot.
14. The method of claim 13, further comprising regenerating a selected
transformed shoot into a soybean plant.
15. The method of claim 1, wherein said soybean seed is a mature seed.
16. The method of claim 1, wherein said soybean seed is an immature seed.
17. The method of claim 1, wherein said soybean seed is a germinated
seed.
18. A method for producing a stably transformed soybean plant,
comprising: (a) preparing an explant from a soybean seed by: (i)
removing a hypocotyl from said soybean seed; (ii) removing one cotyledon
along with its adjacent axillary bud, leaving primary leaves attached to
a remaining cotyledon; and (iii) removing a portion of each primary leaf
from said remaining cotyledon, thereby generating a pair of primary leaf
bases; (b) co-cultivating said explant with Agrobacterium comprising a
nucleic acid of interest to be incorporated into a genome of a soybean
cell; (c) inducing shoot formation from each primary leaf base; (d)
cultivating at least one formed shoot in a medium containing a selection
agent; (e) selecting a transformed shoot; and (f) regenerating a
selected transformed shoot into a soybean plant.
19. A transgenic soybean plant regenerated from soybean cells or tissue
transformed according to the method of claim 1.
20. A transgenic seed produced by the transgenic plant of claim 19.
21. A transgenic soybean plant regenerated from soybean cells or tissue
transformed according to the method of claim 18.
22. A transgenic seed produced by the transgenic plant of claim 21.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/390,562, filed Jun. 22, 2002, the entire contents
of which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to methods for plant
transformation and, more particularly, to methods for transforming
soybean cells or tissues. The invention also relates to methods for
regenerating transgenic soybean plants from transformed soybean cells or
tissues. The invention also relates to transgenic soybean plants and
seeds obtained by such methods.
BACKGROUND
[0003] Soybean is a major food and feed source that is grown on more
acres worldwide than any other dicotyledonous crop. It is reportedly
grown on more than 50 million hectares. Unfortunately, only a few plant
introductions have given rise to the major cultivars grown in the United
States and, as a consequence, this narrow germplasm base has limited
soybean breeding potential. The limited genetic base in domestic soybean
varieties has limited the power of traditional breeding methods to
develop varieties with improved or value-added traits.
[0004] Hence, the use of genetic engineering techniques to modify soybean
can facilitate the development of new varieties with, for example, traits
such as herbicide resistance, disease resistance (such as virus
resistance, for example), and seed quality improvement in a manner that
has been unattainable by traditional breeding methods or tissue-culture
induced variation.
[0005] The development of an efficient transformation system is necessary
for the analysis of gene expression in plants. The requirements for such
a system include a proper target plant tissue that will allow efficient
plant regeneration, a gene delivery vehicle that delivers foreign DNA
efficiently into the target plant cells, and an effective method for
selecting transformed cells. In genetic transformation of dicotyledonous
specics, transformation systems utilizing the bacterium Agrobacterium
tumefaciens have been frequently used as vehicles for gene delivery. The
preferred target tissues for Agrobacterium-mediated transformation
presently include cotyledons, leaf tissues, and hypocotyls. High velocity
microprojectile bombardment offers an alternative method for gene
delivery into dicotyledonous plants.
[0006] Agrobacterium-mediated gene delivery in soybean has been far from
routine. In reports that have been available to the public, meristems and
cotyledon tissues have been frequently mentioned as targets for use in
Agrobacterium-mediated gene delivery. However, reliable and efficient
transformation and regeneration from these two explant sources are often
not accomplished.
[0007] U.S. Pat. No. 5,169,770 and 5,376,543 to Chee et al. discuss a
non-tissue culture method of transforming soybeans to produce transgenic
plants, wherein seeds are germinated and meristematic or mesocotyl cell
tissues are inoculated with bacterial cells, specifically Agrobacterium
strains, which, through infection, transfer DNA into the explants. This
method depends on the growth of preformed shoots.
[0008] Parrott W. A. et al. (1989), "Recovery of primary transformants of
soybean," Plant Cell Reports 7:615-617, report recovery of soybean
transformants from immature cotyledon tissue after co-cultivation with
Agrobacterium. However, the regenerated plants were chimeric, and the
transgenes were not transmitted to the progeny.
[0009] U.S. Pat. No. 5,416,011 (to Hinchee et al.) discusses utilizing a
cotyledon explant, which requires removal of the hypocotyl, saving and
separating the cotyledons and inserting a chimeric gene by inoculation
with Agrobacterium tumefaciens vectors containing the desired gene.
[0010] Yan B. et al. (2000), "Agrobacterium tumefaciens--mediated
transformation of soybean using immature zygotic cotyledon explants,"
Plant Cell Reports 19:1090-1097, report an overall 0.03% transformation
frequency in Agrobacterium-mediated transformation in soybean with
immature cotyledons.
[0011] U.S. Pat. No. 6,384,301 to Martinell et al. describes
Agrobacterium-mediated gene delivery into cells in the meristem of an
isolated soybean embryonic axis. Their method does not involve a
callus-phase tissue culture.
[0012] From the work described above, it is clear that the goal of
establishing a reliable soybean transformation system is seldom
accomplished by the workers involved when meristems and cotyledon tissues
are used as source explants for Agrobacterium-mediated gene delivery.
Therefore, there is a need to continue to exploit new methodology,
including new source explants, in order to develop a more efficient
soybean transformation system.
[0013] It has been demonstrated in soybean tissue culture that plant
regeneration may be achieved from epicotyl tissues and primary leaf
tissues. However, to-date, no successful transformation has been reported
in soybean using these two explant sources as targets for gene delivery.
[0014] Wright M. S. et al. (1987) "Initiation and propagation of Glycine
max L. Merr.: Plants from tissue-cultured epicotyls," Plant Cell Reports
8:83-90, describes successful initiation and proliferation of shoots from
epicotyl tissue of soybean. Explanted epicotyls were induced to form
shoots in Schenk and Hildebrandt medium containing 20 .mu.M kinetin for 5
weeks. Shoot proliferation was maintained on N6 medium containing 2.1 nM
picloram and 0.1 .mu.M benzyladenine.
[0015] Wright M. S. et al. (1987) "Regeneration of soybean (Glycine max
L. Merr.) from cultured primary leaf tissue," Plant Cell Reports 6:83-89,
describes a reproducible method for regeneration of plants from primary
leaf tissue of 27 varieties of soybean They found that while
2,4,5-trichlorophenoxyacetic acid was demonstrated to be essential for
regeneration, addition of benzyadenine (BA) was found to enhance
regeneration.
[0016] Rajasckaren K. et al. (1997) "Somatic embryogenesis from cultured
epicotyls and primary leaves of soybean (Glycine max L. Merr)," In Vitro
Cellular & Developmental Biology 33(2):88-91, describes regeneration of
several varieties of soybean by somatic embryogenesis from cultured
epicotyls and primary leaf tissues of immature seeds from greenhouse
grown plants. They found that somatic embryogenesis was induced from
epicotyls and primary leaves when cotyledon halves with the intact
zygotic embryo axes were cultured on Murashige and Skoog (MS) medium
supplemented with 46.2 .mu.M 2,4-D. In the absence of being cultured with
the cotyledon halves, no embryogenesis was observed from isolated axes,
epicotyls or primary leaves. Rapid multiplication of shoot tips from
germinating somatic embryos was achieved on Cheng's basal medium
containing 11.3 .mu.M benzyladenine.
SUMMARY
[0017] The present invention provides a method for transforming soybean
cells and regeneration of the transformed cells into transformed plants.
The method may be used for transforming many soybean cultivars.
[0018] The invention provides a novel soybean explant that enables
Agrobacterium tumefaciens-mediated gene delivery into soybean cells with
high efficiency.
[0019] In particular, the invention provides a method for transforming
soybean cells or tissue, the method comprising: [0020] (a) preparing
an explant from a soybean seed by: [0021] (i) removing all or a part
of the hypocotyl from said seed; [0022] (ii) removing one cotyledon
along with its adjacent axillary bud from the seed, and leaving one
cotyledon with the epicotyl and primary leaves attached thereto; [0023]
(iii) removing a portion of a primary leaf from the remaining cotyledon,
thereby generating a primary leaf base; and [0024] (b) co-cultivating
the explant with Agrobacterium comprising a nucleic acid of interest to
be incorporated into the genome of the soybean cells.
[0025] In additional embodiments, the method further includes one or more
of the following: inducing shoot formation from the primary leaf base and
the adjacent epicotyl; cultivating the shoot in a medium containing a
selection agent; selecting a transformed shoot; and regenerating a
transformed plant from the transformed shoot.
[0026] In a further embodiment, the invention provides a method for
producing a stably transformed soybean plant, the method comprising:
[0027] (a) preparing an explant from a soybean seed by: [0028] (i)
removing all or a part of the hypocotyl from said seed; [0029] (ii)
removing one cotyledon along with its adjacent axillary bud from the
seed, and leaving one cotyledon with the epicotyl and primary leaves
attached thereto; [0030] (ii) removing a portion of a primary leaf from
the epicotyl, thereby generating at least one primary leaf base;
[0031] (b) co-cultivating the explant with Agrobacterium comprising a
nucleic acid of interest to be incorporated into the genome of the
soybean cells; [0032] (c) inducing shoot formation from the primary
leaf base area; [0033] (d) cultivating a formed shoot in a medium
containing a selection agent,; [0034] (e) selecting a transformed
shoot; and [0035] (f) regenerating a selected transformed shoot into a
soybean plant.
[0036] In another embodiment, a portion of each of the primary leaves of
the explant generated in (a)(ii) is removed, thereby generating a pair of
primary leaf bases.
[0037] The method of the invention may be employed to introduce any
desired nucleic acid into a soybean cell. In one embodiment of the
invention, the nucleic acid comprises a gene that would express a
desirable agronomic trait in soybean.
[0038] In another embodiment of the invention, the nucleic acid comprises
a phosphomannose isomerase gene, which is used as a selectable marker
gene.
[0039] In an additional embodiment of the invention, the co-cultivating
of the explant with Agrobacterium is carried out in the presence of
mannose.
[0040] Both mature and immature seeds may be employed to generate the
explant used in the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 shows a map of plasmid pNOV2105.
[0042] FIG. 2 shows a map of plasmid pNOV2145.
[0043] FIG. 3 shows a map of plasmid pNOV2147.
[0044] FIG. 4 shows an exemplary process for preparing a soybean explant.
Panel A depicts a soybean seed embryo in which a part of the hypocotyl is
removed. Panel B depicts the soybean explant from Panel A in which one
cotyledon is removed along with its adjacent axillary bud. Panel C
depicts the soybean explant from Panel B after removal of the two primary
leaves, generating a break point at the base of each primary leaf.
[0045] FIG. 5 shows a map of plasmid pBSC11234.
[0046] FIG. 6 shows a map of plasmid pBSC11369.
DETAILED DESCRIPTION
[0047] The present invention will now be described more fully hereinafter
with reference to the accompanying figures, in which various embodiments
of the invention are described. This invention may, however, be embodied
in different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are provided so
that this disclosure will be thorough and complete and will fully convey
the scope of the invention to those skilled in the art. The terminology
used in the description of the invention herein is for the purpose of
describing particular embodiments only and is not intended to be limiting
of the invention. As used in the description of the invention and the
appended claims, the singular forms "a", "an" and "the" are intended to
include the plural forms as well, unless the context clearly indicates
otherwise.
[0048] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs.
[0049] Except as otherwise indicated, standard methods may be used for
the production of cloned genes, expression cassettes, vectors (e.g.,
plasmids), proteins and protein fragments, and transformed cells and
plants according to the present invention. Except as otherwise indicated,
standard methods may be used for the production of cloned genes,
expression cassettes, vectors (e.g., plasmids), proteins and protein
fragments according to the present invention. Such techniques are known
to those skilled in the art. See e.g., J. Sambrook et al., Molecular
Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989), and F. M. Ausubel et al.,
Current Protocols In Molecular Biology (Green Publishing Associates, Inc.
and Wiley-Interscience, New York, 1991); J. Draper et al., eds., Plant
Genetic Transformation And Gene Expression: A Laboratory Manual,
(Blackwell Scientific Publications, 1988); and S. B. Gelvin & R. A.
Schilperoort, eds., Introduction, Expression, And Analysis Of Gene
Production In Plants.
[0050] The present invention is drawn to methods and compositions for the
stable transformation of soybean with nucleic acid sequences of interest
and the regeneration of transgenic soybean plants.
[0051] The methods of the invention may be employed to express any
nucleic acid of interest in soybean plants. A gene of interest may be,
for example, a gene for herbicide resistance, disease resistance, or
insect/pest resistance, or is a selectable or scorable marker, and
comprises a plant-operable promoter, a coding region, and a 3' terminator
region. Herbicide resistance genes include the AHAS gene for resistance
to imidazolinone or sulfonyl urea herbicides, the pat or bar gene for
resistance to bialaphos or glufosinate, the EPSP synthase gene for
resistance to glyphosate, etc. Disease resistance genes include genes for
antibiotic synthetic enzymes, e.g., for pyrrolnitrin synthetic enzymes,
plant derived resistance genes, and the like. Insect resistance genes
include genes for insecticidal proteins from Bacillus thuringiensis.
Genes of interest may also encode enzymes involved in biochemical
pathways, the expression of which alters a trait that is important in
food, feed, nutraceutical, and/or pharmaceutical production. The gene of
interest may be located on a plasmid. A plasmid suitable for use in the
present invention may comprise more than one gene of interest and/or the
Agrobacterium may comprise different plasmids having different genes of
interest.
[0052] The present invention provides a method for the transformation of
varieties of soybean, including Glycine max. The method is based on
Agrobacterium-mediated delivery of a desired gene into a soybean cell
followed by regeneration of transformed cell(s) into a transformed
soybean plant. The methods of the invention are cultivar independent.
[0053] In one embodiment of the invention, an explant is prepared by
germinating a soybean mature seed or immature seed collected from a
greenhouse grown plant in a seed germination medium for a period of time,
removing seed coat and, subsequently, a cotyledon from said mature seed
or immature seed. In a preferred embodiment of the invention, a portion
of the exposed primary leaves is then removed, thereby creating a break
point at the primary leaf base (FIG. 4). Agrobacterium-mediated gene
delivery is made into the cells at the primary leaf base or in the area
of the primary leaf break point. Adventitious shoots are induced from the
primary leaf base area of the epicotyl. This induction is achieved by
removing pre-existing meristems (i.e., primary, secondary, and axillary
meristems) and subjecting the explant to a shoot induction medium
containing appropriate growth regulators, The shoot induction process
facilitates the development or regeneration of transformed shoots from
the targeted primary leaf base cells.
[0054] Transformed soybean cells are cultured in the presence of a
selection agent. Preferably, the cells are transformed with a
phosphomannose isomerase (PMI gene, and the transformed cells are
cultivated in the presence of mannose. In a medium that contains mannose
as a selection agent, soybean cells transformed with a PMI gene have a
growth advantage over those that are not so transformed.
[0055] The time required for regenerating a transformed soybean plant
using the method described in this invention is significantly reduced
compared to other Agrobacterium-mediated transformation protocols that
are reported in the literature. A rooted transformed soybean shoot may be
produced 8 to 12 weeks from the initiation of a transformation
experiment. A foreign genetic construct, or transgene, to be inserted
into the soybean genome is created in vitro by normal techniques of
recombinant DNA manipulations. The construct may be comprised of any
heterologous nucleic acid. The genetic construct is transformed into the
Agrobacterium strain for delivery into the soybean cells. The
Agrobacterium is non-oneogenic, and several such strains are now widely
available. The Agrobacterium is preferably selected from A. tumefaciens
and A. rhizogenes.
[0056] The foreign genetic construct preferably comprises a selectable
marker gene. The preferred selectable marker gene is a phosphomannose
isomerase gene. Other suitable selectable marker genes include, but are
not limited to, genes encoding: neomycin. phosphotransferase II (Fraley
et al., CRC Critical Reviews in Plant Science 4, 1 (1986)); cyanamide
hydratase (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88, 4250
(1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et al.,
BioTechnology 11, 715 (1993)); bar gene (Toki et al., Plant Physiol. 100,
1503 (1992); Meagher et al., Crop Sci. 36, 1367 (1996)); tryptophane
decarboxylase (Goddijn et al., Plant Mol. Biol. 22, 907 (1993)); neomycin
phosphotransferase (NEO; Southern et al., J. Mol. Appl. Gen. 1, 327
(1982)); hygromycin phosphotransferase (HPT or HYG; Shimizu et al., Mol.
Cell. Biol. 6, 1074 (1986)); dihydrofolate reductase (DHFR);
phosphinothricin acetyltransferase (DeBlock et al., EMBO J. 6, 2513
(1987)); 2,2- dichloropropionic acid dehalogenase (Buchanan-Wollatron et
al., J. Cell. Biochem. 13D, 330 (1989)); acetohydroxyacid .synthase (U.S.
Pat. No. 4,761,373 to Anderson et al.; Haughn et al., Mol. Gen. Genet.
221, 266 (1988)); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai
et al., Nature 317, 741 (1985)); haloaryinitrilase (WO 87/04181 to
Stalker et al.), acetyl-coenzyme A carboxylase (Parker et al., Plant
Physiol. 92, 1220 (1990)); dihydropteroate synthase (sulI; Guerineau et
al., Plant Mol. Biol. 15, 127 (1990)); and 32 kDa photosystem II
polypeptide (psb A; Hirschberg et al., Science 222, 1346 (1983)).
[0057] Also included are genes encoding resistance to chloramphenicol
(Herrera-Estrella et al., EMBO J. 2, 987 (1983)); methotrexate
(Herrera-Estrella et al., Nature 303, 209 (1983); Meijer et al., Plant
Mol. Biol. 16, 807 (1991)); hygromycin (Waldron et al., Plant Mol. Biol.
5, 103 (1985); Zhijian et al., Plant Science 108, 219 (1995); Meijer et
al., Plant Mol. Bio. 16, 807 (1991)); streptomycin (Jones et al., Mol.
Gen. Genet. 210, 86 (1987)); spectinomycin (Bretagne- Sagnard et al.,
Transgenic Res. 5, 131 (1996)); bleomycin (Hille et al., Plant Mol. Biol.
7, 171 (1986)); sulfonamide (Guerineau et al., Plant Mol. Bio. 15, 127
(1990); bromoxynil (Stalker et al., Science 242, 419 (1988)); 2,4-D
(Streber et al., Biol/Technology 7, 811 (1989)); phosphinothricin
(DeBlock et al., EMBO J. 6, 2513 (1987)); spectinomycin (Bretagne-Sagnard
and Chupeau, Transgenic Research 5, 131 (1996)).
[0058] In one embodiment, the starting material for the transformation
process is a soybean mature seed. In another embodiment, the starting
material can be a soybean immature seed from a growing soybean plant. The
seed is placed on a germination medium and permitted to germinate for a
period of 6-24 hours, preferably for about 6-14 hours, and more
preferably for about 8-12 hours. Seeds may also be allowed to germinate
for a longer period of time, for example, from 2 to 5 days, if desired.
[0059] The seed coat and hypocotyl of the germinating seed is removed.
One cotyledon along with its adjacent axillary shoot bud is also removed.
Afterwards, the primary leaves are substantially removed, thereby
creating an explant comprising the primary leaf base, epicotyl to which
the leaf base is attached, and a cotyledon to which the epicotyl is
attached. Substantially removed means removal of a major portion of
primary leaf tissue.
[0060] For Agrobacterium-mediated gene transfer, wounding of the plant
tissue is known to facilitate gene transfer. Therefore it is preferred,
but not necessary, that a wound is created at the leaf base region.
[0061] The explant, prepared as described above, is then immersed into an
Agrobacterium cell suspension for a few minutes to a few hours, typically
about 0.5-3 hours, and preferably 1-2 hours. Excessive Agrobacterium cell
suspension is removed and the remaining Agrobacterium are permitted to
co-cultivate with the explant on a co-cultivation medium for several
days, typically two to five days, and preferably three to four days,
under 16h light/8h dark conditions at a temperature of about 22.degree.
C..+-.2.degree. C.
[0062] After co-cultivation, the explant is transferred to a medium (or a
series of media) conducive to shoot development and selection of
transformed cells, for 8-12 weeks. Such a medium (or media) generally
contains a shoot-inducing hormone as well as a selection agent. The
regeneration media used in the examples below contain mannose, as the
selection agent, as well as benzylaminopurine (BAP), a shoot-inducing
hormone. The term hormone also includes cell growth regulating compounds
that induce shoot formation, including, but not limited to, auxins (such
as, e.g., IAA, NAA, and indole butyric acid (IBA)), cytokinins (such as,
e.g., thidiazuron, kinetin, and isopentenyl adenine), and/or gibberellic
acids (GA.sub.3).
[0063] When shoots reach about 2 cm and with full trifoliate leaf
formation, shoots are separated from the explant and placed on a rooting
medium to induce root formation. Preferably, the rooting medium also
contains a selection agent to further help identify potential transformed
shoots. Root formation takes approximately 1-2 weeks, following which the
plants can be transferred to soil and grown to full maturity.
[0064] Transgenic plants comprising a heterologous nucleic acid (i.e.,
comprising cells or tissues transformed in accordance with the methods
described herein), as well as the seeds and progeny produced by the
transgenic plants, are an additional aspect of the present invention.
Procedures for cultivating transformed cells to useful cultivars are
known to those skilled in the art. Techniques are known for the in vitro
culture of plant tissue, and in a number of cases, for regeneration into
whole plants. A further aspect of the invention is transgenic plant
tissue, plants, or seeds containing the nucleic acids described above. In
a preferred embodiment, transformed plants produced using the methods
described herein are not chimeric, or only a small proportion of
transformed plants is chimeric. This is preferably achieved by extending
the period of high cytokinin treatment or by increasing the stringency of
mannose selection, or both.
[0065] Thus, the transformed cells of the present invention, identified
by selection or screening and cultured in an appropriate medium that
supports regeneration as provided herein, may then be allowed to mature
into plants. Plants are preferably matured either in a growth chamber or
greenhouse. Plants are regenerated from about 2-6 weeks after a
transformant is identified, depending on the initial tissue. During
regeneration, cells may be grown on solid media in tissue culture
vessels. Illustrative embodiments of such vessels are petri dishes and
Plant Con.RTM.s. After the regenerating plants have reached the stage of
shoot and root development, they may be transferred to a greenhouse for
further growth and testing. As provided above, seeds and progeny plants
of the regenerated plants are an aspect of the present invention.
Accordingly, the term "seeds" is meant to encompass seeds of the
transformed plant, as well as seeds produced from the progeny of the
transformed plants. Plants of the present invention include not only the
transformed and regenerated plants, but also progeny of transformed and
regenerated plants produced by the methods described herein.
[0066] Plants produced by the described methods may be screened for
successful transformation by standard methods described above. Seeds and
progeny plants of regenerated plants of the present invention may be
continuously screened and selected for the continued presence of the
transgenic and integrated nucleic acid sequence in order to develop
improved plant and seed lines, which are another aspect of the present
invention. Desirable transgenic nucleic acid sequences may thus be moved
(i.e., introgressed or inbred) into other genetic lines such as certain
elite or commercially valuable lines or varieties. Methods of
introgressing desirable nucleic acid sequences into genetic plant lines
may be carried out by a variety of techniques known in the art, including
by classical breeding, protoplast fusion, nuclear transfer and chromosome
transfer. Breeding approaches and techniques are known in the art, and
are set forth in, for example, J. R. Welsh, Fundamentals of Plant
Genetics and Breeding (John Wiley and Sons, New York, (1981)); Crop
Breeding (1). R. Wood, ed., American Society of Agronomy, Madison, Wis.,
(1983)); O. Mayo, The Theory of Plant Breeding, Second Edition (Clarendon
Press, Oxford, England (1987)); and Wricke and Weber, Quantitative
Genetics and Selection Plant Breeding (Walter de Gruyter and Co., Berlin
(1986)). Using these and other techniques in the art, transgenic plants
and inbred lines obtained according to the present invention may be used
to produce commercially valuable hybrid plants and crops, which hybrids
are also an aspect of the present invention.
[0067] The foregoing is illustrative of the various embodiments of the
present invention and is not to be construed as limiting thereof.
[0068] The invention will be further described by the following examples,
which are not intended to limit the scope of the invention in any manner.
EXAMPLE 1
Transformation Vectors
[0069] The plasmid pNOV2105 (FIG. 1) is a modification of p Victor, which
is disclosed and described in WO 97/04112 in that the 35S promoter is
replaced with a SMAS promoter, the 35S terminator is replaced with the
Nos terminator, and an additional SMAS promoter is inserted upstream of
the GUSintronGUS sequence, which is flanked on its 3' end by a Nos
terminator. pNOV2105 employed in the methods described herein does not
contain the multicloning site that is found in p Victor. However, it is
well within the skill in the art to add such a cloning site, if desired.
[0070] pNOV2105 (FIG. 1) is a vector for Agrobacterium-mediated plant
transformation and contains the Ti right and left border sequences from
the nopaline type pTiT37 plasmid (Yadav et al. 1982 Proc Natl Acad Sci
79:6322-6326) flanking the genes phosphomannose isomerase (PM) and
beta-glucoronidase (GUS).
[0071] For replication and maintenance in E. coli, the plasmid contains
the origin of replication from the E. coli plasmid pUC19 (pUC19ori)
(Yanish-Perron et al. 1985 Gene 33:103-119), and for replication and
maintenance in Agrobacterium tumefaciens the plasmid further contains the
origin of replication from the Pseudomonas plasmid pVS1 (pVSlori) (Itoh
et al. 1984 Plasmid 11:206-220; Itoh and Haas 1985 Gene 36:27-36). For
selection in E. coli and Agrobacterium tumefaciens, the plasmid contains
the spectinomycin/streptomycin resistance gene (spec/strep) from the
transposon Tn7 encoding the enzyme 3''(9)-O-nucleotidyltransferase (Fling
et al. 1985 Nucleic Acids Res 19:7095-7106). The spec/strep resistance
gene is fused to the tac promoter (see, e.g., Amann et al. 1983 Gene
25(203):167-78) for efficient expression in the bacterium.
[0072] The T-DNA segment between the right and left border harbors the
following genes, which are the only genes transferred to the soybean
plant via the Agrobacterium tumefaciens-mediated transformation.
GUSintronGUS
[0073] beta-glucuronidase (GUS): This segment next to the right border
contains the beta-glucuronidase gene (GUS) from E. coli with an intron in
the coding region to prevent translation by Agrobacterium fused to the
SMAS promoter and Nos terminator. The GUSintronGUS gene was isolated from
plasmid pBISN1. (Narasimhulu et al. 1986 Early transcription of
Agrobacterium DNA in tobacco and maize, Plant Cell 8:873-866).
[0074] phosphomannose isomerase (PMI): This segment next to the left
border is the mannose-6-phosphate isomerases gene from E. coli (Miles and
Guest 1984, Gene 32:41-48 ) fused to the SMAS promoter (Ni M, Cui D,
Einstein J, Narasimhulu S, Vergara C E, Gelvin S B (1995) and Nos
terminator. The phosphomannose isomerase gene is used as a selection
marker to select transgenic shoots on media containing D-mannose as the
carbon source.
[0075] The components and sequence of pNOV2145 (FIG. 2) are set forth in
SEQ ID NO:1. The components and sequence of pNOV2147 (FIG. 3) are set
forth in SEQ ID NO:2.
EXAMPLE 2
Transformation and Regeneration
[0076] Mature dried soybean seeds (Var. S42 H1) were surface sterlized by
releasing chlorine gas inside a desiccator. Seeds were kept in petri
plates and chlorine gas was produced by pouring 100 ml of Clorox.RTM.
into a beaker and slowly adding 8 ml of concentrated HCl. Seeds were
sterilized by at least two gas release treatments each lasting for 8-18
hours.
[0077] Sterilized seeds (approximately 15-20 seeds per plate) were then
placed on a germination medium containing 0.6% agar-solidified MS basal
medium (Murashige and Skoog (1962) A revised medium for rapid growth and
bioassays with tobacco callus cultures. Physiol Plant 15:473-479) and 2%
sucrose. The pH was maintained at 5.8. The petri plates were placed in a
room at 37.degree. C. for overnight growth or imbibition of seeds. The
seed coat was removed, followed by removing part of the hypocotyl,
keeping about 0.5 cm of the hypocotyl. One cotyledon was removed along
with its adjacent axillary shoot bud and was discarded. On the remaining
cotyledon, the primary leaves were broken apart using a scalpel, leaving
the primary leaf bases on the epicotyl. (FIG. 4)
[0078] Agrobacterium strain (LBA 4404). containing the plasmid pNOV 2145
(ZsGreen1and PMI, as described in Example 1) was streaked from frozen
glycerol stocks onto YEP plates (yeast extract 10 g/L, peptone 5 g/L,
NaCl 5g/L, bacto agar 15 g/L) containing appropriate antibiotic (100 mg/L
spectinomycin). Agrobacterium was then incubated at 27.degree. C. for 1-2
days. A scoop of Agrobacterium from plates were grown on 100 ml YEP
liquid medium containing an antibiotic (100 mg/L spectinomycin) for
overnight growth at 27.degree. .C on a shaker. Bacterial suspensions were
centrifuged at about 1500 g for 15 minutes and resuspended to a density
of OD.sub.660=0.2 or 0.65 in a co-cultivation liquid medium (B.sub.5
salts 0.05X (Sigma), B.sub.5 vitamins: (0.05X) (B.sub.5 vitamin
composition (1X): inositol 100 mg/L, nicotinic acid 1 mg/L, pyridoxine
HCl 1 mg/L, thiamine HCl 10 mg/L), acetosyringone 40 mg/L, sucrose 20
g/L, IBAP 2 mg/L, GA.sub.3 0.25 mg/L, MES (Morpholino ethanesulfonic
acid) 3.9 g/L, and pH 5.4.
[0079] The explants containing the target tissue were immersed into
Agrobacterium suspension and incubated for 1-2 hours. The Agrobacterium
suspension was poured off, and the treated explants were placed onto a
filter paper inside co-cultivation plates. The adaxial side of the
explants was kept in contact with the filter paper. The co-cultivation
solid medium was composed of B.sub.5 salts (Sigma, 0.05X), B.sub.5
vitamins (0.05X), 40 mg/L acetosyringone, sucrose 20 g/L, BAP 2 mg/L,
GA.sub.3 0.25 mg/L, MES 3.9 g/L, and pH 5.4. The medium was solidified
with 0.5% purified agar (Sigma).
[0080] The explants were co-cultivated with the Agrobacterium at
20-23.degree. C. for a period of 2-5 days, under 16 h light/8 h dark
conditions. After co-cultivation, the explants were washed in sterile
water containing 250 mg/L cefotaxime, primary and secondary meristems
were removed, and the explants were transferred to regeneration medium
(i.e., REG-1 medium). During the regeneration process, any axillary
shoots adjacent to the cotyledon were also removed to encourage growth
from the area of the primary leaf base.
[0081] REG-1 medium contained MS salts (1X), B.sub.5 vitamins (1X),
KNO.sub.3 1 g/L, BAP 1 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L,
glutamine 250 mg/L, asparagine 50 mg/L, mannose 15-30 g/L, sucrose 0,
0.25, and 1 g/L, pH 5.6, and purified agar 10 g/L. Five explants were
placed in each petri plate in an upright position, such that the epicotyl
end of the explant was inserted into the medium. The plates were kept
inside a plastic container and placed in a culture room at 22-25.degree.
C., under an 18-20 hr light/4-6 hr dark cycle at 60-100 .mu.E m.sup.-2
S.sup.-1. After 2 weeks on REG-1 medium, explants were transferred to
REG-2 medium, which contained MS salts (1X) and B.sub.5 vitamins (1X),
KNO.sub.3 1 g/L, BAP 0.5 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L,
glutamine 250 mg/L, asparagine 50 mg/L, mannose 15 g/L, and sucrose 1
g/L. The media pH was maintained at 5.6, and the media was solidified
with purified agar 10 g/L.
[0082] At 4-6 weeks, the soybean cultures were transferred to REG-3
medium for continuing selection and shoot development. REG-3 medium
contained MS salts (1X), B.sub.5 vitamins (1X), KNO.sub.3 1 g/L, BAP 0.2
mg/L, GA.sub.3 0.5 mg/L, IBA 0.1 mg/L, ticarcillin 300 mg/L, cefotaxime
100 mg/L, glutamine 250 mg/L, asparagine 50 mg/L, mannose 15 g/L, sucrose
1 g/L, pH 5.6, and the medium was solidified with purified agar 10 g/L.
Dcad tissue was removed and explants with regenerating shoots were
subcultured in fresh REG-3 medium every two weeks. Elongated shoots were
continuously harvested from the cultures when they reached about 2-4 cm
in length. At that time, shoots were transferred to a rooting medium,
which contained MS salts (0.5X), B.sub.5 Vitamins (0.5X), glutainine 250
mg/L, asparagine 50 mg/L, KNO.sub.3 1 g/L, cefotaxime 100 mg/L,
ticarcillin 300 mg/L, sucrose 15 g/L, IBA 0.5 mg/L, pH 5.6, and purified
agar 10 g/L.
[0083] Rooted transgenic shoots expressing a fluorescent protein gene
(ZsGreen1) were transferred to 2'' pots which contained moistened Fafard
germinating mix (Conrad Fafard Inc., MA, USA) and were kept covered with
plastic cups for maintaining moisture for approximately 2 weeks. Plants
were acclimatized at 27-29.degree. C. day temperature, 21.degree. C.
night temperature, and a 16h photoperiod (20-40 .mu.E m.sup.-2 S.sup.-1
light intensity). When new leaves began to emerge, plants were
transferred to one-gallon pots which contained a soil mixture composed of
50-55% composted pine bark, 40-45% Peat, 5-10% Perlite (Sungrow
Horticultural Supply, Pine Bluff, Ark.). Acclimatized soybean plants were
grown in the greenhouse at 27-29.degree. C. day temp, 21.degree. C. night
temp, 400-600 .mu.E m.sup.-2 S.sup.-1 light intensity, 70-95% relative
humidity, and a 16 hr photoperiod. The plants were fertilized with
osmocote (Scotts-Sierra Horticultural Products Company, Ohio; 17-6-12)
twice (5-8 g/gallon soil) during the growth period. Transformation was
confirmed by Taqman analysis for the presence of the fluorescent protein
gene as well as the PMI gene in the leaves of the greenhouse grown
plants. Expression of the fluorescent protein gene in the transformed
soybean tissue was also confirmed by visualizing the expression using a
fluorescent microscope.
[0084] Six transgenic plants developed using the gene construct pNOV2145
were confirmed by Southern blot analyses. Progeny analysis of one event
for either the PMI gene or the ZsGreen1 gene revealed one integration
site of the T-DNA into the genome of the transformed soybean, and the
progeny segregated in a 3:1 ratio in the T1 generation.
TABLE-US-00001
TABLE 1
Transformed shoots expressing fluorescent
protein gene (Zsgreen1)
Transformed
Expt No. Gene construct shoots/explant Percent tr. shoots
75 pNOV2145 5/45 11
89 pNOV2145 5/75 7
EXAMPLE 3
[0085] Soybean seeds (Var. S42 H1) were surface sterilized and explants
were prepared as described in Example 2.
[0086] Agrobacterium strain (LBA 4404) carrying the plasmid pNOV2147 was
prepared as described in Example 1. The final bacterial concentration was
adjusted to OD .sub.660=0.60 with a co-cultivation liquid medium. The
conditions for explant preparation, Agrobacterium inoculation, and
co-cultivation were the same as those described in Example 2.
[0087] Following three days of co-cultivation in a solid co-cultivation
medium, excessive Agrobacterium was washed off, primary and secondary
meristems were removed, and the explants were transferred to REG-1
medium. They were cultured at 28-30.degree. C. in 16h light and 8h dark
conditions. After 2 weeks on REG-1 medium, the cultures were transferred
to REG-2 medium. During this regeneration process, only shoots arising
from the base of a primary leaf were kept. At about the 4th week, the
shoot cultures were transferred to REG-3 medium. They were then
transferred to fresh REG-3 medium every 10-14 days. As in REG-1 and REG-2
medium, only the new shoots arising from the base of a primary leaf were
kept while the rest of the shoots were removed. When elongated shoots
reached about 24 cm in length, they were separated from the rest of the
shoot cultures and transferred to rooting medium.
[0088] Five transgenic shoots out of 35 explants were identified as
expressing the cyano fluorescent protein gene (Table 2).
TABLE-US-00002
TABLE 2
Transformed shoots expressing the cyano fluorescent protein gene
Experiment Transformed
No. Gene construct shoots/explants % transformed shoots
92 pNOV2147 5/35 14
EXAMPLE 4
[0089] Mannose Treatment During Co-cultivation
[0090] The gene construct used in this example was pNOV2145 (which
comprises ZsGreen1 and PMI genes, as described in Example 1). The
procedures for preparing the explants, Agrobacteria suspensions, and
inoculation of explants with bacterial suspensions were carried out as
described in Example 2. The final bacterial concentration was adjusted to
OD .sub.660=0.55 or 0.85.
[0091] Following the inoculation step, explants were transferred to a
co-cultivation medium containing either 20 g/L sucrose or 15 g/L mannose
and were kept at 20-23.degree. C. under 16h light and 8h dark conditions.
[0092] After 3-5 days of co-cultivation, expression of the fluorescent
protein gene was visualized using a fluorescent microscope. Explants that
were inoculated with Agrobacterium in a mannose-containing co-cultivation
medium showed at least two-fold the number of fluorescent spots compared
to those co-cultivated in a sucrose-containing co-cultivation medium.
Subsequent shoot regeneration and selection steps were followed as those
described in Example 2.
[0093] A significant increase in the production of transformed shoots was
observed in the experiments where mannose was included in the
co-cultivation medium (Table 3). Five transformed shoots from
co-cultivation medium that included mannose were rooted and transferred
to soil. Subsequent analysis by Taqman as well as Southern blot confirmed
the integration of the transgenes. Transgene expression in the T1 progeny
confirmed the germline transmission of the transgenes.
TABLE-US-00003
TABLE 3
Transformed shoots expressing ZsGreen1 fluorescent
protein gene where explants and Agrobacteria were
co-cultivated in mannose or sucrose
Experi- Transformed %
ment Gene Co-culture in shoots/ Transfor-
No. construct mannose/sucrose explants mation
87 pNOV2145 Sucrose 0/60 0
Mannose 6/80 7.5
102 pNOV2145 Sucrose 1/20 5
Mannose 8/40 20
EXAMPLE 5
[0094] In this example, Agrobacterium EHA101 comprising the plasmid
pNOV2105 (SMAS-PMI SMAS-GUS, as described in Example 1) was used in
soybean transformation. The preparation of the explants, Agrobacteria
suspension, and inoculation of explants with Agrobacteria were the same
as those described in Example 2. The final bacterial concentration was
adjusted to OD .sub.660=0.45 or 0.6.
[0095] Following Agrobacterium inoculation, explants were transferred to
a co-cultivation medium containing either 20 g/L sucrose or 15 g/L
mannose. Co-cultivation was carried out at 20-23.degree. C. under a 16h
light and 8h dark conditions. Following 3-5 days of co-cultivation, GUS
gene expression was visualized using a histochemical gus assay. Explants
co-cultivated in mannose-containing co-cultivation medium showed at least
two-fold the number of GUS spots compared to those co-cultivated in
sucrose-containing co-cultivation medium. Shoot regeneration and
selection were carried out as described in Example 2. A significant
increase in the production of transformed shoots was observed in the
experiment in which mannose was added into the co-cultivation medium.
(Table 4).
TABLE-US-00004
TABLE 4
Transformed shoots expressing GUS gene
Experi- Transformed %
ment Gene Co-cultivation in shoots/ Transfor-
No. construct mannose/sucrose explant mation
63 pNOV2105 sucrose 5/60 8
81 pNOV2105 Sucrose 2/30 7
Mannose 5/30 17
EXAMPLE 6
[0096] In this example, Agrobacterium EHA101 comprising the plasmid
pBSC11234 (FIG. 5) was used in soybean transformation. The components and
sequence of pBSC11234 are set forth in SEQ ID NO:3. pBSC11234 comprises a
CMP-PMI : beta conglycinin-galactosidase gene construct. The preparation
of the explants, Agrobacteria suspension, and inoculation of explants
with Agrobacteria were the same as those described in Example 2. The
final bacterial concentration was adjusted to OD .sub.660=0.6. The
co-cultivation liquid medium contained B.sub.5 salts (0.1X), B.sub.5
vitamins (1X), acetosyringone 80 mg/L, sucrose 20 g/L, BAP 2 mg/L,
GA.sub.3 0.25 mg/L, MES 3.9 g/L, and pH 5.4. Solid co-cultivation medium
was prepared by incorporating 5 g/L purified agar to the liquid
co-cultivation medium.
[0097] Following Agrobacterium inoculation, explants were transferred to
a solid co-cultivation medium and cultured at 20-24.degree. C. under 16h
light and 8h dark conditions. Following 3-5 days of co-cultivation,
primary and secondary shoot meristems were removed and discarded, and the
resulting explants were transferred to REG-4 medium, which contained
B.sub.5 salts (1X), B.sub.5 Vitamins (1X), BAP 1 mg/L, glutamine 50 mg/L,
asparagine 50 mg/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, mannose
15-20 g/L, sucrose 0, 0.25, or 1 g/L, purified agar 10 g/L, and pH at
5.6. After a period of 5-7 days, any shoot grown from the axillary
meristem close to the cotyledon was removed, and the explants were
transferred to REG-5 medium, which contained B.sub.5 salts (1X), B.sub.5
Vitamins (1X), BAP 0.5 mg/L, glutamine 50 mg/L, asparagine 50 mg/L,
cefotaxime 100 mg/L, ticarcillin 300 mg/L, mannose 15 g/L, sucrose 1 g/L,
purified agar 10 g/L, and pH at 5.6. At four weeks, explants were
transferred to REG-6 medium for elongation of shoots. REG-6 medium
contained MS salts (1X), MS Vitamins (1X) (MS vitamin composition:
inositol 100 mg/L, nicotinic acid 0.5 mg/L, pyridoxine HCl 0.5 mg/L,
thiamine HCl 0.1 mg/L, glycine 2 mg/L), myo-inositol 200 mg/L, BAP 0.2
mg/L, zeatin riboside 0.5 mg/L, IBA 0.1 mg/L, GA.sub.3 1 mg/L, glutamine
50 mg/L, asparagine 50 mg/L, ticarcillin 300 mg/L, mannose 15 g/L,
sucrose 5 g/L, silver nitrate 0.8 mg/L, purified agar 10 g/L, and pH 5.6.
Explants were transferred to fresh REG-6 medium every two weeks.
Elongated shoots (24 cm long) were removed and rooted in rooting medium
and transferred to soil. The rooting medium contained MS salts (1X),
B.sub.5 Vitamins (1X), glutamine 100 mg/L, asparagine 100 mg/L, IBA 0.7
mg/L, timentin 100 mg/L, and sucrose 15 g/L. Taqman analysis confirmed
the presence of the transgenes (alpha galactosidase and phosphomannose
isomerase) in leaf samples from two events.
EXAMPLE 7
[0098] In this example, Agrobacterium EHA101 comprising the plasmid
pBSC11369 (FIG. 6) was used in soybean transformation. The components and
sequence of pBSC11369 are set forth in SEQ ID NO:4. pBSC11369 comprises a
CMP-HPT: CMP-ZsGreen1 gene construct. The preparation of the explants,
Agrobacteria suspension, and inoculation of explants with Agrobacteria
were the same as those described in Example 2. The final bacterial
concentration was adjusted to OD .sub.660=0.6. The co-cultivation liquid
medium contained B.sub.5 salts (0.1X), B.sub.5 vitamins (1X),
acetosyringone 80 mg/L, sucrose 20 g/L, BAP 2 mg/L, GA.sub.3 0.25 mg/L,
MES 3.9 g/L, and pH 5.4. Solid co-cultivation medium was prepared by
incorporating 5 g/L purified agar to the liquid co-cultivation medium.
[0099] Following Agrobacterium inoculation, explants wore transferred to
a solid co-cultivation medium and cultured at 20-24.degree. C. under 16h
light and 8h dark conditions. Following 3-5 days of co-cultivation,
explants were transferred to REG-7 medium after removing primary and
secondary meristems from the explants in order to encourage shoot growth
from the primary leaf base area REG-7 medium contained B.sub.5 salts
(1X), B.sub.5 Vitamins (1X), BAP 1 mg/L, glutamine 50 mg/L, asparagine 50
mg/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, sucrose 30 g/L,
hygromycin 2-5 mg/L, purified agar 10 g/L, and pH 5.6. Explants were
placed in an upright position such that the epicotyl end of the explant
was inserted into the medium. After a period of 7-10 days, any shoots
grown from the axillary meristem close to the cotyledon were removed.
Explants were transferred to fresh REG-8 medium, which contained B.sub.5
salts (1X), B.sub.5 Vitamins (1X), BAP 0.5 mg/L, glutamine 50 mg/L,
asparagine 50 mg/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, sucrose 30
g/L, purified agar 10 g/L, and pH at 5.6. After another two weeks,
explants were transferred to REG-9 medium and subcultured thereafter
every two weeks. REG-9 medium contained MS salts (1X), MS Vitamins (1X),
myo-inositol 200 mg/L, BAP 0.2 mg/L, zeatin riboside 0.5 mg/L, IBA 0.1
mg/L, GA.sub.3 1 mg/L, glutamine 50 mg/L, asparagine 50 mg/L, silver
nitrate 0.8 mg/L, ticarcillin 300 mg/L, sucrose 30 g/L, hygromycin
0.1-0.2 mg/L, purified agar 10 g/L, and pH 5.6. Elongated shoots (24 cm
long) were removed, rooted in rooting medium, and then transferred to
soil. The rooting medium contained MS salts (1X), B.sub.5 Vitamins (1X),
glutamine 100 mg/L, asparagine 100 mg/L, IBA 0.7 mg/L, timentin 100 mg/L,
and sucrose 15 g/L. Taqman analysis confirmed the presence of the
transgenes (HPT as well as ZsGreen1) in leaf samples obtained from five
events. Expression of the ZsGreen1 gene in plant parts was confirmed by
visualization under a fluorescent microscope.
[0100] All publications, patents, and patent applications cited herein
are incorporated by reference. While in the foregoing specification this
invention has been described in relation to certain preferred embodiments
thereof, and many details have been set forth for purposes of
illustration, it will be apparent to those skilled in the art that the
invention is susceptible to additional embodiments and that certain of
the details described herein may be varied considerably without departing
from the basic principles of the invention.
Sequence CWU
1
1
4 1 9555 DNA Artificial pNOV2145 1 gatccaccgg tcgccaccat ggcccagtcc
aagcacggcc tgaccaagga gatgaccatg 60 aagtaccgca tggagggctg cgtggacggc
cacaagttcg tgatcaccgg cgagggcatc 120 ggctacccct tcaagggcaa gcaggccatc
aacctgtgcg tggtggaggg cggccccttg 180 cccttcgccg aggacatctt gtccgccgcc
ttcatgtacg gcaaccgcgt gttcaccgag 240 tacccccagg acatcgtcga ctacttcaag
aactcctgcc ccgccggcta cacctgggac 300 cgctccttcc tgttcgagga cggcgccgtg
tgcatctgca acgccgacat caccgtgagc 360 gtggaggaga actgcatgta ccacgagtcc
aagttctacg gcgtgaactt ccccgccgac 420 ggccccgtga tgaagaagat gaccgacaac
tgggagccct cctgcgagaa gatcatcccc 480 gtgcccaagc agggcatctt gaagggcgac
gtgagcatgt acctgctgct gaaggacggt 540 ggccgcttgc gctgccagtt cgacaccgtg
tacaaggcca agtccgtgcc ccgcaagatg 600 cccgactggc acttcatcca gcacaagctg
acccgcgagg accgcagcga cgccaagaac 660 cagaagtggc acctgaccga gcacgccatc
gcctccggct ccgccttgcc ctgagcggcc 720 ctctagatcc ccgaatttcc ccgatcgttc
aaacatttgg caataaagtt tcttaagatt 780 gaatcctgtt gccggtcttg cgatgattat
catataattt ctgttgaatt acgttaagca 840 tgtaataatt aacatgtaat gcatgacgtt
atttatgaga tgggttttta tgattagagt 900 cccgcaatta tacatttaat acgcgataga
aaacaaaata tagcgcgcaa actaggataa 960 attatcgcgc gcggtgtcat ctatgttact
agatcgggaa ttgggtaccg aattcactgg 1020 ccgtcgtttt acaacgtcgt gactgggaaa
accctggcgt tacccaactt aatcgccttg 1080 cagcacatcc ccctttcgcc agctggcgta
atagcgaaga ggcccgcacc gatcgccctt 1140 cccaacagtt gcgcagcctg aatggcgaat
ggcgcctgat gcggtatttt ctccttacgc 1200 atctgtgcgg tatttcacac cgcatatggt
gcactctcag tacaatctgc tctgatgccg 1260 catagttaag ccagccccga cacccgccaa
cacccgctga cgcgccctga cgggcttgtc 1320 tgctcccggc atccgcttac agacaagctg
tgaccgtctc cgggagctgc atgtgtcaga 1380 ggttttcacc gtcatcaccg aaacgcgcga
gacgaaaggg cctcgtgata cgcctatttt 1440 tataggttaa tgtcatgata ataatggttt
cttagacgtc aggtggcact tttcggggaa 1500 atgtgcgcgg aacccctatt tgtttatttt
tctaaataca ttcaaatatg tatccgctca 1560 tgagacaata accctgataa atgcttcaat
ggcgcgccgg taccagcttg catgcctgca 1620 ggtcgactct agaggatcct ggcagacaaa
gtggcagaca tactgtccca caaatgaaga 1680 tggaatctgt aaaagaaaac gcgtgaaata
atgcgtctga caaaggttag gtcggctgcc 1740 tttaatcaat accaaagtgg tccctaccac
gatggaaaaa ctgtgcagtc ggtttggctt 1800 tttctgacga acaaataaga ttcgtggccg
acaggtgggg gtccaccatg tgaaggcatc 1860 ttcagactcc aataatggag caatgacgta
agggcttacg aaataagtaa gggtagtttg 1920 ggaaatgtcc actcacccgt cagtctataa
atacttagcc cctccctcat tgttaaggga 1980 gcaaaatctc agagagatag tcctagagag
agaaagagag caagtagcct agaagtagga 2040 tccccgatca tgcaaaaact cattaactca
gtgcaaaact atgcctgggg cagcaaaacg 2100 gcgttgactg aactttatgg tatggaaaat
ccgtccagcc agccgatggc cgagctgtgg 2160 atgggcgcac atccgaaaag cagttcacga
gtgcagaatg ccgccggaga tatcgtttca 2220 ctgcgtgatg tgattgagag tgataaatcg
actctgctcg gagaggccgt tgccaaacgc 2280 tttggcgaac tgcctttcct gttcaaagta
ttatgcgcag cacagccact ctccattcag 2340 gttcatccaa acaaacacaa ttctgaaatc
ggttttgcca aagaaaatgc cgcaggtatc 2400 ccgatggatg ccgccgagcg taactataaa
gatcctaacc acaagccgga gctggttttt 2460 gcgctgacgc ctttccttgc gatgaacgcg
tttcgtgaat tttccgagat tgtctcccta 2520 ctccagccgg tcgcaggtgc acatccggcg
attgctcact ttttacaaca gcctgatgcc 2580 gaacgtttaa gcgaactgtt cgccagcctg
ttgaatatgc agggtgaaga aaaatcccgc 2640 gcgctggcga ttttaaaatc ggccctcgat
agccagcagg gtgaaccgtg gcaaacgatt 2700 cgtttaattt ctgaatttta cccggaagac
agcggtctgt tctccccgct attgctgaat 2760 gtggtgaaat tgaaccctgg cgaagcgatg
ttcctgttcg ctgaaacacc gcacgcttac 2820 ctgcaaggcg tggcgctgga agtgatggca
aactccgata acgtgctgcg tgcgggtctg 2880 acgcctaaat acattgatat tccggaactg
gttgccaatg tgaaattcga agccaaaccg 2940 gctaaccagt tgttgaccca gccggtgaaa
caaggtgcag aactggactt cccgattcca 3000 gtggatgatt ttgccttctc gctgcatgac
cttagtgata aagaaaccac cattagccag 3060 cagagtgccg ccattttgtt ctgcgtcgaa
ggcgatgcaa cgttgtggaa aggttctcag 3120 cagttacagc ttaaaccggg tgaatcagcg
tttattgccg ccaacgaatc accggtgact 3180 gtcaaaggcc acggccgttt agcgcgtgtt
tacaacaagc tgtaagagct tactgaaaaa 3240 attaacatct cttgctaagc tgggagctct
agatccccga atttccccga tcgttcaaac 3300 atttggcaat aaagtttctt aagattgaat
cctgttgccg gtcttgcgat gattatcata 3360 taatttctgt tgaattacgt taagcatgta
ataattaaca tgtaatgcat gacgttattt 3420 atgagatggg tttttatgat tagagtcccg
caattataca tttaatacgc gatagaaaac 3480 aaaatatagc gcgcaaacta ggataaatta
tcgcgcgcgg tgtcatctat gttactagat 3540 cgggaattgg gtaccatgcc cgggcggcca
gcatggccgt atccgcaatg tgttattaag 3600 ttgtctaagc gtcaatttgt ttacaccaca
atatatcctg ccaccagcca gccaacagct 3660 ccccgaccgg cagctcggca caaaatcacc
actcgataca ggcagcccat cagaattaat 3720 tctcatgttt gacagcttat catcgactgc
acggtgcacc aatgcttctg gcgtcaggca 3780 gccatcggaa gctgtggtat ggctgtgcag
gtcgtaaatc actgcataat tcgtgtcgct 3840 caaggcgcac tcccgttctg gataatgttt
tttgcgccga catcataacg gttctggcaa 3900 atattctgaa atgagctgtt gacaattaat
catccggctc gtataatgtg tggaattgtg 3960 agcggataac aatttcacac aggaaacaga
ccatgaggga agcgttgatc gccgaagtat 4020 cgactcaact atcagaggta gttggcgtca
tcgagcgcca tctcgaaccg acgttgctgg 4080 ccgtacattt gtacggctcc gcagtggatg
gcggcctgaa gccacacagt gatattgatt 4140 tgctggttac ggtgaccgta aggcttgatg
aaacaacgcg gcgagctttg atcaacgacc 4200 ttttggaaac ttcggcttcc cctggagaga
gcgagattct ccgcgctgta gaagtcacca 4260 ttgttgtgca cgacgacatc attccgtggc
gttatccagc taagcgcgaa ctgcaatttg 4320 gagaatggca gcgcaatgac attcttgcag
gtatcttcga gccagccacg atcgacattg 4380 atctggctat cttgctgaca aaagcaagag
aacatagcgt tgccttggta ggtccagcgg 4440 cggaggaact ctttgatccg gttcctgaac
aggatctatt tgaggcgcta aatgaaacct 4500 taacgctatg gaactcgccg cccgactggg
ctggcgatga gcgaaatgta gtgcttacgt 4560 tgtcccgcat ttggtacagc gcagtaaccg
gcaaaatcgc gccgaaggat gtcgctgccg 4620 actgggcaat ggagcgcctg ccggcccagt
atcagcccgt catacttgaa gctaggcagg 4680 cttatcttgg acaagaagat cgcttggcct
cgcgcgcaga tcagttggaa gaatttgttc 4740 actacgtgaa aggcgagatc accaaagtag
tcggcaaata aagctctagt ggatctccgt 4800 acccccgggg gatctggctc gcggcggacg
cacgacgccg gggcgagacc ataggcgatc 4860 tcctaaatca atagtagctg taacctcgaa
gcgtttcact tgtaacaacg attgagaatt 4920 tttgtcataa aattgaaata cttggttcgc
atttttgtca tccgcggtca gccgcaattc 4980 tgacgaactg cccatttagc tggagatgat
tgtacatcct tcacgtgaaa atttctcaag 5040 cgctgtgaac aagggttcag attttagatt
gaaaggtgag ccgttgaaac acgttcttct 5100 tgtcgatgac gacgtcgcta tgcggcatct
tattattgaa taccttacga tccacgcctt 5160 caaagtgacc gcggtagccg acagcaccca
gttcacaaga gtactctctt ccgcgacggt 5220 cgatgtcgtg gttgttgatc taaatttagg
tcgtgaagat gggctcgaga tcgttcgtaa 5280 tctggcggca aagtctgata ttccaatcat
aattatcagt ggcgaccgcc ttgaggagac 5340 ggataaagtt gttgcactcg agctaggagc
aagtgatttt atcgctaagc cgttcagtat 5400 cagagagttt ctagcacgca ttcgggttgc
cttgcgcgtg cgccccaacg ttgtccgctc 5460 caaagaccga cggtcttttt gttttactga
ctggacactt aatctcaggc aacgtcgctt 5520 gatgtccgaa gctggcggtg aggtgaaact
tacggcaggt gagttcaatc ttctcctcgc 5580 gtttttagag aaaccccgcg acgttctatc
gcgcgagcaa cttctcattg ccagtcgagt 5640 acgcgacgag gaggtttatg acaggagtat
agatgttctc attttgaggc tgcgccgcaa 5700 acttgaggca gatccgtcaa gccctcaact
gataaaaaca gcaagaggtg ccggttattt 5760 ctttgacgcg gacgtgcagg tttcgcacgg
ggggacgatg gcagcctgag ccaattccca 5820 gatccccgag gaatcggcgt gagcggtcgc
aaaccatccg gcccggtaca aatcggcgcg 5880 gcgctgggtg atgacctggt ggagaagttg
aaggccgcgc aggccgccca gcggcaacgc 5940 atcgaggcag aagcacgccc cggtgaatcg
tggcaagcgg ccgctgatcg aatccgcaaa 6000 gaatcccggc aaccgccggc agccggtgcg
ccgtcgatta ggaagccgcc caagggcgac 6060 gagcaaccag attttttcgt tccgatgctc
tatgacgtgg gcacccgcga tagtcgcagc 6120 atcatggacg tggccgtttt ccgtctgtcg
aagcgtgacc gacgagctgg cgaggtgatc 6180 cgctacgagc ttccagacgg gcacgtagag
gtttccgcag ggccggccgg catggccagt 6240 gtgtgggatt acgacctggt actgatggcg
gtttcccatc taaccgaatc catgaaccga 6300 taccgggaag ggaagggaga caagcccggc
cgcgtgttcc gtccacacgt tgcggacgta 6360 ctcaagttct gccggcgagc cgatggcgga
aagcagaaag acgacctggt agaaacctgc 6420 attcggttaa acaccacgca cgttgccatg
cagcgtacga agaaggccaa gaacggccgc 6480 ctggtgacgg tatccgaggg tgaagccttg
attagccgct acaagatcgt aaagagcgaa 6540 accgggcggc cggagtacat cgagatcgag
ctagctgatt ggatgtaccg cgagatcaca 6600 gaaggcaaga acccggacgt gctgacggtt
caccccgatt actttttgat cgatcccggc 6660 atcggccgtt ttctctaccg cctggcacgc
cgcgccgcag gcaaggcaga agccagatgg 6720 ttgttcaaga cgatctacga acgcagtggc
agcgccggag agttcaagaa gttctgtttc 6780 accgtgcgca agctgatcgg gtcaaatgac
ctgccggagt acgatttgaa ggaggaggcg 6840 gggcaggctg gcccgatcct agtcatgcgc
taccgcaacc tgatcgaggg cgaagcatcc 6900 gccggttcct aatgtacgga gcagatgcta
gggcaaattg ccctagcagg ggaaaaaggt 6960 cgaaaaggtc tctttcctgt ggatagcacg
tacattggga acccaaagcc gtacattggg 7020 aaccggaacc cgtacattgg gaacccaaag
ccgtacattg ggaaccggtc acacatgtaa 7080 gtgactgata taaaagagaa aaaaggcgat
ttttccgcct aaaactcttt aaaacttatt 7140 aaaactctta aaacccgcct ggcctgtgca
taactgtctg gccagcgcac agccgaagag 7200 ctgcaaaaag cgcctaccct tcggtcgctg
cgctccctac gccccgccgc ttcgcgtcgg 7260 cctatcgcgg ccgctggccg ctcaaaaatg
gctggcctac ggccaggcaa tctaccaggg 7320 cgcggacaag ccgcgccgtc gccactcgac
cgccggcgct gaggtctgcc tcgtgaagaa 7380 ggtgttgctg actcatacca ggcctgaatc
gccccatcat ccagccagaa agtgagggag 7440 ccacggttga tgagagcttt gttgtaggtg
gaccagttgg tgattttgaa cttttgcttt 7500 gccacggaac ggtctgcgtt gtcgggaaga
tgcgtgatct gatccttcaa ctcagcaaaa 7560 gttcgattta ttcaacaaag ccgccgtccc
gtcaagtcag cgtaatgctc tgccagtgtt 7620 acaaccaatt aaccaattct gattagaaaa
actcatcgag catcaaatga aactgcaatt 7680 tattcatatc aggattatca ataccatatt
tttgaaaaag ccgtttctgt aatgaaggag 7740 aaaactcacc gaggcagttc cataggatgg
caagatcctg gtatcggtct gcgattccga 7800 ctcgtccaac atcaatacaa cctattaatt
tcccctcgtc aaaaataagg ttatcaagtg 7860 agaaatcacc atgagtgacg actgaatccg
gtgagaatgg caaaagctct gcattaatga 7920 atcggccaac gcgcggggag aggcggtttg
cgtattgggc gctcttccgc ttcctcgctc 7980 actgactcgc tgcgctcggt cgttcggctg
cggcgagcgg tatcagctca ctcaaaggcg 8040 gtaatacggt tatccacaga atcaggggat
aacgcaggaa agaacatgtg agcaaaaggc 8100 cagcaaaagg ccaggaaccg taaaaaggcc
gcgttgctgg cgtttttcca taggctccgc 8160 ccccctgacg agcatcacaa aaatcgacgc
tcaagtcaga ggtggcgaaa cccgacagga 8220 ctataaagat accaggcgtt tccccctgga
agctccctcg tgcgctctcc tgttccgacc 8280 ctgccgctta ccggatacct gtccgccttt
ctcccttcgg gaagcgtggc gctttctcat 8340 agctcacgct gtaggtatct cagttcggtg
taggtcgttc gctccaagct gggctgtgtg 8400 cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg gtaactatcg tcttgagtcc 8460 aacccggtaa gacacgactt atcgccactg
gcagcagcca ctggtaacag gattagcaga 8520 gcgaggtatg taggcggtgc tacagagttc
ttgaagtggt ggcctaacta cggctacact 8580 agaagaacag tatttggtat ctgcgctctg
ctgaagccag ttaccttcgg aaaaagagtt 8640 ggtagctctt gatccggcaa acaaaccacc
gctggtagcg gtggtttttt tgtttgcaag 8700 cagcagatta cgcgcagaaa aaaaggatct
caagaagatc ctttgatctt ttctacgggg 8760 tctgacgctc agtggaacga aaactcacgt
taagggattt tggtcatgag attatcaaaa 8820 aggatcttca cctagatcct tttgatccgg
aattaattcc tgtggttggc atgcacatac 8880 aaatggacga acggataaac cttttcacgc
ccttttaaat atccgattat tctaataaac 8940 gctcttttct cttaggttta cccgccaata
tatcctgtca aacactgata gtttaaactg 9000 aaggcgggaa acgacaatct gatcatgagc
ggagaattaa gggagtcacg ttatgacccc 9060 cgccgatgac gcgggacaag ccgttttacg
tttggaactg acagaaccgc aacgctgcag 9120 gaattggccg cagcggccat ttaaatcaat
tgggcgcgta cgtagcacta gtgcgcgatc 9180 gcttaattaa gcggcgcgcc taaagcttct
ggcagacaaa gtggcagaca tactgtccca 9240 caaatgaaga tggaatctgt aaaagaaaac
gcgtgaaata atgcgtctga caaaggttag 9300 gtcggctgcc tttaatcaat accaaagtgg
tccctaccac gatggaaaaa ctgtgcagtc 9360 ggtttggctt tttctgacga acaaataaga
ttcgtggccg acaggtgggg gtccaccatg 9420 tgaaggcatc ttcagactcc aataatggag
caatgacgta agggcttacg aaataagtaa 9480 gggtagtttg ggaaatgtcc actcacccgt
cagtctataa atacttagcc cctccctcat 9540 tgttaaggga gcaag
9555 2 9546 DNA Artificial pNOV2147 2
ggatccccga tcatgcaaaa actcattaac tcagtgcaaa actatgcctg gggcagcaaa 60
acggcgttga ctgaacttta tggtatggaa aatccgtcca gccagccgat ggccgagctg 120
tggatgggcg cacatccgaa aagcagttca cgagtgcaga atgccgccgg agatatcgtt 180
tcactgcgtg atgtgattga gagtgataaa tcgactctgc tcggagaggc cgttgccaaa 240
cgctttggcg aactgccttt cctgttcaaa gtattatgcg cagcacagcc actctccatt 300
caggttcatc caaacaaaca caattctgaa atcggttttg ccaaagaaaa tgccgcaggt 360
atcccgatgg atgccgccga gcgtaactat aaagatccta accacaagcc ggagctggtt 420
tttgcgctga cgcctttcct tgcgatgaac gcgtttcgtg aattttccga gattgtctcc 480
ctactccagc cggtcgcagg tgcacatccg gcgattgctc actttttaca acagcctgat 540
gccgaacgtt taagcgaact gttcgccagc ctgttgaata tgcagggtga agaaaaatcc 600
cgcgcgctgg cgattttaaa atcggccctc gatagccagc agggtgaacc gtggcaaacg 660
attcgtttaa tttctgaatt ttacccggaa gacagcggtc tgttctcccc gctattgctg 720
aatgtggtga aattgaaccc tggcgaagcg atgttcctgt tcgctgaaac accgcacgct 780
tacctgcaag gcgtggcgct ggaagtgatg gcaaactccg ataacgtgct gcgtgcgggt 840
ctgacgccta aatacattga tattccggaa ctggttgcca atgtgaaatt cgaagccaaa 900
ccggctaacc agttgttgac ccagccggtg aaacaaggtg cagaactgga cttcccgatt 960
ccagtggatg attttgcctt ctcgctgcat gaccttagtg ataaagaaac caccattagc 1020
cagcagagtg ccgccatttt gttctgcgtc gaaggcgatg caacgttgtg gaaaggttct 1080
cagcagttac agcttaaacc gggtgaatca gcgtttattg ccgccaacga atcaccggtg 1140
actgtcaaag gccacggccg tttagcgcgt gtttacaaca agctgtaaga gcttactgaa 1200
aaaattaaca tctcttgcta agctgggagc tctagatccc cgaatttccc cgatcgttca 1260
aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 1320
atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1380
tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1440
aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 1500
gatcgggaat tgggtaccat gcccgggcgg ccagcatggc cgtatccgca atgtgttatt 1560
aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 1620
gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagaatt 1680
aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag 1740
gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc 1800
gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg 1860
caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt 1920
gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag 1980
tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 2040
tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 2100
atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 2160
accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 2220
ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 2280
ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 2340
ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 2400
cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 2460
ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta 2520
cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 2580
ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 2640
aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 2700
ttcactacgt gaaaggcgag atcaccaaag tagtcggcaa ataaagctct agtggatctc 2760
cgtacccccg ggggatctgg ctcgcggcgg acgcacgacg ccggggcgag accataggcg 2820
atctcctaaa tcaatagtag ctgtaacctc gaagcgtttc acttgtaaca acgattgaga 2880
atttttgtca taaaattgaa atacttggtt cgcatttttg tcatccgcgg tcagccgcaa 2940
ttctgacgaa ctgcccattt agctggagat gattgtacat ccttcacgtg aaaatttctc 3000
aagcgctgtg aacaagggtt cagattttag attgaaaggt gagccgttga aacacgttct 3060
tcttgtcgat gacgacgtcg ctatgcggca tcttattatt gaatacctta cgatccacgc 3120
cttcaaagtg accgcggtag ccgacagcac ccagttcaca agagtactct cttccgcgac 3180
ggtcgatgtc gtggttgttg atctaaattt aggtcgtgaa gatgggctcg agatcgttcg 3240
taatctggcg gcaaagtctg atattccaat cataattatc agtggcgacc gccttgagga 3300
gacggataaa gttgttgcac tcgagctagg agcaagtgat tttatcgcta agccgttcag 3360
tatcagagag tttctagcac gcattcgggt tgccttgcgc gtgcgcccca acgttgtccg 3420
ctccaaagac cgacggtctt tttgttttac tgactggaca cttaatctca ggcaacgtcg 3480
cttgatgtcc gaagctggcg gtgaggtgaa acttacggca ggtgagttca atcttctcct 3540
cgcgttttta gagaaacccc gcgacgttct atcgcgcgag caacttctca ttgccagtcg 3600
agtacgcgac gaggaggttt atgacaggag tatagatgtt ctcattttga ggctgcgccg 3660
caaacttgag gcagatccgt caagccctca actgataaaa acagcaagag gtgccggtta 3720
tttctttgac gcggacgtgc aggtttcgca cggggggacg atggcagcct gagccaattc 3780
ccagatcccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc 3840
gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa 3900
cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc 3960
aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc 4020
gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc 4080
agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg 4140
atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc 4200
agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac 4260
cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac 4320
gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc 4380
tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc 4440
cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc 4500
gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc 4560
acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc 4620
ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga 4680
tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt 4740
ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag 4800
gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca 4860
tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa 4920
ggtcgaaaag gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt 4980
gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg 5040
taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt 5100
attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa 5160
gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt 5220
cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca 5280
gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gctgaggtct gcctcgtgaa 5340
gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 5400
gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc 5460
tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 5520
aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 5580
gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 5640
atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5700
gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 5760
cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 5820
gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc tctgcattaa 5880
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5940
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6000
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6060
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6120
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6180
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6240
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6300
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6360
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6420
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6480
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6540
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6600
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6660
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6720
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6780
aaaaggatct tcacctagat ccttttgatc cggaattaat tcctgtggtt ggcatgcaca 6840
tacaaatgga cgaacggata aaccttttca cgccctttta aatatccgat tattctaata 6900
aacgctcttt tctcttaggt ttacccgcca atatatcctg tcaaacactg atagtttaaa 6960
ctgaaggcgg gaaacgacaa tctgatcatg agcggagaat taagggagtc acgttatgac 7020
ccccgccgat gacgcgggac aagccgtttt acgtttggaa ctgacagaac cgcaacgctg 7080
caggaattgg ccgcagcggc catttaaatc aattgggcgc gtacgtagca ctagtgcgcg 7140
atcgcttaat taagcggcgc gcctaaagct tctggcagac aaagtggcag acatactgtc 7200
ccacaaatga agatggaatc tgtaaaagaa aacgcgtgaa ataatgcgtc tgacaaaggt 7260
taggtcggct gcctttaatc aataccaaag tggtccctac cacgatggaa aaactgtgca 7320
gtcggtttgg ctttttctga cgaacaaata agattcgtgg ccgacaggtg ggggtccacc 7380
atgtgaaggc atcttcagac tccaataatg gagcaatgac gtaagggctt acgaaataag 7440
taagggtagt ttgggaaatg tccactcacc cgtcagtcta taaatactta gcccctccct 7500
cattgttaag ggagcaagga tccaccggtc gccaccatgg ccctgtccaa caagttcatc 7560
ggcgacgaca tgaagatgac ctaccacatg gacggctgcg tgaacggcca ctacttcacc 7620
gtgaagggcg agggcagcgg caagccctac gagggcaccc agacctccac cttcaaggtg 7680
accatggcca acggcggccc cctggccttc tccttcgaca tcctgtccac cgtgttcatg 7740
tacggcaacc gctgcttcac cgcctacccc accagcatgc ccgactactt caagcaggcc 7800
ttccccgacg gcatgtccta cgagagaacc ttcacctacg aggacggcgg cgtggccacc 7860
gccagctggg agatcagcct gaagggcaac tgcttcgagc acaagtccac cttccacggc 7920
gtgaacttcc ccgccgacgg ccccgtgatg gccaagaaga ccaccggctg ggacccctcc 7980
ttcgagaaga tgaccgtgtg cgacggcatc ttgaagggcg acgtgaccgc cttcctgatg 8040
ctgcagggcg gcggcaacta cagatgccag ttccacacct cctacaagac caagaagccc 8100
gtgaccatgc cccccaacca cgtggtggag caccgcatcg ccagaaccga cctggacaag 8160
ggcggcaaca gcgtgcagct gaccgagcac gccgtggccc acatcacctc cgtggtgccc 8220
ttctgagagc tctagatccc cgaatttccc cgatcgttca aacatttggc aataaagttt 8280
cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 8340
cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 8400
gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 8460
ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgggtaccga 8520
attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 8580
atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 8640
atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 8700
tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 8760
ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 8820
gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 8880
tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 8940
gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9000
ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9060
atccgctcat gagacaataa ccctgataaa tgcttcaatg gcgcgccggt accagcttgc 9120
atgcctgcag gtcgactcta gaggatcctg gcagacaaag tggcagacat actgtcccac 9180
aaatgaagat ggaatctgta aaagaaaacg cgtgaaataa tgcgtctgac aaaggttagg 9240
tcggctgcct ttaatcaata ccaaagtggt ccctaccacg atggaaaaac tgtgcagtcg 9300
gtttggcttt ttctgacgaa caaataagat tcgtggccga caggtggggg tccaccatgt 9360
gaaggcatct tcagactcca ataatggagc aatgacgtaa gggcttacga aataagtaag 9420
ggtagtttgg gaaatgtcca ctcacccgtc agtctataaa tacttagccc ctccctcatt 9480
gttaagggag caaaatctca gagagatagt cctagagaga gaaagagagc aagtagccta 9540
gaagta 9546
3 10604 DNA Artificial pBSC11234 3 cgcgcctaaa gcttgcatgc ctgcaggtcg
actctagagg atcctggcag acaaagtggc 60 agacatactg tcccacaaat gaagatggaa
tctgtaaaag aaaacgcgtg aaataatgcg 120 tctgacaaag gttaggtcgg ctgcctttaa
tcaataccaa agtggtccct accacgatgg 180 aaaaactgtg cagtcggttt ggctttttct
gacgaacaaa taagattcgt ggccgacagg 240 tgggggtcca ccatgtgaag gcatcttcag
actccaataa tggagcaatg acgtaagggc 300 ttacgaaata agtaagggta gtttgggaaa
tgtccactca cccgtcagtc tataaatact 360 tagcccctcc ctcattgtta agggagcaaa
atctcagaga gatagtccta gagagagaaa 420 gagagcaagt agcctagaag taggatcccc
gatcatgcaa aaactcatta actcagtgca 480 aaactatgcc tggggcagca aaacggcgtt
gactgaactt tatggtatgg aaaatccgtc 540 cagccagccg atggccgagc tgtggatggg
cgcacatccg aaaagcagtt cacgagtgca 600 gaatgccgcc ggagatatcg tttcactgcg
tgatgtgatt gagagtgata aatcgactct 660 gctcggagag gccgttgcca aacgctttgg
cgaactgcct ttcctgttca aagtattatg 720 cgcagcacag ccactctcca ttcaggttca
tccaaacaaa cacaattctg aaatcggttt 780 tgccaaagaa aatgccgcag gtatcccgat
ggatgccgcc gagcgtaact ataaagatcc 840 taaccacaag ccggagctgg tttttgcgct
gacgcctttc cttgcgatga acgcgtttcg 900 tgaattttcc gagattgtct ccctactcca
gccggtcgca ggtgcacatc cggcgattgc 960 tcacttttta caacagcctg atgccgaacg
tttaagcgaa ctgttcgcca gcctgttgaa 1020 tatgcagggt gaagaaaaat cccgcgcgct
ggcgatttta aaatcggccc tcgatagcca 1080 gcagggtgaa ccgtggcaaa cgattcgttt
aatttctgaa ttttacccgg aagacagcgg 1140 tctgttctcc ccgctattgc tgaatgtggt
gaaattgaac cctggcgaag cgatgttcct 1200 gttcgctgaa acaccgcacg cttacctgca
aggcgtggcg ctggaagtga tggcaaactc 1260 cgataacgtg ctgcgtgcgg gtctgacgcc
taaatacatt gatattccgg aactggttgc 1320 caatgtgaaa ttcgaagcca aaccggctaa
ccagttgttg acccagccgg tgaaacaagg 1380 tgcagaactg gacttcccga ttccagtgga
tgattttgcc ttctcgctgc atgaccttag 1440 tgataaagaa accaccatta gccagcagag
tgccgccatt ttgttctgcg tcgaaggcga 1500 tgcaacgttg tggaaaggtt ctcagcagtt
acagcttaaa ccgggtgaat cagcgtttat 1560 tgccgccaac gaatcaccgg tgactgtcaa
aggccacggc cgtttagcgc gtgtttacaa 1620 caagctgtaa gagcttactg aaaaaattaa
catctcttgc taagctggga gctctagatc 1680 cccgaatttc cccgatcgtt caaacatttg
gcaataaagt ttcttaagat tgaatcctgt 1740 tgccggtctt gcgatgatta tcatataatt
tctgttgaat tacgttaagc atgtaataat 1800 taacatgtaa tgcatgacgt tatttatgag
atgggttttt atgattagag tcccgcaatt 1860 atacatttaa tacgcgatag aaaacaaaat
atagcgcgca aactaggata aattatcgcg 1920 cgcggtgtca tctatgttac tagatcggga
attgggtacc atgcccgggc ggccagcatg 1980 gccgtatccg caatgtgtta ttaagttgtc
taagcgtcaa tttgtttaca ccacaatata 2040 tcctgccacc agccagccaa cagctccccg
accggcagct cggcacaaaa tcaccactcg 2100 atacaggcag cccatcagaa ttaattctca
tgtttgacag cttatcatcg actgcacggt 2160 gcaccaatgc ttctggcgtc aggcagccat
cggaagctgt ggtatggctg tgcaggtcgt 2220 aaatcactgc ataattcgtg tcgctcaagg
cgcactcccg ttctggataa tgttttttgc 2280 gccgacatca taacggttct ggcaaatatt
ctgaaatgag ctgttgacaa ttaatcatcc 2340 ggctcgtata atgtgtggaa ttgtgagcgg
ataacaattt cacacaggaa acagaccatg 2400 agggaagcgt tgatcgccga agtatcgact
caactatcag aggtagttgg cgtcatcgag 2460 cgccatctcg aaccgacgtt gctggccgta
catttgtacg gctccgcagt ggatggcggc 2520 ctgaagccac acagtgatat tgatttgctg
gttacggtga ccgtaaggct tgatgaaaca 2580 acgcggcgag ctttgatcaa cgaccttttg
gaaacttcgg cttcccctgg agagagcgag 2640 attctccgcg ctgtagaagt caccattgtt
gtgcacgacg acatcattcc gtggcgttat 2700 ccagctaagc gcgaactgca atttggagaa
tggcagcgca atgacattct tgcaggtatc 2760 ttcgagccag ccacgatcga cattgatctg
gctatcttgc tgacaaaagc aagagaacat 2820 agcgttgcct tggtaggtcc agcggcggag
gaactctttg atccggttcc tgaacaggat 2880 ctatttgagg cgctaaatga aaccttaacg
ctatggaact cgccgcccga ctgggctggc 2940 gatgagcgaa atgtagtgct tacgttgtcc
cgcatttggt acagcgcagt aaccggcaaa 3000 atcgcgccga aggatgtcgc tgccgactgg
gcaatggagc gcctgccggc ccagtatcag 3060 cccgtcatac ttgaagctag gcaggcttat
cttggacaag aagatcgctt ggcctcgcgc 3120 gcagatcagt tggaagaatt tgttcactac
gtgaaaggcg agatcaccaa agtagtcggc 3180 aaataaagct ctagtggatc tccgtacccc
cgggggatct ggctcgcggc ggacgcacga 3240 cgccggggcg agaccatagg cgatctccta
aatcaatagt agctgtaacc tcgaagcgtt 3300 tcacttgtaa caacgattga gaatttttgt
cataaaattg aaatacttgg ttcgcatttt 3360 tgtcatccgc ggtcagccgc aattctgacg
aactgcccat ttagctggag atgattgtac 3420 atccttcacg tgaaaatttc tcaagcgctg
tgaacaaggg ttcagatttt agattgaaag 3480 gtgagccgtt gaaacacgtt cttcttgtcg
atgacgacgt cgctatgcgg catcttatta 3540 ttgaatacct tacgatccac gccttcaaag
tgaccgcggt agccgacagc acccagttca 3600 caagagtact ctcttccgcg acggtcgatg
tcgtggttgt tgatctaaat ttaggtcgtg 3660 aagatgggct cgagatcgtt cgtaatctgg
cggcaaagtc tgatattcca atcataatta 3720 tcagtggcga ccgccttgag gagacggata
aagttgttgc actcgagcta ggagcaagtg 3780 attttatcgc taagccgttc agtatcagag
agtttctagc acgcattcgg gttgccttgc 3840 gcgtgcgccc caacgttgtc cgctccaaag
accgacggtc tttttgtttt actgactgga 3900 cacttaatct caggcaacgt cgcttgatgt
ccgaagctgg cggtgaggtg aaacttacgg 3960 caggtgagtt caatcttctc ctcgcgtttt
tagagaaacc ccgcgacgtt ctatcgcgcg 4020 agcaacttct cattgccagt cgagtacgcg
acgaggaggt ttatgacagg agtatagatg 4080 ttctcatttt gaggctgcgc cgcaaacttg
aggcagatcc gtcaagccct caactgataa 4140 aaacagcaag aggtgccggt tatttctttg
acgcggacgt gcaggtttcg cacgggggga 4200 cgatggcagc ctgagccaat tcccagatcc
ccgaggaatc ggcgtgagcg gtcgcaaacc 4260 atccggcccg gtacaaatcg gcgcggcgct
gggtgatgac ctggtggaga agttgaaggc 4320 cgcgcaggcc gcccagcggc aacgcatcga
ggcagaagca cgccccggtg aatcgtggca 4380 agcggccgct gatcgaatcc gcaaagaatc
ccggcaaccg ccggcagccg gtgcgccgtc 4440 gattaggaag ccgcccaagg gcgacgagca
accagatttt ttcgttccga tgctctatga 4500 cgtgggcacc cgcgatagtc gcagcatcat
ggacgtggcc gttttccgtc tgtcgaagcg 4560 tgaccgacga gctggcgagg tgatccgcta
cgagcttcca gacgggcacg tagaggtttc 4620 cgcagggccg gccggcatgg ccagtgtgtg
ggattacgac ctggtactga tggcggtttc 4680 ccatctaacc gaatccatga accgataccg
ggaagggaag ggagacaagc ccggccgcgt 4740 gttccgtcca cacgttgcgg acgtactcaa
gttctgccgg cgagccgatg gcggaaagca 4800 gaaagacgac ctggtagaaa cctgcattcg
gttaaacacc acgcacgttg ccatgcagcg 4860 tacgaagaag gccaagaacg gccgcctggt
gacggtatcc gagggtgaag ccttgattag 4920 ccgctacaag atcgtaaaga gcgaaaccgg
gcggccggag tacatcgaga tcgagctagc 4980 tgattggatg taccgcgaga tcacagaagg
caagaacccg gacgtgctga cggttcaccc 5040 cgattacttt ttgatcgatc ccggcatcgg
ccgttttctc taccgcctgg cacgccgcgc 5100 cgcaggcaag gcagaagcca gatggttgtt
caagacgatc tacgaacgca gtggcagcgc 5160 cggagagttc aagaagttct gtttcaccgt
gcgcaagctg atcgggtcaa atgacctgcc 5220 ggagtacgat ttgaaggagg aggcggggca
ggctggcccg atcctagtca tgcgctaccg 5280 caacctgatc gagggcgaag catccgccgg
ttcctaatgt acggagcaga tgctagggca 5340 aattgcccta gcaggggaaa aaggtcgaaa
aggtctcttt cctgtggata gcacgtacat 5400 tgggaaccca aagccgtaca ttgggaaccg
gaacccgtac attgggaacc caaagccgta 5460 cattgggaac cggtcacaca tgtaagtgac
tgatataaaa gagaaaaaag gcgatttttc 5520 cgcctaaaac tctttaaaac ttattaaaac
tcttaaaacc cgcctggcct gtgcataact 5580 gtctggccag cgcacagccg aagagctgca
aaaagcgcct acccttcggt cgctgcgctc 5640 cctacgcccc gccgcttcgc gtcggcctat
cgcggccgct ggccgctcaa aaatggctgg 5700 cctacggcca ggcaatctac cagggcgcgg
acaagccgcg ccgtcgccac tcgaccgccg 5760 gcgctgaggt ctgcctcgtg aagaaggtgt
tgctgactca taccaggcct gaatcgcccc 5820 atcatccagc cagaaagtga gggagccacg
gttgatgaga gctttgttgt aggtggacca 5880 gttggtgatt ttgaactttt gctttgccac
ggaacggtct gcgttgtcgg gaagatgcgt 5940 gatctgatcc ttcaactcag caaaagttcg
atttattcaa caaagccgcc gtcccgtcaa 6000 gtcagcgtaa tgctctgcca gtgttacaac
caattaacca attctgatta gaaaaactca 6060 tcgagcatca aatgaaactg caatttattc
atatcaggat tatcaatacc atatttttga 6120 aaaagccgtt tctgtaatga aggagaaaac
tcaccgaggc agttccatag gatggcaaga 6180 tcctggtatc ggtctgcgat tccgactcgt
ccaacatcaa tacaacctat taatttcccc 6240 tcgtcaaaaa taaggttatc aagtgagaaa
tcaccatgag tgacgactga atccggtgag 6300 aatggcaaaa gctctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 6360 tgggcgctct tccgcttcct cgctcactga
ctcgctgcgc tcggtcgttc ggctgcggcg 6420 agcggtatca gctcactcaa aggcggtaat
acggttatcc acagaatcag gggataacgc 6480 aggaaagaac atgtgagcaa aaggccagca
aaaggccagg aaccgtaaaa aggccgcgtt 6540 gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag 6600 tcagaggtgg cgaaacccga caggactata
aagataccag gcgtttcccc ctggaagctc 6660 cctcgtgcgc tctcctgttc cgaccctgcc
gcttaccgga tacctgtccg cctttctccc 6720 ttcgggaagc gtggcgcttt ctcatagctc
acgctgtagg tatctcagtt cggtgtaggt 6780 cgttcgctcc aagctgggct gtgtgcacga
accccccgtt cagcccgacc gctgcgcctt 6840 atccggtaac tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc 6900 agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 6960 gtggtggcct aactacggct acactagaag
aacagtattt ggtatctgcg ctctgctgaa 7020 gccagttacc ttcggaaaaa gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg 7080 tagcggtggt ttttttgttt gcaagcagca
gattacgcgc agaaaaaaag gatctcaaga 7140 agatcctttg atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg 7200 gattttggtc atgagattat caaaaaggat
cttcacctag atccttttga tccggaatta 7260 attcctgtgg ttggcatgca catacaaatg
gacgaacgga taaacctttt cacgcccttt 7320 taaatatccg attattctaa taaacgctct
tttctcttag gtttacccgc caatatatcc 7380 tgtcaaacac tgatagttta aactgaaggc
gggaaacgac aatctgatca tgagcggaga 7440 attaagggag tcacgttatg acccccgccg
atgacgcggg acaagccgtt ttacgtttgg 7500 aactgacaga accgcaacgc tgcaggaatt
ggccgcagcg gccatttaaa tcaattgggc 7560 gcgtacgtag cactagtgcg cgatcgctta
attaagcggc gcgcctgcag gcggccgcac 7620 aattattata tcaaaatggc aaaaacattt
aatacgtatt atttaagaaa aaaatatgta 7680 ataatatatt tatattttaa tatctattct
tatgtatttt ttaaaaatct attatatatt 7740 gatcaactaa aatattttta tatctacact
tattttgcat ttttatcaat tttcttgcgt 7800 tttttggcat atttaataat gactattctt
taataatcga tcattattct tacatggtac 7860 atattgttgg aaccatatga agtgtccatt
gcatttgact atgtggatag tgttttgatc 7920 caggcctcca tttgccgctt attaattaat
ttggtaacag tccgtactaa tcagttactt 7980 atccttcctc catcataatt aatcttggta
gtctcgaatg ccacaacact gactagtctc 8040 ttggatcata agaaaaagcc aaggaacaaa
agaagacaaa acacaatggg agtatccttt 8100 gcatagcaat gtctaagttc ataaaattca
aacaaaaacg caatcacaca cagtggacat 8160 cacttatcca ctagctgatc aggatcgccg
cgtcaagaaa aaaaaactgg accccaaaag 8220 ccatgcacaa caacacgtac tcacaaaggt
gtcaatcgag cagcccaaaa cattcaccaa 8280 ctcaacccat catgagccca cacatttgtt
gtttctaacc caacctcaaa ctcgtattct 8340 cttccgccac ctcatttttg tttatttcaa
cacccgtcaa actgcatgcc accccgtggc 8400 caaatgtcca tgcatgttaa caagacctat
gactataaat atctgcaatc tcggcccagg 8460 ttttcatcat caagaaccag ttcaatatcc
tagtacaccg tattaaagaa tttaagatat 8520 actccaccgg atccaccatg gccaagctag
ttttttccct ttgttttctg cttttcagtg 8580 gctgctgctt cgctgagatt ttcggcaaga
ccttccgcga gggccgcttc gtgctcaagg 8640 agaagaactt caccgtggag ttcgccgtgg
agaagatcca cctcggctgg aagatatcgg 8700 gccgcgtgaa gggctcgccg ggccgcctcg
aggtgctccg caccaaggcc ccggagaagg 8760 tgctcgtgaa caactggcag tcctggggcc
cgtgccgcgt ggtggacgcc ttctccttca 8820 agccgccgga gatcgacccg aactggcgct
acaccgcatc cgtggtgccg gacgtgctcg 8880 agcgcaacct gcagtccgac tacttcgtgg
ccgaggaggg caaggtgtac ggcttcctct 8940 cctccaagat cgcccacccg ttcttcgcgg
tggaggacgg cgagctggtg gcctacctcg 9000 agtacttcga cgtggagttc gacgacttcg
tgccgctgga gccgctcgtg gtgctcgagg 9060 acccgaacac cccgctcctc ctcgagaagt
acgccgagct ggtgggcatg gagaacaacg 9120 cccgggtgcc gaagcacacg ccgaccggct
ggtgctcctg gtatcactac ttcctcgacc 9180 tcacctggga ggagaccctc aagaacctca
agctcgccaa gaacttcccg ttcgaggtgt 9240 tccagatcga cgacgcctac gagaaggaca
tcggcgactg gctcgtgacc cgcggcgact 9300 tcccgtccgt ggaggagatg gccaaggtga
tcgccgagaa cggcttcatc cccggcatct 9360 ggaccgcccc gttctccgtg tccgagacta
gtgacgtgtt caacgagcac ccggactggg 9420 tggtgaagga gaacggcgag ccgaagatgg
cctaccgcaa ctggaacaag aagatttacg 9480 ccctcgacct ctccaaggac gaggtgctca
actggctctt cgacctcttc tcctccctcc 9540 gcaagatggg ctaccgctac ttcaagatcg
acttcctctt cgcgggcgcc gtgccggggg 9600 agcgcaagaa gaacatcacc ccgatccagg
ccttccgcaa gggcatcgag accatccgca 9660 aggccgtggg ggaggactcc ttcatcctcg
gctgcggctc ccccctcctc ccggccgtgg 9720 gctgcgtgga tggcatgcgc atcggcccgg
acaccgcccc gttctgggga gagcacatcg 9780 aggacaacgg cgccccggcg gcccgctggg
ccctccgcaa cgccatcacc cgctacttca 9840 tgcacgaccg cttctggctc aacgacccgg
actgcctcat cctccgcgag gagaagaccg 9900 acctcaccca gaaggagaag gagctgtact
cctacacctg cggcgttcta gacaacatga 9960 tcatcgagtc cgacgacctc tccctcgtgc
gcgaccacgg caagaaggtg ctcaaggaga 10020 ccctcgagct gctcgggggc aggccgcgcg
tgcagaacat catgtccgag gacctccgct 10080 acgagatcgt gtcctcgggc accctctccg
gcaacgtgaa gatcgtggtg gacctcaact 10140 cccgcgagta ccacctcgag aaggagggca
agtcctccct caagaagcgc gtggtgaagc 10200 gggaggacgg caggaacttc tacttctacg
aggagggcga gcgcgagtga aagcttgacg 10260 tcactagtgc gatcgcgcta gccatggccg
gcctaggcgc ccgggagatc cccgaatttc 10320 cccgatcgtt caaacatttg gcaataaagt
ttcttaagat tgaatcctgt tgccggtctt 10380 gcgatgatta tcatataatt tctgttgaat
tacgttaagc atgtaataat taacatgtaa 10440 tgcatgacgt tatttatgag atgggttttt
atgattagag tcccgcaatt atacatttaa 10500 tacgcgatag aaaacaaaat atagcgcgca
aactaggata aattatcgcg cgcggtgtca 10560 tctatgttac tagatcggga attcctcgag
tctagacctg cagg 10604 4 8757 DNA Artificial pBSC11369
4 aagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa
60 aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac
120 caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac
180 aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcatctt cagactccaa
240 taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac
300 tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccac
360 cggtcgccac catggcccag tccaagcacg gcctgaccaa ggagatgacc atgaagtacc
420 gcatggaggg ctgcgtggac ggccacaagt tcgtgatcac cggcgagggc atcggctacc
480 ccttcaaggg caagcaggcc atcaacctgt gcgtggtgga gggcggcccc ttgcccttcg
540 ccgaggacat cttgtccgcc gccttcatgt acggcaaccg cgtgttcacc gagtaccccc
600 aggacatcgt cgactacttc aagaactcct gccccgccgg ctacacctgg gaccgctcct
660 tcctgttcga ggacggcgcc gtgtgcatct gcaacgccga catcaccgtg agcgtggagg
720 agaactgcat gtaccacgag tccaagttct acggcgtgaa cttccccgcc gacggccccg
780 tgatgaagaa gatgaccgac aactgggagc cctcctgcga gaagatcatc cccgtgccca
840 agcagggcat cttgaagggc gacgtgagca tgtacctgct gctgaaggac ggtggccgct
900 tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgt gccccgcaag atgcccgact
960 ggcacttcat ccagcacaag ctgacccgcg aggaccgcag cgacgccaag aaccagaagt
1020 ggcacctgac cgagcacgcc atcgcctccg gctccgcctt gccctgctct agatcccgaa
1080 tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg
1140 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat
1200 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat
1260 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt
1320 gtcatctatg ttactagatc gggaattggg gaaatttacc ggtgccgaat ttccccgatc
1380 cagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa
1440 aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac
1500 caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac
1560 aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcatctt cagactccaa
1620 taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac
1680 tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccat
1740 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
1800 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
1860 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
1920 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
1980 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
2040 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
2100 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
2160 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
2220 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
2280 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
2340 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
2400 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
2460 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
2520 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
2580 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
2640 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
2700 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
2760 atagggatcc cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
2820 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
2880 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
2940 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
3000 aattatcgcg cgcggtgtca tctatgttac tagatcggga attagcggcc cgaattcact
3060 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
3120 tgcagcacat ccccctttcg ccaggggcgg ccagcatggc cgtatccgca atgtgttatt
3180 aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca
3240 gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagaatt
3300 aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag
3360 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc
3420 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
3480 caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt
3540 gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag
3600 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc
3660 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg
3720 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg
3780 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
3840 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat
3900 ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca
3960 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
4020 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa
4080 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta
4140 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg
4200 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc
4260 aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
4320 ttcactacgt gaaaggcgag atcaccaaag tagtcggcaa ataaagctct agtggatctc
4380 cgtacccggg gatctggctc gcggcggacg cacgacgccg gggcgagacc ataggcgatc
4440 tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt
4500 tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca gccgcaattc
4560 tgacgaactg cccatttagc tggagatgat tgtacatcct tcacgtgaaa atttctcaag
4620 cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac acgttcttct
4680 tgtcgatgac gacgtcgcta tgcggcatct tattattgaa taccttacga tccacgcctt
4740 caaagtgacc gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt
4800 cgatgtcgtg gttgttgatc tagatttagg tcgtgaagat gggctcgaga tcgttcgtaa
4860 tctggcggca aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac
4920 ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc cgttcagtat
4980 cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc
5040 caaagaccga cggtcttttt gttttactga ctggacactt aatctcaggc aacgtcgctt
5100 gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc ttctcctcgc
5160 gtttttagag aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt
5220 acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc tgcgccgcaa
5280 acttgaggca gatccgtcaa gccctcaact gataaaaaca gcaagaggtg ccggttattt
5340 ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatg gcagcctgag ccaattccca
5400 gatccccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg
5460 gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc
5520 atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa
5580 gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac
5640 gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc
5700 atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc
5760 cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt
5820 gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga
5880 taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta
5940 ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc
6000 attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc
6060 ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa
6120 accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca
6180 gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc
6240 atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg
6300 ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc
6360 accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg
6420 gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc
6480 gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt
6540 cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg
6600 aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa
6660 gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt
6720 aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag
6780 ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg
6840 cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg
6900 cgcggacaag ccgcgccgtc gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa
6960 ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag
7020 ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt
7080 gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa
7140 gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt
7200 acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
7260 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
7320 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
7380 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
7440 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctct gcattaatga
7500 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
7560 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
7620 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
7680 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
7740 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
7800 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
7860 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
7920 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
7980 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
8040 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
8100 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
8160 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
8220 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
8280 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
8340 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
8400 aggatcttca cctagatcct tttgatccgg aattaattcc tgtggttggc atgcacatac
8460 aaatggacga acggataaac cttttcacgc ccttttaaat atccgattat tctaataaac
8520 gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg
8580 aaggcgggaa acgacaatct gatcatgagc ggagaattaa gggagtcacg ttatgacccc
8640 cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc aacgctgcag
8700 gaattggccg cagcggccat ttaaatcaat tgggcgcgcc gaattcgagc tcggtac
8757
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