AUXOTROPHIC DIATOMS

DIATOMEES AUXOTROPHES

English Abstract

An auxotrophic strain of Phaeodactylum tricornutum (P. tricornutum) cells
having a loss of
heterozygosity at a gene necessary for the synthesis of an organic compound
necessary for the
growth of P. tricornutum and a method of producing said auxotrophic.

Claims

74
CLAIMS
What is claimed is:
1. An auxotrophic strain of Phaeodactylum tricornutum P. tricornutum) cells,
wherein the strain
has loss of heterozygosity at a gene necessary for the synthesis of an organic
compound necessary
for the growth of P. tricornutum.
2. The auxotrophic strain of P. tricornutum of claim 1, wherein the strain is
complemented with
an auxotrophic selection plasmid comprising an expression cassette comprising
a constitutive
promoter operably linked to an activated gene necessary for the synthesis of
the organic compound.
3. The auxotrophic strain of P. tricornutum according to any one of claims 1
to 2, wherein the
organic compound is histidine.
4. The auxotrophic strain of P. tricornutum according to any one of claims 1
to 2, wherein the
organic compound is uracil.
5. The auxotrophic strain of P. tricornutum according to any one of claims 1
to 2, wherein the 6.
organic compound is tryptophan.
6. The auxotrophic strain of P. tricornutum of claim 1 or claim 3, wherein the
organic compound
is histidine and the gene necessary for the synthesis of the organic compound
comprises SEQ ID
NO: 8 necessary for the synthesis of histidine.
7. The auxotrophic strain of P. tricornutum of claim 2 or claim 3, wherein the
organic compound
is histidine and wherein the gene and the activated gene comprises SEQ ID NO:
8 necessary for
the synthesis of histidine.
8. The auxotrophic strain of P. tricornutum of claim 1 or claim 4, wherein
organic compound is
uracil and the gene comprises SEQ ID NO: 6 necessary for the synthesis of
uracil.
9. The auxotrophic strain of P. tricornutum of claim 6, wherein the
auxotrophic strain of P.
tricornutum survive on an uracil-free medium supplemented with a concentration
of 5-fluoroorotic
acid (5-F0A) that inhibits growth of wild-type P. tricornutum.
10. The auxotrophic strain of P. tricornutum of claim 2 or claim 4, wherein
organic compound is

75
tryptophan uracil and wherein the gene and the activated gene comprises SEQ ID
NO: 6 necessary
for the synthesis of uracil.
11. The auxotrophic strain of P. tricornutum of claim 1, wherein the organic
compound is
tryptophan and the gene comprises SEQ ID NO: 10 necessary for the synthesis of
tryptophan.
12. The auxotrophic strain of P. tricornutum according to any one of claims 1
to 11, wherein the
auxotrophic strain of P. tricornutum is identified by a phenotypic screen or a
genetic screen.
13. The auxotrophic strain of P. tricornutum according to any one of claims 1
to 12, wherein the
auxotrophic selection plasmid further comprises a DNA sequence for encoding a
recombinant
peptide of interest.
14. The auxotrophic strain of P. tricornutum of claim 13, wherein the
recombinant peptide of
interest is an antibody, a protein, an enzyme or a glycoprotein.
15. The auxotrophic strain of P. tricornutum of claim 13 or claim 14, wherein
the auxotrophic
selection plasmid further comprises a constitutive promoter operably linked to
the DNA sequence
for encoding the recombinant peptide of interest.
16. A method for producing auxotrophic strains of Phaeodactylum tricornutum P.
tricornutum)
cells, wherein the strain has loss of heterozygosity at a target gene
necessary for the synthesis of
one of an organic compound necessary for the growth of P. tricornutum, wherein
the method
comprises:
(a) introducing an editing plasmid into a strain of P. tricornutum, the
editing plasmid comprising
a TevCas9 gene (TevCas9 plasmid) or a Cas9 gene (Cas9 plasmid) and a single-
guide RNA
(sgRNA) against a binding site of the target gene, to obtain P. tricornutum
exconjugants,
(b) screening the P. tricornutum exconjugants for colonies of P. tricornutum
edited with the
TevCas9 plasmid or the Cas9 plasmid in the target gene to obtain P.
tricornutum edited colonies,
(c) screening the P. tricornutum edited colonies for target gene knockout P.
tricornutum strains by
growing the P. tricornutum edited colonies on solid media with and without
auxotrophic
supplement including the organic compound, wherein P. tricornutum edited
colonies that do not
grow without auxotrophic supplement and grow with auxotrophic supplement are
target gene
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76
knockout P. tricornutum strains, and
(d) screening for loss of the plasmid from the target gene knockout P.
tricornutum strains thereby
producing the auxotrophic strain of P. tricornutum .
17. The method of claim 16, wherein step (b) comprises screening the P.
tricornutum exconjugants
using a T7EI editing assay.
18. The method of claim 16 or claim 17, wherein the plasmid is resistant to an
antibiotic, and
wherein step (d) comprises screening for loss of the antibiotic-resistant
plasmid by growing the
target gene knockout P. tricornutum strains in media supplemented with the
auxotrophic
supplement and the antibiotic and in media supplemented with the auxotrophic
supplement without
the antibiotic.
19. The method according to any one of claims 16 to 18, wherein the organic
compound is histidine
and the target gene comprises SEQ ID NO: 8 necessary for the synthesis of
histidine.
20. The method of claim 18, wherein the editing plasmid includes SEQ ID NO: 5
(pPtPRAPHCH).
21. The method according to any one of claims 16 to 18, wherein the organic
compound is uracil
and the target gene comprises SEQ ID NO: 6 necessary for the synthesis of
uracil.
22. The method of claim 21, wherein the editing plasmid includes SEQ ID NO: 1
(pPtUMPSA1),
SEQ ID NO: 2 (pPtUMPSA2), SEQ ID NO: 3 (pPtUMPScAl) or SEQ ID NO: 4
(pPtUMPScA2).
23. The method according to any one of claims 16 to 18, wherein the organic
compound is
tryptophan and the target gene comprises SEQ ID NO: 10 necessary for the
synthesis of
tryptophan.
24. The method according to any one of claims 16 to 18, wherein the P.
tricornutum exconjugant
are edited with the Cas9 plasmid.
25. The method according to any one of claims 16 to 18, wherein the P.
tricornutum exconjugant
are edited with the TevCas9 plasmid.
26. A method to produce a cell extract for use in an in vitro protein
expression reaction comprising:
(a) culturing the auxotrophic strain of P. tricornutum according to any one of
claims 1 to 15 in a
growth media comprising the organic compound; and (b) preparing the cell
extract of the culture.
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27. The method of claim 26, wherein the step of preparing the cell extract of
the culture comprises
lysing the auxotrophic strain of P. tricornutum.
28. A method of producing a recombinant peptide comprising:
(a) preparing an auxotrophic strain of Phaeodactylum tricornutum ( P.
tricornutum) cells according
to any one of claims 16 to 26, wherein the editing plasmid further comprises a
DNA sequence
encoding the recombinant peptide;
(b) culturing said auxotrophic strain of P. tricornutum cells under conditions
permitting expression
of the DNA sequence encoding the recombinant peptide; and
d) recovering said recombinant peptide.
.. 29. The method of producing a recombinant peptide of claim 28, wherein the
recombinant peptide
is an antibody, a protein, an enzyme or a glycoprotein.
30. The method of producing a recombinant peptide of claim 28, wherein editing
plasmid further
comprises a constitutive promoter operably linked to the DNA sequence for
encoding the
recombinant peptide.
31. A plasmid, wherein said plasmid comprises SEQ ID NO:1 (pPtUMPSA1), SEQ ID
NO:2
(pPtUMPSA2), SEQ ID NO: 3 (pPtUMPScAl), SEQ ID NO: 4 (pPtUMPScA2) or SEQ ID
NO:5
(pPtPRAPHCH).
Date Recue/Date Received 2021-08-17

Description

1
TITLE OF THE INVENTION
AUXOTROPHIC DIATOMS
FIELD OF THE INVENTION
This disclosure relates to auxotrophic diatoms and methods of producing said
auxotrophic diatoms.
BACKGROUND OF THE INVENTION
Photoautotrophic microalgae and cyanobacteria are emerging as alternative
platforms for synthetic
biology applications [1,2]. One microalgae species of interest is the diploid
marine diatom
Phaeodactylum tricornutum. A variety of plasmid-based genetic tools have been
developed for P.
tricornutum that facilitate basic molecular manipulations and expression of
complex synthetic
pathways [3,4,5,6]. We, and others, have developed plasmid-based and DNA-free
CRISPR
(clustered regularly interspaced palindromic repeats) reagents for targeted
chromosome editing in
P. tricornutum and related diatoms using the Cas9 protein (CRISPR-associated
protein 9)
[6,7,8,9,10,11]. P. tricornutum is diploid, meaning that Cas9-edited cells
must be carefully
screened to determine if knockouts are monoallelic or biallelic and exhibit
loss of heterozygosity.
These plasmid-based tools and synthetic pathways are currently maintained by
available antibiotic-
based selections, including zeocin, phleomycin, nourseothricin, and
blasticidin-S and their
resistance genes, Sh ble, nat, and bsr [12,13,14,15]. Antibiotic-based
selections can be
prohibitively expensive for maintaining large-scale cultures and are
problematic for applications
such as the biosynthesis of products intended for human consumption
[16,17,18].
A viable alternative to antibiotics is the use of auxotrophic selective
markers which require a strain
engineered to have a loss of function mutation in a key enzyme of an essential
biosynthetic
pathway. Examples of commonly used auxotrophic strains in industrial and
academic labs include
uracil, histidine, and tryptophan auxotrophs [19,20,21]. Two approaches have
been taken to
generate P. tricornutum auxotrophs. First, uracil-requiring mutants were
generated by random
.. mutagenesis that resulted in the identification of the bi-functional
uridine monophosphate synthase
(PtUMPS) gene predicted to catalyze the conversion of orotate into uridine
monophosphate (UMP)
[22]. Biolistic transformation and chromosomal integration of the PtUMPs gene
rescued the uracil-
requiring phenotype. Second, Cas9 was used to knockout the PtUMPS gene to
create uracil
auxotrophs and the PtAPT gene encoding a predicted adenine phosphoribosyl
transferase to create
Date Recue/Date Received 2021-08-17

2
adenine auxotrophs [7]. However, direct selection of these auxotrophs via
transformation with the
corresponding complementation marker has not been explored and the generation
of additional
auxotrophic strains would facilitate development of new plasmid-based
complementation markers.
SUMMARY OF THE INVENTION
The present invention relates to a plasmid-based editing strategy to generate
auxotrophic
knockouts in the uracil, histidine, and tryptophan biosynthesis pathways of P.
tricornutum and
plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes complement the
uracil- and
histidine-requiring phenotypes, respectively. Individual auxotrophic strains
of the present
invention are characterized by loss of heterozygosity at the edited alleles,
and large, heterogeneous
deletions up to about 2.7 kb. The auxotrophs of the present invention and
their respective
complementation markers represent alternative to antibiotic-based selection of
plasmids in P.
tricornutum.
In one embodiment, the present invention provides for an auxotrophic strain of
Phaeodactylum
tricornutum (P. tricornutum) cells, wherein the strain has loss of
heterozygosity at a gene
necessary for the synthesis of an organic compound necessary for the growth of
P. tricornutum.
In one embodiment of the auxotrophic strain of P. tricornutum of the present
invention, the strain
is complemented with an auxotrophic selection plasmid having an expression
cassette comprising
a constitutive promoter operably linked to an activated gene necessary for the
synthesis of the
organic compound.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is histidine, uracil or tryptophan.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is histidine, and the gene is PHATR 3140 necessary for the
synthesis of
histidine.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is histidine, and the gene comprises SEQ ID NO: 8 necessary
for the synthesis
of histidine.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
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organic compound is histidine, and the gene and the activated gene comprises
SEQ ID NO: 8
necessary for the synthesis of histidine.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is uracil, and the gene is PtUMPS necessary for the synthesis
of uracil.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
auxotrophic strain of P. tricornutum survive on an uracil-free medium
supplemented with a
concentration of 5-fluoroorotic acid (5-F0A) that inhibits growth of wild-type
P. tricornutum.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is uracil, and the gene and the activated gene is PtUMPS
necessary for the
synthesis of uracil.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is uracil, and the gene comprises SEQ ID NO: 6 necessary for
the synthesis of
uracil.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is uracil, and the gene and the activated gene comprises SEQ
ID NO: 6
necessary for the synthesis of uracil.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is tryptophan, and the gene is PtI3GPS-PRAI necessary for the
synthesis of
tryptophan.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is tryptophan, and the gene comprises SEQ ID NO: 10 necessary
for the
synthesis of tryptophan.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
organic compound is tryptophan, and the gene and the activated gene comprises
SEQ ID NO: 10
necessary for the synthesis of tryptophan.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
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auxotrophic strain of P. tricornutum is identified by a phenotypic screen or a
genetic screen.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
auxotrophic strain of P. tricornutum further carries an expression cassette
comprising a
constitutive promoter operably linked to a gene encoding a recombinant
peptide.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
auxotrophic selection plasmid further comprises a DNA sequence for encoding a
recombinant
peptide of interest.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
recombinant peptide of interest is an antibody, a protein, an enzyme or a
glycoprotein.
In another embodiment of the auxotrophic strain of P. tricornutum of the
present invention, the
auxotrophic selection plasmid further comprises a constitutive promoter
operably linked to the
DNA sequence for encoding the recombinant peptide of interest.
In another embodiment, the present invention provides for a method for
producing auxotrophic
strains of Phaeodactylum tricornutum (P. tricornutum) cells, wherein the
strain has loss of
heterozygosity at a target gene necessary for the synthesis of one of an
organic compound
necessary for the growth of P. tricornutum, wherein the method comprises: (a)
introducing an
editing plasmid into a strain of P. tricornutum, the editing plasmid
comprising a TevCas9 gene
(TevCas9 plasmid) or a Cas9 gene (Cas9 plasmid) and a single-guide RNA (sgRNA)
against a
binding site of the target gene, to obtain P. tricornutum exconjugants, (b)
screening the P.
tricornutum exconjugants for colonies of P. tricornutum edited with the
TevCas9 plasmid or the
Cas9 plasmid in the target gene to obtain P. tricornutum edited colonies, (c)
screening the P.
tricornutum edited colonies for target gene knockout P. tricornutum strains by
growing the P.
tricornutum edited colonies on solid media with and without auxotrophic
supplement including
the organic compound, wherein P. tricornutum edited colonies that do not grow
without
auxotrophic supplement and grow with auxotrophic supplement are target gene
knockout P.
tricornutum strains, and (d) screening for loss of the plasmid from the target
gene knockout P.
tricornutum strains thereby producing the auxotrophic strain of P.
tricornutum.
In one embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of the
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present invention, step (b) comprises screening the P. tricornutum
exconjugants using a T7EI
editing assay.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the plasmid is resistant to an antibiotic, and wherein
step (d) comprises
.. screening for loss of the antibiotic-resistant plasmid by growing the
target gene knockout P.
tricornutum strains in media supplemented with the auxotrophic supplement and
the antibiotic and
in media supplemented with the auxotrophic supplement without the antibiotic.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is histidine and the target gene
is PHATR 3140
necessary for the synthesis of histidine.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is histidine and the target gene
comprises SEQ ID
NO: 8 necessary for the synthesis of histidine.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
.. the present invention, the editing plasmid comprises SEQ ID NO: 5
(pPtPRAPHCH).
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is uracil and the target gene is
UMPS necessary for
the synthesis of uracil.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is uracil and the target gene
comprises SEQ ID NO:
6 necessary for the synthesis of uracil.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the editing plasmid comprises SEQ ID NO: 1 (pPtUMPSA1),
SEQ ID NO:
2 (pPtUMPSA2), SEQ ID NO: 3 (pPtUMPScAl) or SEQ ID NO: 4 (pPtUMPScA2).
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is tryptophan and the target gene
is PtI3GPS-PRAI
necessary for the synthesis of tryptophan.
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In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the organic compound is tryptophan and the target gene
comprises SEQ ID
NO: 10 necessary for the synthesis of tryptophan.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the P. tricornutum exconjugant are edited with the Cas9
plasmid.
In another embodiment of the method for producing auxotrophic strains of P.
tricornutum cells of
the present invention, the P. tricornutum exconjugant are edited with the
TevCas9 plasmid.
In another embodiment, the present invention provides for a method to produce
a cell extract for
use in an in vitro protein expression reaction comprising: (a) culturing the
auxotrophic strain of P.
tricornutum according to an embodiment of the present invention in a growth
media comprising
the organic compound; and (b) preparing the cell extract of the culture.
In one embodiment of the method to produce a cell extract for use in an in
vitro protein expression
reaction of the present invention, the step of preparing the cell extract of
the culture comprises
lysing the auxotrophic strain of P. tricornutum.
In one embodiment, the present invention provides for a method of producing a
recombinant
peptide comprising: (a) preparing an auxotrophic strain of Phaeodactylum
tricornutum (P.
tricornutum) cells according to an embodiment of the present invention,
wherein the editing
plasmid further comprises a DNA sequence encoding the recombinant peptide; (b)
culturing said
auxotrophic strain of P. tricornutum cells under conditions permitting
expression of the DNA
sequence encoding the recombinant peptide; and d) recovering said recombinant
peptide.
In one embodiment of the method of producing a recombinant peptide of the
present invention,
the recombinant peptide is an antibody, a protein, an enzyme or a
glycoprotein.
In one embodiment of the method of producing a recombinant peptide of the
present invention,
the editing plasmid further comprises a constitutive promoter operably linked
to the DNA sequence
for encoding the recombinant peptide.
In another embodiment, the present invention is a plasmid that comprises SEQ
ID NO: 1
(pPtUMPSA1). In another embodiment, the present invention is a plasmid that
comprises SEQ ID
NO:2 (pPtUMPSA2). In another embodiment, the present invention is a plasmid
that comprises
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SEQ ID NO:3 (pPtUMPScA1). In another embodiment, the present invention is a
plasmid that
comprises SEQ ID NO:4 (pPtUMPScA2). In another embodiment, the present
invention is a
plasmid that comprises SEQ ID NO:5 (pPtPRAPHCH).
BRIEF DESCRIPTION OF THE DRAWINGS
The following figures illustrate various aspects and preferred and alternative
embodiments of this
disclosure.
Figs. lA to IF. RISPR-generated knockouts in the predicted P. tricornutum
uracil
biosynthesis pathway. (1A) A portion of the predicted P. tricornutum
biosynthesis pathway for
conversion of carbonic acid to uracil and uridine triphosphate, with the
PtUMPS enzyme
highlighted inside a box. The competitive inhibitor, 5-fluoroorotic acid (5-
F0A), is shown in a
dashed box at the position where it enters the pathway. Abbreviated names for
molecules and
enzymes are indicated in parentheses, and the predicted corresponding P.
tricornutum gene names
are indicated in square brackets. (1B) Example image of T7EI editing assay to
screen exconjugants
for potential editing events in the PtUMPS gene. Substrate indicates PtUMPS
gene fragments
amplified by the PCR, while T7 product indicates exconjugants with evidence of
Cas9 editing.
WT, wild-type P. tricornutum genomic DNA used in the T7EI editing assay. M,
100 bp ladder
with sizes indicated in basepairs (bp).. (1C) Example of phenotypic screening
of one PtUMPS
knockout strain (AUMPS2) plated on Li alone or Li supplemented with uracil at
the indicated
dilution of initial concentration. (1D) Example of screening for loss of the
zeocin-resistant Cas9
editing plasmid in a AUMPS2 knockout strain by plating on Li supplemented with
uracil or Li
supplemented with uracil and zeocin. (1E) Sanger sequencing traces of
characterized PtUMPS
knockouts with the position (below trace) and type of insertion or deletion
(above trace) indicated
for each allele of the three strains. (1F) Graphical map of the position and
extent of indels for each
of the three PtUMPS knockouts relative to the wild-type UMPS gene (shown at
top). Black
rectangles indicate nucleotide deletions, light grey triangles indicate
nucleotide insertions, the
white and dark grey rectangles on the WT gene indicate the position of the
PtUMPS active sites
(orotate phosphoribosyl transferase and orotidine-5'-phosphate decarboxylase),
and the white
rectangles with dashed lines represent introns.
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Figs. 2A to 2E. CRISPR-generated knockouts in the predicted P. tricornutum
histidine
biosynthesis pathway. (2A) A portion of the predicted biosynthesis pathway for
conversion of
ribose-5-phosphate to L-histidine, with the bi-functional PtPRA-PH/CH enzyme
highlighted
inside a box. Abbreviated names for each enzyme are indicated in parentheses,
and the predicted
corresponding P. tricornutum gene names are indicated in square brackets. (2B)
Example image
of T7EI editing assay to screen exconjugants for potential editing events in
the PtPRA-PH/CH
gene. Substrate indicates PtPRA-PH/CH gene fragments amplified by the PCR,
while T7 product
indicates exconjugants with evidence of Cas9 editing. WT, wild-type P.
tricornutum genomic
DNA used in the T7EI editing assay. M, 1 kb ladder with sizes indicated in
basepairs (bp).. (2C)
Example of phenotypic screening of one PtPRA-PH/CH knockout strain
(APtPRAPHCH1)
transformed with or without the complementing PRA-PH/CH plasmid (pPtPRAPHCH)
on Li
solid media alone or Li supplemented with histidine at the indicated dilution
of initial
concentration. WT, wild-type P. tricornutum strain. (2D) Sanger sequencing
traces of
characterized PtPRA-PH/CH knockouts with the position (below trace) and type
of insertion or
deletion (above trace) indicated for each allele. (2E) Graphical map of the
position and extent of
indels for PtPRA-PH/CH knockout relative to the wild-type PtPRA-PH/CH gene
(shown at top).
Black rectangles indicate nucleotide deletions, while the white and dark grey
rectangles on the WT
gene indicated the position of the PRA-PH and PRA-CH active sites.
Figs. 3A to 3F. Large deletions in edited P. tricornutum metabolic genes
captured by Nanopore
amplicon sequencing. For each (3A-3F), the name of the target gene as well as
the editing enzyme
are indicated. The leftmost plot shows normalized read coverage averaged over
a 5-bp window for
the edited sample (black dots) and the wild-type sample (grey dots) relative
to the position in PCR
amplicon. Numbering on the x-axis is relative to the ATG start codon for each
gene, with sequence
upstream indicated by a minus (¨) symbol and sequence downstream indicated by
a plus (+)
symbol. The light grey vertical line indicates the Cas9 or TevCas9 cleavage
site, while the shaded
rectangle indicates the ORF. The middle plot is a density plot of deletions >
50-bp.
The rightmost plot shows the length and position of deletions > 50-bp relative
to their position in
the PCR amplicon, with numbering of the x-axis as in the leftmost panel. Each
horizontal line
indicates a mapped deletion event. Deletions are ordered from longest to
smallest. The light grey
vertical line indicates the Cas9 or TevCas9 cleavage site.
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Figs. 4A to 4D. Phenotypic and metabolomic characterization of PtUMPS
knockouts. (4A)
Spot plating assays of wild type (WT), AUMPS1 and AUMPS2 strains on Li solid
media alone,
Li supplemented with uracil, Li supplemented with 5-F0A, or Li supplemented
with both uracil
and 5-F0A. Indicated dilutions are relative to the initial concentration. (4B)
Liquid growth curves
of wild type (WT), AUMPS1 and AUMPS2 strains in Li liquid media alone, or
supplemented with
uracil or 5-FOA or both. Data points are the mean of three independent
replicates, with error bars
representing the standard error of the mean. (4C) Orotate concentrations were
measured by LC-
MS from cultures grown with and without uracil supplementation. Bars represent
mean values and
error bars represent standard deviation for three biological replicates.
Individual data points are
.. represented as colored dots. Statistical confidence level was calculated by
one-sided t test. p <
0.001 is indicated by an asterisk. (4D) Bar graph showing percent plasmid
retention in
the AUMPS1 and AUMPS2 strains harbouring various PtUMPS constructs after 14
days of
outgrowth. Bars represent the mean ratio of colonies on selective Li +
nourseothricin versus non-
selective Li plates from three independent replicates, with error bars
representing the standard
error of the mean.
Fig. 5. Predicted PtUMPS structure indicating the positions of the ODC (dark
grey) and
OPRT (light grey) domains. Regions containing conserved active site residues
for the ODC
domain are indicated in white and regions containing conserved active residues
for the OPRT
domain are indicated with small arrows. Residue substitutions that
differentiate the two alleles are
labeled with * (allele 1) and A (allele 2). Folding prediction was modeled
using the PHYRE2
Protein Fold Recognition Server.
Figs. 6A to 6D. Assaying AUMPS1 knockout and complemented growth rates in Li
media
supplemented with uracil, 5-F0A, or both. A biallelic PtUMPS mutant (AUMPS1)
is unable to
grow in Li media without uracil supplementation. Reintroducing the gene on a
stably replicating
plasmid restores the WT phenotype in Li media. (6A) Growth rates in Li media.
(6B) Growth
rates in Li media supplemented with uracil. (6C) Growth rates in Li media
supplemented with 5-
FOA. (6D) Growth rates in Li media supplemented with uracil and 5-F0A. WT,
Wild-type P.
tricornutum; AUMPS1, PtUMPS knockout strain 1; AUMP S1 -A 1 , AUMPS1
possessing
pPtUMPSAl; AUMPS1-A2, AUMPS1 possessing pPtUMP SA2; AUMPS1-cAl, AUMPS1
possessing pPtUMPScAl; AUMPS1-cA2, AUMPS1 possessing pPtUMPScA2. Points
represent
Date Recue/Date Received 2021-08-17

10
mean values and error bars represent standard deviation for three replicates.
Figs. 7A to 7D. Assaying AUMPS2 knockout and complemented growth rates in Li
media
supplemented with uracil, 5-F0A, or both. A biallelic PtUMPS mutant (AUMPS2)
is unable to
grow in Li media without uracil supplementation. Reintroducing the gene on a
stably replicating
plasmid restores the WT phenotype in Li media. (7A) Growth rates in Li media.
(7B) Growth
rates in Li media supplemented with uracil. (7C) Growth rates in Li media
supplemented with 5-
FOA. (7D) Growth rates in Li media supplemented with uracil and 5-F0A. WT,
Wild-type P.
tricornutum; AUMPS2, PtUMPS knockout strain 1; AUMPS2-Al, AUMPS2 possessing
pPtUMPSAl; AUMPS2-A2, AUMPS2 possessing pPtUMPSA2; AUMPS2-cAl, AUMPS2
possessing pPtUMPScAl; AUMPS2-cA2, AUMPS2 possessing pPtUMPScA2. Points
represent
mean values and error bars represent standard deviation for three replicates.
Fig. 8. Pre-cropped gel image of T7EI assay to detect editing in the PtUMPS
gene. The area
cropped and shown in Fig. 1B is indicated by a rectangle. Lanes are labeled as
in Fig 1B. M refers
to 100 bp standard ladder.
Fig. 9. Pre-cropped gel image of T7EI assay to detect editing in the PtPRA-
PH/CH gene. The area
cropped and shown in Fig. 2B is indicated by a rectangle. Lanes are labeled as
in Fig 2B. M refers
to 1 kb standard ladder.
DESCRIPTION OF THE INVENTION
Abbreviations
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as
commonly understood by one of ordinary skill in the art to which this
disclosure belongs. Also,
unless indicated otherwise, except within the claims, the use of "or" includes
"and" and vice versa.
Non-limiting terms are not to be construed as limiting unless expressly stated
or the context clearly
indicates otherwise (for example "including", "having" and "comprising"
typically indicate
"including without limitation"). Singular forms including in the claims such
as "a", "an" and "the"
include the plural reference unless expressly stated otherwise. "Consisting
essentially of' means
any recited elements are necessarily included, elements that would materially
affect the basic and
Date Recue/Date Received 2021-08-17

11
novel characteristics of the listed elements are excluded, and other elements
may optionally be
included. "Consisting of' means that all elements other than those listed are
excluded.
Embodiments defined by each of these terms are within the scope of this
disclosure.
All numerical designations, e.g., levels, amounts and concentrations,
including ranges, are
approximations that typically may be varied (+) or (-) by increments of 0.1,
1.0, or 10.0, as
appropriate. All numerical designations may be understood as preceded by the
term "about".
The term "peptide" as used herein is defined as a chain of amino acid
residues, usually having a
defined sequence. As used herein the term peptide is inclusive of the terms
"polypeptides" and
"proteins". The terms also encompass an amino acid polymer that has been
modified. The term
"peptide" includes, for example, proteins, enzymes, antibodies, glycoproteins
and so forth.
Amino acid 3-letter abbreviation 1-letter abbreviation
Alanine Ala A
Arginine Arg
Asparagine Asn
Aspartic acid Asp
Cysteine Cys
Glutamic acid Glu
Glutamine Gln
Glycine Gly
Histidine His
Isoleucine Ile
Leucine Leu
Lysine Lys
Methionine Met
Phenylalanine Phe
Date Recue/Date Received 2021-08-17

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Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
Overview
The marine pennate diatom Phaeodactylum tricornutum (P. tricornutum) is a
genetically tractable
organism with a small, simple genome, a defined liquid growth media with
requirement for light
and oxygen, and scalable bioreactor culturing to volumes exceeding 10,000 L.
The present
invention relates to inventive P. tricornutum auxotrophic knockouts in the
biosynthesis pathway
of an organic compound necessary for the growth of P. tricornutum, to methods
of generating said
P. tricornutum auxotrophic knockouts and to plasmid-based copies of intact
PtUMPS and PtPRA-
PH/CH genes complement the uracil- and histidine-requiring phenotypes,
respectively. Individual
auxotrophic strains of the present invention are characterized by loss of
heterozygosity at the edited
alleles, and large, heterogeneous deletions up to about 2.7 kb. The auxotrophs
and their respective
complementation markers represent alternative to antibiotic-based selection of
plasmids in P.
tricornutum. The present invention also provides for a simple methodology to
cure plasmids from
auxotrophs to enable strain and genome engineering.
In one embodiment, the present invention provides for an auxotrophic strain of
Phaeodactylum
tricornutum (P. tricornutum) cells, wherein the strain has loss of
heterozygosity at a gene
necessary for the synthesis of an organic compound necessary for the growth of
P. tricornutum.
In one embodiment of the auxotrophic strain of P. tricornutum of the present
invention, the strain
is complemented with an auxotrophic selection plasmid having an expression
cassette comprising
a constitutive promoter operably linked to an activated gene necessary for the
synthesis of the
organic compound. In aspects, the organic compound is histidine, uracil or
tryptophan.
In another embodiment, the present invention provides for a method for
producing auxotrophic
Date Recue/Date Received 2021-08-17

13
strains of Phaeodactylum tricornutum (P. tricornutum) cells, wherein the
strain has loss of
heterozygosity at a target gene necessary for the synthesis of one of an
organic compound
necessary for the growth of P. tricornutum. In one embodiment, the method
comprises: (a)
introducing an editing plasmid into a strain of P. tricornutum, the editing
plasmid comprising a
TevCas9 gene (TevCas9 plasmid) or a Cas9 gene (Cas9 plasmid) and a single-
guide RNA
(sgRNA) against a binding site of the target gene, to obtain P. tricornutum
exconjugants, (b)
screening the P. tricornutum exconjugants for colonies of P. tricornutum
edited with the TevCas9
plasmid or the Cas9 plasmid in the target gene to obtain P. tricornutum edited
colonies, (c)
screening the P. tricornutum edited colonies for target gene knockout P.
tricornutum strains by
growing the P. tricornutum edited colonies on solid media with and without
auxotrophic
supplement including the organic compound, wherein P. tricornutum edited
colonies that do not
grow without auxotrophic supplement and grow with auxotrophic supplement are
target gene
knockout P. tricornutum strains, and (d) screening for loss of the plasmid
from the target gene
knockout P. tricornutum strains thereby producing the auxotrophic strain of P.
tricornutum.
The P. tricornutum auxotrophs of the present invention have use as host cells
for the production
of recombinant peptides including proteins, antibodies, glycoproteins and so
forth. In aspects, the
P. tricornutum auxotrophs of the present invention carry one or more plasmids
having an
expression cassette comprising a constitutive promoter operably linked to a
gene encoding the
recombinant peptide. The gene encoding the recombinant peptide is a gene that
can be expressed
(including overexpressed) in the host P. tricornutum auxotrophs to produce the
recombinant
peptide. The recombinant peptide so produced, can then be purified and
isolated.
In one embodiment, the present invention provides for a method of producing a
recombinant
peptide comprising: (a) providing an auxotrophic strain of Phaeodactylum
tricornutum (P.
tricornutum) cells according to the present invention in which the editing
plasmid of the P
tricornutum comprises a DNA sequence encoding the recombinant peptide; (b)
culturing said
auxotrophic strain of P. tricornutum cells under conditions permitting
expression of the DNA
sequence encoding the recombinant peptide; and d) recovering said recombinant
peptide.
In one embodiment of the method of producing a recombinant peptide of the
present invention,
the recombinant peptide is an antibody, a protein, an enzyme or a
glycoprotein.
Date Recue/Date Received 2021-08-17

14
In one embodiment of the method of producing a recombinant peptide of the
present invention,
the editing plasmid further comprises a constitutive promoter operably linked
to the DNA sequence
for encoding the recombinant peptide.
In aspects, the P. tricornutum auxotrophs of the present invention can be used
to produce a cell
.. extract for use in an in vitro protein expression reaction. In one
embodiment, the method includes
(a) culturing P. tricornutum auxotrophs in which a gene that is necessary for
the biosynthesis an
organic compound required for its growth has been inactivated in a growth
media comprising said
organic compound, wherein the P. tricornutum auxotrophs comprises an
auxotrophic selection
plasmid having an expression cassette comprising a constitutive promoter
operably linked to a
gene necessary for the synthesis of said organic compound; and (c) preparing a
P. tricornutum
auxotrophs cell extract of the culture. In aspects of the invention, the
organic compound is
histidine, uracil or tryptophan.
EXAMPLES
METHODS
Microbial strains and growth conditions
Saccharomyces cerevisiae VL6-48 (ATCC MYA-3666: M4Tahis3-6200 trpl-M ura3-
52 1ys2 ade2-1 met14 0) was grown in rich medium (YPD) or complete minimal
medium lacking
histidine (Teknova) supplemented with 60 mg L-1 adenine sulfate. Complete
minimal media used
for spheroplast transformation contained 1 M sorbitol. Escherichia coli
(Epi300, Epicenter) was
grown in Luria Broth (LB) supplemented with appropriate antibiotics
(chloramphenicol 25
mg L-1 or kanamycin 50 mg L-1 or ampicillin 50 mg L-1 or gentamicin 20
mg L-1). Phaeodactylum tricornutum (Culture Collection of Algae and Protozoa
CCAP 1055/1)
was grown in Li medium without silica, with or without uracil (50 mg L-1) or
histidine (200
mg L-1) or 5-FOA (100 mg L-1), supplemented with appropriate antibiotics
zeocin (50 mg L-1) or
nourseothricin (100 mg L-1), at 18 C under cool white fluorescent lights (75
[LE m-2s-1) and a
photoperiod of 16 h light:8 h dark. Li media supplemented with nourseothricin
contained half the
normal amount of aquil salts. P. tricornutum auxotroph genotypes are as
follows. Mutations in
PtUMPS are described in reference to the chromosome 6 sequence (GenBank:
CM000609.1), and
mutations for PtPRA-PH/CH are in reference to the chromosome 3 sequence
(GenBank:
Date Recue/Date Received 2021-08-17

15
CP001142.1). Mutations described for each gene are listed for allele 1
followed by allele 2, and
numbered beginning from the first nucleotide of the start codon for
simplicity. Genotypes of
auxotroph strains generated in this study are listed in Table 5.
Transfer of DNA to P. tricornutum via conjugation from E. coil
Conjugations were performed as previously described [5'6]. Briefly, liquid
cultures (250 [IL) of P.
tricornutum were adjusted to a density of 1.0 x 108 cells mL-1 using counts
from a
hemocytometer, plated on 1/2 x Li 1% agar plates and grown for four days. Li
media (1.5 mL)
was added to the plate and cells were scraped and the concentration was
adjusted to
5.0 x108 cells mL-1. E. coil cultures (50 mL) were grown at 37 C to A600 of
0.8-1.0, centrifuged
for 10 min at 3,000xg and resuspended in 500 [IL of SOC media. Conjugation was
initiated by
mixing 200 [IL of P. tricornutum and 200 [IL of E. coil cells. The cell
mixture was plated on 1/2 x
Li 5% LB 1% agar plates, incubated for 90 min at 30 C in the dark, and then
moved to 18 C in
the light and grown for 2 days. After 2 days, Li media (1.5 mL) was added to
the plates, the cells
scraped, and 300 [IL (20%) plated on 1/2 x Li 1% agar plates supplemented with
zeocin 50
mg L-1 or nourseothricin 200 mg L-1. Colonies appeared after 7-14 days
incubation at 18 C with
light.
Plasmid design and construction
All plasmids (Table 6) were constructed using a modified yeast assembly
protocol [3940].
Plasmids pPtUMPSA1 and pPtUMPSA2 were made from pPtGE31 [P] by replacing the
URA3
element with a PCR fragment consisting of PtUMPS allele 1 or 2 with ¨ 1 kb up-
and down-stream
of the PtUMPS ORF amplified from P. tricornutum genomic DNA (oligonucleotides
are listed in
Table 7). Plasmids pPtUMPScAl and pPtUMPScA2 were made from pPtUMPSA1 and
pPtUMPSA2 by replacing the PtUMPS ORF with a PCR fragment consisting of PtUMPS
allele 1
or 2 amplified from P. tricornutum cDNA. Plasmid pPtUMPS40S was made from
pPtGE31 by
replacing the URA3 element with a cassette consisting of PCR fragments of the
40SRPS8
promoter and terminator [6] flanking a PCR fragment of the PtUMPS allele 1 ORF
amplified
from P. tricornutum genomic DNA. Plasmid pPtPRAPHCH was made from pPtGE31 [P]
by
replacing the URA3 element with a PCR fragment consisting of PtPRA-PH/CH with
¨ 1 kb up-
and downstream of the PtPRA-PH/CH ORF, amplified from P. tricornutum genomic
DNA. Using
Date Recue/Date Received 2021-08-17

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Golden Gate assembly, sgRNAs targeting different regions of the PtUMPS and
PtPRA-PH/CH
genes were cloned into the BsaI sites positioned between the P. tricornutum U6
promoter and
terminator in pPtGE34 and pPtGE35. Plasmid constructs were confirmed by Sanger
sequencing at
the London Regional Genomics Facility.
Generation of PtUMPS 9S and PtPRA-PH/CH knockouts using Cas9 and TevCas9
Plasmids pPtGE34 or pPtGE35, containing no guide RNA or sgRNA.UMPS.1944,
sgRNA.UMPS.1646, sgRNA.UMPS.157, sgRNA.UMPS.311 for the PtUMPS gene, or
sgRNA.PRAPHCH.929 or sgRNA.PRAPHCH.120 for the PtPRA-PH/CH gene, were
conjugated
from E. coli to P. tricornutum and exconjugants were selected on zeocin-
containing media,
.. supplemented with uracil or histidine as appropriate [41]. Ten colonies
from each conjugation were
resuspended in TE buffer and flash frozen at ¨ 80 C followed by heating at 95
C to lyse cells
and extract genomic DNA. The genomic target site of each sgRNA in P.
tricornutum was
amplified by PCR and the products were analyzed by T7EI assay as follows; PCR
products were
denatured at 95 C for 5 min, slowly cooled to 50 C, and flash frozen at ¨ 20
C for 2 min. PCR
products (250 ng) were incubated with 2U of T7EI (NEB) in 1 x NEBuffer 2 for
15 min at 37 C
and analyzed by agarose gel electrophoresis. Colonies that showed editing by
T7EI assay were
grown in liquid culture supplemented with zeocin and uracil or histidine as
appropriate for 2 weeks
and serial dilutions were plated on selective media with uracil or histidine
to isolate sub-clones.
Sub-clones were then screened for homozygous PtUMPS or PtPRA-PH/CH knockout
phenotypes
by replica streaking on minimal Li media and Li media supplemented with uracil
or histidine as
appropriate. Streaks were grown for 5 days before visual identification of
phenotypes. Sub-clones
that were identified as phenotypic knockouts were resuspended in TE buffer and
flash frozen at ¨
80 C followed by heating at 95 C to lyse cells and extract genomic DNA, then
sgRNA target
sites were PCR amplified. Sanger sequencing of PCR products was performed at
the London
Regional Genomics Facility to identify the type and length of indels
generated. Stable bi-allelic
PtUMPS or PtPRA-PH/CH knockout mutant lines were then grown in nonselective Li
media
supplemented with uracil or histidine for 1 week to cure them of plasmids
before plating to obtain
single colonies. Resulting colonies were replica streaked onto nonselective
and zeocin-containing
media supplemented with uracil or histidine to identify colonies which had
successfully been cured
.. of the plasmid.
Date Recue/Date Received 2021-08-17

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Spot plating P. tricornutum
Cultures of P. tricornutum were adjusted to 1 x106 cells mL-1 and serially
diluted 2 X three times.
For uracil auxotrophs, 10 [IL of each adjusted culture and dilutions were spot
plated onto minimal
Li media and Li media supplemented with uracil (50 mg L-1), 5-FOA (100 mg L-
1), or both. For
.. histidine auxotrophs, 10 [IL of each adjusted culture and dilutions were
spot plated onto minimal
Li media and Li media supplemented with histidine (200 mg L-1). Plates were
incubated at 18 C
under cool white fluorescent lights (75 pE m-2s-1) and a photoperiod of 16 h
light:8 h dark for 7-
days.
Measuring P. tricornutum growth rates
10 .. Growth was measured in a Multiskan Go microplate spectrophotometer.
Cultures of each strain
(WT, AUMPS1, AAUMPS1 + pPtUMPS40S, AUMPS1 + pPtUMPSA1, AUMPS1 +
pPtUMPSA2, AUMPS1 + pPtUMPScAl, AUMPS1 + pPtUMPScA2, AUMPS2 +
pPtUMP SA 1 , AUMPS2 + pPtUMP SA2, AUMPS2 + pPtUMP S cA 1 , AUMPS2 + pPtUMP S
cA2)
were adjusted to 5 x 105 cells mL-1 in Li media with and without supplemented
uracil (50 mg L-1),
.. 5-FOA (100 mg L-1), or both. Two hundred microliters of each adjusted
culture was added to three
wells (technical replicates) of a 96-well microplate. The 96-well microplates
were incubated at 18
C under cool white fluorescent lights (75 pE m-2s-1) and a photoperiod of 16 h
light:8 h dark for
10 days, and absorbance at 670 nm (A670) was measured every 24 h. The 96-well
microplates were
shaken briefly to resuspend any settled cells prior to absorbance
measurements. Note that
.. the A670 was not adjusted for path length and light scattering from the
microplate lid and is
therefore not directly comparable to optical density readings measured in a
standard cuvette.
P. tricornutum metabolite extraction
Cultures of P. tricornutum (Wild-type, AUMPS1, and AUMPS2) were grown with and
without
uracil supplementation and harvested during exponential phase as follows
(Note: The AUMPS1
and AUMPS2 cultures were first grown with uracil supplementation, then
switched to minimal Li
media for 1 week prior to harvesting). Cultures (-1 x 109 cells) were pelleted
by centrifugation at
4000xg for 10 min and washed by resuspending in fresh Li media. Cells were
pelleted again,
resuspended in a small volume (¨ 5 mL) of Li media, and transferred to a clean
10 mL syringe
(without needle) with the exit plugged by parafilm. The syringe was placed,
tip-down, into a clean
Date Recue/Date Received 2021-08-17

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50 mL falcon tube and the cells were pelleted as above. The supernatant was
removed and the
pellet was slowly ejected from the syringe into a pre-chilled mortar
containing liquid nitrogen. The
frozen cells were ground to a fine powder and then transferred to a clean pre-
weighed 1.5 mL
Eppendorf tube, suspended half way in liquid nitrogen. Being careful to keep
samples frozen, 50
mg of frozen ground powder was weighed out into a new clean 1.5 mL Eppendorf
tube, pre-cooled
in liquid nitrogen, and 250 uL of cold extraction buffer with internal
standard (IS) (80% methanol
in MilliQ water, 125 jtM 15N2¨uracil) was added. The IS was added to the
samples to compensate
for losses that might occur during preparation of the samples and loss of
sensitivity attributable to
quenching of the signal by coeluting compounds. Samples were then homogenized
by vigorous
vortexing for 30 s in 10 second intervals, between which samples are kept on
ice for ¨ 30 s.
Homogenized samples were then spun down at 4 C for 10 min at 20,000 xg. The
supernatant was
transferred to a new clean 1.5 mL Eppendorf tube and spun down at 20,000xg for
5 min at 4 C.
The supernatant was again transferred to a new clean 1.5 mL Eppendorf tube and
kept at 4 C
overnight prior to LC¨MS analysis.
Chromatographic separation and mass spectrometry
Metabolites were separated at 45 C on a Waters Acquity HSS T3 column [2.1 x
100 mm, 1.8 pm]
in a Waters ACQUITY UPLC I-Class system (Waters, Milford, MA). Solvent A
consisted of water
and solvent B consisted of methanol, both containing 0.1% formic acid. Elution
was performed by
use of a linear gradient, at a flow rate of 0.3 mL/min, as follows: 0-2 min,
100% solvent A to 90%
solvent B; 2.01 min, 100% solvent A to recondition the column. A Waters Xevo
G2-S quadrupole
time of flight mass spectrometer was operated in negative electrospray
ionization (ESI) in
resolution mode. The capillary voltage was set to 1.0 kV, the source
temperature was 150 C,
desolvation temperature was 600 C, the cone gas was 50 L/h and the
desolvation gas was 1000
L/h. Leucine enkephalin was infused as the lock mass with a scan time of 0.3
seconds every 10 s,
and three scans were averaged. Linearity and detection limits for each
compound were established
by injection of calibration mixtures with different concentrations (0, 1, 2,
4, 8, 16, 31.25, 62.5,
125, 250, and 500 jtmol/L). Stable-isotope-labeled uracil (15N2¨uracil) was
used as the IS. The
concentration of each analyte was determined by use of the slope and intercept
of the calibration
curve that was obtained from a least-squares regression for the analyte/IS
peak-area ratio vs the
concentration of the analyte in the calibration mixture.
Date Recue/Date Received 2021-08-17

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P. tricornutum DNA extraction and targeted long-read sequencing
Plasmids pPtGE34 or pPtGE35, containing sgRNAs targeting the PtUMPS, PtUrease,
or PtI3GPS-
PRAT gene were conjugated from E. coil to P. tricornutum and exconjugants were
selected on
zeocin-containing media, supplemented with uracil or tryptophan (100 mg L-1)
as appropriate. For
each transformation, colonies (¨ 1,000) were scraped and pooled in liquid Li
media and genomic
DNA was extracted using a modified akaline lysis protocol as follows: Cells
were pelleted at
4,000xg for 5 min, and resuspended in 250 pL resuspension buffer consisting of
235 pL P1
(Qiagen), 5 pL hemicellulose 100 mg mL-1, 5 pL of lysozyme 25 mg mL-1, and 5
pL zymolyase
solution (200 mg zymolyase 20 T (USB), 9 mL H20, 1 mL 1 M Tris pH 7.5, 10 mL
50% glycerol)
and incubated at 37 C for 30 min. Next, 250 pL of lysis buffer P2 (Qiagen) was
added, followed
by 250 pL of neutralization buffer P3 (Qiagen) and centrifugation at 16,000xg
for 10 min. The
supernatant was transferred to a clean tube, 750 pL isopropanol was added, and
the samples
centrifuged at 16,000xg for 10 min. A 70% Et0H wash was performed, centrifuged
at
16,000xg for 5 min, and pellets briefly dried, resuspended in 50-100 pL of TE
buffer, and
incubated at 37 C for 30-60 min.
The sgRNA target site regions were PCR amplified from sgRNA transformant
genomic DNA
samples, as well as a wild-type sample, with PrimeStar GXL polymerase (Takara)
using primers
positioned ¨ 3 kb up- and downstream of the target site (Table 7). PCR
products were purified
and DNA libraries were prepared, barcoded, and pooled using an Oxford Nanopore
Ligation
Sequencing Kit (SQK L5K109) and Native Barcoding Expansion 1-12 (EXP-NBD104)
kit
according to manufacturer's protocol with the following modification¨all
reactions were scaled
down to half the recommended volume and the end prep incubation times were
extended to 15 min
at 20 C and 15 min at 65 C. The pooled library was then loaded on to a
MinION R9.4.1 flowcell
and sequenced.
Targeted long-read sequencing analysis
After sequencing on an R9.4.1 flowcell, base calling was performed using GPU
Guppy with the
high accuracy configuration file version 3.4.4
(https://community.nanoporetech.com). Reads in
each barcode were filtered using NanoFilt [42] for a minimum average read
quality score of 10
and a minimum read length of 2,000, mapped using minimap2 [43] and filtered
for reads that map
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to within 100 bases of each end of the reference sequence (the unedited 6 kb
PCR product
sequence) to remove short fragments. The filtered reads were mapped using
minimap2
(parameters: -ax map-ont) and outputted in sam format, then converted to bam,
sorted, and indexed
using samtools 1441. The per-base coverage depth for each barcode was
calculated using Mosdepth
[45]. All plots were created in R using the ggp1ot2 package [46].
RESULTS
Identification of Cas9 targets in biosynthetic pathway genes
We examined the KEGG predictions [2324] based on the genome sequence of P.
tricornutum to
identify genes in the uracil and histidine biosynthetic pathways for Cas9
editing. We focused on
these two pathways as uracil and histidine auxotrophy, and counter-selection
strategies are
commonly used in other model organisms. This approach identified the bi-
functional PtUMPS
gene that is predicted to catalyze two steps in the uracil pathway¨conversion
of orotate to
orotidine monophosphate (OMP), and conversion of OMP to uridine monophosphate
(UMP)
(Fig. 1A) [22]. Proteins that are orthologs of characterized enzymes involved
in histidine
biosynthesis were also identified (Fig. 2A). The PHATR 3140 gene, hereafter
called PtPRA-
PH/CH, encodes a predicted bifunctional protein that shares sequence
similarity with the bacterial
protein HisIE, and its plant counterpart HISN 2 [2526]. These proteins possess
two functional
domains that are homologous to the phosphoribosyl-ATP pyrophosphohydrolase
(PRA-PH) and
phosphoribosyl-AMP cyclohydrolase (PRA-CH) enzymes, respectively. PRA-PH and
PRA-CH,
alone or as a bifunctional protein, are predicted to catalyze two successive
steps that occur early
in the histidine biosynthesis pathway (Fig. 2A). The PtIGPS gene encoding
imidazole glycerol
phosphate synthase (a HIS3 homolog) was found to be a duplicated gene in the
P.
tricornutum genome assembly and thus not prioritized as a Cas9 target.
We also identified the PtI3GPS-PRAI gene as a potential target as it encodes a
predicted bi-
functional enzyme that is a fusion of indole-3-glycerol-phosphate synthase
(I3GPS) and
phosphoribosylanthranilate isomerase (PRAT), and would catalyze two successive
steps in the
tryptophan biosynthesis pathway O.
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21
To confirm the genomic target sites, we PCR-amplified and sequenced the PtUMPS
and PtPRA-
PH/CH genes of the P. tricornutum CCAP 1055/1 strain used in our laboratory.
Two distinct
alleles for both the PtUMPS and PtPRA-PH/CH genes were identified. Seven
single-nucleotide
polymorphisms (SNPs) in the PtUMPS alleles result in amino acid substitutions
that differentiate
the two alleles from each other and from the published P. tricornutum genome
(Table 2). All
substitutions are located in non-conserved regions of the PtUMPS protein (Fig.
5). Similarly, an
A to G mutation at base position 1205 in allele 2 of the PtPRA-PH/CH gene was
identified (Table
2). This transversion converts a highly conserved glutamate to a glycine in
the catalytic site of the
PRA-PH domain. The impact of these substitutions on PtUMPS and PtPRA-PH/CH
function is
unknown.
Cas9 and TevCas9 editing of auxotrophic genes is characterized by loss of
heterozygosity
To generate knockouts in uracil and histidine biosynthetic genes, we designed
and individually
cloned Cas9 and TevCas9 single guide RNAs (sgRNAs) against different sites in
the PtUMPS,
PtPRA-PH/CH, and PtI3GPS-PRAI genes (Table 1). The TevCas9 nuclease is a dual
nuclease that
generates a 33-38 base pair deletion between the I-TevI (Tev) and Cas9 cut
sites [27]. The
targeting requirements for a TevCas9 nuclease are an I-TevI 5'-CNNNG-3'
cleavage motif
positioned ¨15-18 base pairs upstream of the 5' end of the sgRNA binding site.
The Cas9 or
TevCas9 editing plasmids were moved into P. tricornutum by bacterial
conjugation and
exconjugants selected on zeocin-containing media.
Table 1 ¨ Summary of sgRNAs used for Cas9 and TevCas9 editing.
Target Platform Guide RNA Exconjugants edited / Number
of
total screened (T7E1) subclones
with
auxotroph
phenotype
screened
sgRNA.UMP S .1944 0/10 N/A
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22
Target Platform Guide RNA Exconjugants edited / Number
of
total screened (T7E1) subclones
with
auxotroph
phenotype
screened
!...01111 1111.
sgRNA.UMPS.1646 0/10 N/A
Cas9
sgRNA.UMPS.157 0/10 N/A
UMPS
sgRNA.UMPS.311 4/10 1/35
sgRNA.UMPS.1944 0/10 N/A
TevCas9 sgRNA.UMPS.1646 4/10 2/35
sgRNA.UMPS.157 0/10 N/A
sgRNA.PRAPHCH.9
2/6 1/28
29
sgRNA.PRAPHCH.1
0/6 N/A
Cas9 20
sgRNA.PRAPHCH.1
1/6 0/28
000
PRA-
PH/CH ___________
sgRNA.PRAPHCH.9
3/6 0/28
29
TevCas9 ______________________________________________________
sgRNA.PRAPHCH.1
0/6 N/A
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23
Target Platform Guide RNA Exconjugants edited / Number
of
total screened (T7E1) subclones
with
auxotroph
phenotype
screened
sgRNA.IGPSPRAI.2
Cas9 0/10 N/A
44
I3GPS-
PRAI
TevCas9 sgRNA.IGPSPRAI.2
3/10 1/35
44
We first assessed editing by screening P. tricornutum exconjugants by T7
endonuclease I (T7EI)
mismatch cleavage assays on PCR products amplified from each target gene
(Figs. 1B, 2B,
Table 1). This assay identified 6 sgRNAs with detectable editing rates based
on screening of
exconjugants. Colonies that showed editing were diluted, plated to obtain
subclones, and
subsequently screened for the corresponding auxotrophic phenotype on solid
media with and
without auxotrophic supplement (uracil or histidine) (Figs. 1C, 2C). To cure
the Cas9-editing
plasmids, knockout strains were grown without zeocin selection for 1 week, and
dilutions were
plated to obtain single colonies. Colonies were streaked onto Li plates with
and without zeocin to
screen for plasmid loss. A representative image demonstrating zeocin
sensitivity due to loss of the
Cas9-editing plasmid is shown in Fig. 1D. For knockout of the PtUMPS gene, we
further
characterized 3 subclones with a uracil-requiring phenotype to determine if
the knockouts were
monoallelic or biallelic. Because the two PtUMPS alleles of P. tricornutum
possessed SNPs
relative to each other, we were able to map allele-specific editing events
(Figs. 1E, 1F). Two of
the strains, AUMPS1 and AUMPS2, were biallelic and exhibited loss of
heterozygosity with one
allele possessing a small deletion (<20 bps) and the other allele possessing a
large deletion (> 610
bp). The third characterized subclone, AUMPS3, was monoallelic and possessed a
homozygous 1-
bp insertion. For the PtPRA-PH/CH knockouts that generated a histidine-
requiring phenotype
(Fig. 2B,C), targeted sequencing of one subclone revealed a biallelic genotype
with an 11-bp
deletion in one allele and a 6-bp deletion in the second allele (Figs. 2D,
2E).
The types of deletions observed in the uracil- and histidine-auxotrophs are
consistent with
Date Recue/Date Received 2021-08-17

24
heterogeneous editing events resulting in loss of heterozygosity [282930]. To
extend these
observations, we used Nanopore sequencing to better assess the spectrum of
large deletions that
are often overlooked in Cas9-editing studies. In addition to the two sgRNAs
that showed robust
editing on the PtUMPS gene, we examined deletion events in exconjugants with
sgRNAs targeted
to the PtUREASE gene [6] and the PtI3GPS-PRAI gene. For each experiment, ¨
1,000
exconjugants were pooled and a ¨ 6 kb PCR product generated for each of the
target genes with
the predicted Cas9 or TevCas9 target sites in the middle of the amplicon. We
focused our attention
on deletions > 50 bp as these deletions are typically under-reported in
targeted amplicon
sequencing. We noted a drop in Nanopore read coverage centered around the
predicted sgRNA
target sites for products amplified from Cas9 and TevCas9 editing experiments
(black dots) as
compared to read coverage for control experiments (orange dots), consistent
with editing at those
sites (Fig. 3, left panels). Mapping the deletion start and end points
revealed that most deletions
were centered on the Cas9 or TevCas9 target site (Fig. 3, right panels), with
deletions extending
up to 2700 bp (Fig. 3, centre panel). The mean deletion length for editing
events examined by
Nanopore sequencing and > 50 bp was 1735 719 bp for Cas9 and 2006 633 bp for
TevCas9.
Collectively, these data shows that Cas9 or TevCas9 editing of biosynthetic
genes can readily
generate P. tricornutum auxotrophs that can be identified by phenotypic or
genetic screens.
Moreover, our data agree with a growing body of evidence revealing that Cas9
editing (and
TevCas9 editing here) generates large deletions that would typically be missed
unless screening
strategies are explicitly designed to look for loss of heterozygosity.
Phenotypic and metabolomic characterization of the PtUMPS knockouts
Two uracil-requiring auxotrophs (AUMPS1 and AUMPS2) were selected for further
characterization by first spot plating onto Li media with and without uracil
and 5-FOA (Fig. 4A).
The PtUMPS knockout strains were only able to survive in the presence of
uracil supplementation.
Additionally, the knockouts survived on 5-FOA concentrations that fully
inhibited the growth of
wild-type P. tricornutum (Fig. 4A). This is consistent with phenotypes
previously observed for P.
tricornutum UMPS knockouts [2'22]. There was a slight growth advantage of
AUMPS1
over AUMPS2 on media supplemented with both 5-FOA and uracil, but not on media
containing
uracil alone. To compare if the observed phenotypes were consistent across
solid and liquid media,
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25
we monitored the growth of these strains over 10 days in liquid media (Fig.
4B) and found that the
growth rates were consistent with those observed on solid media, with one
notable difference
(Supplementary Fig. 6 and Fig. 7, Table 4). The growth advantage of AUMPS1
over AUMPS2
observed on solid media supplemented with both 5-FOA and uracil was not
replicated in liquid
media as the generation times for AUMPS1 and AUMPS2 were very similar (-24 and
¨ 22 h,
respectively).
To investigate the impact of PtUMPS knockouts on uracil metabolism, we
performed targeted
metabolomics on the UMPS substrate orotate using LC¨MS in wild-type and
knockouts strains
(Fig. 4C). We focused on characterizing the orotate intermediate in the uracil
pathway
(Figs. 2A, 4C) predicting that there should be an increase of orotate in
knockout strains relative to
wild type. We were unable to detect orotate in the AUMPS1 strain in the
absence of uracil
supplementation (-uracil), or in the AUMPS2 strain in either the -uracil or
+uracil condition.
A ¨ sixfold increase of cellular orotate levels was observed in the wild-type
strain when Li media
was supplemented with uracil (+uracil) as compared to minimal Li media (-
uracil) (Fig. 4C).
Interestingly, when the AUMPS1 strain was grown with uracil supplementation we
detected
orotate at levels similar to those observed in the wild-type strain grown with
uracil. This result
suggests that allele 1 in the AUMPS1 knockout strain (with an 18-bp in-frame
deletion) retains
UMPS activity that behaves similarly to the wild-type strain. In contrast, the
AUMPS2 strain has
two out-of-frame deletions that likely abolish ODC and OPRT activity. We
speculate that
undetectable levels of orotate in the AUMPS2 strain may be because it is
diverted to another
biosynthetic pathway.
Plasmid complementation of the uracil and histidine auxotrophs
Plasmid-based complementation of P. tricornutum auxotrophs would validate that
the Cas9-
editing event was the cause of the auxotrophic phenotype, as well as providing
alternatives to
antibiotic-based selection methods to maintain episomal vectors. We first
examined
complementation of the uracil-requiring phenotype by cloning both gDNA and
cDNA versions of
each PtUMPS allele with the native promoter and terminator into the
nourseothricin-resistant
pPtGE31 expression plasmid [6]. These plasmids were designated pPtUMPSA1,
pPtUMPSA2,
pPtUMPScAl, and pPtUMPScA2 (Table 4) and moved into the AUMPS1 and AUMPS2
strains
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26
via conjugation. Exconjugants were spot-plated onto solid Li media with and
without uracil and
5-FOA supplementation (Fig. 4A). All complemented strains grew on minimal Li
media, while
the uncomplemented knockouts did not, confirming expression of the UMPS gene
from the
pPtGE31 plasmid. No strain grew on 5-FOA alone. Unexpectedly, some of the
complemented
strains survived on plates supplemented with both 5-FOA and uracil. For
example, when AUMPS2
was transformed with either of the allele 1 complementation plasmids
(pPtUMPSA1 and
pPtUMPScAl), clear resistance to 5-FOA in the presence of uracil was observed.
The phenotypes
observed on solid media were consistent with those observed when the strains
were grown in liquid
media with similar media supplementation (Fig. 6 and Fig. 7).
The growth phenotype of the AUMPS1 and AUMPS2 strains in media supplemented
with uracil
and 5-FOA could be explained by counter-selection against the plasmid carrying
an intact PtUMPS
gene that would metabolize 5-FOA to a toxic intermediate. We thus tested for
plasmid loss in the
complemented strains by plating the AUMPS1 and AUMPS2 strains carrying
different expression
plasmids on solid Li with and without nourseothricin after 14 days of growth.
As shown in
Fig. 4D, plasmid retention, as measured by the ratio of colonies on Li plus
nourseothricin versus
Li plates, was severely reduced in all strains, ranging from ¨ 1 to ¨ 33%.
This observation could
explain why colonies readily appeared on Li media supplemented with 5-FOA and
uracil and
suggest that curing of plasmids carrying the PtUMPS gene from PtUMPS knockout
strains is a
simple matter of growth on the appropriate media.
Similarly, we were able to complement the histidine-requiring phenotype by
cloning a wild-type
copy of the PtPRA-PH/CH gene into an expression vector, and transforming the
plasmid into
the APRAPHCH1 strain by conjugation. The APRAPHCH1 strain with the
complementing
plasmid grew on both solid Li media with and without histidine
supplementation, whereas
the APRAPHCH1 strain without the complementing plasmid only grew on Li media
with histidine
supplementation (Fig. 2C).
Discussion
The available tools for genetic manipulation of P. tricornutum and other
diatoms have grown
substantially in recent years, including the adaptation of TALEN and Cas9
genome-editing
nucleases for targeted knockouts as well as plasmid-based and DNA-free methods
to deliver the
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27
nucleases to cells [6'7'8'9'11'31'32]. Applications of genome-editing
nucleases in P. tricornutum at
this point have mostly been to generate gene knockouts, with a few examples of
reporter construct
knockins. Generation of gene knockouts by Cas9 or other editing enzymes relies
on non-
homologous end-joining repair pathways [33], homologs of which are predicted
to occur in the P.
tricornutum genome [23'24]. A recent study used antisense RNA to knockdown a
predicted DNA
ligase IV homolog (ligIV) in P. tricornutum resulting in an increased rate of
homologous
recombination of a reporter construct [34]. What is not yet known for P.
tricornutum is the balance
between NHEJ and homology directed repair (HDR) pathways that process
endonuclease-
introduced double-strand breaks (see for example [35]). Examination of Cas9 or
TALEN-edited
sites in P. tricornutum revealed small nucleotide insertions or deletions
localized near the editing
site that are consistent with NHEJ repair events. It is becoming increasingly
apparent that repair
of Cas9-edited sites result in heterogenous alleles often characterized by
both small and large
deletions (for example, Fig. 1D) [6'8'36]. Repair events leading to large
indels are often missed by
experimental strategies that examine repair outcomes localized around the
editing site. In contrast,
large deletions visible by long-read sequencing methodologies and our Nanopore
data indicate that
Cas9 and TevCas9 editing events result in deletions up to ¨ 2.7 kb in length.
Cas9 editing with a
single sgRNA in P. tricornutum could achieve the same goal as the paired Cas9
nickase strategy
to specifically introduce large deletions [37], and may be complementary with
recently developed
methods to multiplex sgRNAs on Cas9-editing plasmids for P. tricornutum [10].
Regardless, a
better understanding of DNA repair pathways that operate on Cas9-introduced
double-strand
breaks will better inform strategies to bias repair events depending on the
experimental goal.
The creation of auxotrophic strains of P. tricornutum with plasmid based
rather than
chromosomally integrated complementation markers is critical for a number of
reasons.
Auxotrophic strains expand the available selection schemes beyond traditional
antibiotic markers
and provide a facile method for strain cataloging and validation. Antibiotic-
free selection is also
an advantage when P. tricornutum is used for production of human therapeutics.
In the case of
uracil auxotrophs, complementing plasmids can be cured (or counter selected)
by simple inclusion
of 5-FOA and uracil in the growth media. We have previously shown that
plasmids are lost from P.
tricornutum by passaging cultures over multiple days in the absence of
antibiotic selection required
for maintenance of the plasmid [38]. However, the counter selection method by
5-FOA and uracil
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28
supplementation is more rapid and requires screening significantly fewer
colonies to confirm
plasmid loss. The ability to rapidly cure plasmids will be of tremendous value
to prevent prolonged
expression of Cas9 and possible toxicity issues during strain engineering, to
cure incompatible
plasmids, or to cure reporter or expression plasmids under distinct growth
conditions. We also
.. envision that rapid curing of plasmids would allow recycling of a limited
number of selection
markers for serial transformations needed for strain construction or genomic
engineering.
Table 2 - Wild-type P. tricornutum UMPS sequence analysis. Base positions are
numbered
relative to the first base of the start codon in the genomic PtUMPS sequence.
Underlined SNPs
were not present in the reference genome. SNPs located in intronic sequences
were not included
.. in this table
Base Reference Allele 1 Allele 2 Allele 1 Allele
2
Position Genome Base Base Residue Residue
102 A/G A G A34 A34
127 G/A G A E43 K43
237 TIC T C D79 D79
246 G/A G A M82 182
519 G G A V147 1147
542 G/T G T L154 L154
591 C/A C A R171 S171
599 T/C T C N173 N173
938 G/A G A M286 1286
1003 A/T A T Q308 L308
1048 C/T C T T323 1323
1930 T/A T A 1510 1510
Table 3 ¨ Wild-type P. tricornutum PRA-PH/CH sequence analysis. Base positions
are relative to
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29
the first base of the start codon in the genomic PtPRA-PH/CH sequence.
Underlined SNPs were
not present in the reference genome.
Base Reference Allele 1 Allele 2 Allele 1 Allele
2
Position Genome Base Base Residue Residue
113 T/A T A F38 Y38
203 A/C A C Q68 P68
268 C/T C T L90 F90
270 C/T C T L90 F90
318 T/C T C V106 V106
319 T/C T C L107 L107
354 C/T C T C118 C118
406 A/G A G T136 A136
423 T/G T G F141 L141
623 A/T A T Q208 L208
732 G/A G A R244 R244
817 A/G A G T273 A273
825 C/T C T V275 V275
892 C/T C T L298 L298
978 T/G T G L326 L326
1205 A/G A G E402 G402
1306 G/A G A V436 1436
1323 C/T C T A441 A441
1350 G/A G A R450 R450
Table 4 ¨ Growth rates of WT and UMPS complement strains in minimal Li media.
Generation
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30
times represent the mean value standard deviation for three replicates.
P. tricornutum strain generation time (hours)
WT 29.8 2.6
AUMPS2 + pPtUMPSA1 27.9 2.8
AUMPS2 + pPtUMPSA2 24.8 1.5
AUMPS2 + pPtUMPScAl 28.5 0.9
AUMPS2 + pPtUMPScA2 23.4 0.8
AUMPS1 + pPtUMP SA1 26.5 2.7
AUMPS1 + pPtUMPSA2 29.8 0.7
AUMPS1 + pPtUMPScAl 26.5 4.0
AUMPS1 + pPtUMPScA2 30.7 1.2
Table 5 ¨ List of P. tricornutum auxotroph genotypes.
Strain Name Genotype Description
AUMPS1
PtUMPSg.[1637 1654del];[1050 1661del] P. tricornutum uracil auxotroph
strain 1
AUMPS2 PtUMPSg.[1636 1652del]; [1636 2415delinsT] P. tricornutum
uracil auxotroph strain 2
AUMPS3 PtUMPSg.[309 310insA]; [309 310insA]
P. tricornutum uracil auxotroph strain 3
APRAPHCH1 PtPRA-PH/CHg.[924 934del]; [924 929de1] P. tricornutum histidine
auxotroph strain 1
Table 6 ¨ List of plasmids used in this Example
Plasmid Description
Reference or Source
pPtGE31 P. tricornutum expression vector Slattery, et
al., 2018
pPtGE34 P. tricornutum expression vector, 40SRPS8 promoter and
terminator Slattery, et al., 2018
driving Sh ble, FcpB promoter and FcpA terminator driving Cas9
pPtGE35 P. tricornutum expression vector, 40SRPS8 promoter and
terminator Slattery, et al., 2018
driving Sh ble, FcpB promoter and FcpA terminator driving TevCas9
Date Recue/Date Received 2021-08-17

31
pPtUMPSA1
pPtGE31 encoding PtUMPS allele 1 driven by the PtUMPS promoter This disclosure
and terminator
pPtUMPSA2 pPtGE31 encoding PtUMPS allele 2 driven by the PtUMPS promoter
This disclosure
and terminator
pPtUMPScAl pPtGE31 encoding PtUMPS allele 1 cDNA driven by the PtUMPS
This disclosure
promoter and terminator
pPtUMPScA2 pPtGE31 encoding PtUMPS allele 2 cDNA driven by the PtUMPS
This disclosure
promoter and terminator
pPtPRAPHCH pPtGE31 encoding PtPRA-PH/CH allele 1 driven by the
This disclosure
PtPRA-PH/CH promoter and terminator
pTA-Mob Mobilization helper plasmid required for conjugation
Strand, et al., 2014
Table 7 ¨ List of oligonucleotides used in this Example
Name Sequence (5' to 3') (Priming sequence for PCR) Description
DE3644 tcgaATTAAGTATCGAAACGAATA Top strand sgRNA.UMPS.1944 for PtUMPS
DE3645 aaacTATTCGTTTCGATACTTAAT Bottom strand sgRNA.UMPS.1944
for
PtUMPS
DE3646 tcgaTAAATTGGTCGGGACTTCGT Top strand sgRNA.UMPS.1646 for
PtUMPS
DE3647 aaacACGAAGTCCCGACCAATTTA Bottom strand sgRNA.UMPS.1646 for
PtUMPS
DE3648 tcgaCGACCAACGTTTTGCAAA Top strand sgRNA.UMPS.157 for
PtUMPS
DE3649 aaacTTTGCAAAACGTTGGTCG Bottom strand sgRNA.UMPS.157 for
PtUMPS
DE4236 tcgagATTTTGTTGGATGTCAAGCG Top strand sgRNA.UMPS.311 for PtUMPS
DE4237 aaacCGCTTGACATCCAACAAAATc Bottom strand sgRNA.UMPS.311 for
PtUMPS
DE4162 tcgagCTACTACAGCCGTTCCCGGAA Top strand sgRNA.PRAPHCH.929 for
PtPRA-PH/CH
DE4163 aaacTTCCGGGAACGGCTGTAGTAGc Bottom strand sgRNA.PRAPHCH.929 for
PtPRA-PH/CH
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32
DE4164 tcgagTCGCGAAAGTCAGCGGCCCC Top strand sgRNA.PRAPHCH.120 for
PtPRA-PH/CH
DE4165 aaacGGGGCCGCTGACTTTCGCGAc Bottom strand sgRNA.PRAPHCH.120 for
PtPRA-PH/CH
DE4166 tcgagGGCTCGACAAAGACTGTGAC Top strand sgRNA.PRAPHCH.1000 for
PtPRA-PH/CH
DE4167 aaacGTCACAGTCTTTGTCGAGCCc Bottom strand sgRNA.PRAPHCH.1000 for
PtPRA-PH/CH
DE3650 tcgaGGACTCGGACAACCCCGATT Top strand sgRNA.IGPSPRAI.244
for
PtI3GPS-PRAI
DE3651 aaacAATCGGGGTTGTCCGAGTCC Bottom strand sgRNA.IGPSPRAI.244
for
PtI3GPS-PRAI
DE3728 GGCCTGGCAGCTTTATCAGTAG Rev screening primer for
sgRNA.UMPS.1944 site (pair with DE3646)
DE3726 GGTCAACACCAATTCGCTG Fwd screening primer for
sgRNA.UMPS.1646 site
DE3727 GAAGACTGACAATTGACACGACC Rev screening primer for
sgRNA.UMPS.1646 site
DE3724 GACGAAGTGTCTACTCACAGACAGC Fwd screening primer for sgRNA.UMPS.157
and sgRNA.UMPS.311 sites
DE3725 TCCCATCAGTGGTGAAAGCG Rev screening primer for
sgRNA.UMPS.157
and sgRNA.UMPS.311 sites
DE4176 ACGCCGCCATGGTTGGTC Fwd sgRNA.PRAPHCH.929 screening
primer
DE4177 CTCGTCTTGAAACAGACGTTTCGTG Rev sgRNA.PRAPHCH.929 screening
primer
DE4174 CCGAGTGATACTGTTCGCTTCG Fwd sgRNA.PRAPHCH.120 screening
primer
DE4175 GGAGTCCACTCGCGAGAC Rev sgRNA.PRAPHCH.120 screening
primer
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33
DE4178 GACCGCCTCGCAACTCGG Fwd sgRNA.PRAPHCH.1000 screening
primer
DE4179 CCACGTGTGCCTTCGTATCG Rev sgRNA.PRAPHCH.1000 screening
primer
DE4553 AGCAGGGTTATGCAGCGGAAGATCTATATTAC
CCTGTTATAAAGGCGGCGAATACTTCAT Fwd PtUMPS promoter with pPtGE31
homology
DE4552 TGCAGTCACTCCGCTTTGGTTTCGTAACTATA
ACGGTCTCGATGACTGACAAAGGTATTC Rev PtUMPS terminator with
pPtGE31 homology
DE4622 TGCAGTCACTCCGCTTTGGTTTCGTAACTAT
AACGGTCCTCGCGTTTTACGCGCCGTC Fwd PtPRA-PH/CH promoter with
pPtGE31 homology
DE4623 AGCAGGGTTATGCAGCGGAAGATCTATATT
ACCCTGTTATGCCCGCCTGATGCGTCG Rev PtPRA-PH/CH terminator with
pPtGE31 homology
DE5178 TCCTTTTCCAGTGCTTCGAT Fwd primer to amplify ¨6kb
around
sgRNA.IGPSPRAI.244 site
DE5179 TACCTGCTGCATCAGCTTTG Rev primer to amplify ¨6kb
around
sgRNA.IGPSPRAI.244 site
DE5180 TGGGTGTTGTGCTCTGCTAC Fwd primer to amplify ¨6kb
around
sgRNA.UMPS.1944 site
DE5181 TCTCCAAAGCCCAATTTTTG Rev primer to amplify ¨6kb
around
sgRNA.UMPS.1944 site
DE5182 ACTCAGCGTCACCTCCACTT Fwd primer to amplify ¨6kb around
sgRNA.UMPS.311 site
DE5183 GAATGCACCGATCACAACTG Rev primer to amplify ¨6kb
around
sgRNA.UMPS.311 site
DE5184 TCGAGGGAAGAGGCTAGACA Fwd primer to amplify ¨6kb
around
PtUrease sgRNA.UREASE.1187 site
DE5185 TTCCGTTGCATTGATGTTGT Rev primer to amplify ¨6kb
around PtUrease
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34
sgRNA.UREASE.1187 site
Sequence Listing
SEQ ID NO: Description
1 pPtUMPSA1 (full plasmid)
2 pPtUMPSA2 (full plasmid)
3 pPtUMPScA1 (full plasmid)
4 pPtUMPScA2 (full plasmid)
pPtPRAPHCH (full plasmid)
6 PtUMPS coding sequence and gRNA
target
sites
7 Amino acid sequence of SEQ ID NO:
6
8 PtPRA-PH/CH coding sequence and
gRNA
target sites
9 Amino acid sequence for SEQ ID
NO: 8
PtI3GPS-PRAI coding sequence and gRNA
target sites
5
SEQ ID NO: 1 ¨ pPtUMPSA1
tcgagctggttgccctcgccgctgggctggcggccgtctatggccctgcaaacgcgccagaaacgccgtcgaagccgtg
tgcgagacac
cgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacact
tgaggggcc
gactcacccggcgcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtggagctggccagcctcgcaaa
tcggcgaaa
10
acgcctgattttacgcgagtttcccacagatgatgtggacaagcctggggataagtgccctgcggtattgacacttgag
gggcgcgactact
gacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgagg
ggctgtccaca
ggcagaaaatccagcatttgcaagggtttccgcccgtttttcggccaccgctaacctgtcttttaacctgcttttaaac
caatatttataaaccttg
tttttaaccagggctgcgccctgtgcgcgtgaccgcgcacgccgaaggggggtgcccccccttctcgaaccctcccggt
cgagtgagcga
ggaagcaccagggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccactta
tccacggggatatttt
tataattatttittttatagtttttagatcttcttttttagagcgccttgtaggcctttatccatgctggttctagaga
aggtgttgtgacaaattgcccttt
cagtgtgacaaatcaccctcaaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctcagaa
gaagctgtttittcac
aaagttatccctgcttattgactctttittatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtca
tggcggaaacagcggttat
caatcacaagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgg
gatcaaaaacgt
atgctgtatctgttcgttgaccagatcagaaaatctgatggcaccctacaggaacatgacggtatctgcgagatccatg
ttgctaaatatgctg
aaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggt
ttittatcgccctg
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35
aagaggatgccggcgatgaaaaaggctatgaatct-t-ttccttggt-
ttatcaaacgtgcgcacagtccatccagagggct-ttacagtgtacatat
caacccatatctcattcccttctttatcgggttacagaaccggtttacgcagtttcggcttagtgaaacaaaagaaatc
accaatccgtatgccat
gcgtttatacgaatccctgtgtc agtatcgtaagccggatggctc
aggcatcgtctctctgaaaatcgactggatcatagagcgttaccagctg
cctcaaagttaccagcgtatgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatc aacagc
agaactcc aatgcgcctctc at
acattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggata
gtctgagggttatct
gtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcacagt-t-ttgctgt-
ttccttcagcctgcatggattttctcat
act-tt-ttgaactgtaatttttaaggaagccaaatttgagggcagt-ttgtcacagttgatttccttctct-
ttcccttcgtcatgtgacctgatatcgggg
gttagttcgtcatcattgatgagggttgattatcacagt-
ttattactctgaattggctatccgcgtgtgtacctctacctggagt-ttttcccacggtg
gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttct-
ttgcttcctcgccagttcgctcgctatgctcggttacacggctgc
ggcgagcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaaaaatagaaagtaaa
aaaagaaattaaagaaaa
aatagt-t-tttgt-tttccgaagatgtaaaagactctagggggatcgccaacaaatactacct-
tttaccttgctcttcctgctctcaggtattaatgcc
gaattgtttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttacttatcgttaatcgaat
gtatatctatttaatctgct-ttt
cttgtctaataaatatatatgtaaagtacgct-ttttgttgaaattt-tttaaacct-ttgt-ttattt-
ttttttcttcattccgtaactcttctaccttct-ttatttactt
tctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacga
ggcgcgtgtaagtta
.. caggcaagcgatcctagtacactctatatt-tt-tttatgcctcggtaatgattttcattt-
ttatttccacctagcggatgactct-ttt-tttttcttagcgatt
ggcattatcacataatgaattatacattatataaagtaatgtgatttcttcgaagaatatactaaaaaatgagcaggca
agataaacgaaggcaa
agatgacagagcagaaagccctagtaaagcgtattacaaatgaaaccaagattcagattgcgatctctttaaagggtgg
tcccctagcgata
gagcactcgatcttcccagaaaaagaggcagaagcagtagc agaac
aggccacacaatcgcaagtgattaacgtccacacaggtatagg
gt-
ttctggaccatatgatacatgctctggccaagcattccggctggtcgctaatcgttgagtgcattggtgacttacacat
agacgaccatcac
accactgaagactgcgggattgctctcggtcaagct-t-
ttaaagaggccctaggggccgtgcgtggagtaaaaaggtttggatcaggatttgc
gcct-ttggatgaggcactttccagagcggtggtagatct-
ttcgaacaggccgtacgcagttgtcgaacttggtttgcaaagggagaaagtag
gagatctctcttgcgagatgatcccgcattt-tcttgaaagct-
ttgcagaggctagcagaattaccctccacgttgattgtctgcgaggcaagaat
gatcatcaccgtagtgagagtgcgttcaaggctcttgcggttgccataagagaagccacctcgcccaatggtaccaacg
atgttccctccac
caaaggtgttcttatgtagttttacacaggagtctggacttgacgctagtgataataagtgactgaggtatgtgctctt
cttatctccttttgtagtgt
tgctcttattttaaacaactttgcggifitttgatgactttgcgattttgttgttgct-
ttgcagtaaattgcaagatttaataaaaaaacgcaaagcaat
gattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctggtcatgaaatgacgaaggcta
tcgccattgcac a
gt-
ttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggt-
ttcttctcaggct
atcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggtt
atacaattgaa
caaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttg
ctgcccataaaggtg
gcgt-ttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgt-
tttgctcgtggaaggtaacgacccccaggg
Date Recue/Date Received 2021-08-17

36
aacagcctcaatgtatcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggg
gaaaaggacgatgtc
acttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactg
agttaatgggcaaattt
gatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtca
tagttattgacagcg
cgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtt
tgactacacctccgca
ctgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgc
ttaccaaatacagcaata
gcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacg
tgaaacggatga
agttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggaga
aatgctctttctatttg
ggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgtta
ttccaaaacatacgc
tcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtagg
agtaatggctcgcg
gtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaa
gacctctcgggtatgg
tcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtc
aacagacaccggcg
ttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtg
attatcgtgttctgg
ttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacg
tggtcagcgttatg
caagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattac
ccgctgtatcaacacc
gccaaattgcctaaatcagttgttgctctttittctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaa
aagcctttacagataaa
gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagtta
tcactcttttaactt
ctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgta
taagggcgataaaat
ggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaag
ccagcaccctgatg
cgacctcgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaat
attctctctgggcccactgt
tccacttgtatcgtcggtctgataatcagactgggaccacggtccc
actcgtatcgtcggtctgattattagtctgggaccacggtcccactcgt
atcgtcggtctgattattagtctgggacc acggtcccactcgtatcgtcggtctgataatcagactgggacc
acggtcccactcgtatcgtcgg
tctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgta
tcgtcggtctgattatta
gtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctg
attattagtctgggac
cacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagc
gtgagactacgattcc
atcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgc
ctgctgtggattgct
gctgtgtcctgcttatccacaacattttgcgcacggttatgtggac aaaatacctggttaccc
aggccgtgccggcacgttaaccgggctgc a
tccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgt
attgtctgaagttgtt
tttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctcca
cgcacgttgtgatatgtag
atgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgcta
tttatgaaaattttccg
gtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgaca
ggtgctgaaagcgagctt
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuari5a ele0
UTOSIDITUUSTSUUMMURPSTSMOSSNSOSIDUSSUUSINUMSUOSNSNOSNIUSSSORPUReultuReolutuum
molpuelRe amp:moppet& ouooltSotunautu otne aunpoupRenotSoSup Nam& Sum oS
pumoSoReoolSoopownSompi2op optuimpiSnaelSouSaupantuaelnumuoltuptSoSoltuS
oloSopppSSSuuSnoSupepSop moSueSonSuaeSoReSolReoSooSoptSulReooReaumpoltolSSTS
TetuomeReaumeSpepananSuounpuoReoltuneSSoReStReauSaeutnotSouopueSupeamS
SuolpotuputuuSaumeSami2SoReuotootutuoltoutSulSueSSumuSoSultSpoReneoppeneu cz
uoSoopReSulSomp000ppueuSSSolReounSanSoReautouooputoStoReauSuaeueutoltputo
SmuSSTReSpueSSTSpueStupppoStpoSpeupuReSpopumanutotuSoSunotutolReSSootu
SltuReoSTSSSomoSS000toSttuStoSotunneuSauoupi2ueSSamouSuopoolffeweauntapoS
otouReue toS oaupTSS o otult otu au SIBS oSpio onu ooSoouSaup au o au au olSS
o outopuS auSTS
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ne auS
nuoueopi2SoomonSumoSSuReumooSompoplSoue opSootSaelpp annuReueSTSSIene mewl
utSuiSS000SoupueumopeupSoppueutoopRelpouoSumpoopp_muutReoomeTSSTSSamopmeS
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ci
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otpoomeSoSamoonopeSpeuuotoReootSoltoomenSolReoomoS000taeopSoSuSotpuipaeo
puepReSTReSpepoSTSSSSpoRmultSumpoSueSSooReSoupmu au au ooneu au opS o
oputpueSTSTST
oolutoRepolStu owelS onuoSulemp au oplReppnelSuSuoSue otu one Sp auS olReSupp
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maelnopReSomeSoSSSuppe opeSoupultputReoonauSanuelSuSauSaeolSuompuSSReooSan
TSSSuSueneSoSSuu otottuSSSSReueSoStoRe ooSounepSolppoSSSoSTSSopSonSuuSSSuSpueo
otoRe one ooSone ooS one op oSompueuRenuelSotuRe ame o pupae STSTSS otup
otSuRetaelS
nee Rae onotupeunoStoSSSSoltSSSoSSuSTSSSoRe olSoSouSSSSue oSoonotuSonepuSue
Saul:meow oSuplSouampuloSopopReSoolReuStuemetueSSTSooltputopppomSoltopoStu

38
aaactccgggtatctctgacacaaaacgattcggcttcgcaatttcaacattacggtcaaggctaacgtatctttctcg
gtcaacttcagattatg
ccgattaaattgtcgtagctttcaaggcgttttgagtactgcggcagttgttgaacctgcaaggagaagatctcgacaa
cagaataaagcgaa
aaatgggtctcatgcactaacactcaggcctccctcataatctctgtttgagtttaccaacaacacatatatacatttc
gacaaaatgaccactct
tgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcacc
accgacacc
gtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttcc
ccgacgacga
atcggacgacgaatcggacgacggggaggacggcgacccggactcccggacgttcgtcgcgtacggggacgacggcgac
ctggcgg
gcttcgtggtcgtctcgtactccggctggaaccgccggctgaccgtcgaggacatcgaggtcgccccggagcaccgggg
gcacggggt
cgggcgcgcgttgatggggctcgcgacggagttcgcccgcgagcggggcgccgggcacctctggctggaggtcaccaac
gtcaacgc
accggcgatccacgcgtaccggcggatggggttcaccctctgcggcctggacaccgccctgtacgacggcaccgcctcg
gacggcgag
caggcgctctacatgagcatgccctgcccctgaccgacgccgaccaacaccgccggtccgacgcggcccgacgggtccg
aggcctcg
gagatctgggcccatgcggccgcaac aactacctcgactttggctgggacactttc
agtgaggacaagaagcttcagaagcgtgctatcga
actcaaccagggacgtgcggc acaaatgggcatccttgctctcatggtgc
acgaacagttgggagtctctatccttccttaaaaatttaattttc
attagttgcagtcactccgctttggtttcgtaactataacggtctcgatgactgacaaaggtattcaaaaatgtagctt
gtcacgatttcacaatcg
acaagcctccccgaatcaagatctatttcggcgtgtgctgcatttcgaaggatggtcgaggccgtaagaataggcacct
gcc atttcttggc a
attggcctaccataagatcctttgccacttccaggtggaccgagaatcgtgatatttctcaacaaacggaagctcgtac
ggctcatccgtgtcg
tggcaagattgttgttcggatgcttgaacttttgtgggtgctgacgattttcatggctcaccgtc
aattctgtatgtcatcggatccgcgctctttttt
ggaatcttccacatgaaattatcacggggctatgagaacc
aaggtttittttagttttctgaactgcgcgcgatggcgcc agaaaggtaccgaa
ttacatacacaggcgcatacggaagcagcgaaaatatctaaaccgctctttctccgacgtagaagcacggagacggcac
ccgagctcgat
aagaaatatcacgccggcagatccaattcggtgctactctacaaaatgaatacttcctaaatatgtggaagatctcaga
aaaagacaagccc
atccgtgactattcaccggaaccgcaagccactcatctcgaacaatcataaaaaatttggaaaccactttctctctcag
cgccagtacgaacat
gagggttgtttcgatcgcagttittgcggcaatgcttgctagcgctttcggcatcgacgccaatgttgacaatgcaatg
gacaaccgcgtcttg
caagctgc aagcgatgtaccgagcgatatgccgagcgatatgccgtc
agacattccgagcctcggagggccgactatgcaaccatctatg
actaccagctcgcttcccacaatcattggtgatcggggtggtgtaacgagctcgagtagccctctgctttccgcatgta
tcgctgttcctgctat
ggcaatggcctggcagctttatcagtagagaatcggaagttaaatagagacctggttcgtgccctacaaacgtaacctg
agtctactattccg
tcaaagtctctgttgtgtggaaaacctcgccagagtatttagttgttaaccgaagtattaagtcagtttgatcaaaaag
ttattcattacactccgt
attcgtttcgatacttaattactttttccagcgtttcatcgccgtagtccggacttcgttcgagaaatgtctgtagttg
aggaagactgacaattga
cacgaccaaaagagatagatctcgtgcgactgcttgaacagcggaaatcttgtcctccatagagcgaatttcggctcga
tcgagggcaataa
ctactccaactggcaaagc accc acatcgttgagcaaagtgtgcgactc
acgaatggcggttcccgctgtgattacgtcatctacaatcaga
actcgttttccttccaacgaagtcccgaccaatttaccaccttccccatgatcctttgcctcttttcggtcatacgcaa
aaccgacatctacttcaa
aatcgttatacagagcgcttccaacgacagcacctagagaaatacccttgtatgcaggaccaaaaatcacatcaaaatt
gacttggttgggcc
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe ee/enóej ele0
DO DDD DD DD DO DDDDDDDD DO D DD D DO DD
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L -80-1=ZOZ panpoe apcuari5a ele0
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upaeSSNSTSSSReonaeSolSuSooloSmoupoSoSuRmanael000lolSoReSSReoputReolSouReTSSStuo
u
unoltuonnuaeSp2oSSReploolutupnanTSSoup000StuoomioRetutoSolnaeoReSuoReoplut
oupploanTRe oltuultSpooffeuiSSTSwe olStou poSmoSouSttututotStouReu Soft one
olSotuSo
RelnweoloonutSouSuSaeuReSumutooSoSooluSouRnoneuoReSaeSoaeoluoulReultoReSolpeo
neReuReu o NoSotu op polSou Rem oSnutSoSoSTSSSSSSoonameuloSooRemppe SIT
poReRe oSSS
appoSoNSSTSontutuolneuoSSuoReSaeneoReuReReuSoluReuSoStoSlopoSolooluSaeoptuSTSSo

unoSonaeoSoSopaeSaeSoaeoloStopmetotoonontooSoontoaeSonweoluoSopoStoReSoS

41
tacatgcaatttcgcgccgcatcaacagccacgggccaccatcgacgaatagactcgg
SEQ ID NO: 2- pPtUMPSA2
tcgagctggttgccctcgccgctgggctggcggccgtctatggccctgc aaacgcgcc
agaaacgccgtcgaagccgtgtgcgagacac
cgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacact
tgaggggcc
gactcacccggcgcggcgttgac agatgaggggc aggctcg atttcggccggcgacgtggagctggc c
agcctcgc aaatcggcgaaa
acgcctgatatacgcgagtttc cc ac ag atgatgtggac aagc ctggggataagtgccctgcggtattgac
acttgaggggcgcgactact
gacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgagg
ggctgtccac a
ggc agaaaatcc agc atttgc aagggtttccgcccgtttttcggcc accgctaacctgtcttttaac
ctgcttttaaacc aatatttataaaccttg
ffittaacc agggctgcgcc ctgtgcgcgtgaccgcgc acgccgaaggggggtgcccccc cttctcgaac
cctcccggtcgagtgagcga
ggaagcacc
agggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacgggg
atatttt
tataattatttittttatagffittagatcttatttttagagcgccttgtaggcctttatccatgctggttctagagaa
ggtgttgtgacaaattgcccttt
cagtgtgacaaatc accctc aaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctc
agaagaagctgffitttcac
aaagttatccctgcttattgactatttttatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtcat
ggcggaaacagcggttat
caatcac
aagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgggatcaaa
aacgt
atgctgtatctgttcgttgacc agatc agaaaatctgatggc accctac aggaac
atgacggtatctgcgagatccatgttgctaaatatgctg
aaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagittcgcggggaaggaagtggf
fitttatcgccctg
aagaggatgccggcgatgaaaaaggctatgaatatttccttggtttatcaaacgtgcgcacagtccatccagagggctt
tacagtgtacatat
caaccc
atatctcattcccttctttatcgggttacagaaccggtttacgcagtttcggcttagtgaaacaaaagaaatcacc
aatccgtatgcc at
gcgtttatacgaatccctgtgtc agtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatc
atagagcgttaccagctg
cctcaaagttaccagcgtatgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatc aacagc
agaactcc aatgcgcctctc at
acattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggata
gtctgagggttatct
gtcacagatttgagggtggttcgtcac atttgttctgacctactgagggtaatttgtc ac
agtatgctgtttccttc agc ctgc atggattttctc at
acttittgaactgtaatttttaaggaagcc aaatttgagggc agtttgtc ac
agttgatttccttctctttcccttcgtc atgtgacctgatatcgggg
gttagttcgtcatcattgatgagggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacc
tggagffittcccacggtg
gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctat
gctcggttacacggctgc
ggcgagcatc
acgtgctataaaaataattataatttaaatttittaatataaatatataaattaaaaatagaaagtaaaaaaagaaatt
aaagaaaa
aatagttittgffitccgaagatgtaaaag actctagggggatcgc c aac
aaatactaccttttaccttgctcttcctgctctc aggtattaatgcc
gaattgtttc atcttgtctgtgtagaagac c ac ac acgaaaatcctgtg attttac
attttacttatcgttaatcgaatgtatatctatttaatc tgctttt
cttgtctaataaatatatatgtaaagtacgattttgttgaaatttittaaacattgtttatttitttttcttcattccg
taactcttctaccttctttatttactt
Date Recue/Date Received 2021-08-17

42
tctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacga
ggcgcgtgtaagtta
caggcaagcgatcctagtacactctatatttlittatgcctcggtaatgattttcatttitttittccacctagcggat
gactcttttlitttcttagcgatt
ggcattatcacataatgaattatacattatataaagtaatgtgatttcttcgaagaatatactaaaaaatgagcaggca
agataaacgaaggcaa
agatgacagagcagaaagccctagtaaagcgtattacaaatgaaaccaagattcagattgcgatctctttaaagggtgg
tcccctagcgata
gagcactcgatcttcccagaaaaagaggcagaagc agtagcagaacaggcc
acacaatcgcaagtgattaacgtccacac aggtatagg
gtttctggaccatatgatacatgctctggccaagcattccggctggtcgctaatcgttgagtgcattggtgacttacac
atagacgaccatcac
accactgaagactgcgggattgctctcggtcaagcttttaaagaggccctaggggccgtgcgtggagtaaaaaggtttg
gatcaggatttgc
gcctttggatgaggcactttccagagcggtggtagatcMcgaacaggccgtacgcagttgtcgaacttggtttgcaaag
ggagaaagtag
gagatctctcttgcgagatgatcccgcattttcttgaaagctttgcagaggctagcagaattaccctccacgttgattg
tctgcgaggcaagaat
gatcatcaccgtagtgagagtgcgttcaaggctcttgcggttgccataagagaagccacctcgcccaatggtaccaacg
atgttccctccac
caaaggtgttcttatgtagttttacacaggagtctggacttgacgctagtgataataagtgactgaggtatgtgctctt
cttatctccttttgtagtgt
tgctcttattttaaacaactttgcggifitttgatgactttgcgattttgttgttgctttgcagtaaattgcaagattt
aataaaaaaacgcaaagcaat
gattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctggtcatgaaatgacgaaggcta
tcgccattgcaca
gtttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggtt
tcttctcaggct
atcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggtt
atacaattgaa
caaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttg
ctgcccataaaggtg
gcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaagg
taacgacccccaggg
aacagcctcaatgtatcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggg
gaaaaggacgatgtc
acttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactg
agttaatgggcaaattt
gatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtca
tagttattgacagcg
cgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtt
tgactacacctccgca
ctgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgc
ttaccaaatacagcaata
gcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacg
tgaaacggatga
agttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggaga
aatgctctttctatttg
ggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgtta
ttccaaaacatacgc
tcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtagg
agtaatggctcgcg
gtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaa
gacctctcgggtatgg
tcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtc
aacagacaccggcg
ttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtg
attatcgtgttctgg
ttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacg
tggtcagcgttatg
Date Recue/Date Received 2021-08-17

43
caagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattac
ccgctgtatcaacacc
gccaaattgcctaaatcagttgttgctcttttttctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaa
aagcctttacagataaa
gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagtta
tcactcttttaactt
ctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgta
taagggcgataaaat
..
ggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaag
ccagcaccctgatg
cgacctcgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaat
attctctctgggcccactgt
tccacttgtatcgtcggtctgataatcagactgggaccacggtccc
actcgtatcgtcggtctgattattagtctgggaccacggtcccactcgt
atcgtcggtctgattattagtctgggacc acggtcccactcgtatcgtcggtctgataatcagactgggacc
acggtcccactcgtatcgtcgg
tctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgta
tcgtcggtctgattatta
gtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctg
attattagtctgggac
cacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagc
gtgagactacgattcc
atcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgc
ctgctgtggattgct
gctgtgtcctgcttatccacaacattttgcgcacggttatgtggacaaaatacctggttacccaggccgtgccggcacg
ttaaccgggctgca
tccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgt
attgtctgaagttgtt
tttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctcca
cgcacgttgtgatatgtag
atgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgcta
tttatgaaaattttccg
gtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgaca
ggtgctgaaagcgagctt
tttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtccgagctcatcgctaataacttc
gtatagcatacattatacg
aagttatattcgatgcggccgcaaggggttcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggca
tcagagcagatt
gtactgagagtgcaccatatgcggtgtgaaataccacacagatgcgtaaggagaaaataccgcatcaggcgccattcgc
cattcagctgcg
caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcga
ttaagttgggt
aacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattgtaatacgactcactatagggcgaattc
gagctcggtaccc
ggggatcctctagagtcgacctgcaggc atgcaagcttgagtattctatagtctcacctaaatagcttggcgtaatc
atggtcatagctgtttcc
tgtgtgaaattgttatccgctcacaattccacac aacatacgagccggaagc
ataaagtgtaaagcctggggtgcctaatgagtgagctaact
cacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaa
cgcgaaccccttgc
ggccgcccgggccgtcgaccaattctcatgtttgacagcttatc
atcgaatttctgccattcatccgcttattatcacttattcaggcgtagc aac
caggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattc
attaagcattctgcc
gacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatc
agcaccttgtcgccttgcgtataatatttgcccatggtga
aaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagac
gaaaaacatattc
tcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgcc
ggaaatcgtcgtggt
Date Recue/Date Received 2021-08-17

44
attcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaac actatccc
atatcaccagctcaccgtcttt
cattgccatacgaaattccggatgagcattcatc
aggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtct
ttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttc
tttacgatgccattgg
gatatatc aacggtggtatatc c agtgatttlittctcc
attttagcttccttagctcctgaaaatctcgataactc aaaaaatacgc ccggtagtga
.. tcttatttcattatggtgaaagttggaacctcttacgtgccgatc
aacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagg
gacaccaggatttatttattctgcgaagtgatcttccgtcac
aggtatttattcgcgataagctcatggagcggcgtaaccgtcgc acaggaag
gacagagaaagcgcggatctgggaagtgacggac agaacggtc aggacctggattggggaggcggttgccgc
cgctgctgctgacggt
gtgacgttctctgttccggtcacaccac atacgttccgcc attcctatgcgatgc ac
atgctgtatgccggtataccgctgaaagttctgc aaa
gcctgatgggac ataagtcc atc agttc aacggaagtctac acgaaggtttttgc gctggatgtggctgccc
ggc accgggtgc agtttgtg
atgccggagtctgatgcggttgcgatgctgaaacaattatcctgagaataaatgccttggcctttatatggaaatgtgg
aactgagtggatatg
ctgtttttgtctgttaaac agagaagctggctgttatcc actgagaagcgaacgaaac agtcgggaaaatctccc
attatcgtag agatccgc a
ttattaatctcaggagcctgtgtagcgtttataggaagtagtgttctgtcatgatgcctgcaagcggtaacgaaaacga
tttgaatatgccttcag
gaacaatagaaatcttcgtgcggtgttacgttgaagtggagcggattatgtcagcaatggacagaacaacctaatgaac
acagaaccatgat
gtggtctgtccttttac agcc agtagtgctcgccgc agtcgagcgac agggc gaagccc atc
gatactagcttgattgggatatctcgctc at
.. gtttgtcgcgtgctatgtctttttaggtactttgaacctacgttcgtacttgtataatatgatc
atcgtattatcgtttttcatccgtccagcgcaaaat
gcattagcagctagtcctagcgtgcggagctacctggacaggtgc
atgacggatgcgtgtccttcagtgactttctaattaacagtaacttcttt
acttatgtttcagtttgtaagaagcgggattcgctcgtcgcttgacatctgattggactgcgtcggc
acgtgaaaactacattgtgaaatctgcta
aaactccgggtatctctgacacaaaacgattcggcttcgcaatttcaac
attacggtcaaggctaacgtatctttctcggtcaacttcagattatg
ccgattaaattgtcgtagctttcaaggcgttttgagtactgcggcagttgttgaacctgc aaggagaagatctcgac
aacagaataaagcgaa
..
aaatgggtctcatgcactaacactcaggcctccctcataatctctgtttgagtttaccaacaacacatatatacatttc
gacaaaatgacc actct
tgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcacc
accgacacc
gtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttcc
ccgacgacga
atcggacgacg aatcggacgacggggaggacggcgacccgg actcccgg acgttcgtc
gcgtacggggacgacggcgacctggcgg
gcttcgtggtcgtctcgtactccggctggaaccgc cggctgaccgtcg aggac atcgaggtcgccccggagc
accgggggc acggggt
cgggcgcgcgttgatggggctcgcgacggagttcgcc cgcgagcggggcgccgggc ac ctctggctggaggtc
acc aacgtc aacgc
accggcgatc c acgcgtac cggcggatggggttc ac cctctgcggcctggac
accgccctgtacgacggcaccgcctcggacggcgag
caggcgctctacatgagcatgccctgcccctgaccgacgccgacc
aacaccgccggtccgacgcggcccgacgggtccgaggcctcg
gagatctgggcccatgcggccgc
aacaactacctcgactttggctgggacactttcagtgaggacaagaagcttcagaagcgtgctatcga
actcaaccagggacgtgcggc acaaatgggcatccttgctctcatggtgc
acgaacagttgggagtctctatccttccttaaaaatttaattttc
.. attagttgcagtcactccgctttggtttcgtaactataacggtctcgatgactgacaaaggtattc
aaaaatgtagcttgtcacgatttcacaatcg
Date Recue/Date Received 2021-08-17

45
acaagcctccccgaatcaagatctatttcggcgtgtgctgcatttcgaaggatggtcgaggccgtaagaataggcacct
gcc atttcttggc a
attggcctaccataagatcdttgccacttccaggtggaccgagaatcgtgatatttctcaacaaacggaagctcgtacg
gctcatccgtgtcg
tggcaagattgttgttcggatgcttgaacttttgtgggtgctgacgattttcatggctcaccgtcaattctgtatgtca
tcggatccgcgctctttitt
ggaatcttccacatgaaattatcacggggctatgagaacc
aaggttitttttagttttctgaactgcgcgcgatggcgcc agaaaggtaccgaa
ttacatacacaggcgcatacggaagcagcgaaaatatctaaaccgctctttctccgacgtagaagcacggagacggcac
ccgagctcgat
aagaaatatcacgccggcagatccaattcggtgctactctacaaaatgaatacttcctaaatatgtggaagatctcaga
aaaagacaagccc
atccgtgactattcaccggaaccgcaagccactcatctcgaacaatcataaaaaatttggaaaccactttctctctcag
cgccagtacgaacat
gagggttgtttcgatcgcagtttttgcggcaatgcttgctagcgctttcggcatcgacgccaatgttgacaatgcaatg
gacaaccgcgtcttg
caagctgc aagcgatgtaccgagcgatatgccgagcgatatgccgtc
agacattccgagcctcggagggccgactatgcaaccatctatg
actaccagctcgcttcccacaatcattggtgatcggggtggtgtaacgagctcgagtagccctctgctttccgcatgta
tcgctgttcctgctat
ggcaatggcctggcagctttatcagtagagaatcggaagttaaatagagacctggttcgtgccctacaaacgtaacctg
agtctactattccg
tcaaagtctctgttgtgtggaaaacctcgccagagtatttagttgttaaccgaagtattaagtcagtttgatcaaaaag
ttattcattacactccgt
attcgtttcgatactttattacttittccagcgtttcatcgccgtagtccggacttcgttcgagaaatgtctgtagttg
aggaagactgacaattgac
acgaccaaaagagatagatctcgtgcgactgcttgaacagcggaaatcttgtcctccatagagcgaatttcggctcgat
cgagggcaataac
tactccaactggcaaagcacccacatcgttgagcaaagtgtgcgactcacgaatggcggttcccgctgtgattacgtca
tctacaatcagaa
ctcgttttccttccaacgaagtcccgaccaatttaccaccttccccatgatcdttgcctcttttcggtcatacgcaaaa
ccgacatctacttcaaa
atcgttatacagagcgcttccaacgacagcacctagagaaatacccttgtatgcaggaccaaaaatcacatcaaaattg
acttggttgggccc
agcagctctggaaggaaaaagaaaacgaattttgttgggtaagcctgctcataacaatgatagtcccatagaactaaat
caaaacacagtctc
agaaaatttcgaatcagcgaattggtgttgaccggcatctttccggagaaattaggtgaaagaagaccaaaggagtcat
catcgactatgttg
tttcaagagtcaggccactggaaaaaaatttgctcattgccactcacgtgggggcagatcggtaacacggaccaagtgg
atactcatccttct
cttgaatttttgccgttcagcagagaaaaacacacaaagcacacttacaataattccgaggacatgatagtcgaagcat
aggcMcccaagc
ttgcttaacgcagcgccagaagcaaaaagaccggcgttgaaaaaatatggagagatgcggccgcttttcagcacaaaag
agccgaatttg
agaacacctagagacagactaaattcaagaaactcgcgttgatagagtttaatatcttcgtctgaactUttittatcat
gtgtgcggccacgact
tggtcccgagctttctgaatcctctcctgcaattcttttgcagcctgcgctgggtccgtagctttgctgatacccctag
acacgggaattagcat
gcaagtcccctttgtattcaatccagcctgcgctgcttctagaagatctccaccttgagcaccaacgccgggtgctaga
atccaggtgtcgtc
gcctgcagcctttctcgctttggacaaggccactggatctgtggccccgacaacgagtcccaatgaactctcggtctgc
tgagcccattccg
agccaacaagcttggcaattctttcgtataaacattcgtttgaacttaatcccagagctaaaaaatcgttggatccagg
atttgacgttttgcaaa
gcaaaaatgctcctttgtgaatgtacttttctgtaggagggagtcaattcaacgtatcagatagcacccactcaatcac
tatagatgccaatcctt
tagctacaacttacctgtaacaaagggactgactgagtcccatcccatcagtggtgaaagcgtgacacagtctgcaccc
aaaccatagcac
gcttcggcgtaggccgcagcggtcgagccaatgtcgccgcgcttgacatccaacaaaatcggcacatcatccggtatta
tgttttgacaaac
Date Recue/Date Received 2021-08-17

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p opine oSoSoSTSSSTSSpe SIT ouReuSweSSReuSSSSIT an an ReulReuRe olSouu olRe
000lSSSITSpeuReS
olSuSomuSoltu moo& one ootuTSSoltuStu olReulne ootu oninewe oloSSTS Ian
olSneReSooSt
TeltuoSTSSoltSanoSSIeReouneuReSuSoutSoluoReReSolnowelReulSououuSuotuoReaeSSITSo
Sne
outu Num opoSSReSSounnoneootu000uReStaeolulnanSoSpunweReuoSuStaeSRepoSoSo
uoloouRelmeotneRelSoulutuReSuouReSameSSSooRmemoounweolSoSneoloonanoSttom2 cz
neReoaeSSolSTSSSReonaeSolSuSoolotnoweoSoSuReRmeoul000lolSoReSSRe pout&
olSouReTSSSSI
opuSSoltuonme ouSuSoSSRelolo tweSSani2Sou 0000Stu oomioRetutoSolnou Saw& No
lutoupploanTReoltuultS000ReuTSSTSweolStouooSmoSouSttututoSTSSlouRnSoReoneolSot
uSoRelStReolooSSITSTSouSuSaeuReSneputooSoSooluSouReu oneu oReSouSoae
oulReultoReSou
ou nen Rae ooloS otu 000 olSou oReu ooSnuSTSoSoSTSSSSSSoonameuloSooRemmou SIT
ooReRe o z
SneS000SoolStSontutuolneuonuoReSouneoReuReReuSoluRnSoStot000SolooluSouoota
TSSounoSonou oSoS000uSouSoou oloStopmetotoonontooSoontoaeSoStuu oS000StoS
uSoSo olSou oRetupeSSTS olSonSou wan own& oonolSooStotoouloonoltooSaluonoonoS
SononameneSneSoReReameSouReoollootoultneuomoou000ReoSneuneomeooReuomea
lutoSSITSSoRnoSnetuRemelSonooSoaeloSSooStoneSotolotooweSSSanoloSTSSoStomoS c
onell000lSpeouolloolISTSSSoReSoSoom000nelReutnepneonSooReolSonanoolultnuoomo
01
aultSouSSRanootmeneouppSouoSopploopoomelnommuSoRelmeolSSSSouotoomepoto
SoouoluSupwelSne ammo oSooSonelReutuSoutReuSSSooSmoSolReloReSooSo oRetuwe man
SSuSttunanionounammettoSSTSSSoamen000ltIeSolSounoulotouReumanooluneReS
SoolaelomeouSotoSoluReSuSoulooSoollouppeSnuaeoReSootSoRe000meSmelnoluweooSSTSIT
SS
tolSweounnootoneSooRelneSTReS000meuSonewiSoloReotoSoouSTSSSuooSSOSoSSTSooReu
aeoSololSoS0000lSoaeSSoSou000ReoomeReSoReSp2STSSut_ffineuooltowe oononSotottno
ouSomoReloReopeoSottotoeSol000tnnuoReluSweoopneTSSSITSoneoSutoReSoloSSooutuRel
Wpm Rene otmonnom2SSSSRe ooSSouSne ouReSuRe out& outloae oloSou
olotowelReulloSou
neooSSouSouSSTReameStnelnualuotuneupeuReouReoReSouSunneSSITTSouRanolSuReSSo
SttpuuSpeolSolpeouRelRettoltoRmeuReRe op& oaeSSReRelSoltneuttloweSSSoolututwe
w000loReluttuooSSTSSSSReReuReSolutuoReuouoReSopeSonotouSuReSuReRnaeoSanoouRel
olSnoSTSolomoloReameoSoolSoop000llooSouonooniuneSoReaeolSoSoonmelneuReotmSanoo
uSolSoSuSweoneultSooneoReltuntluoReonnuReSolooSmelooSolSool000moSomeReoSolSol
9t7

47
aacacaattgtggcaatgttcaagttgggcgctggaatggatggttttacctttaccaagatgcgcgtattgatgcatg
ataacaacaatgggc
cctcgttgcgattcgacaagaccggcagccgtgac
aatgtctagattggacaaggtatgttcatttataccggttatgttggctgaacgtcc ag
ttttgtgcagtacggtaacatc actccccgcaagtccaccgttggc ac c gc g atc g ac aagtgc ag
atgtggtattttgtactgtgtgtc ggg a
gacttggtactgtaacac atttgactgaagtgacttggtggtacc
atgaggacggagggaacccgttgatacggtgttgcgtgacgctgcaa
..
gtaccttacgtatgtcgcctggatccatacgtccaacccgatcagaaagatgtgctaggagttccgtttctggtgcaca
atcatgaaaggtatc
cacgggagttccattgttgtcgtccggggctgcgtcgcgactgcc
agttgcatgtacatgcgccatcaagggacgtgccaatccattactaa
ccgggagaaccttagctggggcagccaatccctgaagtatagccttagcatc atctgagagctggttccac
atatctctagggatataaggtc
gttcacggttagtgggcgtactatgatgtcggttattagtacaattctccctgcgggcatgcgcattggcttcatagag
tacggaaggtgac aa
gtcaatattgtagttaacatccggatcagtgtcaaagtctgtaggatggtaagaaagatcagtagaatgaatactacgc
ttttccttgggactac
..
gggaattagagaagttgtttcctttattgtagagtgacgctgaagcaagtagaagactaaggtaactctcgtagctaat
aggattacctcctttg
gctaggtcaagagtggctgtgatcttcacttgacaaagttccggtacattgtggacagcattctccaaaagactaagac
acagttgctttggga
gttgctc agcc attggtac agtattgtggtagatac aaaggtggttcttc c aatgaaggataaatc
cttctgctgtgcctgtcc atgagg atcc at
atttcgccgtagttaggtaacc
aagcgtggtggctgaactgatcttcgcacttgctgattccgtatagtgtttgacaactttacaaaacacttcttg
cgc agttcgctctagc ctggac ag agaagggac aatcttcgtcgttaagactcgtgcg aatagc
aaaagatc ac aaaatagc ac atcggc a
ccgaccaacgattatttccaaggaaaaaaagaatgcttc actacaagaaattgtgtcatccctatac
agagtcttgttactgtgacagaaaattg
atggaagatgtggcggattgcctttacactagcc
aacttgttcgactaattgcagcttcttctgagaggcttcaccgagtaacgcgaagaac a
ccggtgtctcgtac atgctcgtc ggtgaacgctcgtcc aatgac ac cccc c actttgtatc
aatatcccaacttggtagtgaactggaatgata
catgcaatttcgcgccgcatcaacagcc acgggccaccatcgacgaatagactcgg
SEQ ID NO: 3¨ pPtUMPScAl
tcgagctggttgccctcgccgctgggctggcggccgtctatggccctgc aaacgcgcc
agaaacgccgtcgaagccgtgtgcgagacac
cgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacact
tgaggggcc
gactcacccggcgcggcgttgac agatgaggggc aggctcg atttcggccggcgacgtggagctggc c
agcctcgc aaatcggcgaaa
acgcctgattttacgcgagtttc cc ac ag atgatgtggac aagc ctggggataagtgccctgc
ggtattgac acttgaggggcgcgactact
gacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgagg
ggctgtccac a
ggc agaaaatcc agc atttgc aagggtttccgcccgtttttcggcc
accgctaacctgtcttttaacctgcttttaaaccaatatttataaaccttg
tttttaacc agggctgcgcc ctgtgcgcgtgaccgcgc acgccgaaggggggtgcccc
cccttctcgaaccctcccggtc gagtgagcga
ggaagcacc
agggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacgggg
atatttt
tataattatttittttatagtttttagatcttcttttttagagcgccttgtaggcctttatccatgctggttctagaga
aggtgttgtgacaaattgcccttt
cagtgtgacaaatc accctc aaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctc
ag aag aagctgttttttc ac
Date Recue/Date Received 2021-08-17

48
aaagttatccctgcttattgactctttittatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtca
tggcggaaacagcggttat
caatcacaagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgg
gatcaaaaacgt
atgctgtatctgttcgttgaccagatcagaaaatctgatggcaccctacaggaacatgacggtatctgcgagatccatg
ttgctaaatatgctg
aaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggt
ttittatcgccctg
aagaggatgccggcgatgaaaaaggctatgaatcttttccttggtttatcaaacgtgcgcacagtccatccagagggct
ttacagtgtacatat
caacccatatctcattcccttctttatcgggttacagaaccggtttacgcagtttcggcttagtgaaacaaaagaaatc
accaatccgtatgccat
gcgtttatacgaatccctgtgtc agtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatc
atagagcgttaccagctg
cctcaaagttaccagcgtatgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatcaacagcagaactc
caatgcgcctctcat
acattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggata
gtctgagggttatct
gtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcacagttttgctgtttccttca
gcctgcatggattttctcat
actttttgaactgtaatttttaaggaagccaaatttgagggcagtttgtcacagttgatttccttctctttcccttcgt
catgtgacctgatatcgggg
gttagttcgtcatcattgatgagggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacc
tggagtttttcccacggtg
gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctat
gctcggttacacggctgc
ggcgagcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaaaaatagaaagtaaa
aaaagaaattaaagaaaa
aatagtttttgttttccgaagatgtaaaagactctagggggatcgccaacaaatactaccttttaccttgctcttcctg
ctctcaggtattaatgcc
gaattgtttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttacttatcgttaatcgaat
gtatatctatttaatctgctttt
cttgtctaataaatatatatgtaaagtacgcttittgttgaaatttittaaacctttgtttatttlittttcttcattc
cgtaactcttctaccttctttatttactt
tctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacga
ggcgcgtgtaagtta
caggcaagcgatcctagtacactctatatttlittatgcctcggtaatgattttcatttitttittccacctagcggat
gactctttlittttcttagcgatt
ggcattatcacataatgaattatacattatataaagtaatgtgatttcttcgaagaatatactaaaaaatgagcaggca
agataaacgaaggcaa
agatgacagagcagaaagccctagtaaagcgtattacaaatgaaacc
aagattcagattgcgatctctttaaagggtggtcccctagcgata
gagcactcgatcttcccagaaaaagaggcagaagcagtagc agaac
aggccacacaatcgcaagtgattaacgtccacacaggtatagg
gtttctggaccatatgatacatgctctggccaagcattccggctggtcgctaatcgttgagtgcattggtgacttacac
atagacgaccatcac
accactgaagactgcgggattgctctcggtcaagcttttaaagaggccctaggggccgtgcgtggagtaaaaaggtttg
gatcaggatttgc
gcctttggatgaggcactttccagagcggtggtagatcMcgaacaggccgtacgcagttgtcgaacttggtttgcaaag
ggagaaagtag
gagatctctcttgcgagatgatcccgcattttcttgaaagctttgcagaggctagcagaattaccctccacgttgattg
tctgcgaggcaagaat
gatcatcaccgtagtgagagtgcgttcaaggctcttgcggttgccataagagaagccacctcgcccaatggtaccaacg
atgttccctccac
caaaggtgttcttatgtagttttacacaggagtctggacttgacgctagtgataataagtgactgaggtatgtgctctt
cttatctccttttgtagtgt
tgctcttattttaaacaactttgcggifitttgatgactttgcgattttgttgttgctttgcagtaaattgcaagattt
aataaaaaaacgcaaagcaat
gattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctggtcatgaaatgacgaaggcta
tcgccattgcac a
Date Recue/Date Received 2021-08-17

49
gtttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggtt
tcttctcaggct
atcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggtt
atacaattgaa
caaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttg
ctgcccataaaggtg
gcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaagg
taacgacccccaggg
aacagcctcaatgtatcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggg
gaaaaggacgatgtc
acttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactg
agttaatgggcaaattt
gatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtca
tagttattgacagcg
cgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtt
tgactacacctccgca
ctgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgc
ttaccaaatacagcaata
gcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacg
tgaaacggatga
agttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggaga
aatgctctttctatttg
ggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgtta
ttccaaaacatacgc
tcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtagg
agtaatggctcgcg
gtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaa
gacctctcgggtatgg
tcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtc
aacagacaccggcg
ttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtg
attatcgtgttctgg
ttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacg
tggtcagcgttatg
caagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattac
ccgctgtatcaacacc
gccaaattgcctaaatcagttgttgctctttittctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaa
aagcctttacagataaa
gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagtta
tcactcttttaactt
ctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgta
taagggcgataaaat
ggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaag
ccagcaccctgatg
cgacctcgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaat
attctctctgggcccactgt
tccacttgtatcgtcggtctgataatcagactgggaccacggtccc
actcgtatcgtcggtctgattattagtctgggaccacggtcccactcgt
atcgtcggtctgattattagtctgggacc acggtcccactcgtatcgtcggtctgataatcagactgggacc
acggtcccactcgtatcgtcgg
tctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgta
tcgtcggtctgattatta
gtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctg
attattagtctgggac
cacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagc
gtgagactacgattcc
atcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgc
ctgctgtggattgct
gctgtgtcctgcttatccacaacattttgcgcacggttatgtggac aaaatacctggttaccc
aggccgtgccggcacgttaaccgggctgc a
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuari5a ele0
TESTUDDRESUMMESTRETOMEMEREDUSSWEDRENSTeneSSoReSSTReauSaeuSTSSoSTSouoluReSuweanS

Se pp otuputuuSoueue Soup2SoSue otootutuptouSTSpSueSSumuSoSpSiSpoSeSSeoppeneu
oSopuSeSpSomp000ppmeSSSoiSuomeSoueSoSeuSeSpeopuutoStoSueSeSemueutptputo
tupSSTSeSpueSSTSpueStpumooStpoSpeupautoompuountotuSoSuSSotutpSeSSootu
STSmSe oSTSSSom oSS 000toSSTSTeStoSotpuSSue Sou oppSueSSoue ouSu oolSupe
ouSSSIeSpoS
otouSeuetoSoopp2Sootptotu otuSotpoonu ooSoouSope mu au oi2SooutopuSouSTS cz
TSSoutototoSooSooSuSSoneSSSSuuStomne Inoue& ouSSouSTSueSStpuSSoSoSeueSeSemS
SueSSemoSolSompSoSSoSeStupoSepeSoSonumpne oupSompu STSueSotonempuu SSe ouS
SSuoueom2SoomoSSSumoSSuSeumooSompopiSoue puSootSopppoueSSITSeueSTSSIppomeup
uSTSuiSS000S0upeume010epeS0ppueu10010Selp0u0Semp00mmmuSTSuoomp2STSSoueopmeS
StpootammouSweueopoSpueSpapueoSeSneopnemiStoi2SoueSpSeoppepSooneueueu 0z
loiSSournowneuottpuumpSSooSSumputtueSueoSSSoneopmeoSetuSSoppeuSoupooSneo
ci
01
molSom op& opm 000ppe oueSTSSSue oup2ISSoueueStu poSmSe oulSoumetuSoSeSe pope
one
TSSTSpSopueSSooSpeueSulttpepuS oSipp ooSou oupSom opune ooSSepueSSSemoo mem p
onep mueueS ouSe tonne SSSe o o opeueSTSSImeue opupuS ooSSuup o pSuSueSeuS
oSSSSSome
uSTSSIe 000tneppelSotpoSoltpou oSe oSS oSe o oSopetometutu noun au o oSueStu
ouS
ootone Snip onepSutopSe oSpu ooSpooSomoSoppeuemppotouepu o oSSSupuSone o
oSpSone plume opuppSopu ouu ootomueS opuoSeoututuponeumuSolSooSSS000SooSS
Sup o o S Sam o oSS pue tueuu otoSeooSTSoltomueSSSolSeoomoS000Spe
poSoSuSoSuuene o
pupoSeSTSeSpep oSTSSSto oSeupSTSeuep often o oSe S ope me au au owe poS o
oputpueSTSTST
oputoSepoiStu oppS oSSuoSpueup opiSuppupSeSuoSue otu one Sp ouS pSeSupp
puSSSS
000m2SpoSe SoneuSoSSSuppe SopepSuueSTSe ooSSouSoueupSuSou Sou pSe o opluSSSe
ooSoue
TSSSuSuppS 0nm otottuSSSSSeueSoStoSe ooSompoSoupooSSSoSTSSopSoSSSuuSSSITSpueo
Soto& one ooS one ooS one op oSoopeueuSeSSup2 otuSem ompueSTSTSSotup
otSuSetopS
uuSe Sae onotpouppStoSSSSNSTSSSoSSITSTSSSoSe pSoSouSSSSue oSoonotuSoupeuSue
Soul:plump oSuipSoumeppoSoppoSeSopSeuStuemetueSSTSoptputompoluSoltopoStu
uoSeSoSueutoSTSSemSomeneueSeueutpoompueumpputuppepepSouppuSopuSoSSupuS
SoompueutumpoSomiSSSoSpomS oSSoSSSSouune pap puSTSSooppoi2SSopuou mum oppetu
SultpuSTSuSouoSouopooStutuSSTSoutuunetpuppSoSpopeSoupppepuSeotauSueuSopu
uSuSueSpiSuptuutoSpSTSeoupSomoSuSSetuouSSpoSeSoSpoSeSouSoltoSpSTSTSueotuSop
OS

51
gtggtctgtccttttacagccagtagtgctcgccgcagtcgagcgacagggcgaagcccatcgatactagcttgattgg
gatatctcgctcat
gtttgtcgcgtgctatgtctattaggtactttgaacctacgttcgtacttgtataatatgatcatcgtattatcgtatt
catccgtccagcgcaaaat
gcattagcagctagtcctagcgtgcggagctacctggacaggtgcatgacggatgcgtgtccttcagtgactttctaat
taacagtaacttcttt
acttatgtttcagtttgtaagaagcgggattcgctcgtcgcttgacatctgattggactgcgtcggcacgtgaaaacta
cattgtgaaatctgcta
aaactccgggtatctctgacacaaaacgattcggcttcgcaatttcaac
attacggtcaaggctaacgtatctttctcggtcaacttcagattatg
ccgattaaattgtcgtagctttcaaggcgttttgagtactgcggcagttgttgaacctgcaaggagaagatctcgacaa
cagaataaagcgaa
aaatgggtctcatgcactaacactcaggcctccctcataatctctgtttgagtttaccaacaacacatatatacatttc
gacaaaatgaccactct
tgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcacc
accgacacc
gtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttcc
ccgacgacga
atcggacgacgaatcggacgacggggaggacggcgacccggactcccggacgttcgtcgcgtacggggacgacggcgac
ctggcgg
gcttcgtggtcgtctcgtactccggctggaaccgccggctgaccgtcgaggacatcgaggtcgccccggagcaccgggg
gcacggggt
cgggcgcgcgttgatggggctcgcgacggagttcgcccgcgagcggggcgccgggcacctctggctggaggtcaccaac
gtcaacgc
accggcgatccacgcgtaccggcggatggggttcaccctctgcggcctggacaccgccctgtacgacggcaccgcctcg
gacggcgag
caggcgctctacatgagcatgccctgcccctgaccgacgccgaccaacaccgccggtccgacgcggcccgacgggtccg
aggcctcg
gagatctgggcccatgcggccgcaacaactacctcgactttggctgggacactttcagtgaggacaagaagcttcagaa
gcgtgctatcga
actcaaccagggacgtgcggc acaaatgggcatccttgctctcatggtgc
acgaacagttgggagtctctatccttccttaaaaatttaattttc
attagttgcagtcactccgctttggtttcgtaactataacggtctcgatgactgacaaaggtattcaaaaatgtagctt
gtcacgatttcacaatcg
acaagcctccccgaatcaagatctatttcggcgtgtgctgcatttcgaaggatggtcgaggccgtaagaataggcacct
gccatttcttggca
attggcctaccataagatcctttgccacttccaggtggaccgagaatcgtgatatttctcaacaaacggaagctcgtac
ggctcatccgtgtcg
tggcaagattgttgttcggatgcttgaacttttgtgggtgctgacgattttcatggctc accgtc
aattctgtatgtcatcggatccgcgctctttttt
ggaatcttccacatgaaattatcacggggctatgagaaccaaggtattittagttttctgaactgcgcgcgatggcgcc
agaaaggtaccgaa
ttacatacacaggcgcatacggaagcagcgaaaatatctaaaccgctctttctccgacgtagaagcacggagacggcac
ccgagctcgat
aagaaatatcacgccggcagatccaattcggtgctactctacaaaatgaatacttcctaaatatgtggaagatctcaga
aaaagacaagccc
atccgtgactattcaccggaaccgcaagccactcatctcgaacaatcataaaaaatttggaaaccactttctctctcag
cgccagtacgaacat
gagggttgtttcgatcgcagttittgcggcaatgcttgctagcgctttcggcatcgacgccaatgttgacaatgcaatg
gacaaccgcgtcttg
caagctgc aagcgatgtaccgagcgatatgccgagcgatatgccgtc
agacattccgagcctcggagggccgactatgcaaccatctatg
actaccagctcgcttcccacaatcattggtgatcggggtggtgtaacgagctcgagtagccctctgctttccgcatgta
tcgctgttcctgctat
ggcaatggcctggcagctttatcagtagagaatcggaagttaaatagagacctggttcgtgccctacaaacgtaacctg
agtctactattccg
tcaaagtctctgttgtgtggaaaacctcgccagagtatttagttgttaaccgaagtattaagtcagtttgatcaaaaag
ttattcattacactccgt
attcgtttcgatactttattactttttccagcgtttcatcgccgtagtccggacttcgttcgagaaatgtctgtagttg
aggaagactgacaattgac
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuen5a ele0
SSIODUSOSSWENUOSODOSSIORESOSONSMoSetepeSSTSolSoSSSaeolueueoupSneooSSolSooStotoo

upoSSolSpoSetuoSSooSSoSSoSSoneuReSSeSueSoSeSeameSouSeoolpoSpelSTSSeeolepou000Se

oSSSeeneomeooSeuomealapSteSSoSeeoSSSeteSemelSoSSooSooupSSooSSIoneSoSploSpol
ReSSSmeopSTSSonpaeoSonelpoolSpeouolpouSTSSSoSeSoSoom000nelSeeSTSSeleSSeoSSSooSe

olSoSSmeooluTSTSSuooppeSeTSTSouSSSeueootmeneoweleSaeoSompoluoomeTSSommuSoSeleu
uoTSSSSouoSpomeleotoSoolpluSelepeTSSSemeleppoSooSonelSeeteSouSTSeeSSSooSmoSolSe
p cz
SeSooSoae Salem
aeuReSSITSSTSmSSoulpSSouneueueettoSSTSSSomeeSS000lSneSolSouSSoul
oSpeSeueneueooleSSeueSSoolSepineouSotoSoluSeSuSoupoSoolpeppeSneouoSeSooSTSoSe00
0m
uSaeuTSSolupeooSSTSTOStolSpeouneeootoneSooSelneSTSeS000meeSonewiSopSeotoSoouS
TSSSIpoSSeSSoSSTSooSeeouoSopi2oS0000lSoaeSSoSou000SeoomeSeSoSeSuSSTSSutmpeoolSp
le
uoluooSSoSSSotoSTSTSSoaeSoaeoSepSeopeoSottoSpeSopooSTSSeueoSeleteuoopnelSSSIeSo
ne 0z
oSutoSeSoloSSooutuSelSuSmou Rene oSpnompleTSSSSSSe ooSSouSue Beau& BSI&
outpou
ci
01
opSaeopSpeuelSeuuoSouneooSSouSouSSTSeameSSmulSSuuteoteneepeuSeouSeoSeSouSuSS
neSSIelSouSeueolSeReSSoSSTSpmeSpeolSolpeouSelSettoltoSeuReSeSeoluSeoaeSSSeSelSo
ltu
ReSTSuopeSSSoolututpepe000pSweSTSSIeooSSTSSSSSeSeuReSolamoSeemoSeSopeSoSSoSpe
SuSeSe San ou San oae SepTSSSoSTSoppploSe otolSoole 000lp Sae oSSoolppue
Soft olSoS
ooneuelneuReoSpuSmeoaeSolSoSuSpeoneelSTSooneoSeltuSSSueoSeoneueueSopoSaeupoS
oluoopooluooSomeSeoSolSopeueouSmISTemiSSooluoluaeoSSolueuemeooluouSuoSoSooSoltu
eooS
eSolSSoSeoSoonelSoSSouoSouoSeleomeeoomotolSeaeouSTSoSeReSTSSTSeolu000lu000lSeSp
eSpe
SneuemeltompulSoReSTSmoopteueueoSeaeoSpuSoutueSSeooleSSITSolueueRepSeSe000luelS
o
Rapp one aemelSomonee oSSuoSee o oSeSoone
000SetotoTSSoppealue000lSeSmeaeS0000SS
TSpleSSpeooneuaeSSmoSopppoSeoSpoSolSolSTSSeoolueSepSTSSSooSmeomoSetpouoopluSee
SepuotoSoSpoSeoopeonelSpp000lSeuoteoSeneeSSSououSepoomeSpSmoSelSoolSStoSoSpo
SeotmoneuotooppopetomoSeS000lnipeSaeooSSoSTSTSTeoluomppeutolSouolupemSeSeleS
uSoSopeueSeeoneRepeSeouSetpaemeSeSppeSooSeSeuReaeoSeopuoSooSSoSTSSeSeStepeueue
SuSoSSooeSeueueoSeuSeooSoSeoSompSuoSee000moneleoSeeSolSeletuaeSSeSooneneepSeoSe

000SSSuStpetweReoluouopeuReoaeSSeoteltpooupeuSeSepaeoSeaeSmeomoSoSeSeouleuSole
Rae ououpleaeSomeue Soup oi2SomppoSmoolutu0000lpou
oaemeeoaeS000lSeameomoopuSop
Re Se Nue mole olSoune STSpS000uSSoSSIeu Sae ope SoSTSTSeue oSeSuSole ou pope
San Stan Noel
oRepeoSSSeSoluSopSSomeeSoSeSeleoopoltpweeSSoSemeSuoSpeSoSTSopleSeleSeSeueuoaeSo
u
ZS

53
gccgcctgggcggcctgctgaaactctggctcaccgacgacccgcgcacggcgcggttcggtgatgccacgatcctcgc
cctgctggcg
aagatcgaagagaagcaggacgagcttggcaaggtcatgatgggcgtggtccgcccgagggcagagccatgactttttt
agccgctaaaa
cggccggggggtgcgcgtgattgccaagcacgtccccatgcgctccatcaagaagaggcacttcgagctgtaagtacat
caccgacgag
caaggcaagacgatccgcgcctgttttattgagaacgttgttcgtgttggcctcaatggtagcgatgcgtcattcagcg
aagttctggtgctga
tgatgtggttcgctttgccactggtcaatgtggtaagcccgtgtaatgtcagtaacctttttactgatctcagcttgag
cacggtcgctgatgagc
ttatccatggccccacggtaacggatatgatcctctagggcgttgacaaattctttgtcggttttcatggggtagacgt
cagtgaccagggaac
gtctccctacaaaaagttgcgcatactttgctccgttgtcgacggcaggggtgtcggaccagattgtatctgtggcaac
ggcctcattgcgtc
aatggacctttaaagccgggaaacgagacttgaaatgtttacgtagaggtgcattatagacctcacgcgcatattgagt
ggttgcaaaaatgg
ttttgcgaacggtatcactggagacccatgccaggcaaggacggagggcatcataatcatgttcattgcgttggacagc
atgcttgttacacg
taagtatacggtcgagagcatcgtgacgttgagaattacagatggcaacgtgtcggtgcatgtattggccgagattgtc
aaatcgtggctcaa
tatatggcatgccaggtaagtcatggatgtcggtatgccattcagccttcatgtcgatttcgttgtcgagaactgatgg
gtcccagtcaacgtca
gaagtaagaacaacatggggaagggaatgaagttcatgactggtgggtgcgcgcatatccatgtacggaagaccctgtc
gtatgtttagtg
ggatgatgtaatcatccagggtaacaatacgttggttgccgcccacggtacgagaccgatcttgaacacaattgtggca
atgttcaagttggg
cgctggaatggatggttttacctttaccaagatgcgcgtattgatgcatgataacaacaatgggccctcgttgcgattc
gacaagaccggcag
ccgtgacaatgtctagattggacaaggtatgttcatttataccggttatgttggctgaacgtccagttttgtgcagtac
ggtaacatcactccccg
caagtccaccgttggcaccgcgatcgacaagtgcagatgtggtattttgtactgtgtgtcgggagacttggtactgtaa
cacatttgactgaag
tgacttggtggtaccatgaggacggagggaacccgttgatacggtgttgcgtgacgctgcaagtaccttacgtatgtcg
cctggatccatac
gtccaacccgatcagaaagatgtgctaggagttccgtttctggtgcacaatcatgaaaggtatccacgggagttccatt
gttgtcgtccgggg
ctgcgtcgcgactgccagttgcatgtacatgcgccatc aagggacgtgccaatcc
attactaaccgggagaaccttagctggggcagccaa
tccctgaagtatagccttagcatcatctgagagctggttccacatatctctagggatataaggtcgttcacggttagtg
ggcgtactatgatgtc
ggttattagtacaattctccctgcgggcatgcgcattggcttcatagagtacggaaggtgacaagtcaatattgtagtt
aacatccggatcagt
gtcaaagtctgtaggatggtaagaaagatcagtagaatgaatactacgcttttccttgggactacgggaattagagaag
ttgtttcctttattgta
gagtgacgctgaagcaagtagaagactaaggtaactctcgtagctaataggattacctcctttggctaggtcaagagtg
gctgtgatcttcact
tgacaaagttccggtacattgtggacagcattctccaaaagactaagacacagttgctttgggagttgctcagccattg
gtacagtattgtggt
agatacaaaggtggttcttccaatgaaggataaatccttctgctgtgcctgtccatgaggatccatatttcgccgtagt
taggtaaccaagcgtg
gtggctgaactgatcttcgcacttgctgattccgtatagtgtttgacaactttacaaaacacttcttgcgcagttcgct
ctagcctggacagagaa
gggacaatcttcgtcgttaagactcgtgcgaatagcaaaagatcacaaaatagcacatcggcaccgaccaacgattatt
tccaaggaaaaa
aagaatgcttcactacaagaaattgtgtcatccctatacagagtcttgttactgtgacagaaaattgatggaagatgtg
gcggattgcctttaca
ctagccaacttgttcgactaattgcagcttcttctgagaggcttcaccgagtaacgcgaagaacaccggtgtctcgtac
atgctcgtcggtga
acgctcgtccaatgacaccccccactttgtatcaatatcccaacttggtagtgaactggaatgatacatgcaatttcgc
gccgcatcaacagcc
Date Recue/Date Received 2021-08-17

54
acgggccaccatcgacgaatagactcgg
SEQ ID NO: 4¨ pPtUMPScA2
tcgagctggttgccctcgccgctgggctggcggccgtctatggccctgc aaacgcgcc
agaaacgccgtcgaagccgtgtgcgagacac
cgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctcactgac agatgaggggcggacgttgac
acttgaggggcc
gactcacccggcgcggcgttgac agatgaggggc aggctcg atttcggccggcgacgtggagctggc c
agcctcgc aaatcggcgaaa
acgcctgatatacgcgagtttc cc ac ag atgatgtggac aagc ctggggataagtgccctgcggtattgac
acttgaggggcgcgactact
gac agatgaggggcgcgatc cttgac acttgaggggc ag agtgctgac ag atgaggggcgc ac
ctattgac atttgaggggctgtc c ac a
ggc agaaaatcc agc atttgc aagggtttccgcccgtttttcggcc accgctaacctgtcttttaac
ctgcttttaaacc aatatttataaaccttg
ffittaacc agggctgcgcc ctgtgcgcgtgaccgcgc acgccgaaggggggtgcccccc cttctcgaac
cctccc ggtcgagtgagcga
ggaagcacc
agggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacgggg
atatttt
tataattatttittttatagffittagatcttatttttagagcgccttgtaggcctttatccatgctggttctagagaa
ggtgttgtgacaaattgcccttt
cagtgtgacaaatc accctc aaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctc
agaagaagctgffitttcac
aaagttatccctgcttattgactatttttatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtcat
ggcggaaacagcggttat
caatcac
aagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgggatcaaa
aacgt
atgctgtatctgttcgttgacc agatc agaaaatctgatggc accctac aggaac
atgacggtatctgcgagatccatgttgctaaatatgctg
aaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagittcgcggggaaggaagtggf
fitttatcgccctg
aagaggatgccggcgatgaaaaaggctatgaatatttccttggtttatcaaacgtgcgcacagtccatccagagggctt
tacagtgtacatat
caaccc atatctcattcccttctttatcgggttacagaaccggtttacgc agtttcggcttagtgaaac
aaaagaaatcacc aatc cgtatgc c at
gcgtttatacgaatccctgtgtc agtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatc
atagagcgttaccagctg
cctc aaagttac c agcgtatgcctgacttccgccgc cgcttcctgc aggtctgtgttaatgagatc aac
agc agaactcc aatgcgc ctctc at
acattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgac
aggatagtctgagggttatct
gtcacagatttgagggtggttcgtcac atttgttctgacctactgagggtaatttgtc ac
agtatgctgtttccttc agc ctgc atggattttctc at
acttittgaactgtaatttttaaggaagcc aaatttgagggc agtttgtc ac
agttgatttccttctctttcccttcgtc atgtgacctgatatcgggg
gttagttcgtcatcattgatgagggttgattatcacagtttattactctgaattggctatccgcgtgtgtacctctacc
tggagffittcccacggtg
gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttctttgcttcctcgccagttcgctcgctat
gctcggttacacggctgc
ggcgagcatc
acgtgctataaaaataattataatttaaatttittaatataaatatataaattaaaaatagaaagtaaaaaaagaaatt
aaagaaaa
aatagttittgffitccgaagatgtaaaag actctagggggatcgc c aac
aaatactaccttttaccttgctcttcctgctctc aggtattaatgcc
gaattgtttc atcttgtctgtgtagaagac c ac ac acgaaaatcctgtg attttac
attttacttatcgttaatcgaatgtatatctatttaatctgctttt
cttgtctaataaatatatatgtaaagtacgattttgttgaaatttittaaacattgtttatttitttttcttcattccg
taactcttctaccttctttatttactt
Date Recue/Date Received 2021-08-17

55
tctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacga
ggcgcgtgtaagtta
caggcaagcgatcctagtacactctatatttlittatgcctcggtaatgattttcatttitttittccacctagcggat
gactctttlittttcttagcgatt
ggcattatcacataatgaattatacattatataaagtaatgtgatttcttcgaagaatatactaaaaaatgagcaggca
agataaacgaaggcaa
agatgacagagcagaaagccctagtaaagcgtattacaaatgaaaccaagattcagattgcgatctctttaaagggtgg
tcccctagcgata
gagcactcgatcttcccagaaaaagaggcagaagcagtagc agaac
aggccacacaatcgcaagtgattaacgtccacacaggtatagg
gtttctggaccatatgatacatgctctggccaagcattccggctggtcgctaatcgttgagtgcattggtgacttacac
atagacgaccatcac
accactgaagactgcgggattgctctcggtcaagcttttaaagaggccctaggggccgtgcgtggagtaaaaaggtttg
gatcaggatttgc
gcctttggatgaggcactttccagagcggtggtagatcMcgaacaggccgtacgcagttgtcgaacttggtttgcaaag
ggagaaagtag
gagatctctcttgcgagatgatcccgcattttcttgaaagctttgcagaggctagcagaattaccctccacgttgattg
tctgcgaggcaagaat
gatcatcaccgtagtgagagtgcgttcaaggctcttgcggttgccataagagaagccacctcgcccaatggtaccaacg
atgttccctccac
caaaggtgttcttatgtagttttacacaggagtctggacttgacgctagtgataataagtgactgaggtatgtgctctt
cttatctccttttgtagtgt
tgctcttattttaaacaactttgcggifitttgatgactttgcgattttgttgttgctttgcagtaaattgcaagattt
aataaaaaaacgcaaagcaat
gattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgcataaacgctggtcatgaaatgacgaaggcta
tcgccattgcac a
gtttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggtt
tcttctcaggct
atcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggtt
atacaattgaa
caaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttg
ctgcccataaaggtg
gcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaagg
taacgacccccaggg
aacagcctcaatgtatcacggatgggtaccagatcttcatattcatgc
agaagacactctcctgcctttctatcttggggaaaaggacgatgtc
acttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactg
agttaatgggcaaattt
gatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtca
tagttattgacagcg
cgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtt
tgactacacctccgc a
ctgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgc
ttaccaaatacagcaata
gcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacg
tgaaacggatga
agttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggaga
aatgctctttctatttg
ggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgtta
ttccaaaacatacgc
tcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtagg
agtaatggctcgcg
gtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaa
gacctctcgggtatgg
tcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtc
aacagacaccggcg
ttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtg
attatcgtgttctgg
ttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacg
tggtcagcgttatg
Date Recue/Date Received 2021-08-17

56
caagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattac
ccgctgtatcaacacc
gccaaattgcctaaatcagttgttgctcttttttctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaa
aagcctttacagataaa
gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagtta
tcactcttttaactt
ctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgta
taagggcgataaaat
..
ggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaag
ccagcaccctgatg
cgacctcgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaat
attctctctgggcccactgt
tccacttgtatcgtcggtctgataatcagactgggaccacggtcccactcgtatcgtcggtctgattattagtctggga
ccacggtcccactcgt
atcgtcggtctgattattagtctgggacc acggtcccactcgtatcgtcggtctgataatcagactgggacc
acggtcccactcgtatcgtcgg
tctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgta
tcgtcggtctgattatta
gtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctg
attattagtctgggac
cacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagc
gtgagactacgattcc
atcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgc
ctgctgtggattgct
gctgtgtcctgcttatccacaacattttgcgcacggttatgtggac aaaatacctggttaccc
aggccgtgccggcacgttaaccgggctgc a
tccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgt
attgtctgaagttgtt
tttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctcca
cgcacgttgtgatatgtag
atgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgcta
tttatgaaaattttccg
gtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgaca
ggtgctgaaagcgagctt
tttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtccgagctcatcgctaataacttc
gtatagcatacattatacg
aagttatattcgatgcggccgcaaggggttcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggca
tcagagcagatt
gtactgagagtgcacc atatgcggtgtgaaataccacac
agatgcgtaaggagaaaataccgcatcaggcgccattcgccattc agctgcg
caactgttgggaagggcgatcggtgcgggcctcttcgctattacgcc agctggcgaaagggggatgtgctgc
aaggcgattaagttgggt
aacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattgtaatacgactcactatagggcgaattc
gagctcggtaccc
ggggatcctctagagtcgacctgcaggcatgcaagcttgagtattctatagtctcacctaaatagcttggcgtaatcat
ggtcatagctgtttcc
tgtgtgaaattgttatccgctcacaattccacac aacatacgagccggaagc
ataaagtgtaaagcctggggtgcctaatgagtgagctaact
cacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaa
cgcgaaccccttgc
ggccgcccgggccgtcgaccaattctcatgtttgacagcttatcatcgaatttctgccattcatccgcttattatcact
tattcaggcgtagcaac
caggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattc
attaagcattctgcc
gacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatc
agcaccttgtcgccttgcgtataatatttgcccatggtga
aaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagac
gaaaaacatattc
tcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgcc
ggaaatcgtcgtggt
Date Recue/Date Received 2021-08-17

57
attcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcac
cagctcaccgtcttt
cattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgctta
tttttctttacggtct
ttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttc
tttacgatgccattgg
gatatatcaacggtggtatatccagtgatttlittctccattttagcttccttagctcctgaaaatctcgataactcaa
aaaatacgcccggtagtga
tcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggc
ttcccggtatcaacagg
gacaccaggatttatttattctgcgaagtgatcttccgtcac
aggtatttattcgcgataagctcatggagcggcgtaaccgtcgc acaggaag
gacagagaaagcgcggatctgggaagtgacggacagaacggtcaggacctggattggggaggcggttgccgccgctgct
gctgacggt
gtgacgttctctgttccggtcacaccacatacgttccgccattcctatgcgatgcacatgctgtatgccggtataccgc
tgaaagttctgcaaa
gcctgatgggacataagtccatcagttcaacggaagtctacacgaaggiftttgcgctggatgtggctgcccggcaccg
ggtgcagtttgtg
atgccggagtctgatgcggttgcgatgctgaaacaattatcctgagaataaatgccttggcctttatatggaaatgtgg
aactgagtggatatg
ctgtttttgtctgttaaacagagaagctggctgttatccactgagaagcgaacgaaacagtcgggaaaatctcccatta
tcgtagagatccgc a
ttattaatctcaggagcctgtgtagcgtttataggaagtagtgttctgtcatgatgcctgcaagcggtaacgaaaacga
tttgaatatgccttcag
gaacaatagaaatcttcgtgcggtgttacgttgaagtggagcggattatgtcagcaatggacagaacaacctaatgaac
acagaaccatgat
gtggtctgtccttttacagccagtagtgctcgccgcagtcgagcgacagggcgaagcccatcgatactagcttgattgg
gatatctcgctcat
gtttgtcgcgtgctatgtctattaggtactttgaacctacgttcgtacttgtataatatgatcatcgtattatcgtatt
catccgtccagcgcaaaat
gcattagcagctagtcctagcgtgcggagctacctggacaggtgcatgacggatgcgtgtccttcagtgactttctaat
taacagtaacttcttt
acttatgtttcagtttgtaagaagcgggattcgctcgtcgcttgacatctgattggactgcgtcggcacgtgaaaacta
cattgtgaaatctgcta
aaactccgggtatctctgacacaaaacgattcggcttcgcaatttcaacattacggtcaaggctaacgtatctttctcg
gtcaacttcagattatg
ccgattaaattgtcgtagctttcaaggcgttttgagtactgcggcagttgttgaacctgcaaggagaagatctcgacaa
cagaataaagcgaa
aaatgggtctcatgcactaacactcaggcctccctcataatctctgtttgagtttaccaacaacacatatatacatttc
gacaaaatgaccactct
tgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcacc
accgacacc
gtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttcc
ccgacgacga
atcggacgacgaatcggacgacggggaggacggcgacccggactcccggacgttcgtcgcgtacggggacgacggcgac
ctggcgg
gcttcgtggtcgtctcgtactccggctggaaccgccggctgaccgtcgaggacatcgaggtcgccccggagcaccgggg
gcacggggt
cgggcgcgcgttgatggggctcgcgacggagttcgcccgcgagcggggcgccgggcacctctggctggaggtcaccaac
gtcaacgc
accggcgatccacgcgtaccggcggatggggttcaccctctgcggcctggacaccgccctgtacgacggcaccgcctcg
gacggcgag
caggcgctctacatgagcatgccctgcccctgaccgacgccgaccaacaccgccggtccgacgcggcccgacgggtccg
aggcctcg
gagatctgggcccatgcggccgcaacaactacctcgactttggctgggacactttcagtgaggacaagaagcttcagaa
gcgtgctatcga
actcaaccagggacgtgcggc acaaatgggcatccttgctctcatggtgc
acgaacagttgggagtctctatccttccttaaaaatttaattttc
attagttgcagtcactccgctttggtttcgtaactataacggtctcgatgactgacaaaggtattcaaaaatgtagctt
gtcacgatttcacaatcg
Date Recue/Date Received 2021-08-17

58
acaagcctccccgaatcaagatctatttcggcgtgtgctgcatttcgaaggatggtcgaggccgtaagaataggcacct
gcc atttcttggc a
attggcctaccataagatcdttgccacttccaggtggaccgagaatcgtgatatttctcaacaaacggaagctcgtacg
gctcatccgtgtcg
tggcaagattgttgttcggatgcttgaacttttgtgggtgctgacgattttcatggctcaccgtcaattctgtatgtca
tcggatccgcgctctttitt
ggaatcttccacatgaaattatcacggggctatgagaacc
aaggttitttttagttttctgaactgcgcgcgatggcgcc agaaaggtaccgaa
ttacatacacaggcgcatacggaagcagcgaaaatatctaaaccgctctttctccgacgtagaagcacggagacggcac
ccgagctcgat
aagaaatatcacgccggcagatccaattcggtgctactctacaaaatgaatacttcctaaatatgtggaagatctcaga
aaaagacaagccc
atccgtgactattcaccggaaccgcaagccactcatctcgaacaatcataaaaaatttggaaaccactttctctctcag
cgccagtacgaacat
gagggttgtttcgatcgcagtttttgcggcaatgcttgctagcgctttcggcatcgacgccaatgttgacaatgcaatg
gacaaccgcgtcttg
caagctgc aagcgatgtaccgagcgatatgccgagcgatatgccgtc
agacattccgagcctcggagggccgactatgcaaccatctatg
actaccagctcgcttcccacaatcattggtgatcggggtggtgtaacgagctcgagtagccctctgctttccgcatgta
tcgctgttcctgctat
ggcaatggcctggcagctttatcagtagagaatcggaagttaaatagagacctggttcgtgccctacaaacgtaacctg
agtctactattccg
tcaaagtctctgttgtgtggaaaacctcgccagagtatttagttgttaaccgaagtattaagtcagtttgatcaaaaag
ttattcattacactccgt
attcgtttcgatacttaattacttittccagcgtttcatcgccgtagtccggacttcgttcgagaaatgtctgtagttg
aggaagactgacaattga
cacgaccaaaagagatagatctcgtgcgactgcttgaacagcggaaatcttgtcctccatagagcgaatttcggctcga
tcgagggcaataa
ctactccaactggcaaagcacccacatcgttgagcaaagtgtgcgactcacgaatggcggttcccgctgtgattacgtc
atctacaatcaga
actcgttttccttccaacgaagtcccgaccaatttaccaccttccccatgatcdttgcctcttttcggtcatacgcaaa
accgacatctacttcaa
aatcgttatacagagcgcttccaacgacagcacctagagaaatacccttgtatgcaggaccaaaaatcacatcaaaatt
gacttggttgggcc
cagcagctaataattccgaggacatgatagtcgaagcataggattcccaagcttgcttaacgcagcgccagaagcaaaa
agaccggcgtt
gaaaaaatatggagaggtgcggccgcttttcagcacaaaagagccgaatttgagaacaccttgagacagactaaattca
agaaactcgcgt
tgatagagtttaatatcttcgtctgaactcttittcatcatgtgtgcggccacgacttggtcccgagctttctgaatcc
tctcctgcaattcttttgca
gcctgcgctgggtccgtagctttgctgatacccctagacacgggaattagcatgcaagtcccctttgtattcaatccag
cctgcgctgcttcta
gaagatctccaccttgagcaccaacgccgggtgctagaatccaggtgtcgtcgcctgcagcctttctcgctttggacaa
ggccactggatct
gtggccccgacaacgagtcccaatgaactctcggtctgctgagcccattccgagccaacaagcttggcaattctttcgt
ataaacattcatttg
aacgtaatcccagagctaaaaaatcgttggatccaggatttgacgttttgcacagcaaaaatgctcdttgtgaacgtac
ttttctgtaacaaag
ggactgactgagtcccatcccatcagtggtgaaagcgtgacacagtctgcacccaaaccatagcacgcttcggcgtagg
ccgcagcggtc
gagccaatgtcgccgcgcttgacatccaacaaaatcggcacatcatccggtattatgttttgacaaactcgtcgcagaa
ccgctatccctccg
tcgcctaacgcctcgaaaaaggcagcattgggtttgtagcaggccgtgtaaggcaatgttgcgtcgaccaacgttttgc
aaaaggtaaagg
ccgcgtcacagcgattlitttccggcacgccttcccatccgtccgcaaacagctctttctcgtgcgggtctagaccaac
gcacaagagagagt
tgactgcggcgactcgagcttcaagctttgatcgaaaagagggggtggccatggtgatagctcccaataattgtttgat
ccgggaatcttgtg
aatttgtcgtagagggaccagatcagagaaaagctgtctgtgagtagacacttcgtcactgatttttgtggcggaaagt
caaagttcgtatgga
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuen5a ele0
USSIDoSoltulSoulloaelReu oSpSoutSoSuSTSSouleSuSoomeSSReSSounete
oaelStStpetReeSpe
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olumelnouTReottmReoolSoeutoSSuSmiSSoaelemeoutelneuounneReplteuouSTSooReono
B&B aeSoueS SITS opoonSpe melete otanelSoSote Sue oouppoupuSteSSpeStoSonSuS
outueoSttpeaemeSipleSoouReSoulnae000SooSuSSITSoupemelSneooluolueltuteSSSTReu
ltelSolSpoouReeSSoulteoomeoSoSoSTSSSTSSpeteouReaweSSReeSSStememeReelSeuReolSou
cz
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oonolSooStoSpoupo
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SSmeoptSSoStoaeoSonelpoolSpeouolpouSISSSoReSoSoom000nelReetneleneoSSSooReolS
onmeoomtStpoupeReltSouSneueootmeneoweleSaeoSompole oomelnommuSoReleneo
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meleppoSooSonelReeteSoutReeSSSootuoSolRepRe
SooSoaeoRetepemeReSSITSSTSmnoeuonouneueueuttoSSTSSSomeeSS000lt_TeSolSouSSoupS
peReemeReooluneueSSoolSepleleaeSotoSoluReSuSoupoSoolpeppetleou oReSootSoRe
000meS
melnoleweooSttentolSpeounee ootoneSooRelnetReS0000eueSonepuSopReotoSoouSTS
StponeSSoStSooSeuaeoSoplSoS0000lSoaeSSoSou000ReoomeReSoReSuSSTSSutmpeooltolue
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oopum2SteSone o
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ToSaeopSpeuelReelpSoune oonaeSouStReameStuelnualeoteneepeuReouReoReSoutISSu
6S

60
tcc atacgtc caacccgatc agaaagatgtgctaggagttccgtttctggtgc ac aatc
atgaaaggtatccacgggagttccattgttgtcgt
ccggggctgcgtcgcgactgcc agttgc atgtac atgcgcc atc aagggacgtgcc
aatccattactaaccgggagaaccttagctgggg
cagccaatccctgaagtatagccttagcatc atctgagagctggttcc
acatatctctagggatataaggtcgttcacggttagtgggcgtact
atgatgtcggttattagtacaattctccctgcgggcatgcgcattggcttcatagagtacggaaggtgac
aagtcaatattgtagttaacatccg
gatcagtgtcaaagtctgtaggatggtaagaaagatc agtagaatgaatactacgcttttccttgggactac
gggaattagagaagttgtttcct
ttattgtagagtgacgctgaagcaagtagaagactaaggtaactctcgtagctaataggattacctcctttggctaggt
c aagagtggctgtga
tcttcacttgacaaagttccggtacattgtggacagc attctccaaaagactaagac
acagttgctttgggagttgctcagcc attggtacagta
ttgtggtagatacaaaggtggttcttccaatgaaggataaatccttctgctgtgcctgtccatgaggatcc
atatttcgccgtagttaggtaacc a
agcgtggtggctgaactgatcttcgcacttgctgattccgtatagtgtttgacaactttacaaaacacttcttgcgcag
ttcgctctagcctggac
agagaagggac aatcttcgtcgttaagactcgtgcgaatagc
aaaagatcacaaaatagcacatcggcaccgaccaacgattatttccaag
gaaaaaaagaatgcttcactacaagaaattgtgtcatccctatacagagtcttgttactgtgacagaaaattgatggaa
gatgtggcggattgc
ctttacactagcc aacttgttcgactaattgcagcttcttctgagaggcttcaccgagtaacgcgaagaac
accggtgtctcgtacatgctcgtc
ggtgaacgctcgtc caatgac ac ccccc actttgtatc aatatccc aacttggtagtgaactggaatgatac
atgcaatttcgcgccgcatcaa
cagccacgggccacc atcgacgaatagactcgg
SEQ ID NO: 5¨ pPtPRAPHCH
tcgagctggttgccctcgccgctgggctggcggccgtctatggccctgc aaacgcgcc
agaaacgccgtcgaagccgtgtgcgagacac
cgcggccggccgccggcgttgtggatacctcgcggaaaacttggccctc actgac agatgaggggcgg
acgttgacacttgaggggcc
gactcacccggcgcggcgttgac agatgaggggc aggctcg atttcggccggcgacgtggagctggc c
agcctcgc aaatcggcgaaa
acgcctgattttacgcgagtttc cc ac ag atgatgtggac aagc ctggggataagtgccctgcggtattgac
acttgaggggcgcgactact
gacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgagg
ggctgtccac a
ggcagaaaatccagcatttgcaagggtttccgcccgtttttcggcc
accgctaacctgtcttttaacctgcttttaaaccaatatttataaaccttg
ttittaacc agggctgcgcc ctgtgcgcgtgaccgcgc acgccgaaggggggtgcccccc cttctcgaac
cctcccggtcgagtgagcga
ggaagcacc
agggaacagcacttatatattctgcttacacacgatgcctgaaaaaacttcccttggggttatccacttatccacgggg
atatttt
tataattattattttatagttittagatcttcttitttagagcgccttgtaggcctttatccatgctggttctagagaa
ggtgttgtgacaaattgcccttt
cagtgtgacaaatc accctc aaatgacagtcctgtctgtgacaaattgcccttaaccctgtgacaaattgccctc
agaagaagctgttitttc ac
aaagttatccctgcttattgactUttittatttagtgtgacaatctaaaaacttgtcacacttcacatggatctgtcat
ggcggaaacagcggttat
caatcac
aagaaacgtaaaaatagcccgcgaatcgtccagtcaaacgacctcactgaggcggcatatagtctctcccgggatcaaa
aacgt
atgctgtatctgttcgttgacc agatc agaaaatctgatggc accctac aggaac
atgacggtatctgcgagatccatgttgctaaatatgctg
aaatattcggattgacctctgcggaagccagtaaggatatacggcaggcattgaagagtttcgcggggaaggaagtggt
titttatcgccctg
Date Recue/Date Received 2021-08-17

61
aagaggatgccggcgatgaaaaaggctatgaatct-t-ttccttggt-
ttatcaaacgtgcgcacagtccatccagagggct-ttacagtgtacatat
caacccatatctcattcccttct-ttatcgggttacagaaccggt-
ttacgcagtttcggcttagtgaaacaaaagaaatcaccaatccgtatgccat
gcgtttatacgaatccctgtgtc agtatcgtaagccggatggctcaggcatcgtctctctgaaaatcgactggatc
atagagcgttaccagctg
cctcaaagttaccagcgtatgcctgacttccgccgccgcttcctgcaggtctgtgttaatgagatc aacagc
agaactcc aatgcgcctctc at
acattgagaaaaagaaaggccgccagacgactcatatcgtattttccttccgcgatatcacttccatgacgacaggata
gtctgagggttatct
gtcacagatttgagggtggttcgtcacatttgttctgacctactgagggtaatttgtcacagttttgctgtttccttca
gcctgcatggattttctcat
act-tt-ttgaactgtaatttttaaggaagccaaatttgagggcagt-ttgtcacagttgatttccttctct-
ttcccttcgtcatgtgacctgatatcgggg
gttagttcgtcatcattgatgagggttgattatcacagt-
ttattactctgaattggctatccgcgtgtgtacctctacctggagt-ttttcccacggtg
gatatttcttcttgcgctgagcgtaagagctatctgacagaacagttcttct-
ttgcttcctcgccagttcgctcgctatgctcggttacacggctgc
ggcgagcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaaaaatagaaagtaaa
aaaagaaattaaagaaaa
aatagt-t-tttgt-tttccgaagatgtaaaagactctagggggatcgccaacaaatactacct-
tttaccttgctcttcctgctctcaggtattaatgcc
gaattgt-
ttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttacttatcgttaatcgaatgtatatc
tatttaatctgctt-tt
cttgtctaataaatatatatgtaaagtacgct-ttttgttgaaattt-tttaaacct-ttgt-ttatt-tt-
tttttcttcattccgtaactcttctaccttct-ttatttactt
tctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacga
ggcgcgtgtaagtta
caggcaagcgatcctagtacactctatatt-tt-
tttatgcctcggtaatgattttcattffitttttccacctagcggatgactct-tt-ttt-tttcttagcgatt
ggcattatcacataatgaattatacattatataaagtaatgtgatttcttcgaagaatatactaaaaaatgagcaggca
agataaacgaaggcaa
agatgacagagcagaaagccctagtaaagcgtattacaaatgaaaccaagattcagattgcgatctctttaaagggtgg
tcccctagcgata
gagcactcgatcttcccagaaaaagaggcagaagcagtagc agaac
aggccacacaatcgcaagtgattaacgtccacacaggtatagg
gt-
ttctggaccatatgatacatgctctggccaagcattccggctggtcgctaatcgttgagtgcattggtgacttacacat
agacgaccatcac
accactgaagactgcgggattgctctcggtcaagct-t-
ttaaagaggccctaggggccgtgcgtggagtaaaaaggtttggatcaggatttgc
gcct-ttggatgaggcactttccagagcggtggtagatct-
ttcgaacaggccgtacgcagttgtcgaacttggtttgcaaagggagaaagtag
gagatctctcttgcgagatgatcccgcattt-tcttgaaagct-
ttgcagaggctagcagaattaccctccacgttgattgtctgcgaggcaagaat
gatcatcaccgtagtgagagtgcgttcaaggctcttgcggttgccataagagaagccacctcgcccaatggtaccaacg
atgttccctccac
caaaggtgttcttatgtagttttacacaggagtctggacttgacgctagtgataataagtgactgaggtatgtgctctt
cttatctccttttgtagtgt
.. tgctcttattttaaacaactttgcggifitttgatgactttgcgattttgttgttgct-
ttgcagtaaattgcaagatttaataaaaaaacgcaaagcaat
gattaaaggatgttcagaatgaaactcatggaaacacttaaccagtgc
ataaacgctggtcatgaaatgacgaaggctatcgccattgcac a
gt-
ttaatgatgacagcccggaagcgaggaaaataacccggcgctggagaataggtgaagcagcggatttagttggggt-
ttcttctcaggct
atcagagatgccgagaaagcagggcgactaccgcacccggatatggaaattcgaggacgggttgagcaacgtgttggtt
atacaattgaa
caaattaatcatatgcgtgatgtgtttggtacgcgattgcgacgtgctgaagacgtatttccaccggtgatcggggttg
ctgcccataaaggtg
gcgtttacaaaacctcagtttctgttcatcttgctcaggatctggctctgaaggggctacgtgttttgctcgtggaagg
taacgacccccaggg
Date Recue/Date Received 2021-08-17

62
aacagcctcaatgtatcacggatgggtaccagatcttcatattcatgcagaagacactctcctgcctttctatcttggg
gaaaaggacgatgtc
acttatgcaataaagcccacttgctggccggggcttgacattattccttcctgtctggctctgcaccgtattgaaactg
agttaatgggcaaattt
gatgaaggtaaactgcccaccgatccacacctgatgctccgactggccattgaaactgttgctcatgactatgatgtca
tagttattgacagcg
cgcctaacctgggtatcggcacgattaatgtcgtatgtgctgctgatgtgctgattgttcccacgcctgctgagttgtt
tgactacacctccgca
..
ctgcagtttttcgatatgcttcgtgatctgctcaagaacgttgatcttaaagggttcgagcctgatgtacgtattttgc
ttaccaaatacagcaata
gcaatggctctcagtccccgtggatggaggagcaaattcgggatgcctggggaagcatggttctaaaaaatgttgtacg
tgaaacggatga
agttggtaaaggtcagatccggatgagaactgtttttgaacaggccattgatcaacgctcttcaactggtgcctggaga
aatgctctttctatttg
ggaacctgtctgcaatgaaattttcgatcgtctgattaaaccacgctgggagattagataatgaagcgtgcgcctgtta
ttccaaaacatacgc
tcaatactcaaccggttgaagatacttcgttatcgacaccagctgccccgatggtggattcgttaattgcgcgcgtagg
agtaatggctcgcg
gtaatgccattactttgcctgtatgtggtcgggatgtgaagtttactcttgaagtgctccggggtgatagtgttgagaa
gacctctcgggtatgg
tcaggtaatgaacgtgaccaggagctgcttactgaggacgcactggatgatctcatcccttcttttctactgactggtc
aacagacaccggcg
ttcggtcgaagagtatctggtgtcatagaaattgccgatgggagtcgccgtcgtaaagctgctgcacttaccgaaagtg
attatcgtgttctgg
ttggcgagctggatgatgagcagatggctgcattatccagattgggtaacgattatcgcccaacaagtgcttatgaacg
tggtcagcgttatg
caagccgattgcagaatgaatttgctggaaatatttctgcgctggctgatgcggaaaatatttcacgtaagattattac
ccgctgtatcaacacc
..
gccaaattgcctaaatcagttgttgctctttittctcaccccggtgaactatctgcccggtcaggtgatgcacttcaaa
aagcctttacagataaa
gaggaattacttaagcagcaggcatctaaccttcatgagcagaaaaaagctggggtgatatttgaagctgaagaagtta
tcactcttttaactt
ctgtgcttaaaacgtcatctgcatcaagaactagtttaagctcacgacatcagtttgctcctggagcgacagtattgta
taagggcgataaaat
ggtgcttaacctggacaggtctcgtgttccaactgagtgtatagagaaaattgaggccattcttaaggaacttgaaaag
ccagcaccctgatg
cgacctcgttttagtctacgtttatctgtctttacttaatgtcctttgttacaggccagaaagcataactggcctgaat
attctctctgggcccactgt
tccacttgtatcgtcggtctgataatcagactgggaccacggtccc
actcgtatcgtcggtctgattattagtctgggaccacggtcccactcgt
atcgtcggtctgattattagtctgggacc acggtcccactcgtatcgtcggtctgataatcagactgggacc
acggtcccactcgtatcgtcgg
tctgattattagtctgggaccatggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgta
tcgtcggtctgattatta
gtctggaaccacggtcccactcgtatcgtcggtctgattattagtctgggaccacggtcccactcgtatcgtcggtctg
attattagtctgggac
cacgatcccactcgtgttgtcggtctgattatcggtctgggaccacggtcccacttgtattgtcgatcagactatcagc
gtgagactacgattcc
atcaatgcctgtcaagggcaagtattgacatgtcgtcgtaacctgtagaacggagtaacctcggtgtgcggttgtatgc
ctgctgtggattgct
gctgtgtcctgcttatccacaacattttgcgcacggttatgtggac
aaaatacctggttacccaggccgtgccggcacgttaaccgggctgca
tccgatgcaagtgtgtcgctgtcgacgagctcgcgagctcggacatgaggttgccccgtattcagtgtcgctgatttgt
attgtctgaagttgtt
tttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctcca
cgcacgttgtgatatgtag
atgataatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgcta
tttatgaaaattttccg
gtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctctgaaaagaaaggaaacgaca
ggtgctgaaagcgagctt
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuari5a ele0
UTOSIDITUUSTSUUMMURPSTSMOSSNSOSIDUSSUUSINUMSUOSNSNOSNIUSSSORPUReultuReolutuum
molpuelReanneuppputSuolpoltSotunautu otne aunpoupRenotSoSup Nam& Sum oS
pumoSoReoolSoopownSompi2op optuwepiSnaelSouSaupaeutuaelnumuoltuptSoSoltuS
popSoppluSSSuutpRepupSop000SueSoSSReauSoReSolReoSooSoptSulReooReaumpoltolSSTS
TetuomeReaumeSpupanaeuReauStuuoReoltuneSSoReSSTReauSaeutnotSouopueSupeamS
SuolpotuputuuSaeueuSanTSSoReuoSpotutuoltoutSulSueSSumuSoReltSpoReneoppeneu cz
oSoopReSulSomp000ppueuSSSolReounSanSoReuReSpeooputonpReauSuaeueutoltputo
SmuSSTReSpueSSTSpeuStupppoStpoSpeupuReSpompumeaptuSoSuSSotutolSuSSootu
SltuReoSTSSSomoSS000toSttuStoSotunneuSauoupi2ueSSamouSuopoolffeweauntutooS
otouReue toS o aupTSS o otult otu au SIBS oSpio onu ooSoouSaup au o au au
olSS o outopuS auSTS
TSSoutototoSooSooSuSS oSSOSSSuunpaene Inoue& auSSoutSueSSSpIeSSoSoReueSuReauS
z
Sueneau oS olSomelS oSSoSu Stu opReupSoS onumulne au olSoolpp STSueSotonemume
ne auS
ci
01
nuoueopi2SoomonSumoSSuReumooSompoplSoue opSootSaelpp annuReueSTSSIene mewl
utSuiSS000SoupueueuopeupSoppueutoopRelpouoSumpoopmpuutSuoomeTSSTSSamopmeS
StpootaaemouSweueopoSpueSpetanoReSuuoulnepuStolnanSpReooppulSooneueueu
TolnaupppmenoSltpueuepnoonumputtueSueonSonu op me oRe tunooneuuSoup ootp o
molSom op& opp 000ppe auuSTSSSue anTSTSSaumeStu optuRe omSaeueutuSoSuRe pope
one
TSSTSolSopueSSootaeueSulttuppeS oSipp au ooSau ouelSom mune oonepueSSSuppo
anupe
onepounueSouRetonneSSReooaeopmetStanue opeumS au oonnep oolSuSueSuuSoSSSSSaeue
utStu000tnepwelSotpoSoltpaeoRe op on oRe o oSopeSpaeututu onanu au o oSueStu
auS
ootoneoSuenuoneult_itaelReoSopopeooSpooSomoSounememelpotampuomoSnuemSoneo
oSulSone oneme ompuoSoop one ootoppeS opuoReaututuompuoaeSolSooSSS000SooSS
otp o o oueS oS o ope Spew otoReootSoltoomenSolReoomoS000tau opSoSuSotpune au
o
puepReSTReSpepoSTSSSSpoReueltSumpoSueSSooReSoupmu au au o oneu au opS o
oputpueSTSTST
oolutoRepolStu owelSonuoSuiumpaeoplReppnelSuSuoSueotu one Sp auS olReSupp
Nunn
maelnopReSoneuSoSSReppeopeSoupultputReoonauSanuelSuSauSaeolSuompuSSReooSan
TSSSITSueneSonueotottuSSSSSueuSoStoReooSounepSolppoSSSoSTSSopSoSSSuuSSSuSpueo
otoRe one ooSone ooS one op oSompueuRenuelSotuRe aum o pupae STSTSS otup
otSuRetaelS
nee Rae onoSpianuoStonnoTSTSSSoSSITSTSSSoRe olSoSouSSSSue oSoonotuSonepuSue
Somme aup oSwelSouampupS opReSoolReuStuemetueSSTSooltputopppomSoltopoStu
9

64
aaactccgggtatctctgacacaaaacgattcggcttcgcaatttcaacattacggtcaaggctaacgtatctttctcg
gtcaacttcagattatg
ccgattaaattgtcgtagctttcaaggcgttttgagtactgcggcagttgttgaacctgcaaggagaagatctcgacaa
cagaataaagcgaa
aaatgggtctcatgcactaacactcaggcctccctcataatctctgtttgagtttaccaacaacacatatatacatttc
gacaaaatgaccactct
tgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcacc
accgacacc
gtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttcc
ccgacgacga
atcggacgacgaatcggacgacggggaggacggcgacccggactcccggacgttcgtcgcgtacggggacgacggcgac
ctggcgg
gcttcgtggtcgtctcgtactccggctggaaccgccggctgaccgtcgaggacatcgaggtcgccccggagcaccgggg
gcacggggt
cgggcgcgcgttgatggggctcgcgacggagttcgcccgcgagcggggcgccgggcacctctggctggaggtcaccaac
gtcaacgc
accggcgatccacgcgtaccggcggatggggttcaccctctgcggcctggacaccgccctgtacgacggcaccgcctcg
gacggcgag
caggcgctctacatgagcatgccctgcccctgaccgacgccgaccaacaccgccggtccgacgcggcccgacgggtccg
aggcctcg
gagatctgggcccatgcggccgcaacaactacctcgactttggctgggacactttcagtgaggacaagaagcttcagaa
gcgtgctatcga
actcaaccagggacgtgcggc acaaatgggcatccttgctctcatggtgc
acgaacagttgggagtctctatccttccttaaaaatttaattttc
attagttgcagtcactccgctttggtttcgtaactataacggtcctcgcgttttacgcgccgtcagtttcttggcccac
gaactggattcccacttg
ttcatggtgtcggcttccgtccaggccttgagcagactcttttgacgagcgtttcgctgaatatcgaccttcaggtcgg
tcagcgcgaggcgtt
tgtaggaaataacttgtcgggtgactcccgtgatagattctgggccgtcaatcagacacttgttctggtcgacaatgtc
aataatggtggcgag
cttgccagcctccggtccgtagttgatcaaaaccacgcgacccaattcaacgtatcggtgaaacggcatggttgttaaa
gagattgaaaatg
agagagaagtggtatgtgtatgtaaatgtgtgggtgatcctacgcaaaaacgagtttcgaagtagcagcgacacacgag
atggagtgtagtt
tgttgttggtcaaaggacacggcgaggggcccaccacgagttgtccgtagtccgtttttgtcgggtaatttcgcatgct
agagacggagcac
ggggttccgcgagcggcggatacccgcgtgcggtgatgcgggccgagaattttagtaaaccggtaatggagggttgaat
agggtagggc
gggaaaagaccaccaacctaccccggaaacgataggacatgtcgtagcggacatttaaaagaagagaatttcgttggat
atacctacttctt
ggatttacatcacacaaccagtagaagataccgggtagagtaggtactcttcttcgacaccacacaaaacaccaaccaa
tacactctggaaa
agacacttctaacgtgaaaggagatcatcgtcccc
acaatttgccgagtgatattgttcgcttcgcaacaacctagcagactctgctggtgtc a
cattttcggacacattttcgtctgccaaggccgcgtacac acacgcac
atattcataaaactcgttcggaaatcgtttcatgtcgagttttccgta
tcccgcctttctccaccaccgtccggtggacgccgcggcgcagcgtgccgcactcgcggccgttggtcgttgccgcatc
gtccctacatcg
tcctttcctggggccgctgactttcgcgagattgtccaaatgaacgtggcacaagcacacgtcggactcacggacgaga
ccgacggtgcg
gatcaggaaacgctccagaccacacgcaaggcccggatgaaacagggcatcgccatggccaagacgtatctcgtcgaaa
ccttacagtt
ggatgtcacggtcgtggatgtggaagtcgttttggatgctcgggatgtgattcaatcggacggctgtctcgcgagtgga
ctcctcgatgcgg
gttgtgacaaaattgtcgtggacggcaccaatcgggacgcttttgagactgcacaaattccgaaagcgagactgatggg
ccactttgtgtatc
cccataccgattcacgaacccgaaacgggagtctcgccgtggcctttgccgaagtccaacccttttgcgattccgtcag
tgtcgagctatcg
aatcctgaagccgtcacggaaagtgtcatccaggaaattgtgcaagcggcaaaagcccagcaattgcaaatttcaatcg
tctacaatcccaa
Date Recue/Date Received 2021-08-17

L -80-1=ZOZ panpoe apcuen5a ele0
MOODOSSIBoomioRetaloSolnaeoReSuoReoplapeumpanTReoltuultSpooReuTSSTSweolnpeop
0
SmoSouSttutaloSTSSIouRnSoReonuolSotuSoRelnweolooSSITSTSouSuSaeuReSumutooSoSo
oluSouRnonnoReSouSoaeoluoulReultoReSolpeoneReuRaeopooloSoSp0000lSouoRnooSuutSoS

oSTSSSSSSoonameuloSooRemmoutuooReReoSSReS000SoolSSTSontutuolneuoSSuoReSouneo
ReuReRnSoluRnSoStot000SolooluSaeootuSTSSounoSonaeoSoSooaeSouSoaeoloStopmetot
oonontooSoontoaeSoStReoluoS000StoReSoSoolSouoRetupeSSTSNSoSSSaeopuReouloSSReo
cz
onolSooStotoouloonoltooSaluonoononononameneSneSoReReameSouReoollootouTS
lneuoluloae000ReoSneuneomeooRnomeututoSSITSSoReuoSSRetuReani2oSSooSoaeloSSooS
toneSotolotooluuSSSanoloSTSSoStomoSonen000lSpeououoouSTSSSoReSoSoom000nelRea
TSRepneoSSSooReolSonanoolultnuooppeReltSouSSRanootmuneoumeSaeoSopmoopoolu
TelnommuSoRelmeolSSSSonotoomepotoSoouoluRemeuTSSReanpoSSSoneoluoSouSopeloReo
oz
oom000lotReltuonnoSolopeuoRetuoutuomootoSoltuoonetuututtnneantoRetuolS
ci
ltotuaeopumwellotuSSRmelSamSooSutuRe000lSneouSuoltolototoSolopoSSanSSSTReSol
StIonSuReSSRe000lnoRepoulSooSSoRnotoononanSoSuSuRnweltoRnomeulnanowneu
toloolRanouStouReSolooSoSSSutopeomoReoppeStouReReSoultneSolneuoltloSoltReolSS
uoSoltRameoluSonotolSoaeutuSuSoReolSupetnneSSSSSoSoluultumoluoltooSouReSSReSS
oRmeRenetuSoluanoaeSSSTSoRnoluuSoneuStuuttuoomuuntuSoSuReooRelouSool000ut
ouSonelSneop000luReSSolopolReameooStououRetuStaameoluloRnoolSoluReamentpoolot
ReaelopoSonoReutuRelouooluopeSoolouTSSReoRnSoaeloltpeReoolSSSSooutloouRepuoSouS
TS
TSSoRelototulome000lReSneoollotSoneotanuootoReneSouSoSuounannooluimaetae000
oiReputoSutonuoiSSweSoiSnmeluSoReSSoReoSuSTReSuoluaelnuooltomeaeoRewenepSSu01
oolooweneloweSSoupi2oneReweanoSanoRelSolReoSoRammetweloReuSTSSSoReotweSomoo
SuRelSweSoneuReSouSoSouSTRameSoSoSSReloneReloSuReoSolSootutuStuffeolReStonnolSS

oSSRetStuootuoulolooltunoStaeannoSSTSouaeoneuSoweSooReuSSoltweneoSoReanS
olStoRnaeSSSoSuSpeouReSouRnolutolSameSaeoulooluneuSSooloSSoltSopeSoouRnotoSou
ReRnStneouSoSuoneReSolSouTSSStoSTSmelolouwetpeootmuoSooSl000aeoneuRnopeolno
uSTSSSoSmotuSSSSouttoanuouSoloSSoaeououlneoppeounoolSououSoSneuoSoSSTSneSSou
un000uSooReoulouloTSTSSSSolStSweStaeoSooSolSnelolSolSouooloopeloltloSSSSpeoffeu
tuuou
uoSoloultolSolSomoaemoloi2SouSouSSSoouSometpololtSonoSoulooSououSSolanoSolooSoa
eSoo
pSooutlooSSITSSoolanoSneuRe000lloSuReootulSoSolnuStuooSooSouolutStooluuReSoupeS
o
S9

66
ggtaacggatatgatcctctagggcgttgac aaattctttgtcggttttc atggggtagacgtc
agtgaccagggaacgtctccctacaaaaa
gttgcgcatactttgctccgttgtcgacggc
aggggtgtcggaccagattgtatctgtggcaacggcctcattgcgtcaatggacctttaaagc
cgggaaacgagacttgaaatgtttacgtagaggtgcattatagacctc
acgcgcatattgagtggttgcaaaaatggttttgcgaacggtatc
actggagacccatgccaggc aaggacggagggcatcataatc atgttc attgcgttggacagc
atgcttgttacacgtaagtatacggtcga
gagcatcgtgacgttgagaattacagatggcaacgtgtcggtgcatgtattggccgagattgtc
aaatcgtggctcaatatatggcatgccag
gtaagtcatggatgtcggtatgcc attcagccttcatgtcgatttcgttgtcgagaactgatgggtcccagtc
aacgtc agaagtaagaacaac
atggggaagggaatgaagttc atgactggtgggtgcgcgcatatcc
atgtacggaagaccctgtcgtatgtttagtgggatgatgtaatcatc
cagggtaacaatacgttggttgccgccc
acggtacgagaccgatcttgaacacaattgtggcaatgttcaagttgggcgctggaatggatgg
ttttacctttaccaagatgcgcgtattgatgcatgataacaacaatgggccctcgttgcgattcgacaagaccggcagc
cgtgac aatgtctag
attggacaaggtatgttcatttataccggttatgttggctgaacgtccagttttgtgcagtacggtaac
atcactccccgcaagtccaccgttgg
caccgcgatcgacaagtgc agatgtggtattttgtactgtgtgtcgggagacttggtactgtaac ac
atttgactgaagtgacttggtggtacc
atgaggacggagggaacccgttgatacggtgttgcgtgacgctgc aagtaccttacgtatgtc gcctggatcc
atacgtcc aac ccgatc a
gaaagatgtgctaggagttccgtttctggtgcacaatcatgaaaggtatccacgggagttcc
attgttgtcgtccggggctgcgtcgcgactg
cc agttgc atgtac atgcgcc atc aagggacgtgcc
aatccattactaaccgggagaaccttagctggggcagcc aatccctgaagtatag
ccttagcatc
atctgagagctggttccacatatctctagggatataaggtcgttcacggttagtgggcgtactatgatgtcggttatta
gtacaat
tctccctgcgggc atgc gc attggcttc atagagtacggaaggtgac aagtc aatattgtagttaac
atccgg atc agtgtc aaagtctgtag
gatggtaagaaagatc
agtagaatgaatactacgcttttccttgggactacgggaattagagaagttgtttcctttattgtagagtgacgctgaa

gcaagtagaagactaaggtaactctcgtagctaataggattacctcattggctaggtcaagagtggctgtgatcttc
acttgacaaagttccg
gtacattgtggacagcattctccaaaagactaagacacagttgctttgggagttgctcagccattggtac
agtattgtggtagatacaaaggtg
gttcttccaatgaaggataaatccttctgctgtgcctgtccatgaggatccatatttcgccgtagttaggtaaccaagc
gtggtggctgaactga
tcttcgc acttgctgattccgtatagtgtttgac aactttac aaaac acttcttgcgc
agttcgctctagcctggac agag aagggac aatcttcg
tcgttaagactcgtgcgaatagcaaaagatcacaaaatagcac atcggcaccgaccaacgattatttcc
aaggaaaaaaagaatgcttc act
acaagaaattgtgtcatccctatac
agagtcttgttactgtgacagaaaattgatggaagatgtggcggattgcctttacactagccaacttgttc
gactaattgcagcttcttctgagaggcttcaccgagtaacgcgaagaacaccggtgtctcgtacatgctcgtcggtgaa
cgctcgtcc aatga
.. c acccc cc actttgtatc aatatccc aacttggtagtgaactggaatgatacatgc aatttcgcgccgc
atcaacagccacgggccaccatcg
acgaatagactcgg
SEQ ID NO: 6 - The PtUMPS coding sequence and gRNA target sites.
[] used to indicate TevCas9 target sites
Date Recue/Date Received 2021-08-17

67
() used to indicate ODC
{} used to indicate OPRT
atggccaccccctcttttcgatcaaagcttgaagctcgagtcgccgcagtc
aactctctcttgtgcgttggtctagacccgc acgagaaagag
ctg [tttgcggacggatggg aaggcgtgccgg]
aaaaaaatcgctgtgacgcggcctttaccttttgcaaaacgttggtcgacgcaacattg
ccttacacggcctgctacaaaccc
aatgctgcctttttcgaggcgttaggcgacggagggatagcggttctgcgacgagtttgtc aaaac at
aataccggatgatgtgccgattttgttg(gatgtcaagcgcggcgacattggctcgacc)gctgcggcctacgccgaag
cgtgctatggtttg
ggtgcagactgtgtcacgctttcaccactgatgggatgggactcagtcagtccctttgttacagaaaagtacgttcaca
aaggagcatttttgc
tgtgcaaaacgtc aaatcctggatcc aacgattttttagctctgggattacgttc
aaatgaatgtttatacgaaagaattgcc aagcttgttggct
cggaatgggctcagc agaccgagagttc attgggactcgttgtcggggcc ac agatcc agtggccttgtc c
aaagcgag aaaggctgc a
ggcgacgacacctggattctagc acccggcgttggtgctcaaggtggagatcttctagaagc
agcgcaggctggattgaatacaaagggg
acttgcatgctaattcccgtgtctaggggtatcagc
aaagctacggacccagcgcaggctgcaaaagaattgcaggagaggattcagaaa
gctcgggaccaagtcgtggccgc acacatgatgaaaaagagttc agacgaagatattaaactctatc
aacgcgagtttcttgaatttagtctgt
ctcaaggtgttctcaaattcggctcttttgtgctgaaaagcggccgcacctctcc
atatttittcaacgccggtctttttgcttctggcgctgcgtta
agcaagcttgggaaagcctatgcttcgactatcatgtcctcggaattattagctgctgggcccaaccaagtc
aattttgatgtgatttttggtcct
gcatacaagggtatttctctaggtgctgtcgttggaagcgctctgtataacgattttgaagtagatgtcggttttgcgt
atgaccgaaaagagg [
caaaggatc atggggaaggtggtaaattggtc gggacttcgt]tggaaggaaaacg a
{gttctgattgtagatgacgtaatc ac agcggga
accgcc} attcgtgagtcgc ac actttgctc aacgatgtgggtgctttgcc agttggagtagttattgccctc
gatcgagccgaaattcgctct
atggaggacaagatttccgctgttcaagcagtcgcacgagatctatctcttttggtcgtgtcaattgtc
agtcttcctc aactacagacatttctc
gaacgaagtccggactacgg [cgatgaaacgctggaaaaagtaattaagtatcgaaacgaata]cggagtgtaa
SEQ ID NO: 7 ¨ Amino acid sequence for SEQ ID NO: 6 (1-letter abbreviation)
MATP SFRSKLEARVAAVNSLLCVGLDPHEKELFADGWEGVPEKNRCDAAFTF CKTLVD
ATLPYTACYKPNAAFFEALGDGGIAVLRRVCQNIIPDDVPILLDVKRGDIGSTAAAYAEA
CY GL GADCVTL SPLMGWD SVSPFVTEKYVHKGAFLLCKTSNP GSNDFLAL GLRSNECL
YERIAKLVGSEWAQQTESSLGLVVGATDPVAL SKARKAAGDDTWILAPGVGAQGGDLL
EAAQAGLNTKGTCMLIPVSRGISKATDPAQAAKELQERIQKARDQVVAAHMMKKSSDE
DIKLYQREFLEF SL SQGVLKF GSFVLKSGRTSPYFFNAGLFASGAAL SKLGKAYASTIMS
SELLAAGPNQVNFDVIFGPAYKGISLGAVVGSALYNDFEVDVGFAYDRKEAKDHGEGG
KLVGTSLEGKRVLIVDDVITAGTAIRESHTLLNDVGALPVGVVIALDRAEIRSMEDKISA
VQAVARDLSLLVVSIVSLPQLQTFLERSPDYGDETLEKVIKYRNEYGV
Date Recue/Date Received 2021-08-17

68
SEQ ID NO: 8 ¨ PtPRA-PH/CH coding sequence and gRNA target sites
[] used to indicate TevCas9 target sites
() used to indicate ODC
{} used to indicate OPRT
atgtcgagttttccgtatcccgc ctttctcc ace accgtccggtggacgccgcggcgc agcgtgc cgc
actcgcggc cgttggtcgttgccg
catcgtccctacatcgtcctttcct[ggggccgctgactttcgcgagattgtccaaatgaacgtgg]c
acaagcacacgtcggactcacggac
gagaccgacggtgcggatc
aggaaacgctccagaccacacgcaaggcccggatgaaacagggcatcgccatggccaagacgtatctc
gtcgaaaccttac
agttggatgtcacggtcgtggatgtggaagtcgttttggatgctcgggatgtgattcaatcggacggctgtctcgcgag
t
ggactcctcgatgcgggttgtgac aaaattgtcgtggacggc acc aatcgggacgcttttgagactgc ac
aaattccgaaagc gagactg a
tgggccactttgtgtatccccataccgattcacgaacccgaaacgggagtctcgccgtggcctttgccgaagtccaacc
cttttgcgattccgt
cagtgtcgagctatcgaatcctgaagccgtc acggaaagtgtc atccaggaaattgtgc aagcggc aaaagccc
agc aattgc aaatttc a
atcgtctac aatc cc aacg actac gagaatcctggtgatc acgccgcc atggttggtcgcgtatgcc
agagcttccc agaagggc aagtcc
ggttggccttgaccgatccgaccgcctcgc aactcggac acgcctacgcggcgtgtctc
aaaaccgaccgggacgacggtctctttacc a
ccgtcgtctgtactcgcaacaatgaagcactggggcttgtctactcctccacgtcgtctattgtcgccg
[cactggaatgtggtcggggtgtct
ac(tacagccgttcccggaa]cggattgtggcgcaagggcgacacgtccggac
actttcaggtacttcaccggctcgacaaagactgtgac
ggggat)gctttgcgggtgacggtcactcaaaaaggcacccctgccgcattttgccacttgaatactctaacgtgctgg
ggtacgtcgagag
gcttgcgac atttggaagaaacgctgc aagaccg actcgtgtcggctc cggaaggatcctac acg
aaacgtctgtttc aagacgagac act
gttgcgggacaagctggtc
{gaagaagcgcaggaattgtcggaagccgatacgaaggcacacgtggcggaagaactggcggat}gtcc
tctactttgccatggtgagggcggtcaaggctggagtcagtttggatgatgccgtcgcagagctagattctagggcgcg
aaaagtgacgcg
acgaaaaggcgatagtaaagcctttcgaattgc agcgggtg aagctattttgaaaaaagcgc agtcgtag
SEQ ID NO: 9 ¨ amino acid sequence of SEQ ID NO: 8 (1-letter abbreviation)
MSSFPYPAFLHHRPVDAAAQRAALAAVGRCRIVPTSSFPGAADFREIVQMNVAQAHVG
LTDETDGADQ ETL QTTRKARMKQGIAMAKTYLVETL QLDVTVVDVEVVLDARDVIQ S
DGCLA S GLLDAGCDKIVVDGTNRDAFETAQIPKARLMGHFVYPHTD SRTRNGSLAVAF
AEVQPF CD SVSVELSNPEAVTESVIQEIVQAAKAQQLQISIVYNPNDYENPGDHAAMVG
RVCQ SFPEGQVRLALTDPTASQLGHAYAACLKTDRDDGLFTTVVCTRNNEALGLVYSS
TSSIVAALECGRGVYYSRSRNGLWRKGDTSGHF QVLHRLDKDCDGDALRVTVTQKGTP
AAF CHLNTLTCWGTSRGLRHLEETLQDRLVSAPEGSYTKRLF QDETLLRDKLVEEAQEL
Date Recue/Date Received 2021-08-17

69
SEADTKAHVAEELADVLYFAMVRAVKAGVSLDDAVAELDSRARKVTRRKGDSKAFRI
AAGEAILKKAQS
SEQ ID NO: 10- The PtI3GPS-PRAI coding sequence and gRNA target sites. The
coding regions
for the I3GPS and PRAI catalytic residues are highlighted. Residues that
differ between the two
alleles are indicated by boxes.
A. A AA:A.
,'.".1.._,TAAATCTCTG14.12CVJGGTTITGGTCTCGCCAATTGCCTCAITATOCCCOGAAAZTOTCOATTACCA
GGALIµ1,GGIOGTCCCACCIGTTOGCCGMTCOCAGGGACGGAGGAIWOCAAGGC
--- ,.,-11 11 1 1 -
11'11111111 1 1 1 .1 , 1 .. I. 1. c .1. 140
.,04CV1.64 01 401C1. MIAPFINCRL POT 818 111.FAVVP07 EEA11 A
2 3 4 5 11 i i 1 ilt ii it it ii is it it le it lo il it is is is ti iv it ii
36 ii 31/ 3.3 3.6 36 ili 71 29 4 4 42.1 4 4.3 4 ,i6 4 4.1
f __________________ "I'' .I""I '61 ' 'I 'I' l'' ' l''' 6-11.4I,,-11,,-
III'iIIIII'I'
a- moR16.8.101411411.244-=
661000111161APP VI 8001(1EAL 011. W0111)111)11161.1141111Y01012
48 10 SO 61 62 13 64 U 56 51 68 69 611 61 62 63 64 GI 66 6.1 68 69 TO 71 /2 13
14 7s /6 rr 22, /9 80 81 82 83 84 85 16 17 10 * 90 91 91 13
CTICTCACMATTGAZTATGCTTCAAACGATTACAGCAKOCGTCTACTOGACTATCAATCCCMTOGNIAGTAIGGACGTC
TTOITTIGOCAAGT1C7GGATGA.ACTAGCCCAAGATIOCATIAATCC1111130CTTTGATCAT
PSHAL 61/1.011111.47111.10142917ESST SeLP91.021.ACIEAK SF ATOM
91 91 A is ii it tit tit a tit Ilia tis tit tit lie lit lit iii six ,13 1st mi
.16 tit 14 ITS 120 44 422 43 lir sis tit 2/2 lis lis ii= lit fie 2i3 214 232
ii$ lir lis li2 2.4
OGAGCCATATTGJUITTTOCAGACTGTCATCCAATCOCAAGCTCCTOGGATGGCL11-1,
TTOCTACCCATCTGANIVOCOGTGAGCAAGCOGTCAAGTATAC
AV
i, 11560
66111110TV, CISOAPR 1.411.
GO 1 A71111161104 VKY T
lit ii2 113 144 116 116 1,17 66 in no 111 1* 113 tu 116 116 117 tie 1
tit lit lit lit Its tit 111 till tit tit lit tit Ito tit iis lit lir
rttts...-4GGGGGAGCCAILTATC1111 ___________________________
29.104TATTGACCGENTCCUICTGGTTTAAAGGGAGTCTOGACGACATGACACAAGOCAGACICIMIAIICATCIANI
MGAMYTYCCGAXGACIOACGCCCGGCTATTTTACGCAAAM
.EII0.. 1 I '1 l= 1
'1 1 I. 1. 1 .1 I I. .1 ,1.. 1 1 1 1
1 1 1 1 1 1.= I 1 700
A010011.1. 166 811.T ES/1428081.00MT OAR LETS 10EIVIIODSRPAT.I.RIC
108 I 190 191 192 IN IN SS 14 IN IN NO 281
282 203 204 205 24 202 218 289 NO 21,1 212 213 214 VI 211 1I7 21I 24 VS 222
223 NI NS 228 21/ 2211 no no 231 232 723
ATITO2TGATCAPCGAATAGATGATINCIXAGGCGCNCGCMAANCITECOGATACT6TOCINTTWATCGTCOCTSTECT
ACCJIMAC1102TACTCACTO2ATTARTI2GGGRIEMTOBTTOCCICOGGATGGACCCACTA
= ! = I =I .1 = I = =I I 1 . 1
__ 1 1 . 1 = 1= 1= -=1 =1 =1===-.1 .1=-=-= 1
. . .1....1....1 .. 1. I. 1 ...1 eoo
OF1/114111.41A1AAAK0AD761.1.1 VAVI.P0111,11.7111.16F(112/31.13141PL
114 216 *1 2* 2* 2* 260 34 252 tit tit 715 2* 217 248 219 210 611 212 213 *4
266 216 217 tie tit tio tit tit tit tit is tit tir its tie 270 221 24 221 224
tit zit zit tie zit zit
I3GPS
w,,,,,,,,,,,,,,,,,,,,,,,,' 77T
GCACACCITCCAAATGGATTTGGGGACATCIGAGCGTGTGGCTAGAGAGCTTGAIWCW3
0,11.6711011DL G7 SLR VAREL 1 AR
396 42 14 399 399 41 42 34 354 MS 14 sir 14 313 321 321 322 i3 124 325 926 ir
OGOATTGTETTATOCCCACJACIZAtTECCD2CGOGGCATh
ATGGACGTGGA00GTTATCGTCEGGTOGGTCTTGGTATG2GTTTAATOGGTGAGAGITTGATGCGTG
Gil is VA 11101CAA T TV C ST A MOVOR VR OV01.011111.102 ST MR
3211 321 3i0 3it 332 3/1 tit 326 3.26 337 lie 3* 6* 341 612 343 tit tit tit
tit tit tit tits 326 197 396 14 398 34 292 41 34 is tic tit tit tit 320 121
tit tit
pRA1
OCACTGATCOGGGACMOCGATTGCTGC2CTATOTTTGCATCCAG21.CGATTICOCTGTOCGTATGGCMGGGIITGGTO
TCOOCGGEGCCTA1A000GOOGTACTCAAGTOGMAAACTATCTT3GCATAACAAGCTCTCAA
1 1 = 1 = 1 = 1 I. 1 1 1 I=
____________ I I = I I - I = I I I= I-. =I=
I=-==1====-==-1-,=====,-1====1.-=-, 1,...11.-'1,1 1260
.9 T OP004.0 4AI CI.MPDO 4
GAY YT OGIOT vTvOCCI /SSE
34 326 326 34 34 34 34 392 302 34 34 34 *6 311 3/8 3* 310 311 312 313 *4 39.
356 957 lie 359 Mk Ail 422 46 411 416 416 411 *6 4141 450 451 44 453 44 411
454 44 14 44 41
GICGCACTEGTGOCCTGTCAAOCAGGAGCCAATTLGETTIOGGGITATTTTPGCODCAAAGTOGGCGCGTAAAGTracT
,CTGAGCAACCAAAGGCAGTTGTGCAGGCAGTOOGGAACTITGGAGAGCGCCACCAGGATCA
.. __ 1 .... 1 1400
O4.6 VACOA OAN4 0 Ovi F VIP PE OAK A V10161114 GER 110D11
421 422 411 44 496 426 42/ 421 44 420 41 42 42) 44 s96 498 422 499 49 oio 41
4;2 4;3 44 46 46 44 44 451 410 4/1 452 413 454 165 4/8 sir mi. .6 04 451 412
453 olo 416 44 44
CACCTICAAACCTOCGAAOGIICOASTMCCGCTACCTCATITE0114721.20TTCACGACAGTTGGEGGEGGCTICAA
GAC TTC,C7CGT2CGTGCGGGAAATOGTTG
$ .. 1 .1 .1"t= 1 1==1. I 1.--.I .1==1==-=1=
=1.--1.--.I.= 1 =i----.1= l.-'1 I . I = I. I. 11-, t 1610
1 1 1( PPIODESPl P111481111/01,AEASRRPI.VV *VS 0NODS611111/64
44 411 *0 421 at? tit tit 6i4 tit 4t1 44 tit tit tit 482 43 44 494 496 492 44
49 400 44 tit tit tit *is tit tit tit *it sio sit six sio sot sit sit sin sio
699 64 401680
622 623
ATT022GTEGGTTGGATCTAZTOCAOCTTCATGGACEAGWIOGCTTCGCAGCAGOCANIGTTGAAAGTTGTOGEGI
___ 49.9.CGCGATTCGAGTOGTGGACATTGTCGTOGECTCOGAATCTGGTAGAGCAGCAGAGARTGCT
O /3801.01.v01.010302641428 vESCOVP/I A V VOI VvOSES-011 A12111
S14 46 626 64 64 41 sio sis six sit sit sis sit sii sit) sit sit sil tit tit
sit sis $it sit sis sit sio sit si: sit $it sis sit sit sio sit sio sis sit
sit sit sis sit sit sit sits 550
GTAGA/1110171TTCTGGAATCCATTACAAGCGATCOGATTC,CTATTTTACTCGACACAGOCATCATIGIXTV.117
111/171.01,AGGGGGAGGAACCTCCOGATOGITTGATTOGACCATMCAGANUUTGTOCAAGOCGCTGGTTT
1 . 1 .5 1 1 1 1 .. , 1 =666.16.66.-e1 , 1
= 1 I I I I = I= = I I I I I I I I I
I I 1620
/E711.181113041 A I I, 10117114601111360781191 OWT1AEKV0AAOL
si, ,i2 ,i, si, si, 4, ,i, sii, si, sio 4, 02 '
, , 4 4211 64 590 64 692 99) 44 696 696 sit sit sit sie sit sit sit sit
696 46 64 690 sit sit Gil tit sit sit tit tit sir
A,
1960
P VI I A GGL.SP ENV ISCA VA 0 1 RP F CV 04553T EASPGRK OHCIn ,PDF
40 NS 6.0 ell 622 64 64 416 64 sir sit 49 silt oii 122 429 44 sis sis six sit
sit sit sis sit oil sit its sit sir sit *9 6* 6it 612 613 114 645, 8* ai1 6; t
til t 55 6.2 C;9
TACAAAKTGOGAAOCAAGOGGCOGTGGAT1GCMACAAAGGAITTMA
/ONAK OA AVEA N11.31
sit eis eitt tit tie sit sio oil tit sit 44 64 606 04 sit sit
Date Recue/Date Received 2021-08-17

70
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Through the embodiments that are illustrated and described, the currently
contemplated best mode
of making and using the disclosure is described. Without further elaboration,
it is believed that one
of ordinary skill in the art can, based on the description presented herein,
utilize the present
disclosure to the full extent.
Although the description above contains many specificities, these should not
be construed as
limiting the scope of the disclosure, but as merely providing illustrations of
some of the presently
embodiments of this disclosure.
Date Recue/Date Received 2021-08-17