U.S. patent application number 15/096164 was filed with the patent office on 2016-10-13 for novel method for the cheap, efficient, and effective production of pharmaceutical and therapeutic api's intermediates, and final products.
The applicant listed for this patent is Sher Ali Butt, Jacob Michael Vogan. Invention is credited to Sher Ali Butt, Jacob Michael Vogan.
Application Number | 20160298151 15/096164 |
Document ID | / |
Family ID | 57112506 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160298151 |
Kind Code |
A1 |
Butt; Sher Ali ; et
al. |
October 13, 2016 |
Novel Method for the cheap, efficient, and effective production of
pharmaceutical and therapeutic api's intermediates, and final
products
Abstract
The present invention is a method for the biosynthesis of
hundreds of compounds, mainly found in the cannabis plant. The
starting material for these compounds can be any biological
compound that is used/produced in a biological organism from the
sugar family starting materials or other low cost raw materials
processed via enzymes or within organisms to give final products.
These final products include, but are not limited to: cannabinoids,
terpenoids, stilbenoids, flavonoids, phenolic amides, lignanamides,
spermidine alkaloids, and phenylpropanoids. Specifically, the
present invention relates to the regular/modified/synthetic gene(s)
of select enzymes are processed and inserted into an expression
system (vector, cosmid, BAC, YAC, phage, etc.) to produce modified
hosts. The modified host is then optimized for efficient production
and yield via manipulation, silencing, and amplifying inserted or
other genes in the host, leading to an efficient system for
product.
Inventors: |
Butt; Sher Ali; (San Diego,
CA) ; Vogan; Jacob Michael; (Oakland, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Butt; Sher Ali
Vogan; Jacob Michael |
San Diego
Oakland |
CA
CA |
US
US |
|
|
Family ID: |
57112506 |
Appl. No.: |
15/096164 |
Filed: |
April 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62145430 |
Apr 9, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 13/02 20130101;
C12P 7/42 20130101; C12P 17/06 20130101; C12N 15/52 20130101; C12P
7/22 20130101 |
International
Class: |
C12P 13/02 20060101
C12P013/02; C12P 7/22 20060101 C12P007/22; C12P 13/00 20060101
C12P013/00; C12P 17/06 20060101 C12P017/06 |
Claims
1) Process of biosynthesis of compounds of interest (Table 1);
using a cheap and readily available starting materials in any
organism or cell-free expression system (Table 2).
2) Process of genetically engineering micro-organisms that produce
compounds of interest in large/industrial-scale quantities.
3) Process of altering genes in organisms to allow better yields
for compounds of interest.
4) Process of genetically engineering food plants that are
optimized for production of compounds of interest.
5) Using s. cerevisaie, e coli, p. pastorisis, n. crassa, s. pombe,
r. palmatum, c. longa, o. sativa, a. arborescens, a. terrus, c.
sativa, s. griseus, s. erythera, s. coelicolor, s. toxytruini, s.
cellulosum, p. floresceus, a. orientalis, s. livedans, a.
vinelandii, b. subtilis, m. tuberculosis, m. xanthus, a. oryzae, a.
niger, r. palmatum, h. serrata, r. rubra, l. erythrhizon, l.
acetobutylicum, c. utilis, a. niger, c. glutamicum, b. spp., and
other large-scale biotechnology organisms to biosynthesize a total
pathway using cheap and readily available starting materials to
make our compounds of interest.
6) Process of claims 1 to 5, wherein the products are those listed
in table 1.
7) Process of claims 1 to 6, wherein the starting materials are
those listed in table 2.
8) Process of claims 1 to 7, wherein certain steps are performed
outside or inside an organism or cell free expression system using
various techniques, while other steps may use biosynthesis
techniques.
9) Producing compositions (e.g. tinctures, balms, capsules,
tablets, concentrates, edibles, topicals) that use claims 1 to 8,
whether they are classified as pharmaceuticals or not.
10) Producing compositions that contain combinations of certain
compounds for certain therapeutic and non-therapeutic applications,
as listed in table 1.
11) To claim 10, compositions that have added cofactors for better
efficacy, absorption, etc, for our compounds of interest. These
include terpenes, sequiterpenes, vitamins, and other cofactors.
12) cDNA sequences of enzymes and organisms in the pathways and
processes listed in FIGS. 1-7 for biosynthesis of compounds of
interest using cheap and readily available starting materials,
including mRNA's, tRNA's, vectors, and other genetic coding
molecules needed for process of claims 1 to 8.
13) A method for the efficient and cheap biosynthesis of CBGA,
THCA, CBDA, CBCA, CBDVA, THCVA, CBGVA, and CBCVA utilizing
manipulation of any ERG, IDI, gal80p, upc2-1, HMGR, tHMGR, ALD6,
DPP1, and ADH2 genes that result in exogenous sterol mutations for
the uptake of exogenous sterols, more permeable cell membrane for
easy inward flow of exogenous materials while easy outward flow of
our compounds of interest, and carbon flux manipulation and
blocking of other pathways for many fold higher yields in S.
Cerevisiae and P. Pastoris.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. Provisional Patent Application Ser. No. 62/145,430, entitled
"A Novel Method for the Cheap, Efficient, and Effective Production
of Pharmaceutical and Therapeutic API's, Intermediate, and Final
Products", filed Apr. 9, 2015, and currently co-pending.
FIELD OF THE INVENTION
[0002] The present invention is in the technical field of large
scale production of pharmaceutical and supplemental products for
various common illnesses, medical conditions, and general
industrial use. More particularly, the present invention is in the
technical field of bio-synthesis of cannabinoids, terpenoids,
stilbenoids, flavonoids, phenolic amides, lignanamides, spermidine
alkaloids, and phenylpropanoids; compounds found in cannabis
sativa, along with various combinations and specialized
formulations which are beneficial in ailments ranging from cancer
to glaucoma. The final product(s) can be an intermediate or a
compound of interest. The core concept of the invention is based on
the idea of cheaper and more efficient production, along with novel
products and applications.
BACKGROUND OF THE INVENTION
[0003] Cannabinoids from cannabis have been used for thousands of
years for treatment of various ailments and conditions in many
different cultures around the world. However, most of various types
of cannabinoids in cannabis are at a very low concentration in the
plant. Therefore, most patients/users never get a threshold dosage
for any kind of relief from anything other than
tetrahydrocannabinolic acid (THC/A), cannabinolic-acid (CBD/A), and
cannabinol (CBN). There are a few strains or concentrates available
that have a rare cannabinoid, but are usually very highly
concentrated in tetrahydrocannabinol (THC) or cannabidiol (CBD) to
have any pronounced effect by the rare cannabinoid.
[0004] In other words, the pharmaceutical industry has not tapped
into the real potential of the cannabis plant. With time, more
research is being conducted into the different kinds of
cannabinoids and their medicinal applications. Researchers are
finding that many of the other cannabinoids also have unique
medicinal properties.
SUMMARY OF THE INVENTION
[0005] Biosynthesis of important molecules can be used for
therapeutic applications, bulk substance production, intermediate
API biosynthesis, and various other novel formulations and
applications for such substances, as known to those skilled in the
art. Many biological molecules can be changed/converted into
molecules of importance by using enzymes and other processes. This
process can be utilized by employing methods for transforming a
range of starting materials into final products to be used in
pharmaceuticals and supplements as active ingredients, or donating
a significant portion of their structure to the final active
ingredients. The final products can also be used in other
industries and applications, such as food, beverage, and other
goods production. For example, table sugar, starch, and cellulose
can be converted to glucose, creating a molecule that can readily
be utilized by any organism as an energy source. Therefore,
depending on the specific compound(s) being manufactured, and the
kind(s) of starting materials available, along with the host and
production technique(s) any kind of host engineering, various
expression systems and methods, and varying materials, a spectrum
of different methods and products is possible.
[0006] The advantages of the present invention include, without
limitation, creation of hundreds of compounds from readily
available biological molecules that can be produced and harvested
from virtually all known sources of plants and other energy
producing organisms. Since sugar producing plants and organisms,
biomass, and carbon based industrial waste products are very
abundant, our "raw material" will be very cheap and easy to obtain
anywhere in the world. After scaling up the given methods, hundreds
of compounds with medicinal properties can be produced at a very
low cost, allowing the widespread distribution and aiding of
millions of people.
[0007] Another advantage is that there is no need or use of growing
any illegal plants. For example, no marijuana, poppy, or other
plant production is necessary. This is advantageous as it will lead
to drastically cutting down the production, consumption, and
trafficking of many unregulated substances.
[0008] The most important advantage of the present invention is
that we can make and use many compounds that are virtually so low
in concentration in the cannabis plant, that there is no effect in
using cannabis if we are only after the therapeutic effects of
these compounds. For example, patients using marijuana can only
benefit from tetrahydrocannabinolic acic (THCA), THC, cannabidiolic
acid (CBDA), CBD, CBN, and a few other compound class families, as
the concentrations of the other compounds is so low that it has no
effect. This invention will allow the production of hundreds of
compounds in pure form, leading to many new medical discoveries and
applications.
BRIEF DESCRIPTION OF THE FIGURES
[0009] The nature, objects, and advantages of the present invention
will become more apparent to those skilled in the art after
considering the following detailed description in connection with
the accompanying figures, in which like reference numerals
designate like parts throughout, and wherein:
[0010] FIG. 1 is a diagram of the pathway for the biosynthesis of
all molecules of interest via the conversion of starting materials
to glucose and then to final products;
[0011] FIG. 2 is a diagram of the pathway for the biosynthesis of
cannabinoids;
[0012] FIG. 3 is a diagram of the pathway for the biosynthesis of
stilbenoids;
[0013] FIG. 4 is a diagram of the pathway for the biosynthesis of
phenylpropanoids and flavonoids;
[0014] FIG. 5 is a diagram of the pathway for the biosynthesis of
phenolic amides and ligananamides;
[0015] FIG. 6 is a diagram of the pathway for the biosynthesis of
spermidine alkaloids;
[0016] FIG. 7 is a diagram of the combined biosynthetic pathways of
FIGS. 1-6; and
[0017] FIG. 8 is diagram of the genetic modification of certain
genes for higher product yield in Saccharomyces cerevisiae
yeast.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention is a method for the biosynthesis of
hundreds of compounds, mainly found in the cannabis plant. The
starting material for these compounds can be any biological
compound that is used/produced in a biological organism from the
sugar family starting materials or other low cost raw materials
processed via enzymes or within organisms to give final products.
These final products include, but are not limited to: cannabinoids,
terpenoids, stilbenoids, flavonoids, phenolic amides, lignanamides,
spermidine alkaloids, and phenylpropanoids (collectively, "final
products").
DEFINITIONS, TERMS, ELEMENTS
[0019] The following are a list of terms and their definitions:
[0020] Genetic engineering: targeted manipulation of a cell's
genetic information [0021] Rational Metabolic Engineering:
engineering of enzymes, transporters, or regulatory proteins based
on available information about enzymes, pathways, and their
regulation. [0022] Evolutionary engineering: encompasses all
methods for empirical strain improvement (mutagenesis [natural or
induced] and recombination and/or shuffling of genes, pathways, and
even whole cells; usually performed in cycles or sequentially
[0023] Cannabinoids: compounds that are terpenophenolic with 22
carbons (21 carbons for neutral forms), found in cannabis [0024]
Terpenoids: also known as isoprenoids, class of organic compounds
[0025] Stilbenoids: hydroxylated derivatives of stilbene [0026]
Flavonoids/phenylpropanoids: compounds derived from or using
phenylalanine as a precursor [0027] Lignanamides/phenolic amides:
compounds produced through tyramine pathways [0028] Spermidine
alkaloids: compounds produced through glutamic acid pathways [0029]
Starting material/reactant/excipient: compounds used for the
initial step of biosynthesis, which are cheap and readily available
[0030] Intermediate: products that are formed within the
biosynthesis pathways, which can further be processed to make final
products, or can, themselves, be utilized as a final product [0031]
Final product/product/end product/compounds of interest:
cannabinoids, terpenoids, stilbenoids, flavonoids, phenolic amides,
lignanamides, spermidine alkaloids, and phenylpropanoids [0032]
In-vivo: inside the cell [0033] In-vitro: outside the cell [0034]
BAC: bacterial artificial chromosome, carrier of DNA of interest
into host [0035] YAC: yeast artificial chromosome, carrier of DNA
of interest into host [0036] Vector/cosmid/phage: carrier of DNA of
interest into host
Starting Materials
[0037] All biological organisms produce organic molecules that are
processed in many different processes in the organism. The present
invention utilizes starting materials that are either: [0038] 1)
Readily available and relatively pure [0039] 2) Cheap to produce or
buy [0040] 3) Easily modified (via enzymes, catalysts, or other
methods)
[0041] Based on the above criteria, there are multiple groups and
families of compounds that would fit one or all three of the above
criteria. These groups and families of compounds include, but are
not limited to: ligno-cellulosic biomass, forest biomass,
energy/food production waste, commonly available sugar-based
substrates, food and feed grains.
[0042] Sugars and metabolic intermediates from cellular processes
can be used as starting materials. Sugars can be found in abundance
in many substances, including, but not limited to the following:
rice, soya/rape, cereals (maize), wheat, beans, sugar beet (sugar
cane), plant biomass (wood), grasses, and various other sources.
Starch, cellulose, fructose, ethanol, and saccharose in the
aforementioned substances can be enzymatically converted to
glucose, which, after filtration and purification steps, can be
used as a raw material for the final products.
[0043] Subsequent steps can also be performed on the
lignocellulose, which further makes hemicellulose and cellulose,
both which make glucose. An advantage of this method is that there
are by-products generated which can be sold as raw material to make
hydrocarbons, biogas, and other fuel sources. Whole crops or parts
of crops, or waste matter from crop products can be used and
incorporated into this system, yielding an "eco-friendly" facility.
Products made from these raw materials can use any of the starting
materials listed in Table 2.
[0044] Within the realm of readily available non-biomass/crop bulk
material, HFCS (high fructose corn syrup) is a cost effective syrup
made with fruit sources that contains anywhere from 30-90%
fructose, along with some other sugars. Plants that make molasses,
HFCS, and other sugars can be genetically modified to enhance the
production of sugar, leading to better yields of starting material
from the crop. Other products from these plants can also be
incorporated into compounds of interest production via slight
system modification. Biodiesel, ethanol, glycerol, lactic acid,
whey and glucose are a few others. These work due to the fact that
any of these products can be converted into starting material for
our own purposes using enzymatic or physiochemical tools.
[0045] Plants also have their own innate levels of metabolites that
can be harvested into the process from a plant biomass source.
Processes can be crafted that utilize most of the metabolites and
biomass for API production giving the maximum efficiency and
usability per amount of starting material used. (Enzyme
combinations or chambers that utilize most intermediates, sugars,
oils, etc. in each biomass load).
[0046] Biorefineries can be custom designed that cater to specific
raw material (plant biomass for harvesting lignocellulose which is
further processed and refined into a simple carbohydrate used in
the API manufacturing processes). During certain steps throughout
the process, thermochemical and other processing can be used for
higher efficiencies which are not possible with biochemical
processing alone.
[0047] Another group of cheap starting materials is agricultural
residue, grass, aquatic biomass, and water hyacinth. Products such
as oils and alcohols can be made with these bulk materials. These
materials can be converted enzymatically and chemically into
starting materials that can readily by injected into our API
production system.
[0048] Specifically, biorefineries can be designed to be extremely
efficient, using all parts of the raw material. For example,
concerning plant biomass, the biomass can be step-wise processed so
we are able to harvest all individual components. The first step
can be using solvent to extract terpenes, alkaloids, etc. Other
methods can be used to extract steroids, triglycerides, and other
valuable metabolites. Finally the biomass can be treated with
cellulases to give glucose, which is one of the primary raw
materials of choice.
Production Roadmap Summary
[0049] The present invention is a method that covers the
bio-synthesis of hundreds of compounds, mainly found in the
cannabis plant. The starting material for these compounds can be
any biological compound that is used/produced in a biological
organism from the sugar family starting materials or other low cost
raw materials processed via enzymes or within organisms to give
final products. Information related to the starting materials were
detailed in the previous section.
[0050] Most sugars and related compounds can be inter-changed using
various enzyme systems. For example, we can convert glucose to
fructose using Fructose 6-Phosphate (F-6-P) as an intermediate.
[0051] Apart from starting materials, we can either: [0052] 1) Make
enzymes via vectors in bacteria (e.g. E. coli) or yeast (e.g. S.
cerevisiae), extract enzymes, and create in vitro models for making
cannabinoids. [0053] 2) Make enzymes via protein synthesizing
systems (Protein Synth. Robot, Cell Free Expression Systems, etc.)
[0054] 3) Make final products (compounds of interest) in bacteria
or yeast via vectors, plasmids, cosmids, mRNA, various RNA, etc;
feed them substrate and purify product. [0055] 4) Genetically
engineer strains of bacteria and yeast that specialize in
cannabinoid production, or intermediate production, or substrate
production, etc. [0056] 5) Use organic chemistry for certain parts
of the above processes. [0057] 6) Use various plant starting
material for large quantities of substrates or intermediates.
[0058] 7) Genetically engineer various plants to produce
cannabinoids. (e.g. Tomatoes or celery that naturally produce
cannabinoids, or algae that produces cannabinoids) [0059] 8) Using
bioengineered or unengineered C. sativa or any other plant/algae
cell lines for enzyme/substrate/intermediates/product(s)
production. [0060] 9) Protein engineering on the various proteins
involved in the processes; engineering will enhance the
functionality, ruggedness, and efficiency of the enzymes, and
altering them into a novel protein, one not found to be covered in
any of the above prior art patents. [0061] 10) Genetically engineer
various plant species to produce higher yielding raw material
(sugars) to be used in production of the products. A possibility is
to have an indoor/grow for different plants to be used as raw
material producers.
[0062] After the final product is made, a purification system will
filter and concentrate the target molecules. Examples include large
scale filtration systems such as chromatography. Once a pure
product, we can utilize liquid solutions, caps, sprays, and other
delivery systems.
[0063] As many of these final products are made, their applications
can be seen from glaucoma to cancer, or general well-being. Certain
cofactors can be combined with certain final products for more
efficacy against specific medical conditions (e.g. combine certain
vitamins or other therapeutic compounds with certain compounds of
interest). We can also make final products that have certain
combinations of compounds of interest with other cofactors as well
(e.g. combine THCA/CBDANitamin C, or CBDVA/CBD). This patent covers
all the products above and also ones discovered in the future based
on the same principles and methods.
DETAILED DESCRIPTION OF THE FIGURES
[0064] Referring now to the invention in more detail, in FIG. 1
there is shown a family of sugars and other common derivatives.
Along each arrow for each reaction, the number denotes a specific
enzyme that catalyzes the reaction. Starting with any sugar in
Figurel (list of starting materials in Table 1), we can convert it
to glucose to incorporate it into the reaction using the
appropriate enzyme, as known to those skilled in the art. An
unlimited number of ways are possible when dealing with any
starting material, as described above. Enzymes needed for each kind
of substrate can be made in vivo or in vitro just as we will be
doing for the enzymes in the final product or intermediate
production. The final sugar that enters our mechanism will be
either glucose or fructose. Through the glycolysis pathway, the
sugar will be converted into Acetyl-CoA with the addition of ATP
and CoA (shown in FIG. 1). From this point on, the intermediate can
follow a variety of paths that can lead to hundreds of products.
There are many alternative ways of doing this. We can use the DOX,
MEP or MVA pathways to get IPP and DMAPP, which give us GPP and
NPP. For a reaction with Olivetolic Acid or Divarinolic Acid, we
get many cannabinoids as final products.
[0065] The generalized pathway for the production of cannabinoids
once the starting material is converted to glucose is the
following, using appropriate enzymes as known by those skilled in
the art: [0066]
Glucose.fwdarw.Fructose.fwdarw.F-6-P.fwdarw.FI:6BP.fwdarw.3-P-Glyceraldeh-
yde.fwdarw.1,3-BPG.fwdarw.3PGA.fwdarw.2-PGA.fwdarw.PEP.fwdarw.Pyruvate.fwd-
arw.Acetyl-CoA.fwdarw.Acetoacetyl
CoA.fwdarw.HMG-CoA.fwdarw.MVA.fwdarw.Mevalonic
Acid.fwdarw.Mevalonate-5-P.fwdarw.Mevalonate-5-PP.fwdarw.Isopentyl-5-PP.f-
wdarw.Dimethylallyl-PP.fwdarw.NPP/GPP.fwdarw.GPP
[0067] This general pathway is outlined in FIG. 1. From this point
on, the pathway can utilize Olivetolic Acid or Divarinolic Acid
with GPP, yielding CBGA or CBGVA, which can further yield other
cannabinoids, as shown in FIG. 2.
[0068] The pathways for stilbenoids, phenylpropanoids, and
flavonoids work in a similar fashion. Phenylalanine is generated
from sugars, which is then further processed into other compounds
using enzymes to final compounds, as shown in FIG. 3 and FIG.
4.
[0069] Phenolic amides and lignanamide pathways are derived from
tyramine molecules reacting with other compounds, as shown in FIG.
5. Tyramine can also be synthesized in our cells of interest as
most living organisms contain the pathway to synthesize tyramine on
their own. Same is the case for spermidine alkaloid production, as
most cells already produce glutamic acid, which can be further
processed by enzymes into the final components, as shown in FIG.
6.
[0070] FIG. 7 is the total pathway overview, showing how all the
different classes of compounds can be made, and the general paths
they take for being biosynthesized in the cell.
Overview of Procedure
[0071] A general scheme of the work flow is as follows: [0072] 1)
Regular/modified/synthetic gene(s) of select enzymes are processed
and inserted into an expression system (vector, cosmid, BAC, YAC,
phage, etc.) to produce modified hosts. [0073] 2) Mod host is then
optimized for efficient production and yield via manipulation,
silencing, and amplifying inserted or other genes in the host,
leading to an efficient system for product. It is important to
remember that every organism is different, and to get a specific
compound each optimization will also be different. [0074] 3) Mod
host can produce enzymes and final products/intermediates, or be
further modified using host engineering techniques. (Host
engineering Can also be performed before insertion of exp. System)
[0075] 4) Mod and engineering hosts produce products and
intermediates. [0076] 5) Product is purified and can be further
modified/processed.
[0077] In Table 1, different final products are listed along with
possible uses. This list is by no means exhaustive, and as such
this patent covers any molecules that are made this way. Table 2
lists all possible starting materials that can be utilized for a
cheap and efficient biosynthesis.
[0078] In more detail, referring to the inter-conversion of sugars,
we employ enzymes readily available in the market. Pure enzyme
stock can be diluted and added to a solution with the substrates.
Once the reaction is complete, we can filter out the enzyme via
dialysis tubing, by precipitation out of the solution,
chromatography, or other industrial methods for filtration and
purification. Each step in FIGS. 1 to 7 will give work with this
strategy, leading us up to the final products or key intermediate
molecules. Certain steps in the process can be worked on by using
chemical and physical methods as well. For example, prenylation of
certain compounds can be done outside the cell, as it may be
advantageous to do so since unprenylated compounds are also high
value compounds. Small batches can be prenylated accordingly to
demand via a chemical process.
[0079] There are also commercially available cell free expression
systems, which are able to produce proteins without the need of any
host. With appropriate optimization steps, it is possible to get a
cheap and efficient process for production of these compounds using
identified starting molecules.
Application Techniques
[0080] Referring to bacterial, yeast, plant, and algae
incorporation of genes, there are a number of strategies that can
be applied to achieve this. We can: [0081] 1) Add genes for 1-10
enzymes in various commercially available vectors, cosmids,
plasmids, etc. Only need 1-10 enzymes added, as others are already
built in most living organisms. For example, glycolysis pathway and
related enzymes are already present in most hosts. [0082] 2)
Bioengineer genes for better yield and suitability in the host.
[0083] 3) Bioengineer strains of bacteria and yeast that are
specialized in producing important molecules. Many metabolic
strategies exist, with identification by appropriate screening
methods: [0084] 1) Rational metabolic engineering: engineering
pathways using available information [0085] 2) Evolutionary
engineering: using random genetic perturbations and/or mutations
(via random mutagenesis in whole genome and/or parts) [0086] 3)
Transposon mutagenesis & gene overexpression libraries:
overexpression and/or deletion of single or multiple genes; [0087]
4) Global transcription machinery engineering: basal transcription
factors mutagenesis causing a global reprogramming of gene
transcription and/or translation [0088] One strategy is to suppress
any pathway that is not essential to our goals or the survival of
the host.
[0089] Another is to enhance our key pathways, or mixing and
matching the two methods. The second strategy is through rapid
directed evolution, possible by producing many generations so
eventually we get a generation of host that has evolved with our
genes/functions of interest. [0090] 4) Bioengineer custom basic
life forms that are specifically making our products, using another
organism or using synthetic/modifications. Components from other
hosts and system to make a custom organism. [0091] 5) Bioengineer
bacteria and yeast to have enzyme genes in their chromosomes, and
make intermediates or final products inside the host. The product
of this process can further be modified. [0092] 6) Propagate
various colonies of organisms which co-exist symbiotically, with
the first making our starting material after utilizing a precursor,
and the other colonies making our final product. This process can
also be incorporated into an ecosystem type setup of different
chambers, each holding different organisms that use specific parts
of the raw material to produce intermediates or final products that
can be modified post-manufacturing.
[0093] Referring to the extraction of enzymes once they have been
produced in the host, there are many ways to isolate and purify our
enzymes. Many organisms have the ability to excrete proteins, which
can be collected much easier than cell lysis, as known by those
skilled in the art. This technique is the preferred method.
[0094] Another method is to lyse the host culture and purify with
traditional biochemistry methods (gels, centrifugation, ammonium
sulfate precipitation, etc.), use a specialized nickel column with
a prep HPLC (need to add a HIS tag to our proteins; remove HIS tag
after purification), etc.
Example
Bacterial
[0095] Bacteria (E. Coli, etc.) are inserted with exp. system
giving us a modified host. The mod host can either be further
processed or it can generate products. Products/intermediates are
made in the host, and may be either enzymes that are further
extracted and used in vitro, or we add substrates into the
bacterial culture so they use the enzymes produced in them to make
the substrate. Either way (protein or prod production),
purification is carried out to get final products, or intermediates
that can be further processed in vitro to give final products.
Throughout this procedure, host engineering can be carried out at
any step of any process to get better yields.
Example 2
Plants
[0096] Plant tissue can be used as a starting material to get a
tissue culture going. Appropriate expression vectors/systems carry
our interest genes into the cells. Alternatively, cell engineering
can lead to many combinations that may have similar or different
outcomes. The culture can be grown into full plants, and products
are ingested by consuming the plants (e.g. tomatoes with certain
cannabinoids produced within, etc.). The second way uses the cell
culture in a synthetic environment to produce final
products/intermediates. Finally, product is purified and used.
Example 3
Algae
[0097] Algae are modified with the usual techniques used for host
engineering. Once completed, the mod host can be embedded into a
system similar to biofuel production from algae. Using sunlight and
some nutrients, the algae produces final products/intermediates,
which is appropriately filtered from the bulk. Other products
generated can be further processed to get biofuels or other
important compounds that can readily be sold in the market.
Example 4
Fungi
[0098] Fungi modified with the techniques can: [0099] 1) Use
plastic to produce final products/intermediates. Plastic needs to
be processed and broken down into components before being used in
this process via chemical and biological processes, known by those
skilled in the art. [0100] 2) Clean up waste, whilst producing
final products/intermediates at the same time. [0101] 3) Produce
beer and wine with fungi that also makes final prod/intermediates.
Beer and wine will contain our compounds of interest. [0102] 4) Use
fungi cultures to produce compounds of interest. [0103] 5) Genes
for s. cerevisiae strains to be modified for better yields of final
products: [0104] tHMGR [0105] upc2-1 (allows higher uptake of
exogenous sterol five-fold from medium) [0106] ERG genes (ERG6,
ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG9, ERG10, ERG13, ERG12,
ERGS, ERG19, ERG20) [0107] HMGR1 and HMGR2 [0108] IDI genes [0109]
Gal80p [0110] DPP1, ADH2, and ALD6 genes [0111] FPP/GPP synthase
(chose avian FPP synthase as it exhibits higher catalytic turnover
rates and lower Kms for substrates than other
prenyltransferases)
[0112] Manipulation, deletion, overexpression, and other
modifications to the genes listed above will produce strains that
are highly efficient for the production of our compounds of
interest. These strains have an exogenous sterol uptake, as the
internal sterol pathway has been disabled by manipulations so that
all the carbon flux can be directed toward the production of our
compounds of interest. Example of genetic pathway regulation in
yeast is shown in FIG. 8.
[0113] Our initial strategy in S. Cerevisiae was to increase the
carbon flux of our pathways of interest, while decreasing or
eliminating pathways that led carbon flux away from our pathways as
well. We also focused on exogenous sterol uptake for higher
production and secretion levels, cell permeability for more
efficient and cheaper production, along with focusing the pathways
on utilizing the cheapest sugars. Dynamic control over ergosterol
regulation can increase yields as well. Overall result is a strain
that is has increased yield many fold, while making the overall
production more stable and cheaper. [0114] 1) Perform EMS
mutagenesis on yeast strains (BY4741, BY4742, CEN.PK, CEN.PK2,
EPY300) to get colonies with a SUE (sterol uptake exogenous)
mutation. This enables us to provide exogenous sterol to the yeast
while cancelling out the gene that diverts carbon flux towards
ergosterol, thereby increasing total carbon flux. Without the SUE
mutation, the cell diverts lots of carbon flux toward manufacturing
sterols, thereby diverting the pools of intermediates away from our
compounds and interest leading to very low yields. [0115] 2)
Perform ERG1 and ERG9 gene knockouts. ERG1 knockout stops the
activity of conversion of squalene to squalene epoxide, thereby
complementing the SUE mutation and allowing higher uptake of
exogenous ergosterol, while ERG9 knockout takes out the cells
ability to divert carbon flux towards other metabolites. [0116] 3)
On some lines, we can perform a DPP1 knockout. DPP1 knockout
ensures that FPP/GPP are not converted to FOH, thereby blocking the
pathway towards FOH products in the cell. [0117] 4) Perform ERG2,
ERG3, or ERG6 mutations in different cell lines, while performing
upregulation mutation on upc2-1 gene (general transcription factor)
on all three lines. This helps increase cell membrane permeability
for better excretion of our compounds without the need for cell
lysis and having the ability to use two-phase or continuous
fermentation. This also allows the cells to uptake more fatty
acids, thereby increasing the yield many fold. [0118] 5)
Overexpression of ERG10, ERG13, HMGR1/2 or tHMGR, ERG12, ERG8,
IDI11, HFA1 genes in yeast inserted via vectors. By overexpression
of these genes, we are amplifying the enzymes of the MVA pathway
from the sugars to our compounds, thereby amplifying the
intermediates and final products. [0119] 6) Modification of avian
and/or salmonella ERG20 gene encoded FPP synthase (ERG20p). Some
cells lines can also be modified using the Erg20p(F96C) mutations.
This allows for higher Kms and increased catalytic turnover
compared to endogenous GPP synthase, while the engineering itself
allows for production of GPP. [0120] 7) Gal80p gene deletion so we
do not need to use galactose sugar when inducing promoter
expression. This is important since others have used galactose
promoters, which need expensive galactose sugars for production. By
deleting this gene, the cells bypass the need for galactose to
express enzymes, leading to cheaper and more efficient
biosynthesis. [0121] 8) Adding ADH2p promoter to induce strong
transcription under conditions with low glucose. This promoter is
more efficient than the GAL promoter, and has best results while
using non-glucose sugars (ethanol, fructose, etc.) which are
cheaper. [0122] 9) On some lines, we also overexpress ADH2 and ALD6
genes, along with overexpression of an acetyl-CoA
C-acetyltransferase to increase efficiency of the system, while
also gaining the ability to convert ethanol to acetate efficiently.
[0123] 10) Adding and overexpressing enzymes for the production of
CBDA (OS-OAC fusion enzyme, CsPti, CBDA Synthase), constructed in a
single vector. These enzymes are codon optimized. [0124] 11) Grow
colonies while adding free fatty acids, and hexanoic acid (for
THCA, CBDA, CBGA, CBCA) or butyric acid (for THCVA, CBDVA, CBGVA,
CBCVA). [0125] 12) For production of THCA/THCVA, use THCA synthase
in step 10 instead of CBDA synthase. For production of CBGA/CBGVA,
follow step 10 but don't use CBDA synthase in vector construct. For
production of CBCA/CBCVA, use CBC synthase in step 10 instead.
[0126] Our strategy for Pichia pastoris (Pichia Pink 1, 2, 3 from
Invitrogen) yeast was similar to S. Cerevisiae, except for the
following differences: [0127] 1) Each enzyme, vector, and primer
were optimized for insertion into pichia cells instead of s.
cerevisiae. [0128] 2) Methanol is used to supplement cells in
addition to free fatty acid, hexanoic acid, and butyric acid,
thereby reducing the total cost of production many fold, while
eliminating any contamination issues from other species. [0129] 3)
No EMS mutagenesis is performed. [0130] 4) Knockouts of pep4
(encoding Proteinase A), prb1 (encoding Proteinase B), and YPS1
genes are also introduced. These knockouts allow for the
integration of high copy plasmids leading to higher yields. [0131]
5) Steps 7, 8, and 9 from the S. cerevisiae strategy above are not
to be performed in pichia cells.
Example 5
Cell Free Expression Systems
[0132] Vectors are introduced into cell free expression systems,
and make either enzymes or intermediate/final products. Further
processing or steps are needed to get purified final products.
Procedures
EMS Mutagensis (S. Cere.; BY4741, BY4742, CEN.PK, CEN.Pk2,
BY300)
[0133] 1) Cells incubated overnight @30 C in 5 mL TPD medium while
shaking @200 rpm to establish 200 mL YPD shake flask culture.
[0134] 2) When OD600 of yeast culture reaches 1.0, cells are spun
down by centrifugation (12 mins at 4,000 g), washed twice with 20
mL 0.1M sodium phosphate buffer, pH7.0. [0135] 3) Cells
concentrated by centrifugation again, re-suspended in 1 mL 0.1M
sodium phosphate buffer, transferred to 30 mL FALCON tubes, treated
with 300 uL EMS (1.2 g/mL). [0136] 4) Cells are incubated at 30 C
for 1 hr while shaking. [0137] 5) Stop mutagenesis by adding 8 mL
of sterile 5% sodium thiosulfate to yeast cells. [0138] 6) Cells
are pelleted, washed with 8 mL sterile water, concentrated by
centrifugation, re-suspended in 1 mL sterile water and 100 uL
aliquots plated into YPD-NCS agar plate (YPD+50 mg/L each of
cholesterol, nystatin, sqalestatin, and 2% Bacto-agar). [0139] 7)
In some instances, washed cells were resuspended in 1 mL YPDE
liquid media for overnight recovery before plating to YPD-NCS agar
medium. [0140] 8) Incubate cultures for up to two weeks at 30 C
until distinct colonies are visible.
Bacteria & Yeast Culturing
[0140] [0141] 1) Grown using standard culture practices. [0142] 2)
YPD media without selection consisted of 1% Bacto-yeast extract, 2%
Bacto-peptone, and 2% glucose. [0143] 3) Add 40 mg/L ergosterol to
YPD media to get YPDE media. [0144] 4) Add 40 mg/L each of
nystatin, cholesterol, and squalestatin to YPD media to get TPDNCS
media. [0145] 5) Add 40 mg/L each of ergosterol and squalestatin to
YPD media to get YPDSE media. [0146] 6) Prepare minimal media, SCE
(pH5.3), by adding 0.67% Bacto-yeast nitrogen base (without amino
acids), 2% dextrose, 0.6% succinic acid, 0.14% Sigma yeast dropout
soln (-his, -leu, -ura, -trp), uracil (300 mg/L), L-tryptophan (150
mg/L), L-histidine (250 mg/L), L-methionine (200 mg/L), L-leucine
(I g/L), and 40 mg/L of ergosterol. [0147] 7) Cholesterol and
ergosterol stocks are 10 mg/mL in 50% Triton X-100, 50% ethanol and
kept at -20 C. [0148] 8) Selection media prepared similarly except
without supplementation of media with indicated reagent based on
the yeast auxotrophic markers. [0149] 9) All solid media plates are
prepared with 2% Bacto-agar.
Yeast Transformation & Culture Performance
[0149] [0150] 1) Used FROZEN-EZ Yeast Transformation II Kit from
Zymo Research, Orange, CA, according to manufacturer's
recommendations. [0151] 2) lug of plasmid was used per
transformation, followed by selection on agar plates of SCE medium
lacking specified amino acids for auxotrophic markers, or YPDE
containing 300 mg/L hygromycin B for screening erg9 knockout at 30
C. [0152] 3) Colonies are picked and used to start 3 mL cultures in
minimal media to characterize their terpene production
capabilities. (6 days incubation at 30 C while shaking) [0153] 4)
Best cultures are chosen to move further, using 30 mL shake flask
cultures. [0154] 5) Cultures are grown to saturation in minimal
media, inoculated into 30 mL SCE media and 1 mL aliquots are taken
out daily for 15 days. [0155] 6) Cell growth is monitored via
change in optical density at 600 nm every two days using dilutions
at later stages of growth. [0156] 7) Production of terpenes is
determined via testing.
ERG9 Knockout Mutations
[0156] [0157] 1) Primers ERG9PS1 and ERG9-250downS2 used to amplify
hygromycin resistance gene, hphNT1, from the pFA6-hph-NT1 vector.
[0158] 2) Simulataneously add 42 bp nucleotide sequences homologous
to regions surrounding ERG9 gene in yeast genome. [0159] 3)
Purified PCR fragment is transformed into various cell lines
identified in phase 2 with the ability to accumulate farnesol and
selected on YPDE plates containing 300 mg/L hygromycin. [0160] 4)
Independent single colonies are picked for ergosterol dependent
test, PCR confirmation of recombination with hphF and ERG9 450DWR
primer. [0161] 5) Farnesol production analysis done by
GC-MS/LC-MS.
ERG1 Knockout Mutations
[0161] [0162] 1) Primers ERG1F and ERG1R used to amplify the
sqalene epoxidase synthase ERG1 gene by using Takara high fidelity
Primerstar taq polymerase. [0163] 2) Obtained PCR fragment is gel
purified, A tailed and ligated into the pGEM-Teasy vector. [0164]
3) Obtained vector is used as template to run second PCR with
primers Erg1-splitF and EGR1-splitR to obtain PCR fragment with
deletion of 8916 bp CDS in the middle, yet containing 310 bp at 5'
end region and 291 bp at 3' end region of ERG1 gene which are the
target homologous recombination sequence for ERG1 knockout. [0165]
4) After digestion with BamHI, self-ligation, and transformation to
DH5alpha competent cells, resulting vector is pGEM-ERG1-split.
[0166] 5) Padh-Kanmx4-Tcyc-LoxP antibiotic selection marker
cassette is constructed by assembly PCR of three fragments. [0167]
6) Padh promoter is PCR amplified with Padh-loxP-ManHIF and
Padh-Kanmx4R primers using Yep352 vector as a template. [0168] 7)
Kanmx4 selection gene is PCR amplified using Padh-kanmx4F and
Tcyc-kanmx4R primers using PYM-N14 plasmid as a template. [0169] 8)
Tcyc terminator was PCR amplified with Padh-loxP-BamHIF and
Padh-Kanmx4R primers using Pesc vector as a template. [0170] 9) 3
PCR fragments containing homologous regions with each other were
gel purified and 250 ng of each fragment were mixed together to
serve as template for the secondary assembly PCR reaction to yield
pAdh-Kanmx4-Tcyc-LoxP cassette. [0171] 10) Cassette is digested and
inserted into pGEM-ERG1-split vector, and used as template to run
PCR with ERG1F and ERG1R to get PCR fragment used to generate cell
lines. [0172] 11) Pgpd-tHMGR-Tadh fragment was amplified from
Pesc-Gpd-leu-tHMGR vector with primers GPD-BamHIP and Tadh-XholIR.
[0173] 12) Insert fragment into pGEM-ERG1-split vector containing
kanmx4 cassette. [0174] 13) Use construct as template to amplify
with ERG1F and EGR1R primers to gain the fragment for building
slightly different cell lines, which include integration of one
copy of tHMGR into the ERG1 gene.
TABLE-US-00001 [0174] Primer name Primer Sequence ERG9pS1
GTACATTTCATAGCCCATCTTCAACAACAATACCGACTTACCCGTACG (SEQ ID NO 1)
CTGCAGGTCGAC ERG9 250dwS2
CAGATTGACGGAGAGAGGGCCACATTGTTTGTCGGAATAAATCGAT (SEQ ID NO 2)
GAATTCGAGCTCG Hph F ATGGGTAAAAAGCCTGAACTCA (SEQ ID NO 3) Hph R
TTATTCCTTTGCCCTCGGACGAG (SEQ ID NO 4) ERG9 450dwR
AGATGCTAGTCAATGGCAGAAG (SEQ ID NO 5) WEG9p300upF
TGCTTACACAGAGTGAACCTGC (SEQ ID NO 6) ERG9 300R CTCGTGGAAGTGACGCAAC
(SEQ ID NO 7) pGPD-BAMHI F cgGGATCCagtttatcattatcaatactcgcc (SEQ ID
NO 8) pGPD-NotIR gggGCGGCCGCgagctcagtttatcattatc (SEQ ID NO 9)
tHMGR-NotIF GGGGCGGCCGCAAAACAATGTTGTCACGACTTTTCCGTATGC (SEQ ID NO
10) tHMGR-SpeIR GACTAGTTCAAGCTGACTTCTTGGTGCACGTTCCTTG (SEQ ID NO
11) ERG1F ATGTCTGCTGTTAACGTTGCACCTG (SEQ ID NO 12) ERG1R
TTAACCAATCAACTCACCAAAC (SEQ ID NO 13) ERG1-split F CGGGATCCCTCGAG
TTGTTCGCTGCTGACAGCGATAAC (SEQ ID NO 14) ERG1-splitR
CGGGATCCGCTAGCGGTACCACATGGGTCCTTTATATTGACACG (SEQ ID NO 15) ERG1
90up F ATCAGAACAATTGTCCAGTATTG (SEQ ID NO 16) ERG1100dwR
AATGTACTATACAAGCCTTCC (SEQ ID NO 17) bSQS-NotIF
GGGGCGGCCGCAAAACAATGGGGATGCTTCGCTGGGGAGT (SEQ ID NO 18) bSQS-SpeIR
GACTAGTTTAGCTCCTCAATTCGTCAAAGGT (SEQ ID NO 19) Cre-NotIF
GGGGCGGCCGCAAAACAATGGACATGTTCAGGGATCGCCAGG (SEQ ID NO 20) Cre-SpeIR
GACTAGTCTAATCGCCATCTTCCAGCAGGCG (SEQ ID NO 21) Padb-Loxp-
CGGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATGTGGAA BamHIF
TATTTCGGATAT (SEQ ID NO 22) Padh-Kanmx4F
GCATACAATCAACTAAGCTAAGCTAAAACAATGGGTAAGGAAAAGACT (SEQ ID NO 23)
CACGTTTC Padh-Hanmx4R
GAAACGTGAGTCTTTTCCTTACCCATTGTTTTAGCTTAGCTTAGTTGA (SEQ ID NO 24)
TTGTATGC Kanmx4-TcycF
CATTTGATGCTCGATGAGTTTTTCTAAATCCGCTCTAACCGAAAAGGA (SEQ ID NO 25)
AGGAG Kanmx4-TcycR CTCCTTCCTTTTCGGTTAGAGCGGATTTAGAAAAACTCATCGAGCATC
(SEQ ID NO 26) AAATG Tcyc-LoxP-NheIR
GGGGCTAGCATAACTTCGTATAATGTATGCTATACGAAGTTATCTTCG (SEQ ID NO 27)
AGCGTCCCAAAA Gpd-GamHIF CGGGATCCAGTTTATCATTATCAATACTCG (SEQ ID NO
28) Tadh-XohI GGGCTCGAG GAGCGACCTCATGCTATACCTG (SEQ ID NO 29)
Kanmx4R TTAGAAAAACTCATCGAGCATC (SEQ ID NO 30)
Expression of Enzymes for Cannabinoid Production
OLS 5' FWD
SEQ ID NO 31
Length: 55
Type: DNA
Organism: Artificial Sequence
Notes: Primer
TABLE-US-00002 [0175] Gcatagcaatctaatctaagtttaaa
atgaatcatttgagagcagaa gggcctgc
CB 5' FWD
SEQ ID NO 32
Length: 56
Type: DNA
Organism: Artificial Sequence
Notes: Primer
TABLE-US-00003 [0176] caccagaacttagtttcgacggataaa
atggaaaccggtttgtcctcgg tttgcac
All REV
SEQ ID NO 33
Length: 58
Type: DNA
Organism: Artificial Sequence
Notes: Primer
TABLE-US-00004 [0177]
cataactaattacatgatttaaccTAAACATCAGATTCAATAGAGCCGCC TCCACTG
Backbone|CBGA synthase|Flexible spacer|CBD synthase|target
peptide
SEQ ID NO 34
Length:
Type: DNA
[0178] Organism: artificial sequence Notes: Codon optimized
TABLE-US-00005 1 ggttaaatca tgtaattagt tatgtcacgc ttacattcac
gccctccccc cacatccgct 61 ctaaccgaaa aggaaggagt tagacaacct
gaagtctagg tccctattta tttttttata 121 gttatgttag tattaagaac
gttatttata tttcaaattt ttcttttttt tctgtacaga 181 cgcgtgtacg
catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 241
aaggctttaa tttgcggccc ctcacctgca cgcaaaatag gataattata ctctatttct
301 caacaagtaa ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag
aaatatcttg 361 accgcagtta actgtgggaa tactcaggta tcgtaagatg
caagagttcg aatctcttag 421 caaccattat ttttttcctc aacataacga
gaacacacag gggcgctatc gcacagaatc 481 aaattcgatg actggaaatt
ttttgttaat ttcagaggtc gcctgacgca tatacctttt 541 tcaactgaaa
aattgggaga aaaaggaaag gtgagagcgc cggaaccggc ttttcatata 601
gaatagagaa gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta
661 tttaaggacc tattgttttt tccaataggt ggttagcaat cgtcttactt
tctaactttt 721 cttacctttt acatttcagc aatatatata tatatatttc
aaggatatac cattctaatg 781 tctgccccta agaagatcgt cgttttgcca
ggtgaccacg ttggtcaaga aatcacagcc 841 gaagccatta aggttcttaa
agctatttct gatgttcgtt ccaatgtcaa gttcgatttc 901 gaaaatcatt
taattggtgg tgctgctatc gatgctacag gtgttccact tccagatgag 961
gcgctggaag cctccaagaa ggctgatgcc gttttgttag gtgctgtggg tggtcctaaa
1021 tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa
agaacttcaa 1081 ttgtacgcca acttaagacc atgtaacttt gcatccgact
ctcttttaga cttatctcca 1141 atcaagccac aatttgctaa aggtactgac
ttcgttgttg tcagagaatt agtgggaggt 1201 atttactttg gtaagagaaa
ggaagatgat ggtgatggtg tcgcttggga tagtgaacaa 1261 tacaccgttc
cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat 1321
gagccaccat tgcctatttg gtccttggat aaagctaatg ttttggcctc ttcaagatta
1381 tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa
ggttcaacat 1441 caattgattg attctgccgc catgatccta gttaagaacc
caacccacct aaatggtatt 1501 ataatcacca gcaacatgtt tggtgatatc
atctccgatg aagcctccgt tatcccaggt 1561 tccttgggtt tgttgccatc
tgcgtccttg gcctctttgc cagacaagaa caccgcattt 1621 ggtttgtacg
aaccatgcca cggttctgct ccagatttgc caaagaataa ggtcaaccct 1681
atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa
1741 ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggcatcag
aactggtgat 1801 ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg
tcgccgaaga agttaagaaa 1861 atccttgctt aaaaagattc tcttttttta
tgatatttgt acataaactt tataaatgaa 1921 attcataata gaaacgacac
gaaattacaa aatggaatat gttcataggg taacgctatg 1981 atccaatatc
aaaggaaatg atagcattga aggatgagac taatccaatt gaggagtggc 2041
agcatataga acagctaaag ggtagtgctg aaggaagcat acgatacccc gcatggaatg
2101 ggataatatc acaggaggta ctagactacc tttcatccta cataaataga
cgcatataag 2161 tacgcattta agcataaaca cgcactatgc cgttcttctc
atgtatatat atatacaggc 2221 aacacgcaga tataggtgcg acgtgaacag
tgagctgtat gtgcgcagct cgcgttgcat 2281 tttcggaagc gctcgtttCc
ggaaacgctt tgaagttcct attccgaagt tcctattctc 2341 tagaaagtat
aggaacttca gagcgctttt gaaaaccaaa agcgctctga agtcgcactt 2401
tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta aaatattgcg aataccgctt
2461 ccacaaacat tgctcaaaag tatctctttg ctatatatct ctgtgctata
tccctatata 2521 acctacccat ccacctttcg ctccttgaac ttgcatctaa
actcgacctc tacatttttt 2581 atgtttatct ctagtattac tctttagaca
aaaaaattgt agtaagaact attcatagag 2641 tgaatcgaaa acaatacgaa
aatgtaaaca tttcctatac gtagtatata gagacaaaat 2701 agaagaaacc
gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt 2761
tcacaaagta tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga
2821 aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag
tcaggctttt 2881 tttatggaag agaaaataga caccaaagta gccttcttct
aaccttaacg gacctacagt 2941 gcaaaaagtt atcaagagac tgcattatag
agcgcacaaa ggagaaaaaa agtaatctaa 3001 gatgctttgt tagaaaaata
gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta 3061 tagattcttt
gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag 3121
ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg
3181 aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt
tctgtaaaaa 3241 tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
gttgcatttt tgttctacaa 3301 aatgaagcac agatgcttcg ttcaggtggc
acttttcggg gaaatgtgcg cggaacccct 3361 atttgtttat ttttctaaat
acattcaaat atgtatccgc tcatgagaca ataaccctga 3421 tattggtcag
aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa 3481
tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
3541 gaaaaaggaa gaatatgagc catattcaac gggaaacgtc gaggccgcga
ttaaattcca 3601 acatggatgc tgatttatat gggtataaat gggctcgcga
taatgtcggg caatcaggtg 3661 cgacaatcta tcgcttgtat gggaagcccg
atgcgccaga gttgtttctg aaacatggca 3721 aaggtagcgt tgccaatgat
gttacagatg agatggtcag actaaactgg ctgacggaat 3781 ttatgccact
tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca 3841
ccactgcgat ccccggaaaa acagcgttcc aggtattaga agaatatcct gattcaggtg
3901 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcactcgatt
cctgtttgta 3961 attgtccttt taacagcgat cgcgtatttc gcctcgctca
ggcgcaatca cgaatgaata 4021 acggtttggt tgatgcgagt gattttgatg
acgagcgtaa tggctggcct gttgaacaag 4081 tctggaaaga aatgcataaa
cttttgccat tctcaccgga ttcagtcgtc actcatggtg 4141 atttctcact
tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg 4201
gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
4261 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
aatcctgata 4321 tgaataaatt gcaatttcat ttgatgctcg atgagttttt
ctaactcatg accaaaatcc 4381 cttaacgtga gttacgcgcg cgtcgttcca
ctgagcgtca gaccccgtag aaaagatcaa 4441 aggatcttct tgagatcctt
tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 4501 accgctacca
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 4561
aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
4621 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
tcctgttacc 4681 agtggctgct gccagtggcg ataagtcgtg tcttaccggg
ttggactcaa gacgatagtt 4741 accggataag gcgcagcggt cgggctgaac
ggggggttcg tgcacacagc ccagcttgga 4801 gcgaacgacc tacaccgaac
tgagatacct acagcgtgag ctatgagaaa gcgccacgct 4861 tcccgaaggg
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4921
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
4981 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
tatggaaaaa 5041 cgccagcaac gcggcctttt tacggttcct ggccttttgc
tggccttttg ctcacatgtt 5101 ctttcctgcg ttatcccctg attctgtgga
taaccgtatt accgcctttg agtgagctga 5161 taccgctcgg ggtcgtgcag
gtagtttatc attatcaata ctcgccattt caaagaatac 5221 gtaaataatt
aatagtagtg attttcctaa ctttatttag tcaaaaaatt agccttttaa 5281
ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat atataacatc
5341 gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
agcccgctCt 5401 ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa
aatattgttt tcttcaccaa 5461 ccatcagttc ataggtccat tctcttagcg
caactacaga gaacaggggc acaaacaggc 5521 aaaaaacggg cacaacctca
atggagtgat gcaaccagcc tggagtaaat gatgacacaa 5581 ggcaattgac
ccacgcatgt atctatctca ttttcttaca ccttctatta ccttctgctc 5641
tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa attattcccc
5701 tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
aaatctattt 5761 cttaaacttc ttaaattcta cttttatagt tagtcttttt
tttagtttta aaacaccaga 5821 acttagtttc gacggataaa atggaaaccg
gtttgtcctc ggtttgcact ttctccttcc 5881 aaacaaacta tcatacactc
ctgaacccgc acaataacaa tcccaaaact tccctgctgt 5941 gttataggca
cccaaagaca ccaatcaaat actcctacaa taactttcca tctaagcatt 6001
gtagcacaaa aagtttccat ttgcaaaata agtgttccga atctctgtcc atcgccaaaa
6061 attccattag ggctgccact actaatcaaa ctgaaccacc agagtctgat
aatcattctg 6121 tcgccacaaa gattctgaat tttgggaagg cttgttggaa
gttacaaaga ccatatacaa 6181 ttattgcctt tacctcttgt gcctgtggtt
tatttggtaa ggaactgttg cataatacaa 6241 atttaatatc ttggtcattg
atggaaacgt tcaaagcatt ttttttctta gtcgctatcc 6301 tttgtattgc
ttctttcacc accactatca accagattta cgacttacat attgacagaa 6361
ttaacaagcc agatttgcca ctggcttcgg gcgagatttc cgtcaatact gcctggatca
6421 tggaaacttc tattattgtt gccttgtttg gattgataat caccataaaa
atggaaacta 6481 agggtggtcc attgtatatt ttcggttact gttttggtat
cttcgggggc atcgtctact 6541 ctgttcctcc attcagatgg aaacaaaatc
cttccacagc attccttttg aacttcctgg 6601 cgcacattat aaccaacttt
actttttatt atgcctccag agccgccctg gggctgccct 6661 ttgaattacg
cccctccttt acatttttac tggccttcat ggagaccaag tccatggaga 6721
ctggttctgc tctcgcgttg atcaaagatg cttccgatgt ggaaggtgac accaaatttg
6781 gtatatccac tttggccagc aagtatggtt ccaggaattt gaccctattt
tgttctggta 6841 tcgtgctgct gtcttatgtt gcagccatct tggctggcat
catttggcca caggctttca 6901 attcaaatgt tatggagacg ctgctctcgc
atgctatttt ggcattttgg ttgattctac 6961 agacaagaga ttttgcttta
accaattatg acccagaagc tggtagaaga ttttacgaat 7021 ttatggaaac
atggaaatta tactatgctg aatatttagt gtacgttttc attgggggcg 7081
gctccagcgc cggcggcggc tcttctgcgg gcggttggtc tcatccacaa tttgagaaag
7141 gtgggtcgtc tggcggcggc agcgggggcg ggtccggcgg ggggagcggc
ggtatgaaat 7201 gttcgacctt ctctttttgg tttgtctgta aaataatttt
ttttttcttc agctttaaca 7261 ttcaaaccag cattgcaaat ccaagagaaa
atttcttgaa atgcttttca caatatatcc 7321 ccaataatgc tactaacttg
aagctagttt atactcaaaa caaccctttg tacatgtccg 7381 tgctcaactc
caccattcac aacctaagat tcacttcaga cactacccca aaaccattag 7441
ttattgtgac accttctcac gtttcacata tccaaggtac tattttatgc
tccaagaagg
7501 tcggcctgca aattagaact agatctggag gtcatgattc agaaggaatg
tcttacatct 7561 ctcaagttcc atttgtgatt gtcgatttaa gaaatatgag
gagcattaag atcgatgttc 7621 actcccaaac ggcatgggtt gaagccggtg
ccaccttggg cgaagtttac tactgggtca 7631 acgagaagaa tgaaaactta
tcactagccg caggttattg tccaactgtt tgtgctggtg 7741 gccatttcgg
aggcggcggc tacggtcctc taatgagaaa ctacggctta gctgctgaca 7801
atatcatcga cgctcacttg gttaacgttc atggtaaagt tttagataga aaatctatgg
7861 gtgaggatct tttctgggct ttgagaggtg gcggcgcaga atcatttggc
attatcgttg 7921 cttggaagat cagattggtg gctgtcccca agtctacaat
gttttctgtg aagaaaatta 7981 tggaaatcca tgaattggtc aaactggtga
ataaatggca aaacatagct tacaagtacg 8041 ataaagactt gctgttaatg
acacatttta ttaccaggaa catcactgat aaccaaggca 8101 agaacaagac
tgcaattcat acttattttt cctccgtttt tttgggtggt gtcgactccc 8161
tcgtggatct gatgaataaa tcattccctg aactaggtat taaaaaaacc gattgtagac
8221 aattgagttg gattgatacc atcatattct acagtggtgt tgttaattat
gatactgaca 8281 acttcaacaa agaaatactg ctggaccgtt ccgccggcca
gaatggtgct tttaaaatca 8341 agttggatta tgtgaaaaag cctattccag
aatccgtatt tgttcaaata ttggaaaagc 8401 tgtatgaaga agacattggt
gcaggcatgt acgctcttta tccttatggc ggcataatgg 8461 atgaaatttc
tgaaagtgcc attcctttcc cacatagggc cgggatcctg tacgagttat 8521
ggtacatttg ttcatgggaa aagcaagaag ataatgaaaa acatttaaat tggataagaa
8581 atatttataa ttttatgact ccatacgtct ccaaaaaccc acgcctggca
tatttgaatt 8641 acagagacct ggatattggc atcaatgatc ctaaaaaccc
aaataattac actcaggcaa 8701 gaatatgggg tgaaaaatat ttcggcaaaa
attttgatag gctggtcaag gttaaaacac 8761 tggttgatcc aaacaatttc
tttagaaacg aacaatctat cccacctctg cctagacata 8821 gacacggcgg
tggaagcagt ggaggcggct ctattgaatc tgatgtttaa tga
[0179] Backbone|OLS|Flexible spacer|OAC|target peptide
SEQ ID NO 35
Length:
Type: DNA
[0180] Organism: artificial sequence Notes: Codon optimized
TABLE-US-00006 1 ggttaaatca tgtaattagt tatgtcacgc ttacattcac
gccctccccc cacatccgct 61 ctaaccgaaa aggaaggagt tagacaacct
gaagtctagg tccctattta tttttttata 121 gttatgttag tattaagaac
gttatttata tttcaaattt ttcttttttt tctgtacaga 181 cgcgtgtacg
catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 241
aaggctttaa tttgcggccc ctcacctgca cgcaaaaagc ttttcaattc aattcatcat
301 ttttttttta ttcttttttt tgatttcggt ttctttgaaa tttttttgat
tcggtaatct 361 ccgaacagaa ggaagaacga aggaaggagc acagacttag
attggtatat atacgcatat 421 gtagtgttga agaaacatga aattgcccag
tattcttaac ccaactgcac agaacaaaaa 481 ccagcaggaa acgaagataa
atcatgtcga aagctacata taaggaacgt gctgctactc 541 atcctagtcc
tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt 601
gtgcttcatt ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc
661 ccaaaatttg tttactaaaa acacatgtgg atatcttgac tgatttttcc
atggagggca 721 cagttaagcc gctaaaggca ttatccgcca agtacaattt
tttactcttc gaagatagaa 781 aatttgctga cattggtaat acagtcaaat
tgcagtactc tgcgggtgta tacagaatag 841 cagaatgggc agacattacg
aatgcacacg gtgtggtggg cccaggtatt gttagcggtt 901 tgaagcaggc
ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat 961
tgtcatgcaa gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga
1021 aaagcgacaa agattttgtt atcggcttta ttgctcaaag agacatgggt
ggaagagatg 1081 aaggttacga ttggttgatt atgacacccg gtgtgggttt
agatgacaag ggagatgcat 1141 tgggtcaaca gtatagaacc gtggatgatg
ttgtctctac aggatctgac attattattg 1201 ttggaagagg actatttgca
aagggaaggg atgctaaggt agagggtgaa cgttacagaa 1261 aagcaggctg
ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat 1321
aagtaaatgc atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat
1381 tacccacgct atgatccaat atcaaaggaa atgatagcat tgaaggatga
gactaatcca 1441 attgaggagt ggcagcatat agaacagcta aagggtagtg
ctgaaggaag catacgatac 1501 cccgcatgga atgggataat atcacaggag
gtactagact acctttcatc ctacataaat 1561 agacgcatat aagtacgcat
ttaagcataa acacgcacta tgccgttctt ctcatgtata 1621 tatatataca
ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca 1681
gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga
1741 agttcctatt ctctagaaag tataggaact tcagagcgct tttgaaaacc
aaaagcgctc 1801 tgaagtcgca ctttcaaaaa accaaaaacg caccggactg
taacgagcta ctaaaatatt 1861 gcgaataccg cttccacaaa cattgctcaa
aagtatctct ttgctatata tctctgtgct 1921 atatccctat ataacctacc
catccacctt tcgctccttg aacttgcatc taaactcgac 1981 ctctacattt
tttatgttta tctctagtat tactctttag acaaaaaaat tgtagtaaga 2041
actattcata gagtgaatcg aaaacaatac gaaaatgtaa acatttccta tacgtagtat
2101 atagagacaa aatagaagaa accgttcata attttctgac caatgaagaa
tcatcaacgc 2161 tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg
tatagaatat aatcggggat 2221 gcctttatct tgaaaaaatg cacccgcagc
ttcgctagta atcagtaaac gcgggaagtg 2281 gagtcaggct ttttttatgg
aagagaaaat agacaccaaa gtagccttct tctaacctta 2341 acggacctac
agtgcaaaaa gttatcaaga gactgcatta tagagcgcac aaaggagaaa 2401
aaaagtaatc taagatgctt tgttagaaaa atagogctct cgggatgcat ttttgtagaa
2461 caaaaaagaa gtatagattc tttgttggta aaatagcgct ctcgcgttgc
atttctgttc 2521 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc
gctctcgcgt tgcatttttg 2581 ttttacaaaa atgaagcaca gattcttcgt
tggtaaaata gcgctttcgc gttgcatttc 2641 tgttctgtaa aaatgcagct
cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat 2701 ttttgttcta
caaaatgaag cacagatgct tcgttcaggt ggcacttttc ggggaaatgt 2761
gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
2621 acaataaccc tgatattggt cagaattggt taattggttg taacactgac
ccctatttgt 2881 ttatttttct aaatacattc aaatatgtat ccgctcatga
gacaataacc ctgataaatg 2941 cttcaataat attgaaaaag gaagaatatg
agtattcaac atttccgtgt cgcccttatt 3001 cccttttttg cggcattttg
ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 3061 aaagatgctg
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 3121
ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
3131 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
actcggtcgc 3241 cgcatacact attctcagaa tgacttggtt gagtactcac
cagtcacaga aaagcatctt 3301 acggatggca tgacagtaag agaattatgc
agtgctgcca taaccatgag tgataacact 3361 gcggccaact tacttctgac
aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 3421 aacatggggg
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 3481
ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta
3541 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
gatggaggcg 3601 gataaagttg caggaccact tctgcgctcg gcccttccgg
ctggctggtt tattgctgat 3661 aaatccggag ccggtgagcg tggttctcgc
ggtatcatcg cagcgctggg gccagatggt 3721 aagccctccc gtatcgtagt
tatctacacg acggggagtc aggcaactat ggatgaacga 3781 aatagacaga
tcgctgagat aggtgcctca ctgattaagc attggtaact catgaccaaa 3841
atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
3901 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
caaacaaaaa 3961 aaccaccgct accagcggtg gtttgtttgc cggatcaaga
gctaccaact ctttttccga 4021 aggtaactgg cttcagcaga gcgcagatac
caaatactgt tcttctagtg tagccgtagt 4081 tagcccacca cttcaagaac
tctgtagcac cgcctacata cctcgctctg ctaatcctgt 4141 taccagtggc
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 4201
agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
4261 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
gaaagcgcca 4321 cgcttcccga agggagaaag gcggacaggt atccggtaag
cggcagggtc ggaacaggag 4381 agcgcacgag ggagcttcca gggggaaacg
cctggtatct ttatagtcct gtcgggtttc 4441 gccacctctg acttgagcgt
cgatttttgt gatgctcgtc aggggggcgg agcctatgga 4501 aaaacgccag
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 4561
tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
4621 ctgataccgc tcggggtcgt gcaggtatag cttcaaaatg tttctactcc
ttttttactc 4681 ttccagattt tctcggactc cgcgcatcgc cgtaccactt
caaaacaccc aagcacagca 4741 tactaaattt cccctctttc ttcctctagg
gtgtcgttaa ttacccgtac taaaggtttg 4801 gaaaagaaaa aagtgaccgc
ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4861 ttatcacgtt
tctttttctt gaaaattttt ttttttgatt tttttctctt tcgatgacct 4921
cccattgata tttaagttaa taaacggact tcaatctctc aagtttcagt ttcatttttc
4981 ttgttctatt acaacttttt ttacttcttg ctcattagaa agaaagcata
gcaatctaat 5041 ctaagtttaa aatgaatcat ttgagagcag aagggcctgc
ttccgtgctg gctattggta 5101 ccgccaatcc agaaaatatc ctgctgcagg
acgaattccc agattactat tttagggtca 5161 ccaaatctga acatatgaca
caattgaaag agaaattcag aaagatttgt gacaagtcca 5221 tgattaggaa
aagaaattgt tttttgaatg aagaacactt gaagcaaaat cctcgcctgg 5281
tggagcatga aatgcaaact ttggatgcta gacaagacat gttggtggtg gaagttccaa
5341 agctggggaa ggatgcctgt gccaaggcca ttaaagaatg gggccaacca
aaatccaaaa 5401 ttacccacct gattttcacc tccgcctcca ccactgatat
gccaggtgca gactatcatt 5461 gtgctaaatt gttgggtttg tccccctccg
tgaagagagt tatgatgtat caattaggtt 5521 gttatggcgg cggcaccgtt
ctgagaattg ccaaagacat tgctgaaaac aataaaggtg 5581 cgcgcgtttt
ggctgtttgt tgtgatatta tggcatgttt atttagaggt ccaagtgaaa 5641
gtgacttgga attgctagtg ggccaggcca tatttggtga tggtgccgct gctgtgatcg
5701 ttggtgctga gcctgatgaa tctgtcggtg aaagaccaat ttttgaactg
gtttccactg 5761 gtcaaaccat tttgccaaat tcagaaggta ctattggcgg
ccatatcaga gaagctggtt 5821 taatctttga tttgcacaag gatgtcccaa
tgttaatttc caataatatt gaaaaatgtt 5881 tgatcgaagc atttaccccc
atcggtattt ctgattggaa ttccatcttc tggattacac 5941 atcctggcgg
taaagctatc ttagataaag ttgaggagaa gttgcattta aagtctgaca 6001
aatttgttga ttcaagacat gtcctgtctg agcacggtaa tatgtcttcc tcgaccgtct
6061 tgtttgtcat ggatgagttg aggaagaggt ccctggaaga aggcaagagc
accaccggtg 6121 acggttttga gtggggggtc ctctttggat ttgggccagg
cctgaccgta gaaagggttg 6181 ttgtccgctc ggtgccaatc aaatatggtg
gggggtccag cgccggtggc gggagctccg 6241 cgggcggttg gtctcaccca
caatttgaaa agggtggcag cagcggcggc ggctctggcg 6301 gaggctccgg
cgggggctcg gggggtatgg ctgtcaagca tctgatcgtg ctgaagttca 6361
aagatgaaat tactgaagcc caaaaggagg aatttttcaa gacatatgtt aatttggtta
6421 acatcattcc agcaatgaaa gatgtttatt ggggtaagga cgttactcaa
aaaaataagg 6481 aagagggtta cactcatatt gttgaagtca ctttcgaatc
cgtcgaaaca attcaagatt 6541 atattattca tccagctcat gttgggtttg
gcgatgtgta cagatcattt tgggaaaaat 6601 tattgatttt tgactacaca
ccaagaaaag gcggtggaag cagtggaggc ggctctattg 6661 aatctgatgt
ttaatag
Overexpression of ERG8, HFA1, ERG 10, ERG13, tHMGR, HMGR, ERG12,
ERG8, IDI Genes (for Higher Levels of Intermediates) Same process
as expression of Synthase expression, but with 3 copies expressed
in yeast cells. Backbone|GGPS1|2a protease|HMG-CoA
reductase|flexible spacer|IDI1
SEQ ID NO 36
Length:
Type: DNA
[0181] Organsm: artificial sequence Notes: Codon optimized
TABLE-US-00007 1 atggagaaga ctcaagaaac agtccaaaga attcttctag
aaccctataa atacttactt 61 cagttaccag gtaaacaagt gagaaccaaa
ctttcacagg catttaatca ttggctgaaa 121 gttccagagg acaagctaca
gattattatt gaagtgacag aaatgttgca taatgccagt 181 ttactcatcg
atgatattga agacaactca aaactccgac gtggctttcc agtggcccac 241
agcatctatg gaatcccatc tgtcatcaat tctgccaatt acgtgtattt ccttggcttg
301 gagaaagtct taacccttga tcacccagat gcagtgaagc tttttacccg
ccagcttttg 361 gaactccatc agggacaagg cctagatatt tactggaggg
ataattacac ttgtcccact 421 gaagaagaat ataaagctat ggtgctgcag
aaaacaggtg gactgtttgg attagcagta 481 ggtctcatgc agttgttctc
tgattacaaa gaagatttaa aaccgctact taatacactt 541 gggctctttt
tccaaattag ggatgattat gctaatctac actccaaaga atatagtgaa 601
aacaaaagtt tttgtgaaga tctgacagag ggaaagttct catttcctac tattcatgct
661 atttggtcaa ggcctgaaag cacccaggtg cagaatatct tgcgccagag
aacagaaaac 721 atagatataa aaaaatactg tgtacattat cttgaggatg
taggttcttt tgaatacact 761 cgtaataccc ttaaagagct tgaagctaaa
gcctataaac agattgatgc acgtggtggg 841 aaccctgagc tagtagcctt
agtaaaacac ttaagtaaga tgttcaaaga agaaaatgaa 901 ggcggttctg
gcagcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 961
aatcccggcc ctaggtctgg cagcggagag ggcagaggaa gtcttctaac atgcggtgac
1021 gtggaggaga atcccggccc taggacacaa aagaaagtcc cagacaattg
ttgtagacgt 1081 gaacctatgc tggtcagaaa taaccagaaa tgtgattcag
tagaggaaga gacagggata 1141 aaccgagaaa gaaaagttga ggttataaaa
cccttagtgg ctgaaacaga taccccaaac 1201 agagctacat ttgtggttgg
taactcctcc ttactcgata cttcatcagt actggtgaca 1261 caggaacctg
aaattgaact tcccagggaa cctcggccta atgaagaatg tctacagata 1321
cttgggaatg cagagaaagg tgcaaaattc cttagtgatg ctgagatcat ccagttagtc
1351 aatgctaagc atatcccagc ctacaagttg gaaactctga tggaaactca
tgagcgtggt 1441 gtatctattc gccgacagtt actttccaag aagctttcag
aaccttcttc tctccagtac 1501 ctaccttaca gggattataa ttactccttg
gtgatgggag cttgttgtga gaatgttatt 1561 ggatatatgc ccatccctgt
tggagtggca ggaccccttt gcttagatga aaaagaattt 1621 caggttccaa
tggcaacaac agaaggttgt cttgtggcca gcaccaatag aggctgcaga 1681
gcaataggtc ttggtggagg tgccagcagc cgagtccttg cagaCgggat gactcgtggc
1741 ccagttgtgc gtcttccacg tgcttgtgac tctgcagaag tgaaagcctg
gctcgaaaca 1801 tctgaagggt tcgcagtgat aaaggaggca tttgacagca
ctagcagatt tgcacgtcta 1861 cagaaacttc atacaagtat agctggacgc
aacctttata tccgtttcca gtccaggtca 1921 ggggatgcca tggggatgaa
catgatttca aagggtacag agaaagcact ttcaaaactt 1981 cacgagtatt
tccctgaaat gcagattcta gccgttagtg gtaactattg tactgacaag 2041
aaacctgctg ctataaattg gatagaggga agaggaaaat ctgttgtttg tgaagctgtc
2101 attccagcca aggttgtcag agaagtatta aagactacca cagaggctat
gattgaggtc 2161 aacattaaca agaatttagt gggctctgcc atggctggga
gcataggagg ctacaacgcc 2221 catgcagcaa acattgtcac cgccatctac
attgcctgtg gacaggatgc agcacagaat 2281 gttggtagtt caaactgtat
tactttaatg gaagcaagtg gtcccacaaa tgaagattta 2341 tatatcagct
gcaccatgcc atctatagag ataggaacgg tgggtggtgg gaccaaccta 2401
ctacctcagc aagcctgttt gcagatgcta ggtgttcaag gagcatgcaa agataatcct
2461 ggggaaaatg cccggcagct tgcccgaatt gtgtgtggga ccgtaatggc
tggggaattg 2521 tcacttatgg cagcattggc agcaggacat cttgtcaaaa
gtcacatgat tcacaacagg 2581 tcgaagatca atttacaaga cctccaagga
gcttgcacca agaagacagc cggctcagga 2641 ggttcttcag gactggaagt
gctgtttcag ggcccgggtg gatctggcat gatgcctgaa 2701 ataaacacta
accacctcga caagcaacag gttcaactcc tggcagagat gtgtatcctt 2761
attgatgaaa atgacaataa aattggagct gagaccaaga agaattgtca cctgaacgag
2821 aacattgaga aaggattatt gcatcgagct tttagtgtct tcttattcaa
caccgaaaat 2881 aagcttctgc tacagcaaag atcagatgct aagattacct
ttccaggttg ttttacgaat 2941 acgtgttgta gtcatccatt aagcaatcca
gccgagcttg aggaaagtga cgcccttgga 3001 gtgaggcgag cagcacagag
acggctgaaa gctgagctag gaattccctt ggaagaggtt 3061 cctccagaag
aaattaatta tttaacacga attcactaca aagctcagtc tgatggtatc 3121
tggggtgaac atgaaattga ttacattttg ttggtgagga agaatgtaac tttgaatcca
3181 gatcccaatg agattaaaag ctattgttat gtgtcaaagg aagaactaaa
agaacttctg 3241 aaaaaagcag ccagtggtga aattaagata acgccatggt
ttaaaattat tgcagcgact 3301 tttctcttta aatggtggga taacttaaat
catttgaatc agtttgttga ccatgagaaa 3361 atatacagaa tg
ERG2, ERG3, and ERG6 mutations/deletions for increased membrane
permeability
[0182] Same process as ERG9 knockout, but targeting ERG2, ERG3, and
ERG6 genes.
ERG20p Modification
[0183] We experimented with a few types of ERG20 genes, (avian,
salmon entrica, and human). Currently we are still trying to see
which is the best by engineering the ERG20p gene into a FPP
synthase, thereby creating a new enzyme that can create GPP instead
at high rates.
Gal80p Deletion/Mutation for not Needing Expensive Galactose to
Induce Promoter
[0184] Induce mutation in Gal80 gene by site directed
mutagenesis.
Using ADH2p (Glucose Repressible Promoter) Induces Strong
Transcription with No Glucose, Better than GAL Promoter
[0185] Same process as Gal80p deletion.
Overexpression of ADH2 and ALD6 (Ethanol to Acetate), as Well as
Overexpression of an Acetyl-CoA C-Acetyltransferase
[0186] Same process as IDI and HMGR overexpression, but with genes
for ADH2 and ALD6.
Tables
[0187] Below is a table of various cannabinoids, along with their
structure and variants and main pharmacological characteristics as
well as a table listing potential starting materials.
TABLE-US-00008 TABLE 1 Compounds Pharmacological Characteristics
Cannabinoids (FIG. 1 and 2) Cannabigerolic acid (CBGA) Antibiotic
(1) Cannabigerolic acid monomethylether (CBGAM) Cannabigerol (CBG)
Antibiotic, antifungal, anti-inflammatory, analgesic (1) Partial
agonist at CB1/CB2 receptors (2) Cannabigerovarinic acid (CBGVA)
Cannabigerovarin (CBGV) Cannabichromenic acid (CBCA)
Cannabichromene (CBC) Anti-inflammatory, antibiotic, antifungal,
analgesic (1) Cannabichromevarinic acid (CBCVA) Cannabichromevarin
(CBCV) Cannabidiolic acid (CBDA) Antibiotic Cannabidiol (CBD)
Anxiolytic, antipsychotic, analgesic, anti-inflammatory,
antioxidant, antispasmodic (1) Ant schizophrenic, antiepileptic,
sleep-promoting, anti-oxidizing, anti-inflammatory,
immunomodulation properties (2) Cannabidiol monomethylether (CBDM)
Cannabidiol-C4 (CBD-C4) Cannabidivarinic acid (CBDVA)
Cannabidivarin (CBDV) Cannabidiorcol (CBD-C1)
Tetrahydrocannabinolic acid A (THCA-A) Tetrahydrocannabinolic acid
B (THCA-B) Delta-9-tetrahydrocannabinol Euphoriant, analgesic,
anti- (THC) inflammatory, antioxidant, antiemetic (1)
Delta-9-tetrahydrocannabinolic acid-C4 (THCA-C4)
Delta-9-tetrahydrocannabinol-C4 (THC-C4)
Delta-8-tetrahydrocannabivarin Exhibit in vitro pharma (D8-THCV)
properties similar to THCV, and both can antagonize THC; behave as
agonists or antagonists in dose dependent manner (2)
Delta-9-tetrahydrocannabivarinic acid (THCVA)
Delta-9-tetrahydrocannabivarin Analgesic, euphoriant (1) (THCV)
Strong antagonist of anandamide (due to interactions with non-
CB1/2 receptors), neuromodulator (in animal and human organs), some
affects due to interaction with non CB1/CB2 receptors (2)
Delta-9-tetrahydrocannabiorcolic acid (THCA-C1)
Delta-9-tetrahydrocannabiorcol (THC-C1) Delta-7-cis-iso-
tetrahydrocannabivarin (D7-THCV) Delta-8- tetrahydrocannabinolic
acid (D8-THCA) Delta-8-tetrahydrocannabinol Similar to THC (1)
(D8-THC) Several 1-O-methyl- and 1-deoxy-delta-8- THC analogs have
high CB2 receptor affinity[JWH133, JWH359, trans-
(6aR,10aR)-3-(1,1- dimethylhexyl)-1-O- methyl-delta-8-THC];
antiemetic effects similar to THC (2) Cannabicyclolic acid (CBLA)
Cannabicyclol (CBL) Cannabicyclovarin (CBLV) Cannabielsoic acid A
(CBEA-A) Cannabielsoic acid B (CBEA-B) Cannabielsoin (CBE)
Cannabinolic acid (CBNA) Cannabinol (CBN) Sedative, antibiotic,
anticonvulsant, anti- inflammatory (1) Cannabinol methylether
(CBNM) Cannabinol-C4 (CBN-C4) Cannabivarin (CBV) Cannabinol-C2
(CBN-C2) Cannabinol-C1 (CBN-C1) Cannabinodiol (CBND)
Cannabinodivarin (CBVD) Cannabitriol (CBT) 10-Ethoxy-9-hydroxy-
delta-6a-tetrahydrocannabinol 8,9-Dihydroxy-delta-6a-
tetrahydrocannabinol Cannabitriolvarin (CBTV)
Ethoxy-cannabitriolvarin (CBTVE) Dehydrocannabifuran (DCBF)
Cannabifuran (CBF) Cannabichromanon (CBCN) Cannabicitran (CBT)
10-oxo-delta-6a- tetrahydrocannabinol (OTHC) Delta-9-cis-
tetrahydrocannabinol (Cis-THC) 3,4,5,6-Tetrahydro-7-hydroxy-
alpha-alpha-2-trimethyl-9-n- propyl-2,6-methano-2H-1-
benzoxocin-5-methanol (OH-iso-HHCV) Cannabiripsol (CBR)
Trihydroxy-delta-9- tetrahydrocannabinol (triOH-THC)
Terpeses/Terpenoids Beta-Myrcene Analgesic, anti-inflammatory,
antibiotic, antimutagenic d-Limonene Immune potentiator,
antidepressant, antimutagenic Linalool Sedative, antidepressant,
anxiolytic, immune potentiator Trans-Ocimene Beta-Pinene
Alpha-Pinene Anti-inflammatory, bronchodilator, stimulant,
antibiotic, antineoplastic, AChE inhibitor Beta-Caryophyllene
Anti-inflammatory, cytoprotective, antimalarial, CB2 agonist
Delta-3-Carene Pulegone AChE inhibitor, sedative,
Trans-gamma-Bisabolene antipyretic Trans-alpha-Farnesene
Beta-Fenchol Beta-Phellandrene Alpha-Humulene Guajol Alpha-Gualene
Alpha-Eudesmol Terpinolene Alpha-Selinene Alpha-Terpineol Sedative,
antibiotic, AChE inhibitor, antioxidant, antimalarial Fenchone
Camphene Cis-Sabinene hydrate Cis-Ocimene Beta-Eudesmol
Beta-Selinene Alpha-trans-Bergamolene Gamma-Eudesmol Borneol
Cis-beta-Farnescene Gamma-Curcumene Cis-gamma-Bisabolene
Alpha-Thujene Epi-alpha-Bisabolol Ipsdienol Alpha-Yiangene
Beta-Elemene Alpha-cis-Bergamontene Gamma-Muurolene Alpha-Cadinene
Alpha-Longipinene Caryophyllene oxide Spermidine Alkaloids (FIG. 6)
(+)-Cannabisativine Palustridine Palustrine Spermidine
Anhydrocannabisativine Phenolic Amides and Lignanamides (FIG. 5)
N-trans-Feruloyltyramine N-p-Coumaroyltyramine
N-trans-Caffeoyltyramine Grossamide Cannabisin-A Cannabisin-B
Cannabisin-C Cannabisin-D Cannabisin-E Cannabisin-F Cannabisin-G
Phenylpropanoids and Flavonoids (FIG. 4) Apigenin Luteolin
Kaempferol Quercetin Orientin Vitexin Cannflavin A Inhibit
prostaglandin E2 in human rheumatoid synovial cells Cannflavin B
Inhibit prostaglandin E2 in human rheumatoid synovial cells
Stilbenoids (FIG. 3) Cannabispiran Isocannabispiran
Cannabistilbene-IIa Cannabistilbene-IIb Cannithrene-1 Cannithrene-2
Acetyl cannabispirol Alpha-cannabisporanol Canniprene
Cannabispirone
TABLE-US-00009 TABLE 2 (Starting Materials) Sugar based
concentrates (High Hemicellulose Glycerol Fructose Corn Syrup,
Molasses) Glucose Xylose Whey Sucrose Methanol Biodiesel Cellulose
Lactic Acid Citrate Ethanol Lignin Fructose Succinic Acid Arabinose
Biofuels Biomass Saccharose Starch based products Agricultural
residue Water hyacinth Aquatic biomass
Sequence CWU 1
1
36160DNAArtificial SequencePrimer 1gtacatttca tagcccatct tcaacaacaa
taccgactta cccgtacgct gcaggtcgac 60260DNAArtificial SequencePrimer
2cagattgacg gagagagggc cacattgttt gtcggcaata aatcgatgaa ttcgagctcg
60322DNAArtificial SequencePrimer 3atgggtaaaa agcctgaact ca
22423DNAArtificial SequencePrimer 4ttattccttt gccctcggac gag
23522DNAArtificial SequencePimer 5agatgctagt caatggcaga ag
22622DNAArtificial SequencePimer 6tgcttacaca gagtgaacct gc
22719DNAArtificial SequencePrimer 7ctcgtggaag tgacgcaac
19832DNAArtificial SequencePrimer 8cgggatccag tttatcatta tcaatactcg
cc 32931DNAArtificial SequencePrimer 9ggggcggccg cgagctcagt
ttatcattat c 311042DNAArtificial SequencePrimer 10ggggcggccg
caaaacaatg ttgtcacgac ttttccgtat gc 421137DNAArtificial
SequencePrimer 11gactagttca agctgacttc ttggtgcacg ttccttg
371225DNAArtificial SequencePrimer 12atgtctgctg ttaacgttgc acctg
251322DNAArtificial SequencePrimer 13ttaaccaatc aactcaccaa ac
221438DNAArtificial SequencePrimer 14cgggatccct cgagttgttc
gctgctgaca gcgataac 381544DNAArtificial SequencePrimer 15cgggatccgc
tagcggtacc acatgggtcc tttatattga cacg 441623DNAArtificial
SequencePrimer 16atcagaacaa ttgtccagta ttg 231721DNAArtificial
SequencePrimer 17aatgtactat acaagccttc c 211840DNAArtificial
SequencePrimer 18ggggcggccg caaaacaatg gggatgcttc gctggggagt
401931DNAArtificial SequencePrimer 19gactagttta gctcctcaat
tcgtcaaagg t 312042DNAArtificial SequencePrimer 20ggggcggccg
caaaacaatg gacatgttca gggatcgcca gg 422131DNAArtificial
SequencePrimer 21gactagtcta atcgccatct tccagcaggc g
312260DNAArtificial SequencePrimer 22cgggatccat aacttcgtat
agcatacatt atacgaagtt atgtggaata tttcggatat 602356DNAArtificial
SequencePrimer 23gcatacaatc aactaagcta agctaaaaca atgggtaagg
aaaagactca cgtttc 562456DNAArtificial SequencePrimer 24gaaacgtgag
tcttttcctt acccattgtt ttagcttagc ttagttgatt gtatgc
562553DNAArtificial SequencePrimer 25catttgatgc tcgatgagtt
tttctaaatc cgctctaacc gaaaaggaag gag 532653DNAArtificial
SequencePrimer 26ctccttcctt ttcggttaga gcggatttag aaaaactcat
cgagcatcaa atg 532760DNAArtificial SequencePrimer 27ggggctagca
taacttcgta taatgtatgc tatacgaagt tatcttcgag cgtcccaaaa
602830DNAArtificial SequencePrimer 28cgggatccag tttatcatta
tcaatactcg 302931DNAArtificial SequencePrimer 29gggctcgagg
agcgacctca tgctatacct g 313022DNAArtificial SequencePrimer
30ttagaaaaac tcatcgagca tc 223155DNAArtificial SequencePrimer, OLS
5' FWD 31gcatagcaat ctaatctaag tttaaaatga atcatttgag agcagaaggg
cctgc 553256DNAArtificial SequencePrimer, CB 5' FWD 32caccagaact
tagtttcgac ggataaaatg gaaaccggtt tgtcctcggt ttgcac
563358DNAArtificial SequencePrimer, ALL REV 33cataactaat tacatgattt
aaccttaaac atcagattca atagagccgc ctccactg 58348933DNAArtificial
SequenceCodon Optimized 34ggttaaatca tgtaattagt tatgtcacgc
ttacattcac gccctccccc cacatccgct 60ggttaaatca tgtaattagt tatgtcacgc
ttacattcac gccctccccc cacatccgct 120ctaaccgaaa aggaaggagt
tagacaacct gaagtctagg tccctattta tttttttata 180gttatgttag
tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
240cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt
gggacgctcg 300aaggctttaa tttgcggccc ctcacctgca cgcaaaatag
gataattata ctctatttct 360caacaagtaa ttggttgttt ggccgagcgg
tctaaggcgc ctgattcaag aaatatcttg 420accgcagtta actgtgggaa
tactcaggta tcgtaagatg caagagttcg aatctcttag 480caaccattat
ttttttcctc aacataacga gaacacacag gggcgctatc gcacagaatc
540aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca
tatacctttt 600tcaactgaaa aattgggaga aaaaggaaag gtgagagcgc
cggaaccggc ttttcatata 660gaatagagaa gcgttcatga ctaaatgctt
gcatcacaat acttgaagtt gacaatatta 720tttaaggacc tattgttttt
tccaataggt ggttagcaat cgtcttactt tctaactttt 780cttacctttt
acatttcagc aatatatata tatatatttc aaggatatac cattctaatg
840tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga
aatcacagcc 900gaagccatta aggttcttaa agctatttct gatgttcgtt
ccaatgtcaa gttcgatttc 960gaaaatcatt taattggtgg tgctgctatc
gatgctacag gtgttccact tccagatgag 1020gcgctggaag cctccaagaa
ggctgatgcc gttttgttag gtgctgtggg tggtcctaaa 1080tggggtaccg
gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa
1140ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga
cttatctcca 1200atcaagccac aatttgctaa aggtactgac ttcgttgttg
tcagagaatt agtgggaggt 1260atttactttg gtaagagaaa ggaagatgat
ggtgatggtg tcgcttggga tagtgaacaa 1320tacaccgttc cagaagtgca
aagaatcaca agaatggccg ctttcatggc cctacaacat 1380gagccaccat
tgcctatttg gtccttggat aaagctaatg ttttggcctc ttcaagatta
1440tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa
ggttcaacat 1500caattgattg attctgccgc catgatccta gttaagaacc
caacccacct aaatggtatt 1560ataatcacca gcaacatgtt tggtgatatc
atctccgatg aagcctccgt tatcccaggt 1620tccttgggtt tgttgccatc
tgcgtccttg gcctctttgc cagacaagaa caccgcattt 1680ggtttgtacg
aaccatgcca cggttctgct ccagatttgc caaagaataa ggtcaaccct
1740atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt
gcctgaagaa 1800ggtaaggcca ttgaagatgc agttaaaaag gttttggatg
caggcatcag aactggtgat 1860ttaggtggtt ccaacagtac caccgaagtc
ggtgatgctg tcgccgaaga agttaagaaa 1920atccttgctt aaaaagattc
tcttttttta tgatatttgt acataaactt tataaatgaa 1980attcataata
gaaacgacac gaaattacaa aatggaatat gttcataggg taacgctatg
2040atccaatatc aaaggaaatg atagcattga aggatgagac taatccaatt
gaggagtggc 2100agcatataga acagctaaag ggtagtgctg aaggaagcat
acgatacccc gcatggaatg 2160ggataatatc acaggaggta ctagactacc
tttcatccta cataaataga cgcatataag 2220tacgcattta agcataaaca
cgcactatgc cgttcttctc atgtatatat atatacaggc 2280aacacgcaga
tataggtgcg acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat
2340tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt
tcctattctc 2400tagaaagtat aggaacttca gagcgctttt gaaaaccaaa
agcgctctga agtcgcactt 2460tcaaaaaacc aaaaacgcac cggactgtaa
cgagctacta aaatattgcg aataccgctt 2520ccacaaacat tgctcaaaag
tatctctttg ctatatatct ctgtgctata tccctatata 2580acctacccat
ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt
2640atgtttatct ctagtattac tctttagaca aaaaaattgt agtaagaact
attcatagag 2700tgaatcgaaa acaatacgaa aatgtaaaca tttcctatac
gtagtatata gagacaaaat 2760agaagaaacc gttcataatt ttctgaccaa
tgaagaatca tcaacgctat cactttctgt 2820tcacaaagta tgcgcaatcc
acatcggtat agaatataat cggggatgcc tttatcttga 2880aaaaatgcac
ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt
2940tttatggaag agaaaataga caccaaagta gccttcttct aaccttaacg
gacctacagt 3000gcaaaaagtt atcaagagac tgcattatag agcgcacaaa
ggagaaaaaa agtaatctaa 3060gatgctttgt tagaaaaata gcgctctcgg
gatgcatttt tgtagaacaa aaaagaagta 3120tagattcttt gttggtaaaa
tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag 3180ctcagattct
ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg
3240aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt
tctgtaaaaa 3300tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
gttgcatttt tgttctacaa 3360aatgaagcac agatgcttcg ttcaggtggc
acttttcggg gaaatgtgcg cggaacccct 3420atttgtttat ttttctaaat
acattcaaat atgtatccgc tcatgagaca ataaccctga 3480tattggtcag
aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa
3540tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
caataatatt 3600gaaaaaggaa gaatatgagc catattcaac gggaaacgtc
gaggccgcga ttaaattcca 3660acatggatgc tgatttatat gggtataaat
gggctcgcga taatgtcggg caatcaggtg 3720cgacaatcta tcgcttgtat
gggaagcccg atgcgccaga gttgtttctg aaacatggca 3780aaggtagcgt
tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
3840ttatgccact tccgaccatc aagcatttta tccgtactcc tgatgatgca
tggttactca 3900ccactgcgat ccccggaaaa acagcgttcc aggtattaga
agaatatcct gattcaggtg 3960aaaatattgt tgatgcgctg gcagtgttcc
tgcgccggtt gcactcgatt cctgtttgta 4020attgtccttt taacagcgat
cgcgtatttc gcctcgctca ggcgcaatca cgaatgaata 4080acggtttggt
tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
4140tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
actcatggtg 4200atttctcact tgataacctt atttttgacg aggggaaatt
aataggttgt attgatgttg 4260gacgagtcgg aatcgcagac cgataccagg
atcttgccat cctatggaac tgcctcggtg 4320agttttctcc ttcattacag
aaacggcttt ttcaaaaata tggtattgat aatcctgata 4380tgaataaatt
gcaatttcat ttgatgctcg atgagttttt ctaactcatg accaaaatcc
4440cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag
aaaagatcaa 4500aggatcttct tgagatcctt tttttctgcg cgtaatctgc
tgcttgcaaa caaaaaaacc 4560accgctacca gcggtggttt gtttgccgga
tcaagagcta ccaactcttt ttccgaaggt 4620aactggcttc agcagagcgc
agataccaaa tactgttctt ctagtgtagc cgtagttagc 4680ccaccacttc
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
4740agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
gacgatagtt 4800accggataag gcgcagcggt cgggctgaac ggggggttcg
tgcacacagc ccagcttgga 4860gcgaacgacc tacaccgaac tgagatacct
acagcgtgag ctatgagaaa gcgccacgct 4920tcccgaaggg agaaaggcgg
acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4980cacgagggag
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
5040cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
tatggaaaaa 5100cgccagcaac gcggcctttt tacggttcct ggccttttgc
tggccttttg ctcacatgtt 5160ctttcctgcg ttatcccctg attctgtgga
taaccgtatt accgcctttg agtgagctga 5220taccgctcgg ggtcgtgcag
gtagtttatc attatcaata ctcgccattt caaagaatac 5280gtaaataatt
aatagtagtg attttcctaa ctttatttag tcaaaaaatt agccttttaa
5340ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
atataacatc 5400gtaggtgtct gggtgaacag tttattcctg gcatccacta
aatataatgg agcccgcttt 5460ttaagctggc atccagaaaa aaaaagaatc
ccagcaccaa aatattgttt tcttcaccaa 5520ccatcagttc ataggtccat
tctcttagcg caactacaga gaacaggggc acaaacaggc 5580aaaaaacggg
cacaacctca atggagtgat gcaaccagcc tggagtaaat gatgacacaa
5640ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
ccttctgctc 5700tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca
gttccctgaa attattcccc 5760tacttgacta ataagtatat aaagacggta
ggtattgatt gtaattctgt aaatctattt 5820cttaaacttc ttaaattcta
cttttatagt tagtcttttt tttagtttta aaacaccaga 5880acttagtttc
gacggataaa atggaaaccg gtttgtcctc ggtttgcact ttctccttcc
5940aaacaaacta tcatacactc ctgaacccgc acaataacaa tcccaaaact
tccctgctgt 6000gttataggca cccaaagaca ccaatcaaat actcctacaa
taactttcca tctaagcatt 6060gtagcacaaa aagtttccat ttgcaaaata
agtgttccga atctctgtcc atcgccaaaa 6120attccattag ggctgccact
actaatcaaa ctgaaccacc agagtctgat aatcattctg 6180tcgccacaaa
gattctgaat tttgggaagg cttgttggaa gttacaaaga ccatatacaa
6240ttattgcctt tacctcttgt gcctgtggtt tatttggtaa ggaactgttg
cataatacaa 6300atttaatatc ttggtcattg atggaaacgt tcaaagcatt
ttttttctta gtcgctatcc 6360tttgtattgc ttctttcacc accactatca
accagattta cgacttacat attgacagaa 6420ttaacaagcc agatttgcca
ctggcttcgg gcgagatttc cgtcaatact gcctggatca 6480tggaaacttc
tattattgtt gccttgtttg gattgataat caccataaaa atggaaacta
6540agggtggtcc attgtatatt ttcggttact gttttggtat cttcgggggc
atcgtctact 6600ctgttcctcc attcagatgg aaacaaaatc cttccacagc
attccttttg aacttcctgg 6660cgcacattat aaccaacttt actttttatt
atgcctccag agccgccctg gggctgccct 6720ttgaattacg cccctccttt
acatttttac tggccttcat ggagaccaag tccatggaga 6780ctggttctgc
tctcgcgttg atcaaagatg cttccgatgt ggaaggtgac accaaatttg
6840gtatatccac tttggccagc aagtatggtt ccaggaattt gaccctattt
tgttctggta 6900tcgtgctgct gtcttatgtt gcagccatct tggctggcat
catttggcca caggctttca 6960attcaaatgt tatggagacg ctgctctcgc
atgctatttt ggcattttgg ttgattctac 7020agacaagaga ttttgcttta
accaattatg acccagaagc tggtagaaga ttttacgaat 7080ttatggaaac
atggaaatta tactatgctg aatatttagt gtacgttttc attgggggcg
7140gctccagcgc cggcggcggc tcttctgcgg gcggttggtc tcatccacaa
tttgagaaag 7200gtgggtcgtc tggcggcggc agcgggggcg ggtccggcgg
ggggagcggc ggtatgaaat 7260gttcgacctt ctctttttgg tttgtctgta
aaataatttt ttttttcttc agctttaaca 7320ttcaaaccag cattgcaaat
ccaagagaaa atttcttgaa atgcttttca caatatatcc 7380ccaataatgc
tactaacttg aagctagttt atactcaaaa caaccctttg tacatgtccg
7440tgctcaactc caccattcac aacctaagat tcacttcaga cactacccca
aaaccattag 7500ttattgtgac accttctcac gtttcacata tccaaggtac
tattttatgc tccaagaagg 7560tcggcctgca aattagaact agatctggag
gtcatgattc agaaggaatg tcttacatct 7620ctcaagttcc atttgtgatt
gtcgatttaa gaaatatgag gagcattaag atcgatgttc 7680actcccaaac
ggcatgggtt gaagccggtg ccaccttggg cgaagtttac tactgggtca
7740acgagaagaa tgaaaactta tcactagccg caggttattg tccaactgtt
tgtgctggtg 7800gccatttcgg aggcggcggc tacggtcctc taatgagaaa
ctacggctta gctgctgaca 7860atatcatcga cgctcacttg gttaacgttc
atggtaaagt tttagataga aaatctatgg 7920gtgaggatct tttctgggct
ttgagaggtg gcggcgcaga atcatttggc attatcgttg 7980cttggaagat
cagattggtg gctgtcccca agtctacaat gttttctgtg aagaaaatta
8040tggaaatcca tgaattggtc aaactggtga ataaatggca aaacatagct
tacaagtacg 8100ataaagactt gctgttaatg acacatttta ttaccaggaa
catcactgat aaccaaggca 8160agaacaagac tgcaattcat acttattttt
cctccgtttt tttgggtggt gtcgactccc 8220tcgtggatct gatgaataaa
tcattccctg aactaggtat taaaaaaacc gattgtagac 8280aattgagttg
gattgatacc atcatattct acagtggtgt tgttaattat gatactgaca
8340acttcaacaa agaaatactg ctggaccgtt ccgccggcca gaatggtgct
tttaaaatca 8400agttggatta tgtgaaaaag cctattccag aatccgtatt
tgttcaaata ttggaaaagc 8460tgtatgaaga agacattggt gcaggcatgt
acgctcttta tccttatggc ggcataatgg 8520atgaaatttc tgaaagtgcc
attcctttcc cacatagggc cgggatcctg tacgagttat 8580ggtacatttg
ttcatgggaa aagcaagaag ataatgaaaa acatttaaat tggataagaa
8640atatttataa ttttatgact ccatacgtct ccaaaaaccc acgcctggca
tatttgaatt 8700acagagacct ggatattggc atcaatgatc ctaaaaaccc
aaataattac actcaggcaa 8760gaatatgggg tgaaaaatat ttcggcaaaa
attttgatag gctggtcaag gttaaaacac 8820tggttgatcc aaacaatttc
tttagaaacg aacaatctat cccacctctg cctagacata 8880gacacggcgg
tggaagcagt ggaggcggct ctattgaatc tgatgtttaa tga
8933356677DNAArtificial SequenceCondon Optimized 35ggttaaatca
tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 60ctaaccgaaa
aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
120gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
tctgtacaga 180cgcgtgtacg catgtaacat tatactgaaa accttgcttg
agaaggtttt gggacgctcg 240aaggctttaa tttgcggccc ctcacctgca
cgcaaaaagc ttttcaattc aattcatcat 300ttttttttta ttcttttttt
tgatttcggt ttctttgaaa tttttttgat tcggtaatct 360ccgaacagaa
ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat
420gtagtgttga agaaacatga aattgcccag tattcttaac ccaactgcac
agaacaaaaa 480ccagcaggaa acgaagataa atcatgtcga aagctacata
taaggaacgt gctgctactc 540atcctagtcc tgttgctgcc aagctattta
atatcatgca cgaaaagcaa acaaacttgt 600gtgcttcatt ggatgttcgt
accaccaagg aattactgga gttagttgaa gcattaggtc 660ccaaaatttg
tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca
720cagttaagcc gctaaaggca ttatccgcca agtacaattt tttactcttc
gaagatagaa 780aatttgctga cattggtaat acagtcaaat tgcagtactc
tgcgggtgta tacagaatag 840cagaatgggc agacattacg aatgcacacg
gtgtggtggg cccaggtatt gttagcggtt 900tgaagcaggc ggcagaagaa
gtaacaaagg aacctagagg ccttttgatg ttagcagaat 960tgtcatgcaa
gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga
1020aaagcgacaa agattttgtt atcggcttta ttgctcaaag agacatgggt
ggaagagatg 1080aaggttacga ttggttgatt atgacacccg gtgtgggttt
agatgacaag ggagatgcat 1140tgggtcaaca gtatagaacc gtggatgatg
ttgtctctac aggatctgac attattattg 1200ttggaagagg actatttgca
aagggaaggg atgctaaggt agagggtgaa cgttacagaa 1260aagcaggctg
ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat
1320aagtaaatgc atgtatacta aactcacaaa ttagagcttc aatttaatta
tatcagttat 1380tacccacgct atgatccaat atcaaaggaa atgatagcat
tgaaggatga gactaatcca 1440attgaggagt ggcagcatat agaacagcta
aagggtagtg ctgaaggaag catacgatac 1500cccgcatgga atgggataat
atcacaggag gtactagact acctttcatc ctacataaat 1560agacgcatat
aagtacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata
1620tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg
tatgtgcgca 1680gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg
ctttgaagtt cctattccga 1740agttcctatt ctctagaaag tataggaact
tcagagcgct tttgaaaacc aaaagcgctc 1800tgaagtcgca ctttcaaaaa
accaaaaacg caccggactg taacgagcta ctaaaatatt 1860gcgaataccg
cttccacaaa cattgctcaa aagtatctct ttgctatata tctctgtgct
1920atatccctat ataacctacc catccacctt tcgctccttg aacttgcatc
taaactcgac 1980ctctacattt tttatgttta tctctagtat tactctttag
acaaaaaaat tgtagtaaga 2040actattcata gagtgaatcg aaaacaatac
gaaaatgtaa acatttccta tacgtagtat 2100atagagacaa aatagaagaa
accgttcata attttctgac caatgaagaa tcatcaacgc 2160tatcactttc
tgttcacaaa gtatgcgcaa tccacatcgg tatagaatat aatcggggat
2220gcctttatct tgaaaaaatg cacccgcagc ttcgctagta atcagtaaac
gcgggaagtg 2280gagtcaggct ttttttatgg aagagaaaat agacaccaaa
gtagccttct tctaacctta 2340acggacctac agtgcaaaaa gttatcaaga
gactgcatta tagagcgcac aaaggagaaa 2400aaaagtaatc taagatgctt
tgttagaaaa atagcgctct cgggatgcat ttttgtagaa 2460caaaaaagaa
gtatagattc tttgttggta aaatagcgct ctcgcgttgc atttctgttc
2520tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt
tgcatttttg 2580ttttacaaaa atgaagcaca gattcttcgt tggtaaaata
gcgctttcgc gttgcatttc 2640tgttctgtaa aaatgcagct cagattcttt
gtttgaaaaa ttagcgctct cgcgttgcat 2700ttttgttcta caaaatgaag
cacagatgct tcgttcaggt ggcacttttc ggggaaatgt 2760gcgcggaacc
cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
2820acaataaccc tgatattggt cagaattggt taattggttg taacactgac
ccctatttgt 2880ttatttttct aaatacattc aaatatgtat ccgctcatga
gacaataacc ctgataaatg 2940cttcaataat attgaaaaag gaagaatatg
agtattcaac atttccgtgt cgcccttatt 3000cccttttttg cggcattttg
ccttcctgtt tttgctcacc
cagaaacgct ggtgaaagta 3060aaagatgctg aagatcagtt gggtgcacga
gtgggttaca tcgaactgga tctcaacagc 3120ggtaagatcc ttgagagttt
tcgccccgaa gaacgttttc caatgatgag cacttttaaa 3180gttctgctat
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
3240cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
aaagcatctt 3300acggatggca tgacagtaag agaattatgc agtgctgcca
taaccatgag tgataacact 3360gcggccaact tacttctgac aacgatcgga
ggaccgaagg agctaaccgc ttttttgcac 3420aacatggggg atcatgtaac
tcgccttgat cgttgggaac cggagctgaa tgaagccata 3480ccaaacgacg
agcgtgacac cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta
3540ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
gatggaggcg 3600gataaagttg caggaccact tctgcgctcg gcccttccgg
ctggctggtt tattgctgat 3660aaatccggag ccggtgagcg tggttctcgc
ggtatcatcg cagcgctggg gccagatggt 3720aagccctccc gtatcgtagt
tatctacacg acggggagtc aggcaactat ggatgaacga 3780aatagacaga
tcgctgagat aggtgcctca ctgattaagc attggtaact catgaccaaa
3840atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc
gtagaaaaga 3900tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
ctgctgcttg caaacaaaaa 3960aaccaccgct accagcggtg gtttgtttgc
cggatcaaga gctaccaact ctttttccga 4020aggtaactgg cttcagcaga
gcgcagatac caaatactgt tcttctagtg tagccgtagt 4080tagcccacca
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
4140taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
tcaagacgat 4200agttaccgga taaggcgcag cggtcgggct gaacgggggg
ttcgtgcaca cagcccagct 4260tggagcgaac gacctacacc gaactgagat
acctacagcg tgagctatga gaaagcgcca 4320cgcttcccga agggagaaag
gcggacaggt atccggtaag cggcagggtc ggaacaggag 4380agcgcacgag
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
4440gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
agcctatgga 4500aaaacgccag caacgcggcc tttttacggt tcctggcctt
ttgctggcct tttgctcaca 4560tgttctttcc tgcgttatcc cctgattctg
tggataaccg tattaccgcc tttgagtgag 4620ctgataccgc tcggggtcgt
gcaggtatag cttcaaaatg tttctactcc ttttttactc 4680ttccagattt
tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca
4740tactaaattt cccctctttc ttcctctagg gtgtcgttaa ttacccgtac
taaaggtttg 4800gaaaagaaaa aagtgaccgc ctcgtttctt tttcttcgtc
gaaaaaggca ataaaaattt 4860ttatcacgtt tctttttctt gaaaattttt
ttttttgatt tttttctctt tcgatgacct 4920cccattgata tttaagttaa
taaacggact tcaatttctc aagtttcagt ttcatttttc 4980ttgttctatt
acaacttttt ttacttcttg ctcattagaa agaaagcata gcaatctaat
5040ctaagtttaa aatgaatcat ttgagagcag aagggcctgc ttccgtgctg
gctattggta 5100ccgccaatcc agaaaatatc ctgctgcagg acgaattccc
agattactat tttagggtca 5160ccaaatctga acatatgaca caattgaaag
agaaattcag aaagatttgt gacaagtcca 5220tgattaggaa aagaaattgt
tttttgaatg aagaacactt gaagcaaaat cctcgcctgg 5280tggagcatga
aatgcaaact ttggatgcta gacaagacat gttggtggtg gaagttccaa
5340agctggggaa ggatgcctgt gccaaggcca ttaaagaatg gggccaacca
aaatccaaaa 5400ttacccacct gattttcacc tccgcctcca ccactgatat
gccaggtgca gactatcatt 5460gtgctaaatt gttgggtttg tccccctccg
tgaagagagt tatgatgtat caattaggtt 5520gttatggcgg cggcaccgtt
ctgagaattg ccaaagacat tgctgaaaac aataaaggtg 5580cgcgcgtttt
ggctgtttgt tgtgatatta tggcatgttt atttagaggt ccaagtgaaa
5640gtgacttgga attgctagtg ggccaggcca tatttggtga tggtgccgct
gctgtgatcg 5700ttggtgctga gcctgatgaa tctgtcggtg aaagaccaat
ttttgaactg gtttccactg 5760gtcaaaccat tttgccaaat tcagaaggta
ctattggcgg ccatatcaga gaagctggtt 5820taatctttga tttgcacaag
gatgtcccaa tgttaatttc caataatatt gaaaaatgtt 5880tgatcgaagc
atttaccccc atcggtattt ctgattggaa ttccatcttc tggattacac
5940atcctggcgg taaagctatc ttagataaag ttgaggagaa gttgcattta
aagtctgaca 6000aatttgttga ttcaagacat gtcctgtctg agcacggtaa
tatgtcttcc tcgaccgtct 6060tgtttgtcat ggatgagttg aggaagaggt
ccctggaaga aggcaagagc accaccggtg 6120acggttttga gtggggggtc
ctctttggat ttgggccagg cctgaccgta gaaagggttg 6180ttgtccgctc
ggtgccaatc aaatatggtg gggggtccag cgccggtggc gggagctccg
6240cgggcggttg gtctcaccca caatttgaaa agggtggcag cagcggcggc
ggctctggcg 6300gaggctccgg cgggggctcg gggggtatgg ctgtcaagca
tctgatcgtg ctgaagttca 6360aagatgaaat tactgaagcc caaaaggagg
aatttttcaa gacatatgtt aatttggtta 6420acatcattcc agcaatgaaa
gatgtttatt ggggtaagga cgttactcaa aaaaataagg 6480aagagggtta
cactcatatt gttgaagtca ctttcgaatc cgtcgaaaca attcaagatt
6540atattattca tccagctcat gttgggtttg gcgatgtgta cagatcattt
tgggaaaaat 6600tattgatttt tgactacaca ccaagaaaag gcggtggaag
cagtggaggc ggctctattg 6660aatctgatgt ttaatag
6677363372DNAArtificial SequenceCodon Optimized 36atggagaaga
ctcaagaaac agtccaaaga attcttctag aaccctataa atacttactt 60cagttaccag
gtaaacaagt gagaaccaaa ctttcacagg catttaatca ttggctgaaa
120gttccagagg acaagctaca gattattatt gaagtgacag aaatgttgca
taatgccagt 180ttactcatcg atgatattga agacaactca aaactccgac
gtggctttcc agtggcccac 240agcatctatg gaatcccatc tgtcatcaat
tctgccaatt acgtgtattt ccttggcttg 300gagaaagtct taacccttga
tcacccagat gcagtgaagc tttttacccg ccagcttttg 360gaactccatc
agggacaagg cctagatatt tactggaggg ataattacac ttgtcccact
420gaagaagaat ataaagctat ggtgctgcag aaaacaggtg gactgtttgg
attagcagta 480ggtctcatgc agttgttctc tgattacaaa gaagatttaa
aaccgctact taatacactt 540gggctctttt tccaaattag ggatgattat
gctaatctac actccaaaga atatagtgaa 600aacaaaagtt tttgtgaaga
tctgacagag ggaaagttct catttcctac tattcatgct 660atttggtcaa
ggcctgaaag cacccaggtg cagaatatct tgcgccagag aacagaaaac
720atagatataa aaaaatactg tgtacattat cttgaggatg taggttcttt
tgaatacact 780cgtaataccc ttaaagagct tgaagctaaa gcctataaac
agattgatgc acgtggtggg 840aaccctgagc tagtagcctt agtaaaacac
ttaagtaaga tgttcaaaga agaaaatgaa 900ggcggttctg gcagcggaga
gggcagagga agtcttctaa catgcggtga cgtggaggag 960aatcccggcc
ctaggtctgg cagcggagag ggcagaggaa gtcttctaac atgcggtgac
1020gtggaggaga atcccggccc taggacacaa aagaaagtcc cagacaattg
ttgtagacgt 1080gaacctatgc tggtcagaaa taaccagaaa tgtgattcag
tagaggaaga gacagggata 1140aaccgagaaa gaaaagttga ggttataaaa
cccttagtgg ctgaaacaga taccccaaac 1200agagctacat ttgtggttgg
taactcctcc ttactcgata cttcatcagt actggtgaca 1260caggaacctg
aaattgaact tcccagggaa cctcggccta atgaagaatg tctacagata
1320cttgggaatg cagagaaagg tgcaaaattc cttagtgatg ctgagatcat
ccagttagtc 1380aatgctaagc atatcccagc ctacaagttg gaaactctga
tggaaactca tgagcgtggt 1440gtatctattc gccgacagtt actttccaag
aagctttcag aaccttcttc tctccagtac 1500ctaccttaca gggattataa
ttactccttg gtgatgggag cttgttgtga gaatgttatt 1560ggatatatgc
ccatccctgt tggagtggca ggaccccttt gcttagatga aaaagaattt
1620caggttccaa tggcaacaac agaaggttgt cttgtggcca gcaccaatag
aggctgcaga 1680gcaataggtc ttggtggagg tgccagcagc cgagtccttg
cagatgggat gactcgtggc 1740ccagttgtgc gtcttccacg tgcttgtgac
tctgcagaag tgaaagcctg gctcgaaaca 1800tctgaagggt tcgcagtgat
aaaggaggca tttgacagca ctagcagatt tgcacgtcta 1860cagaaacttc
atacaagtat agctggacgc aacctttata tccgtttcca gtccaggtca
1920ggggatgcca tggggatgaa catgatttca aagggtacag agaaagcact
ttcaaaactt 1980cacgagtatt tccctgaaat gcagattcta gccgttagtg
gtaactattg tactgacaag 2040aaacctgctg ctataaattg gatagaggga
agaggaaaat ctgttgtttg tgaagctgtc 2100attccagcca aggttgtcag
agaagtatta aagactacca cagaggctat gattgaggtc 2160aacattaaca
agaatttagt gggctctgcc atggctggga gcataggagg ctacaacgcc
2220catgcagcaa acattgtcac cgccatctac attgcctgtg gacaggatgc
agcacagaat 2280gttggtagtt caaactgtat tactttaatg gaagcaagtg
gtcccacaaa tgaagattta 2340tatatcagct gcaccatgcc atctatagag
ataggaacgg tgggtggtgg gaccaaccta 2400ctacctcagc aagcctgttt
gcagatgcta ggtgttcaag gagcatgcaa agataatcct 2460ggggaaaatg
cccggcagct tgcccgaatt gtgtgtggga ccgtaatggc tggggaattg
2520tcacttatgg cagcattggc agcaggacat cttgtcaaaa gtcacatgat
tcacaacagg 2580tcgaagatca atttacaaga cctccaagga gcttgcacca
agaagacagc cggctcagga 2640ggttcttcag gactggaagt gctgtttcag
ggcccgggtg gatctggcat gatgcctgaa 2700ataaacacta accacctcga
caagcaacag gttcaactcc tggcagagat gtgtatcctt 2760attgatgaaa
atgacaataa aattggagct gagaccaaga agaattgtca cctgaacgag
2820aacattgaga aaggattatt gcatcgagct tttagtgtct tcttattcaa
caccgaaaat 2880aagcttctgc tacagcaaag atcagatgct aagattacct
ttccaggttg ttttacgaat 2940acgtgttgta gtcatccatt aagcaatcca
gccgagcttg aggaaagtga cgcccttgga 3000gtgaggcgag cagcacagag
acggctgaaa gctgagctag gaattccctt ggaagaggtt 3060cctccagaag
aaattaatta tttaacacga attcactaca aagctcagtc tgatggtatc
3120tggggtgaac atgaaattga ttacattttg ttggtgagga agaatgtaac
tttgaatcca 3180gatcccaatg agattaaaag ctattgttat gtgtcaaagg
aagaactaaa agaacttctg 3240aaaaaagcag ccagtggtga aattaagata
acgccatggt ttaaaattat tgcagcgact 3300tttctcttta aatggtggga
taacttaaat catttgaatc agtttgttga ccatgagaaa 3360atatacagaa tg
3372
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