U.S. patent application number 13/135966 was filed with the patent office on 2011-12-08 for selection of host cells expressing protein at high levels.
This patent application is currently assigned to ChromaGenics B.V.. Invention is credited to Theodorus Hendrikus Jacobus Kwaks, Arie Pieter Otte, Richard George Antonius Bernardus Sewalt, Henricus Johannes Maria Van Blokland.
Application Number | 20110300580 13/135966 |
Document ID | / |
Family ID | 46324389 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110300580 |
Kind Code |
A1 |
Otte; Arie Pieter ; et
al. |
December 8, 2011 |
Selection of host cells expressing protein at high levels
Abstract
Described is a DNA molecule comprising an open reading frame
sequence that encodes a selectable marker polypeptide, wherein the
DNA molecule in the coding strand comprises a translation start
sequence for the selectable marker polypeptide having a GTG start
codon or a TTG start codon, and wherein the ORF sequence that
encodes the selectable marker protein has been mutated to replace
at least half of its CpG dinucleotides as compared to the native
ORF sequence that encodes the selectable marker protein. Further
provided are such DNA molecules wherein the ORF sequence that
encodes a selectable marker polypeptide is part of a multicistronic
transcription unit that further comprises an open reading frame
sequence encoding a polypeptide of interest. Also described are
methods for obtaining host cells expressing a polypeptide of
interest, wherein the host cells comprise the DNA molecules
described herein. Further provided is the production of
polypeptides of interest, comprising culturing host cells
comprising the DNA molecules described herein.
Inventors: |
Otte; Arie Pieter;
(Amersfoort, NL) ; Van Blokland; Henricus Johannes
Maria; (Wijdewormer, NL) ; Kwaks; Theodorus Hendrikus
Jacobus; (Amsterdam, NL) ; Sewalt; Richard George
Antonius Bernardus; (Arnhem, NL) |
Assignee: |
ChromaGenics B.V.
|
Family ID: |
46324389 |
Appl. No.: |
13/135966 |
Filed: |
July 18, 2011 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11416490 |
May 2, 2006 |
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13135966 |
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11269525 |
Nov 7, 2005 |
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11416490 |
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11359953 |
Feb 21, 2006 |
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11269525 |
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11269525 |
Nov 7, 2005 |
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11359953 |
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12226706 |
Oct 24, 2008 |
8039230 |
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PCT/EP2007/053984 |
Apr 24, 2007 |
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11269525 |
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60626301 |
Nov 8, 2004 |
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60696610 |
Jul 5, 2005 |
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Current U.S.
Class: |
435/70.3 ;
435/243; 435/252.33; 435/320.1; 435/352; 435/354; 435/358; 435/366;
435/369; 435/455; 435/471; 435/71.2; 536/23.5 |
Current CPC
Class: |
C12N 2830/46 20130101;
C12N 15/67 20130101; C12N 2840/50 20130101; C12Y 207/01095
20130101; C12N 9/1205 20130101; C12N 15/85 20130101; C12N 2840/203
20130101; C07K 14/505 20130101; C12N 2840/20 20130101; C12N
2840/206 20130101 |
Class at
Publication: |
435/70.3 ;
536/23.5; 435/320.1; 435/352; 435/455; 435/358; 435/369; 435/354;
435/366; 435/243; 435/252.33; 435/471; 435/71.2 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 15/85 20060101 C12N015/85; C12N 15/63 20060101
C12N015/63; C12N 1/00 20060101 C12N001/00; C12N 1/21 20060101
C12N001/21; C07H 21/04 20060101 C07H021/04; C12N 5/10 20060101
C12N005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2004 |
EP |
04105593.0 |
May 2, 2006 |
EP |
06113354.2 |
Claims
1.-92. (canceled)
93. A DNA molecule comprising an open reading frame encoding a
selectable marker polypeptide, wherein the DNA molecule in the
coding strand comprises a translation start sequence for the
selectable marker polypeptide selected from the group consisting
of: a) a GTG start codon; and b) a TTG start codon; and wherein the
open reading frame encoding the selectable marker protein has been
mutated to replace at least half of its CpG dinucleotides as in
comparison to the native open reading frame encoding the selectable
marker protein.
94. The DNA molecule of claim 93, wherein the translation start
sequence for the selectable marker polypeptide comprises a TTG
start codon.
95. The DNA molecule of claim 94, wherein the open reading frame
encoding the selectable marker polypeptide has no ATG sequence in
the coding strand.
96. The DNA molecule of claim 95, wherein the selectable marker
polypeptide provides resistance against ZEOCIN.RTM. antibiotic or
against neomycin.
97. The DNA molecule of claim 96, comprising an open reading frame
encoding a polypeptide that provides resistance against ZEOCIN.RTM.
antibiotic, wherein the DNA molecule comprises a sequence selected
from the group consisting of: a) SEQ ID NO:92, with the proviso
that at least half of the CpG dinucleotides has been replaced
without mutating the amino acid sequence that is encoded, and with
the further proviso that the start codon is either GTG or TTG; and
b) SEQ ID NO:92 wherein nucleotide A at position 280 is replaced by
T, and with the proviso that at least half of the CpG dinucleotides
has been replaced without mutating the amino acid sequence that is
encoded, and with the further proviso that the start codon is
either GTG or TTG.
98. (canceled)
99. The DNA molecule of claim 96, comprising an open reading frame
encoding a polypeptide that provides resistance against neomycin,
wherein the DNA molecule comprises a sequence selected from the
group consisting of: a) SEQ ID NO:128, with the proviso that at
least half of the CpG dinucleotides has been replaced without
mutating the amino acid sequence that is encoded, and with the
further proviso that the start codon is either GTG or TTG; and b)
SEQ ID NO:118, with the proviso that at least half of the CpG
dinucleotides of the coding strand has been replaced without
mutating the amino acid sequence that is encoded, and with the
further proviso that the start codon is either GTG or TTG; and c)
SEQ ID NO:128 or SEQ ID NO:118, with the proviso that it contains a
mutation to encode either of the following polypeptide variants as
in comparison to the polypeptide encoded by the native sequences:
(i) substitution of valine at position 201 into glycine
(201V>G), or (ii) substitution of glutamic acid at position 185
into aspartic acid (185E>D), or (iii) a combination of both
mutations (i) and (ii) (185E>D and 201V>G), with the further
proviso that at least half of the CpG dinucleotides of the coding
strand has been replaced without further mutating the amino acid
sequence that is encoded beyond the mutation indicated under
(i)-(iii), and with the further proviso that the start codon is
either GTG or TTG.
100. The DNA molecule of claim 99, comprising SEQ ID NO:130, with
the proviso that nucleotide A at position 555 is replaced by C, and
that nucleotide T at position 602 is replaced by G and that
nucleotide G at position 603 is replaced by T, and with the further
proviso that the start codon is either GTG or TTG.
101. The DNA molecule of claim 93, wherein the open reading frame
encoding a selectable marker polypeptide is part of a
multicistronic transcription unit that further comprises an open
reading frame polynucleotide encoding a polypeptide of
interest.
102. The DNA molecule of claim 101, wherein the open reading frame
encoding the selectable marker polypeptide is upstream of the open
reading frame encoding the polypeptide of interest, and wherein the
open reading frame encoding the selectable marker polypeptide has
no ATG sequence in the coding strand.
103. The DNA molecule of claim 101, wherein the open reading frame
encoding the polypeptide of interest is upstream of the open
reading frame encoding the selectable marker polypeptide, and
wherein the open reading frame encoding the selectable marker
polypeptide is operably linked to an internal ribosome entry site
(IRES).
104. An expression cassette comprising the DNA molecule of claim
103, the expression cassette comprising a promoter upstream of the
multicistronic expression unit and a transcription termination
sequence downstream of the multicistronic expression unit, wherein
the expression cassette is functional in a eukaryotic host cell for
initiating transcription of the multicistronic expression unit.
105. The expression cassette of claim 104, further comprising at
least one chromatin control element selected from the group
consisting of matrix or scaffold attachment regions (MAR/SAR), and
anti-repressor (STAR) sequences.
106. The expression cassette of claim 105, wherein the at least one
chromatin control element is an anti-repressor molecule selected
from the group consisting of any one of SEQ ID NO:1 through SEQ ID
NO:66 and the complement of any one of SEQ ID NO:1 through SEQ ID
NO:66.
107. The expression cassette of claim 106, wherein the expression
cassette comprises SEQ ID NO:66 positioned upstream of the promoter
that drives transcription of the multicistronic expression
unit.
108. The expression cassette of claim 107, wherein the
multicistronic expression unit is flanked on both sides by at least
one anti-repressor molecule selected from the group consisting of
any one of SEQ ID NO:1 through SEQ ID NO:65 and the complement of
any one of SEQ ID NO:1 through SEQ ID NO:65.
109. A host cell comprising the DNA molecule of claim 103.
110. A host cell comprising the expression cassette of claim
108.
111. A method of generating a host cell able to express a
polypeptide of interest, the method comprising the steps of: a)
introducing into a plurality of precursor cells a DNA molecule
according to claim 103, and b) culturing the plurality of precursor
cells under conditions suitable for expression of the selectable
marker polypeptide, and c) selecting at least one host cell
expressing the polypeptide of interest.
112. A method of generating a host cell able to express a
polypeptide of interest, the method comprising the steps of: a)
introducing into a plurality of precursor cells an expression
cassette according to claim 104, and b) culturing the plurality of
precursor cells under conditions suitable for expression of the
selectable marker polypeptide, and c) selecting at least one host
cell expressing the polypeptide of interest.
113. A method of expressing a polypeptide of interest, comprising
culturing a host cell comprising the expression cassette of claim
104, and expressing the polypeptide of interest from the expression
cassette.
114. The method according to claim 113, further comprising
harvesting the polypeptide of interest.
115.-140. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/416,490, filed May 2, 2006, which is a
continuation-in-part of co-pending U.S. patent application Ser. No.
11/269,525, filed Nov. 7, 2005, which application claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application Ser. No. 60/626,301, filed Nov. 8, 2004, and U.S.
Provisional Patent Application Ser. No. 60/696,610, filed Jul. 5,
2005. U.S. patent application Ser. No. 11/269,525 also claims the
benefit of EP 04105593.0, filed Nov. 8, 2004. This application is
further a continuation-in-part of co-pending U.S. patent
application Ser. No. 11/359,953, filed Feb. 21, 2006, and which
itself is a continuation-in-part of the aforementioned co-pending
U.S. patent application Ser. No. 11/269,525, filed Nov. 7, 2005.
This application is also a continuation of co-pending U.S. Ser. No.
12/226,706, filed Oct. 24, 2008, which is the national stage of PCT
International Patent Application No. PCT/EP2007/053984, filed on
Apr. 24, 2007, designating the United States of America, and
published, in English, as PCT International Publication No. WO
2007/128685 A1 on Nov. 15, 2007, and claims priority to U.S. Ser.
No. 11/416,490, filed May 2, 2006, and EP 06113354.2, also filed on
May 2, 2006. The contents of each of the preceding applications are
incorporated herein by this reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn.1.52(e)(5)-SEQUENCE LISTING
SUBMITTED ON COMPACT DISC
[0002] Pursuant to 37 C.F.R. .sctn.1.52(e)(1)(ii), a compact disc
containing an electronic version of the Sequence Listing has been
submitted concomitant with this application, the contents of which
are hereby incorporated by reference. A second compact disc is
submitted and is an identical copy of the first compact disc. The
discs are labeled "copy 1" and "copy 2," respectively, and each
disc contains one file entitled "2578-7784US seq list.txt" which is
239 KB and created on May 2, 2006.
TECHNICAL FIELD
[0003] The invention relates to the field of molecular biology and
biotechnology. More specifically the invention relates to means and
methods for improving the selection of host cells that express
proteins at high levels.
BACKGROUND
[0004] Proteins can be produced in various host cells for a wide
range of applications in biology and biotechnology, for instance as
biopharmaceuticals. Eukaryotic and particularly mammalian host
cells are preferred for this purpose for expression of many
proteins, for instance when such proteins have certain
posttranslational modifications such as glycosylation. Methods for
such production are well established, and generally entail the
expression in a host cell of a nucleic acid (also referred to as
"transgene") encoding the protein of interest. In general, the
transgene together with a selectable marker gene is introduced into
a precursor cell, cells are selected for the expression of the
selectable marker gene, and one or more clones that express the
protein of interest at high levels are identified, and used for the
expression of the protein of interest.
[0005] One problem associated with the expression of transgenes is
that it is unpredictable, stemming from the high likelihood that
the transgene will become inactive due to gene silencing (McBurney
et al., 2002), and therefore many host cell clones have to be
tested for high expression of the transgene.
[0006] Methods to select recombinant host cells expressing
relatively high levels of desired proteins are known.
[0007] One method describes the use of selectable marker proteins
with mutations in their coding sequence that diminish, but not
destroy the function of the marker (e.g., WO 01/32901). The
rationale is that higher levels of the mutant marker expression are
required when selection conditions are employed and therefore
selection for high expression of the marker is achieved, therewith
concomitantly selecting host cells that also express the gene of
interest at high levels.
[0008] Another method makes use of a selection marker gene under
control of a promoter sequence that has been mutated such that the
promoter has an activity level substantially below that of its
corresponding wild type (U.S. Pat. No. 5,627,033).
[0009] Another method describes the use of an impaired dominant
selectable marker sequence, such as neomycin phosphotransferase
with an impaired consensus Kozak sequence, to decrease the number
of colonies to be screened and to increase the expression levels of
a gene of interest that is co-linked to the dominant selectable
marker (U.S. Pat. Nos. 5,648,267 and 5,733,779). In certain
embodiments therein, the gene of interest is placed within an
(artificial) intron in the dominant selectable marker. The gene of
interest and the dominant selectable marker are in different
transcriptional cassettes and each contains its own eukaryotic
promoter in this method (U.S. Pat. Nos. 5,648,267 and
5,733,779).
[0010] Another method uses the principle of a selectable marker
gene containing an intron that does not naturally occur within the
selectable gene, wherein the intron is capable of being spliced in
a host cell to provide mRNA encoding a selectable protein and
wherein the intron in the selectable gene reduces the level of
selectable protein produced from the selectable gene in the host
cell (European Patent 0724639 B1).
[0011] In yet another method, DNA constructs are used comprising a
selectable gene positioned within an intron defined by a 5' splice
donor site comprising an efficient splice donor sequence such that
the efficiency of splicing an mRNA having the splice donor site is
between about 80-99%, and a 3' splice acceptor site, and a product
gene encoding a product of interest downstream of 3' splice
acceptor site, the selectable gene and the product gene being
controlled by the same transcriptional regulatory region (U.S. Pat.
No. 5,561,053).
[0012] In certain methods, use is made of polycistronic expression
vector constructs. An early report of use of this principle
describes a polycistronic expression vector, containing sequences
coding for both the desired protein and a selectable protein, which
coding sequences are governed by the same promoter and separated by
a translational stop and start signal codons (U.S. Pat. No.
4,965,196). In certain embodiments in U.S. Pat. No. 4,965,196, the
selectable marker is the amplifiable DHFR gene. In a particularly
preferred embodiment of the system described in U.S. Pat. No.
4,965,196, the sequence coding for the selectable marker is
downstream from that coding for the desired polypeptide, such that
procedures designed to select for the cells transformed by the
selectable marker will also select for particularly enhanced
production of the desired protein.
[0013] In further improvements based on the concept of
multicistronic expression vectors, bicistronic vectors have been
described for the rapid and efficient creation of stable mammalian
cell lines that express recombinant protein. These vectors contain
an internal ribosome entry site (IRES) between the upstream coding
sequence for the protein of interest and the downstream coding
sequence of the selection marker (Rees et al., 1996). Such vectors
are commercially available, for instance the pIRES1 vectors from
Clontech (CLONTECHniques, October 1996). Using such vectors for
introduction into host cells, selection of sufficient expression of
the downstream marker protein then automatically selects for high
transcription levels of the multicistronic mRNA, and hence a
strongly increased probability of high expression of the protein of
interest is envisaged using such vectors.
[0014] Preferably in such methods, the IRES used is an IRES which
gives a relatively low level of translation of the selection marker
gene, to further improve the chances of selecting for host cells
with a high expression level of the protein of interest by
selecting for expression of the selection marker protein (see,
e.g., PCT International Publication WO 03/106684).
[0015] The invention aims at providing improved means and methods
for selection of host cells expressing high levels of proteins of
interest.
DISCLOSURE
[0016] U.S. patent application Ser. No. 11/269,525 (hereinafter the
'525 application) and International Patent Application No.
PCT/EP2005/055794, both incorporated in their entirety by reference
herein, disclose a concept for selecting host cells expressing high
levels of polypeptides of interest, the concept referred to therein
as "reciprocal interdependent translation." In that concept, a
multicistronic transcription unit is used wherein a sequence
encoding a selectable marker polypeptide is upstream of a sequence
encoding a polypeptide of interest, and wherein the translation of
the selectable marker polypeptide is impaired by mutations therein,
whereas translation of the polypeptide of interest is very high
(see, e.g., FIG. 2 herein for a schematic view).
[0017] U.S. patent application Ser. No. 11/359,953 (hereinafter the
'953 application), incorporated in its entirety by reference
herein, discloses alternative means and methods for selecting host
cells expressing high levels of polypeptide. The '953 application
is based on a similar principle as the '525 application, this
principle also using multicistronic transcription units and
impairment of the translation initiation of the selectable marker
polypeptide by mutation of the start codon thereof. The main
difference between the means and methods disclosed in the '525
application and the '953 application is in the order of the
sequences encoding the selectable marker polypeptide and the
sequence encoding the polypeptide of interest in the multicistronic
transcription units.
[0018] Both the '525 application and the '953 application thus
provide means and methods for selecting host cells with very high
expression levels of a polypeptide of interest. The invention
provides further advantageous embodiments and improvements to the
means and methods disclosed in the incorporated '525 and '953
applications.
[0019] In one aspect, provided is a DNA molecule comprising an open
reading frame sequence that encodes a selectable marker
polypeptide, wherein the DNA molecule in the coding strand
comprises a translation start sequence for the selectable marker
polypeptide chosen from the group consisting of: a) a GTG start
codon; and b) a TTG start codon; and wherein the open reading frame
sequence that encodes the selectable marker protein has been
mutated to replace at least 10% of its CpG dinucleotides as
compared to the native open reading frame sequence that encodes the
selectable marker protein.
[0020] The translation start sequence in the coding strand for the
selectable marker polypeptide may comprise a GTG or TTG start
codon, most preferably a TTG start codon, flanked by sequences
providing for relatively good recognition of the non-ATG sequences
as start codons, such that at least some ribosomes start
translation from these start codons, i.e., the translation start
sequence may comprise the sequence ACC[GTG or TTG start codon]G or
GCC[GTG or TTG start codon]G.
[0021] In certain embodiments, the selectable marker protein
provides resistance against lethal and/or growth-inhibitory effects
of a selection agent, such as an antibiotic. In certain
embodiments, the selectable marker polypeptide provides resistance
against ZEOCIN.TM. antibiotic or against neomycin.
[0022] In certain embodiments, the DNA molecule comprises
comprising an open reading frame sequence that encodes a
polypeptide that provides resistance against neomycin, wherein the
DNA molecule comprises a sequence chosen from the group consisting
of: a) SEQ ID NO:128, with the proviso that at least half of the
CpG dinucleotides has been replaced without mutating the amino acid
sequence that is encoded, and with the further proviso that the
start codon is either GTG or TTG; and b) SEQ ID NO:118, with the
proviso that at least half of the CpG dinucleotides of the coding
strand has been replaced without mutating the amino acid sequence
that is encoded, and with the further proviso that the start codon
is either GTG or TTG; and c) SEQ ID NO:128 or SEQ ID NO:118, with
the proviso that it contains a mutation to encode either of the
following polypeptide variants as compared to the polypeptide
encoded by the native sequences: (i) substitution valine at
position 201 into glycine (201V>G), or (ii) subtitution of
glutamic acid at position 185 into aspartic acid (185E>D), or
(iii) a combination of both mutations (i) and (ii) (185E>D and
201V>G), with the further proviso that at least half of the CpG
dinucleotides of the coding strand has been replaced without
further mutating the amino acid sequence that is encoded beyond the
mutation indicated under (i)-(iii), and with the further proviso
that the start codon is either GTG or TTG. In one advantageous
embodiment hereof, the DNA molecule comprises SEQ ID NO:130, with
the proviso that nucleotide A at position 555 is replaced by C to
encode the encode the 185E>D mutation, and that nucleotide T at
position 602 is replaced by G and that nucleotide G at position 603
is replaced by T to encode the 201V>G mutation, and with the
further proviso that the start codon is either GTG or TTG.
[0023] In certain embodiments, the DNA molecule comprises an open
reading frame sequence that encodes a polypeptide that provides
resistance against zeocin, wherein the DNA molecule comprises a
sequence chosen from the group consisting of: a) SEQ ID NO:92, with
the proviso that at least half of the CpG dinucleotides has been
replaced without mutating the amino acid sequence that is encoded,
and with the further proviso that the start codon is either GTG or
TTG; and b) SEQ ID NO:92 wherein nucleotide A at position 280 is
replaced by T, and with the proviso that at least half of the CpG
dinucleotides has been replaced without mutating the amino acid
sequence that is encoded, and with the further proviso that the
start codon is either GTG or TTG. In one advantageous embodiment
hereof, the DNA sequence comprises SEQ ID NO:132.
[0024] In another aspect, provided is a DNA molecule comprising an
open reading frame sequence that encodes a selectable marker
polypeptide, wherein the selectable marker polypeptide is chosen
from the group consisting of: (i) tryptophan synthesizing enzyme
(trp); (ii) histidine synthesizing enzyme (his); and (iii) 5,6,7,8
tetrahydrofolate synthesizing enzyme (dhfr); and wherein the DNA
molecule in the coding strand comprises a translation start
sequence for the selectable marker polypeptide chosen from the
group consisting of: a) a GTG start codon; and b) .sub.a TTG start
codon.
[0025] In certain embodiments, the DNA molecule comprises an open
reading frame sequence that encodes trp, wherein the DNA molecule
comprises a sequence chosen from the group consisting of SEQ ID
NO:134 and SEQ ID NO:136, with the proviso that the first three
nucleotides (the start codon) are either GTG or TTG.
[0026] In certain embodiments, the DNA molecule comprises an open
reading frame sequence that encodes his, wherein the DNA molecule
comprises a sequence chosen from the group consisting of SEQ ID
NO:138 and SEQ ID NO:140, with the proviso that the first three
nucleotides (the start codon) are either GTG or TTG.
[0027] In certain embodiments, the DNA molecule comprises an open
reading frame sequence that encodes dhfr, wherein the DNA molecule
comprises a sequence chosen from the group consisting of SEQ ID
NO:98 and SEQ ID NO:122, with the proviso that the first three
nucleotides (the start codon) are either GTG or TTG.
[0028] The coding sequence of the polypeptide of interest may
comprises an optimal translation start sequence.
[0029] In certain embodiments, the open reading frame sequence that
encodes the selectable marker polypeptide has no ATG sequence in
the coding strand.
[0030] In certain embodiments, the open reading frame sequence that
encodes a selectable marker polypeptide is part of a multicistronic
transcription unit that further comprises an open reading frame
sequence encoding a polypeptide of interest.
[0031] In certain embodiments thereof, the open reading frame that
encodes the selectable marker polypeptide is upstream of the open
reading frame encoding the polypeptide of interest, and the open
reading frame that encodes the selectable marker polypeptide has no
ATG sequence in the coding strand. In alternative embodiments, the
open reading frame that encodes the polypeptide of interest is
upstream of the open reading frame that encodes the selectable
marker polypeptide, and the open reading frame that encodes the
selectable marker polypeptide is operably linked to an internal
ribosome entry site (IRES).
[0032] Further provided are expression cassettes comprising a DNA
molecule hereof, which expression cassettes further comprise a
promoter upstream of the multicistronic expression unit and being
functional in a eukaryotic host cell for initiation transcription
of the multicistronic expression unit, and the expression cassettes
further comprising a transcription termination sequence downstream
of the multicistronic expression unit.
[0033] In certain embodiments thereof, such expression cassettes
further comprise at least one chromatin control element chosen from
the group consisting of a matrix or scaffold attachment region
(MAR/SAR), an insulator sequence, a ubiquitous chromatin opener
element (UCOE), and an anti-repressor sequence. Anti-repressor
sequences are most preferred in this aspect, and in certain
embodiments, the anti-repressor sequences are chosen from the group
consisting of: a) any one SEQ ID NO:1 through SEQ ID NO:66; b)
fragments of any one of SEQ ID NO:1 through SEQ ID NO:66, wherein
the fragments have anti-repressor activity; c) sequences that are
at least 70% identical in nucleotide sequence to a) or b) wherein
the sequences have anti-repressor activity; and d) the complement
to any one of a) to c). In certain certain embodiments, the
anti-repressor sequences are chosen from the group consisting of:
STAR67 (SEQ ID NO:66), STAR7 (SEQ ID NO:7), STAR9 (SEQ ID NO:9),
STAR17 (SEQ ID NO:17), STAR27 (SEQ ID NO:27), STAR29 (SEQ ID
NO:29), STAR43 (SEQ ID NO:43), STAR44 (SEQ ID NO:44), STAR45 (SEQ
ID NO:45), STAR47 (SEQ ID NO:47), STAR61 (SEQ ID NO:61), and
functional fragments or derivatives of these STAR sequences. In
certain embodiments, the expression cassette comprises STAR67, or a
functional fragment or derivative thereof, positioned upstream of
the promoter driving expression of the multicistronic gene. In
certain embodiments, the multicistronic gene is flanked on both
sides by at least one anti-repressor sequence. In certain
embodiments, expression cassettes are provided according to the
invention, comprising in 5' to 3' order: anti-repressor sequence
A-anti-repressor sequence B-[promoter-multicistronic transcription
unit hereof (encoding the functional selectable marker protein
{from a sequence with a GTG or TTG start codon} and upstream or
downstream thereof the polypeptide of interest)-transcription
termination sequence]-anti-repressor sequence C, wherein A, B and C
may be the same or different.
[0034] In certain embodiments, the polypeptide of interest is a
part of a multimeric protein, for example a heavy or light chain of
an immunoglobulin.
[0035] Also provided are host cells comprising DNA molecules
hereof.
[0036] Further provided are methods for generating host cells
expressing a polypeptide of interest, such a method comprising the
steps of: introducing into a plurality of precursor host cells an
expression cassette hereof, culturing the cells under conditions
selecting for expression of the selectable marker polypeptide, and
selecting at least one host cell producing the polypeptide of
interest.
[0037] Further provided are methods for producing a polypeptide of
interest, the methods comprising culturing a host cell, the host
cell comprising an expression cassette hereof, and expressing the
polypeptide of interest from the expression cassette. In certain
embodiments, the polypeptide of interest is harvested from the host
cells and/or from the host cell culture medium.
[0038] In certain embodiments, if the selectable marker polypeptide
is trp, the host cell in advantageous embodiments is cultured in a
culture medium that contains indole and which culture medium is
essentially devoid of tryptophan. In other embodiments, if the
selectable marker polypeptide is his, the host cell in advantageous
embodiments is cultured in a culture medium that contains
histidinol and which culture medium is essentially devoid of
histidine. In other embodiments, if the selectable marker
polypeptide is dhfr, the host cell in advantageous embodiments is
cultured in a culture medium that contains folate and which culture
medium is essentially devoid of glycine, hypoxanthine and
thymidine.
[0039] In further aspects, provided is RNA molecules having the
sequence of a transcription product of a DNA molecule hereof.
Further, provided is selectable marker polypeptides that are the
translation product of a DNA molecule of the invention.
[0040] In another aspect, further provided is a DNA molecule
comprising an expression cassette comprising a multicistronic
transcription unit, the multicistronic transcription unit
comprising a sequence coding for a polypeptide of interest, a
sequence coding for a first selectable marker polypeptide, and a
sequence coding for a second selectable marker polypeptide, wherein
the sequence encoding the first selectable marker polypeptide in
the coding strand comprises a translation start sequence chosen
from the group consisting of a GTG start codon and a TTG start
codon, and wherein the second selectable marker polypeptide is
chosen from the group consisting of: (i) tryptophan synthesizing
enzyme (trp); (ii) histidine synthesizing enzyme (his); and (iii)
5,6,7,8 tetrahydrofolate synthesizing enzyme (dhfr), and wherein
the expression cassette further comprises a promoter upstream of
the multicistronic expression unit and a transcription termination
sequence downstream of the multicistronic expression unit, wherein
the expression cassette is functional in a eukaryotic host cell for
initiating transcription of the multicistronic expression unit, and
wherein the DNA molecule further comprises at least one chromatin
control element selected from the group consisting of matrix
attachment regions (MAR), and anti-repressor (STAR) sequences.
[0041] In one embodiment thereof, the sequence encoding the first
selectable marker polypeptide is upstream of the sequence encoding
the polypeptide of interest and the sequence encoding the first
selectable marker polypeptide in the coding strand is devoid of the
sequence ATG, and the sequence encoding the second selectable
marker polypeptide is downstream of the polypeptide of interest and
is operably linked to an IRES.
[0042] In another embodiment, the sequence encoding the polypeptide
of interest is upstream of the sequences encoding the first and
second selectable marker polypeptide, and the sequence encoding the
first selectable marker polypeptide is operably linked to an IRES,
and the sequence encoding the second selectable marker polypeptide
is operably linked to an IRES.
[0043] In certain embodiments, the first selectable marker
polypeptide confers resistance against lethal or growth-inhibitory
effects of a selection agent chosen from the group consisting of
ZEOCIN.RTM. and neomycin antibiotics.
[0044] In certain embodiments, a chromatin control element is an
anti-repressor sequence chosen from the group consisting of any one
of SEQ ID NO:1 through SEQ ID NO:66, and the complement of any of
these.
[0045] Further provided is host cells comprising such DNA
molecules.
[0046] Further provided is a method for expressing a polypeptide of
interest, comprising culturing a host cell that comprises a DNA
molecule of the invention, and expressing the polypeptide of
interest form the expression cassette, and wherein: a) if the
second selectable marker polypeptide is trp, the host cell is
cultured in a culture medium that contains indole and which culture
medium is essentially devoid of tryptophan; b) if the second
selectable marker polypeptide is his, the host cell is cultured in
a culture medium that contains histidinol and which culture medium
is essentially devoid of histidine; c) if the second selectable
marker polypeptide is dhfr, the host cell is cultured in a culture
medium that contains folate and which culture medium is essentially
devoid of glycine, hypoxanthine and thymidine. In certain
embodiments, the method further comprises harvesting the
polypeptide of interest, from the host cell, from the culture
medium, or from both the host cell and the culture medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1. Schematic representation of the use of a selection
marker gene (ZEOCIN.RTM.-resistance gene) of the incorporated '525
application. A. wild-type zeocin-resistance gene, having its normal
translation initation site (ATG start codon) and one internal ATG
codon, which codes for methionine. B. mutant zeocin-resistance
gene, wherein the internal ATG has been mutated into a codon for
leucine; this mutant is a functional ZEOCIN.RTM.-resistance gene.
C. same as B, but comprising a mutated translation initiation site,
wherein the context of the ATG start codon has been mutated to
decrease the translation initiation. D. same as B, but comprising a
mutated start codon (GTG). E. same as B, but with a TTG start
codon. The numbers under the Figures C-E schematically indicate a
relative amount of initiation frequency (under the start codon) and
"scan-through" frequency (under the coding sequence) by the
ribosomes, but only in a semi-quantitative manner, i.e., they
indicate the efficiency of translation initiation compared to each
other, but the qualitative numbers may differ completely: the
numbers only serve to explain the invention. See, Example 1 for
details.
[0048] FIG. 2. Schematic representation of a multicistronic
transcription unit according to the invention of the incorporated
'525 application, with more or less reciprocal interdependent
translation efficiency. Explanation as for FIG. 1, but now a dEGFP
gene (here exemplifying a gene of interest) has been placed
downstream of the selectable marker polypeptide coding sequence.
The ZEOCIN.RTM.-resistance gene comprises the internal
Met.fwdarw.Leu mutation (see FIG. 1B). See, Example 2 for
details.
[0049] FIG. 3. Results of selection systems according to the
invention of the incorporated '525 application, with and without
STAR elements. A. ZEOCIN.RTM.-resistance gene with ATG start codon
in bad context (referred to as "ATGmut" in the picture, but
including a spacer sequence behind the ATG in the bad context, so
in the text generally referred to as "ATGmut/space"). B.
ZEOCIN.RTM.-resistance gene with GTG start codon. C.
ZEOCIN.RTM.-resistance gene with TTG start codon. d2EGFP signal for
independent colonies is shown on the vertical axis. See, Example 2
for details.
[0050] FIG. 4. Results of selection system according to the
invention of the incorporated '525 application in upscaled
experiment (A), and comparison with selection system according to
prior art using an IRES (B). d2EGFP signal for independent colonies
is shown on the vertical axis. See, Example 3 for details.
[0051] FIG. 5. Results of selection system with multicistronic
transcription unit according to the invention of the incorporated
'525 application, using blasticidin as a selectable marker. A.
blasticidin resistance gene mutated to comprise a GTG start codon.
B. blasticidin resistance gene mutated to comprise a TTG start
codon. The blasticidin resistance gene has further been mutated to
remove all internal ATG sequences. d2EGFP signal for independent
colonies is shown on the vertical axis. See, Example 4 for
details.
[0052] FIG. 6. Stability of expression of several clones with a
multicistronic transcription unit according to the invention
(including a ZEOCIN.RTM. with TTG start codon) of the incorporated
'525 application. Selection pressure (100 .mu.g/ml zeocin) was
present during the complete experiment. d2EGFP signal for
independent colonies is shown on the vertical axis. See, Example 5
for details.
[0053] FIG. 7. As FIG. 6, but ZEOCIN.RTM. antibiotic concentration
was lowered to 20 .mu.g/ml after establishment of clones.
[0054] FIG. 8. As FIG. 6, but ZEOCIN.RTM. antibiotic was absent
from culture medium after establishment of clones.
[0055] FIG. 9. Expression of an antibody (anti-EpCAM) using the
selection system with the multicistronic transcription unit
according to the invention of the incorporated '525 application.
The heavy chain (HC) and light chain (LC) are the polypeptide of
interest in this example. Each of these is present in a separate
transcription unit, which are both on a single nucleic acid
molecule in this example. The HC is preceded by the
zeocin-resistance gene coding for a selectable marker polypeptide,
while the LC is preceded by the blasticidin resistance gene coding
for a selectable marker polypeptide. Both resistance genes have
been mutated to comprise an ATG start codon in a non-optimal
context ("mutATG" in Figure, but including a spacer sequence, and
hence in the text generally referred to as "ATGmut/space"). Each of
the multicistronic transcription units is under control of a CMV
promoter. Constructs with STAR sequences as indicated were compared
to constructs without STAR sequences. The antibody levels obtained
when these constructs were introduced into host cells are given on
the vertical axis in pg/cell/day for various independent clones.
See, Example 6 for details.
[0056] FIG. 10. As FIG. 9, but both the selection marker genes have
been provided with a GTG start codon. See, Example 6 for
details.
[0057] FIG. 11. As FIG. 9, but both the selection marker genes have
been provided with a TTG start codon. See, Example 6 for
details.
[0058] FIG. 12. Stability of expression in sub-clones in the
absence of selection pressure (after establishing colonies under
selection pressure, some colonies where sub-cloned in medium
containing no zeocin). See, Example 5 for details.
[0059] FIG. 13. Copy-number dependency of expression levels of an
embodiment of the invention of the incorporated '525 application.
See, Example 5 for details.
[0060] FIG. 14. As FIG. 1, but for the blasticidin resistance gene.
None of the 4 internal ATGs in this gene are in frame coding for a
methionine, and therefore the redundancy of the genetic code was
used to mutate these ATGs without mutating the internal amino acid
sequence of the encoded protein.
[0061] FIG. 15. Coding sequence of the wild-type zeocin-resistance
gene (SEQ ID NO:92). Bold ATGs code for methione. The first bold
ATG is the start codon.
[0062] FIG. 16. Coding sequence of the wild-type blasticidin
resistance gene (SEQ ID NO:94). Bold ATGs code for methione. The
first bold ATG is the start codon. Other ATGs in the sequence are
underlined: these internal ATGs do not code for methionine, because
they are not in frame.
[0063] FIG. 17. Coding sequence of the wild-type puromycin
resistance gene (SEQ ID NO:96). Bold ATGs code for methione. The
first bold ATG is the start codon.
[0064] FIG. 18. Coding sequence of the wild-type mouse DHFR gene
(SEQ ID NO:98). Bold ATGs code for methione. The first bold ATG is
the start codon. Other ATGs in the sequence are underlined: these
internal ATGs do not code for methionine, because they are not in
frame.
[0065] FIG. 19. Coding sequence of the wild-type hygromycin
resistance gene (SEQ ID NO:100). Bold ATGs code for methione. The
first bold ATG is the start codon. Other ATGs in the sequence are
underlined: these internal ATGs do not code for methionine, because
they are not in frame.
[0066] FIG. 20. Coding sequence of the wild-type neomycin
resistance gene (SEQ ID NO:102). Bold ATGs code for methione. The
first bold ATG is the start codon. Other ATGs in the sequence are
underlined: these internal ATGs do not code for methionine, because
they are not in frame.
[0067] FIG. 21. Coding sequence of the wild-type human glutamine
synthase (GS) gene (SEQ ID NO:104). Bold ATGs code for methione.
The first bold ATG is the start codon. Other ATGs in the sequence
are underlined: these internal ATGs do not code for methionine,
because they are not in frame.
[0068] FIG. 22. Schematic representation of some further modified
zeocin-resistance selection marker genes with a GTG start codon
according to the invention, allowing for further fine-tuning of the
selection stringency. See, Example 7 for details.
[0069] FIG. 23. Results with expression systems containing the
further modified zeocin-resistance selection marker genes. See,
Example 7 for details. Dots indicate individual data points; lines
indicate the average expression levels; used constructs (see also
FIG. 22) are indicated on the horizontal axis (the addition of
7/67/7 at the end of the construct name indicates the presence of
STAR sequences 7 and 67 upstream of the promoter and STAR7
downstream of the transcription termination site), and
schematically depicted above the graph; vertical axis indicates
d2EGFP signal.
[0070] FIG. 24. Schematic representation of some further modified
zeocin-resistance selection marker genes with a TTG start codon
according to the invention, allowing for further fine-tuning of the
selection stringency. See, Example 8 for details.
[0071] FIG. 25. Results with expression systems containing the
further modified zeocin-resistance selection marker genes. See,
Example 8 for details. Dots indicate individual data points; lines
indicate the average expression levels; used constructs are
indicated on the horizontal axis, and schematically depicted above
the graph; vertical axis indicates d2EGFP signal.
[0072] FIG. 26. As FIG. 1, but for the puromycin resistance gene.
All three internal ATGs code for methione (panel A), and are
replaced by CTG sequences coding for leucine (panel B). See,
Example 9 for details.
[0073] FIG. 27. Results with expression constructs containing the
puromycin resistance gene with a TTG start codon and no internal
ATG codons. See, Example 9 for details. Dots indicate individual
data points; lines indicate the average expression levels; used
constructs are indicated on the horizontal axis, and schematically
depicted above the graph; vertical axis indicates d2EGFP
signal.
[0074] FIG. 28. As FIG. 1, but for the neomycin resistance gene.
See, Example 10 for details. A. wild-type neomycin resistance gene;
ATG sequences are indicated, ATGs coding for methionine are
indicated by Met above the ATG. B. neomycin resistance gene without
ATG sequences, and with a GTG start codon. C. neomycin resistance
gene without ATG sequences, and with a TTG start codon.
[0075] FIG. 29. As FIG. 1, but for the dhfr gene. See, Example 11
for details. A. wild-type dhfr gene; ATG sequences are indicated,
ATGs coding for methionine are indicated by Met above the ATG. B.
dhfr gene without ATG sequences, and with a GTG start codon. C.
dhfr gene without ATG sequences, and with a TTG start codon.
[0076] FIG. 30. Results with expression constructs
(zeocin-selectable marker) according to the invention of the
incorporated '525 application in PER.C6.RTM. cells. See, Example 12
for details. Dots indicate individual data points lines indicate
the average expression levels; used constructs are indicated on the
horizontal axis, and schematically depicted above the graph;
vertical axis indicates d2EGFP signal.
[0077] FIG. 31. Results with expression constructs (blasticidin
selectable marker) according to the invention of the incorporated
'525 application in PER.C6.RTM. cells. See, Example 12 for details.
Dots indicate individual data points; lines indicate the average
expression levels; used constructs are indicated on the horizontal
axis, and schematically depicted above the graph; vertical axis
indicates d2EGFP signal.
[0078] FIG. 32. Results with expression constructs according to the
invention of the incorporated '525 application, further comprising
a transcription pause (TRAP) sequence. See, Example 13 for details.
Dots indicate individual data points; lines indicate the average
expression levels; used constructs are indicated on the horizontal
axis, and schematically depicted above the graph; vertical axis
indicates d2EGFP signal.
[0079] FIG. 33. Copy-number dependency of expression of an antibody
using transcription units according to the invention of the
incorporated '525 application. See, Example 14 for details.
[0080] FIG. 34. Antibody expression from colonies containing
expression constructs according to the invention of the
incorporated '525 application, wherein the copy number of the
expression constructs is amplified by methotrexate. See, Example 15
for details. White bars: selection with ZEOCIN.RTM. and
blasticidin; black bars: selection with zeocin, blasticidin and
methotrexate (MTX). Numbers of tested colonies are depicted on the
horizontal axis.
[0081] FIG. 35. Results with different promoters. See, Example 16
for details. Dots indicate individual data points; lines indicate
the average expression levels; used constructs are indicated on the
horizontal axis, and schematically depicted above the graph;
vertical axis indicates d2EGFP signal.
[0082] FIG. 36. Results with different STAR elements. See, Example
17 for details. Dots indicate individual data points; lines
indicate the average expression levels; used constructs are
indicated on the horizontal axis, and schematically depicted above
the graph; vertical axis indicates d2EGFP signal.
[0083] FIG. 37. Results with other chromatin control elements. See,
Example 18 for details. Dots indicate individual data points; lines
indicate the average expression levels; used constructs are
indicated on the horizontal axis, and schematically depicted above
the graph (black triangles indicate different tested chromatin
control elements); vertical axis indicates d2EGFP signal.
[0084] FIG. 38. Results with expression constructs according to the
invention of the incorporated '953 application. The expression
construct contains the sequence encoding the polypeptide of
interest (exemplified here by d2EGFP) upstream of an IRES, which is
upstream of the sequence encoding the selectable marker according
to the invention (exemplified here by the zeocin-resistance gene,
with a TTG start codon (TTG Zeo) (or in controls with its normal
ATG start codon (ATG Zeo)). See, Example 19 for details. Dots
indicate individual data points; lines indicate the average
expression levels; used constructs are indicated on the horizontal
axis, and schematically depicted above the graph; vertical axis
indicates d2EGFP signal.
[0085] FIG. 39. Erythropoietin (EPO) expression with expression
constructs of the invention. See, Example 20 for details.
[0086] FIG. 40. Results with different STAR elements in the
CHO-DG44 cell line. Dots indicate individual data points; lines
indicate the average expression levels; vertical axis indicates
d2EGFP signal. The construct is schematically shown above the
graph, while the STAR elements tested in the construct are
indicated below the horizontal axis. See, Example 21 for
details.
[0087] FIG. 41. Results with a zeocin-resistance marker with
reduced CpG content in CHO-K1 cells. Dots indicate individual data
points; lines indicate the average expression levels; vertical axis
indicates d2EGFP signal. See, Example 22 for details.
[0088] FIG. 42. As in FIG. 41, but now in CHO-DG44 cells. See,
Example 22 for details.
[0089] FIG. 43. Results with "CpG poor" neomycin resistance marker
having different mutations. Dots indicate individual data points;
lines indicate the average expression levels; vertical axis
indicates d2EGFP signal. See, Example 23 for details.
[0090] FIG. 44. Schematic drawing of constructs with tryptophane
synthesizing enzyme (trp) as selectable marker polypeptide
according to the invention. See, Example 24 for details.
[0091] FIG. 45. Schematic drawing of constructs with histidine
synthesizing enzyme (his) as selectable marker polypeptide
according to the invention. See, Example 25 for details.
[0092] FIG. 46. Schematic drawing of constructs with dhfr as
selectable marker polypeptide according to the invention. See,
Example 26 for details.
[0093] FIG. 47. Schematic drawing of constructs having
multicistronic transcription units with two selectable marker
polypeptides and one polypeptide of interest (HC: heavy chain; LC:
light chain), the first selectable marker polypeptide providing
resistance to an antibiotic and having a TTG (or GTG, not shown)
start codon in the coding sequence and the second selectable marker
polypeptide being trp or dhfr and being under control of an IRES.
See, Example 27 for details.
DETAILED DESCRIPTION
[0094] In one aspect, provided is a DNA molecule comprising an open
reading frame sequence that encodes a selectable marker
polypeptide, wherein the DNA molecule in the coding strand
comprises a translation start sequence for the selectable marker
polypeptide chosen from the group consisting of: a) a GTG start
codon; and b) a TTG start codon; and wherein the open reading frame
sequence that encodes the selectable marker protein has been
mutated to replace at least 10% of its CpG dinucleotides (any "CG"
in the sequence) as compared to the native open reading frame
sequence that encodes the selectable marker protein. Such a DNA
molecule can be used according to the invention for obtaining
eukaryotic host cells expressing high levels of the polypeptide of
interest, by selecting for the expression of the selectable marker
polypeptide. Subsequently or simultaneously, one or more host
cell(s) expressing the polypeptide of interest can be identified,
and further used for expression of high levels of the polypeptide
of interest.
[0095] It is shown herein that the reduction of the CpG content of
the selectable marker gene of the invention, i.e., having a TTG or
GTG start codon, can lead to improved expression of a polypeptide
of interest that is translated from a multicistronic transcription
unit from which also the selectable marker polypeptide is
translated. Without wishing to be bound by theory, it is believed
that reduction of the CpG content may reduce the possibility for
silencing of transcription, because CpG dinucleotides can be
methylated and silenced in eukaryotes. Selectable marker
polypeptides that are encoded by genes with a relatively high CpG
content, often derived from bacterial sequences, for instance,
ZEOCIN.TM. antibiotic and neomycin, may benefit from the reduction
of the CpG content. In certain embodiments, CpG dinucleotides are
removed from a sequence encoding a selectable marker polypeptide
without changing the encoded amino acid sequence. This can be done
by taking advantage of the redundancy of the genetic code, as is
well known and routine to the person skilled in the art of
molecular biology.
[0096] In certain embodiments, in particular when the selectable
marker polypeptide coding sequence is to be used upstream of the
coding sequence of a polypeptide of interest in a multicistronic
transcription unit described herein, the coding sequence of the
selectable marker polypeptide is devoid of ATG sequences.
[0097] It is expected that a positive effect of removing CpG
dinucleotides will be apparent when at least 10% of the CpG
dinucleotides in the coding sequence of the selectable marker gene
have been replaced. It is expected that removal of more CpG
dinucleotides will increase the effect, and hence in certain
embodiments, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70% or at least 80% of the CpG
dinucleotides are mutated compared to the native open reading frame
sequence that encodes the selectable marker protein. In certain
advantageous embodiments, at least half of the CpG dinucleotides of
the open reading frame sequence that encodes the selectable marker
polypeptide have been replaced as compared to the native open
reading frame sequence that encodes the selectable marker
polypeptide.
[0098] A native open reading frame sequence that encodes the
selectable marker polypeptide that provides resistance to neomycin
is given as SEQ ID NO:128 (containing internal ATGs) and as SEQ ID
NO:118 (lacking internal ATGs). In advantageous embodiments, these
sequences may contain one or more further mutations so that the
encoded polypeptide has a mutation of valine at position 201 to
glycine (201V>G), of glutamic acid at position 185 to aspartic
acid (185E>D), or both (185E>D, 201V>G).
[0099] A native open reading frame sequence that encodes the
selectable marker polypeptide that provides resistance to
ZEOCIN.TM. antibiotic is given as SEQ ID NO:92 (containing internal
ATGs), and mutation of A at position 280 into T in this sequence
gives a sequence lacking internal ATGs, and wherein the internally
encoded methionine at position 94 is replaced by leucine. For the
DNA sequences of the invention, the start codon (first three
nucleotides of the DNA sequences) is mutated into a GTG or into a
TTG start codon.
[0100] In certain advantageous embodiments, the selectable marker
polypeptide provides resistance against ZEOCIN.TM. antibiotic. In
certain embodiments thereof, the DNA molecule comprises SEQ ID
NO:92, wherein at least half of the CpG dinucleotides has been
replaced without mutating the amino acid sequence that is encoded,
with the proviso that the start codon (first three nucleotides in
the sequence) is replaced by a start codon chosen from GTG or TTG.
In an alternative embodiment, the DNA molecule comprises SEQ ID
NO:92 wherein nucleotide A at position 280 is replaced by T, such
that encoded amino acid 94 (methionine) is replaced by leucine, and
wherein at least half of the CpG dinucleotides has been replaced
without further mutating the amino acid sequence that is encoded,
with the proviso that the start codon (first three nucleotides in
the sequence) is replaced by a start codon chosen from GTG or TTG.
This embodiment lacks ATG sequences in the coding sequence for the
ZEOCIN.TM. antibiotic-resistance gene, and is therefore suitable in
the multicistronic transcription units of the invention wherein the
coding sequence for the selectable marker polypeptide is upstream
of the coding sequence for the polypeptide of interest. In one
preferred embodiment hereof, the DNA molecule comprises SEQ ID
NO:132.
[0101] In other advantageous embodiments, the selectable marker
polypeptide provides resistance against neomycin. In certain
embodiments thereof, the DNA molecule comprises a sequence chosen
from the group consisting of any one of: a) SEQ ID NO:128, with the
proviso that at least half of the CpG dinucleotides has been
replaced without mutating the amino acid sequence that is encoded,
and with the further proviso that the start codon (the first ATG
sequence) is replaced by either GTG or TTG; b) SEQ ID NO:118, with
the proviso that at least half of the CpG dinucleotides has been
replaced without mutating the amino acid sequence that is encoded,
and with the further proviso that the start codon (the first ATG
sequence) is replaced by either GTG or TTG; and c) SEQ ID NO:128 or
SEQ ID NO:118, containing a mutation to encode a neomycin
resistance protein variant as compared to the sequences encoded by
the indicated sequences, the variant having glycine at position 201
in the encoded protein (201G variant), or aspartic acid at position
185 (185D variant), or both glycine at position 201 and aspartic
acid at position 185 (185D, 201G variant), with the proviso that at
least half of the CpG dinucleotides in the given DNA sequence has
been replaced without further mutating the amino acid sequence that
is encoded, and with the further proviso that the start codon (the
first ATG sequence) is replaced by either GTG or TTG. The 185D
variant is for instance obtained by replacing the codon from
position 553-555 in the provided nucleic acid sequences with the
sequence GAC, and the 201G variant is for instance obtained by
replacing the codon from position 601-603 in the provided nucleic
acid sequence with GGT. In one preferred embodiment, the DNA
molecule comprises SEQ ID NO:130, with the proviso that nucleotide
A at position 555 is replaced by C (to encode the 185E>D
variant), and that nucleotide T at position 602 is replaced by G
and that nucleotide G at position 603 is replaced by T (to encode
the 201V>G variant), and with the further proviso that the start
codon (ATG at positions 1-3) is replaced by either GTG or TTG. It
will be clear to the skilled person that further variations can be
prepared by the skilled person without departing from the teaching
of the invention, and such further variations are encompassed with
the invention as long as the start codon is not ATG and the encoded
protein provides resistance against neomycin (or G418). The 185D
and 201G variants further improve the selection stringency
according to the invention.
[0102] The term "monocistronic gene" is defined as a gene capable
of providing a RNA molecule that encodes one polypeptide. A
"multicistronic transcription unit," also referred to as
multicistronic gene, is defined as a gene capable of providing an
RNA molecule that encodes at least two polypeptides. The term
"bicistronic gene" is defined as a gene capable of providing a RNA
molecule that encodes two polypeptides. A bicistronic gene is
therefore encompassed within the definition of a multicistronic
gene. A "polypeptide" as used herein comprises at least five amino
acids linked by peptide bonds, and can for instance be a protein or
a part, such as a subunit, thereof. Mostly, the terms polypeptide
and protein are used interchangeably herein. A "gene" or a
"transcription unit" as used in the invention can comprise
chromosomal DNA, cDNA, artificial DNA, combinations thereof, and
the like. Transcription units comprising several cistrons are
transcribed as a single mRNA.
[0103] A multicistronic transcription unit according to the
invention can for instance be a bicistronic transcription unit
coding from 5' to 3' for a selectable marker polypeptide and for a
polypeptide of interest, or for instance a bicistronic
transcription unit coding from 5' to 3' for a polypeptide of
interest and for a selectable marker polypeptide. In the former
case, the coding sequence for the selectable marker polypeptide is
preferably devoid of ATG sequences in the coding starnd. In the
latter case, the polypeptide of interest is encoded upstream from
the coding sequence for the selectable marker polypeptide and an
internal ribosome entry site (IRES) is operably linked to the
sequence encoding the selectable marker polypeptide, and hence the
selectable marker polypeptide is dependent from (also referred to
as "operably linked to") the IRES for its translation.
[0104] One may use separate transcription units for the expression
of different polypeptides of interest, also when these form part of
a multimeric protein (see, e.g., Example 6: the heavy and light
chain of an antibody each are encoded by a separate transcription
unit, each of these expression units being a bicistronic expression
unit).
[0105] The DNA molecules described herein can be present in the
form of double stranded DNA, having with respect to the selectable
marker polypeptide and the polypeptide of interest a coding strand
and a non-coding strand, the coding strand being the strand with
the same sequence as the translated RNA, except for the presence of
T instead of U. Hence, an AUG start codon is coded for in the
coding strand by an ATG sequence, and the strand containing this
ATG sequence corresponding to the AUG start codon in the RNA is
referred to as the coding strand of the DNA. It will be clear to
the skilled person that start codons or translation initiation
sequences are in fact present in an RNA molecule, but that these
can be considered equally embodied in a DNA molecule coding for
such an RNA molecule; hence, wherever the invention refers to a
start codon or translation initation sequence, the corresponding
DNA molecule having the same sequence as the RNA sequence but for
the presence of a T instead of a U in the coding strand of the DNA
molecule is meant to be included, and vice versa, except where
explicitly specified otherwise. In other words, a start codon is
for instance an AUG sequence in RNA, but the corresponding ATG
sequence in the coding strand of the DNA is referred to as start
codon as well in the invention. The same is used for the reference
of "in frame" coding sequences, meaning triplets (3 bases) in the
RNA molecule that are translated into an amino acid, but also to be
interpreted as the corresponding trinucleotide sequences in the
coding strand of the DNA molecule.
[0106] The selectable marker polypeptide and the polypeptide of
interest encoded by the multicistronic gene each have their own
translation initation sequence, and therefore each have their own
start codon (as well as stop codon), i.e., they are encoded by
separate open reading frames.
[0107] The term "selection marker" or "selectable marker" is
typically used to refer to a gene and/or protein whose presence can
be detected directly or indirectly in a cell, for example a
polypeptide that inactivates a selection agent and protects the
host cell from the agent's lethal or growth-inhibitory effects
(e.g., an antibiotic resistance gene and/or protein). Another
possibility is that the selection marker induces fluorescence or a
color deposit (e.g., green fluorescent protein (GFP) and
derivatives (e.g d2EGFP), luciferase, lacZ, alkaline phosphatase,
etc.), which can be used for selecting cells expressing the
polypeptide inducing the color deposit, e.g., using a fluorescence
activated cell sorter (FACS) for selecting cells that express GFP.
Preferably, the selectable marker polypeptide according to provided
is resistance against lethal and/or growth-inhibitory effects of a
selection agent. The selectable marker polypeptide is encoded by
the DNA described herein. The selectable marker polypeptide
described herein is functional in a eukaryotic host cell, and thus
able to be selected for in eukaryotic host cells. Any selectable
marker polypeptide fulfilling this criterion can in principle be
used. Such selectable marker polypeptides are well known in the art
and routinely used when eukaryotic host cell clones are to be
obtained, and several examples are provided herein. In certain
embodiments, a selection marker used is ZEOCIN.RTM. antibiotic. In
other embodiments, blasticidin is used. The person skilled in the
art will know that other selection markers are available and can be
used, e.g., neomycin, puromycin, bleomycin, hygromycin, etc. In
other embodiments, kanamycin is used. In yet other embodiments, the
DHFR gene is used as a selectable marker, which can be selected for
by methotrexate, especially by increasing the concentration of
methotrexate cells can be selected for increased copy numbers of
the DHFR gene. Similarly, the glutamine synthetase (GS) gene can be
used, for which selection is possible in cells having insufficient
GS (e.g., NS-0 cells) by culturing in media without glutamine, or
alternatively in cells having sufficient GS (e.g., CHO cells) by
adding an inhibitor of GS, methionine sulphoximine (MSX). Other
selectable marker genes that could be used, and their selection
agents, are for instance described in table 1 of U.S. Pat. No.
5,561,053, incorporated by reference herein; see also Kaufman,
Methods in Enzymology, 185:537-566 (1990), for a review of
these.
[0108] Other selectable marker polypeptides that can be used are
enzymes involved in metabolic pathways. For instance, mammalian
cells lack enzymes that are part of the metabolic pathway to create
the amino acids tryptophan or histidine. Hence, these amino acids
need to present in the culture medium when mammalian cell lines are
to be cultured. However, providing the genetic information (which
can be derived from the sequences present in bacteria) encoding the
enzymes to the mammalian cells and that are essential for the
synthesis of the respective amino acid can be used for selection
purposes, by growing the cells in a culture medium lacking the
respective amino acid, and containing certain precursors for the
amino acid which precursor can then be converted into the amino
acid by the encoded metabolic enzyme, if this is expressed in the
mammalian cell. For example, tryptophan synthesizing enzyme (trp)
can be used as a selection marker, by omitting tryptophan from the
culture medium and including indol into the culture medium (Hartman
and Mulligan, 1988). The trp (trpB) gene can be derived from E.
coli, and can be used according to the invention, preferably by
providing it with a GTG or TTG start codon (see SEQ ID NO:134 for
the sequence of the trp gene, and SEQ ID NO:136 for the sequence of
the trp gene wherein all internal ATG sequences have been removed).
As another example histindine synthesizing enzyme (his) can be used
as a selection marker, by omitting histidine from the culture
medium and including histidinol into the culture medium (Hartman
and Mulligan, 1988). The his gene can be derived from
S.typhimurium, and can be used according to the invention,
preferably by providing it with a GTG or TTG start codon (see, SEQ
ID NO:138 for the sequence of the his gene, and SEQ ID NO:140 for
the sequence of the his gene wherein all internal ATG sequences
have been removed). As another example, the mammalian 5,6,7,8
tetrahydrofolate synthesizing enzyme dihydrofolate reductase (dhfr)
can be used as a selection marker in cells that have a dhfr.sup.-
phenotype (e.g., CHO-DG44 cells), by omitting glycine, hypoxanthine
and thymidine from the culture medium and including folate (or
(dihydro)folic acid) into the culture medium (Simonsen et al.,
1988). The dhfr gene can for instance be derived from the mouse
genome or mouse cDNA and can be used, preferably by providing it
with a GTG or TTG start codon (see SEQ ID NO:98 for the sequence of
the dhfr gene, and SEQ ID NO:122 for the sequence of the dhfr gene
wherein all internal ATG sequences have been removed). In all these
embodiments, by "omitting from the culture medium" is meant that
the culture medium has to be essentially devoid of the indicated
component(s), meaning that there is insufficient of the indicated
component present to sustain growth of the cells in the culture
medium, so that a good selection is possible when the genetic
information for the indicated enzyme is expressed in the cells and
the indicated precursor component is present in the culture medium.
For instance, the indicated component is present at a concentration
of less than 0.1% of the concentration of that component that is
normally used in the culture medium for a certain cell type.
Preferably, the indicated component is absent from the culture
medium. A culture medium lacking the indicated component can be
prepared according to standard methods by the skilled person or can
be obtained from commercial media suppliers. A potential advantage
of the use of these types of metabolic enzymes as selectable marker
polypeptides is that they can be used to keep the multicistronic
transcription units under continuous selection, which may result in
higher expression of the polypeptide of interest.
[0109] In another aspect, the invention uses the trp, his, or dhfr
metabolic selection markers as an additional selection marker in a
multicistronic transcription unit hereof. In such embodiments,
selection of host cell clones with high expression is first
established by use of, for instance, an antibiotic selection
marker, e.g., ZEOCIN.TM. antibiotic, neomycin, etc, the coding
sequences of which will have a GTG or TTG start codon according to
the invention. After the selection of suitable clones, the
antibiotic selection is discontinued, and now continuous or
intermittent selection using the metabolic enzyme selection marker
can be performed by culturing the cells in the medium lacking the
appropriate identified components described supra and containing
the appropriate precursor components described supra. In this
aspect, the metabolic selection markers are operably linked to an
IRES, and can have their normal ATG content, and the start codon
can be suitably chosen from ATG, GTG or TTG. The multicistronic
transcription units in this aspect are at least tricistronic.
[0110] When two multicistronic transcription units are to be
selected for in a single host cell, each one preferably contains
the coding sequence for a different selectable marker, to allow
selection for both multicistronic transcription units. Of course,
both multicistronic transcription units may be present on a single
nucleic acid molecule or alternatively each one may be present on a
separate nucleic acid molecule.
[0111] The term "selection" is typically defined as the process of
using a selection marker/selectable marker and a selection agent to
identify host cells with specific genetic properties (e.g., that
the host cell contains a transgene integrated into its genome). It
is clear to a person skilled in the art that numerous combinations
of selection markers are possible. One antibiotic that is
particularly advantageous is ZEOCIN.TM., because the ZEOCIN.RTM.
antibiotic-resistance protein (zeocin-R) acts by binding the drug
and rendering it harmless. Therefore, it is easy to titrate the
amount of drug that kills cells with low levels of zeocin-R
expression, while allowing the high-expressors to survive. All
other antibiotic-resistance proteins in common use are enzymes, and
thus act catalytically (not 1:1 with the drug). Hence, the
antibiotic ZEOCIN.TM. is a preferred selection marker. However, the
invention also works with other selection markers.
[0112] A selectable marker polypeptide described herein is the
protein that is encoded by the nucleic acid of the invention, which
polypeptide can be detected, for instance because it provides
resistance to a selection agent such as an antibiotic. Hence, when
an antibiotic is used as a selection agent, the DNA encodes a
polypeptide that confers resistance to the selection agent, which
polypeptide is the selectable marker polypeptide. DNA sequences
coding for such selectable marker polypeptides are known, and
several examples of wild-type sequences of DNA encoding selectable
marker proteins are provided herein (FIGS. 15-21). It will be clear
that mutants or derivatives of selectable markers can also be
suitably used according to the invention, and are therefore
included within the scope of the term "selectable marker
polypeptide," as long as the selectable marker protein is still
functional.
[0113] For convenience and as generally accepted by the skilled
person, in many publications as well as herein, often the gene and
protein encoding the resistance to a selection agent is referred to
as the "selectable agent (resistance) gene" or "selection agent
(resistance) protein," respectively, although the official names
may be different, e.g., the gene coding for the protein conferring
restance to neomycin (as well as to G418 and kanamycin) is often
referred to as neomycin (resistance) (or neo.sup.r) gene, while the
official name is aminoglycoside 3'-phosphotransferase gene.
[0114] It is beneficial to have low levels of expression of the
selectable marker polypeptide, so that stringent selection is
possible. In the invention this is brought about by using a
selectable marker coding sequence with a non-optimal translation
efficiency. Upon selection, only cells that have nevertheless
sufficient levels of selectable marker polypeptide will be
selected, meaning that such cells must have sufficient
transcription of the multicistronic transcription unit and
sufficient translation of the selectable marker polypeptide, which
provides a selection for cells where the multicistronic
transcription unit has been integrated or otherwise present in the
host cells at a place where expression levels from this
transcription unit are high.
[0115] In certain embodiments, the DNA molecules hereof have the
coding sequence for the selectable marker polypeptide upstream of
the coding sequence for the polypeptide of interest, to provide for
a multicistronic transcript (disclosed in detail in the
incorporated '525 application). Hence, such a multicistronic
transcription unit comprises in the 5' to 3' direction (both in the
transcribed strand of the DNA and in the resulting transcribed RNA)
the coding sequence for the selectable marker polypeptide and the
sequence encoding the polypeptide of interest. In such embodiments,
the open reading frame sequence that encodes the selectable marker
polypeptide has no ATG sequences in the coding strand.
[0116] In alternative embodiments (disclosed in detail in the
incorporated '953 application), the DNA molecules according to the
invention have the coding sequence for the selectable marker
polypeptide downstream of the coding sequence for the polypeptide
of interest. Hence, the multicistronic transcription unit comprises
in the 5' to 3' direction (both in the transcribed strand of the
DNA and in the resulting transcribed RNA) the sequence encoding the
polypeptide of interest and the coding sequence for the selectable
marker polypeptide. In such embodiments, an IRES is upstream of and
operably linked to the coding sequence for the selectable marker
polypeptide.
[0117] To decrease translation of the selectable marker cistron,
according to the invention the nucleic acid sequence coding for the
selectable marker polypeptide comprises a mutation in the start
codon (or in the context thereof) that decreases the translation
initiation efficiency of the selectable marker polypeptide in a
eukaryotic host cell. Preferably, a GTG start codon or more
prefereably a TTG start codon is engineered into the selectable
marker polypeptide. The translation efficiency is lower than that
of the corresponding wild-type sequence in the same cell, i.e., the
mutation results in less polypeptide per cell per time unit, and
hence less selectable marker polypeptide. This can be detected
using routine methods known to the person skilled in the art. For
instance, in the case of antibiotic selection, the mutation will
result in less resistance than obtained with the sequence having no
such mutation and hence normal translation efficiency, which
difference can easily be detected by determining the number of
surviving colonies after a normal selection period, which will be
lower when a translation efficiency decreasing mutation is present.
As is well known to the person skilled in the art there are a
number of parameters that indicate the expression level marker
polypeptide such as, the maximum concentration of selection agent
to which cells are still resistant, number of surviving colonies at
a given concentration, growth speed (doubling time) of the cells in
the presence of selection agent, combinations of the above, and the
like.
[0118] The mutation that decreases the translation initiation
efficiency according to the invention is established by providing
the selectable marker polypeptide coding sequence with a
non-optimal translation start sequence.
[0119] For example, the translation initiation efficiency of the
selectable marker gene in eukaryotic cells can be suitably
decreased according to the invention by mutating the start codon
and/or the nucleotides in positions -3 to -1 and +4 (where the A of
the ATG start codon is nt +1), for instance in the coding strand of
the corresponding DNA sequence, to provide a non-optimal
translation start sequence. A translation start sequence is often
referred to in the field as "Kozak sequence," and an optimal Kozak
sequence is RCCATGG, the start codon underlined, R being a purine,
i.e., A or G (see Kozak M, 1986, 1987, 1989, 1990, 1997, 2002).
Hence, besides the start codon itself, the context thereof, in
particular nucleotides -3 to -1 and +4, are relevant, and an
optimal translation startsequence comprises an optimal start codon
(i.e., ATG) in an optimal context (i.e., the ATG directly preceded
by RCC and directly followed by G). A non-optimal translation start
sequence is defined herein as any sequence that gives at least some
detectable translation in a eukaryotic cell (detectable because the
selection marker polypeptide is detectable), and not having the
consensus sequence RCCATGG (start codon underlined). Translation by
the ribosomes is most efficient when an optimal Kozak sequence is
present (see Kozak M, 1986, 1987, 1989, 1990, 1997, 2002). However,
in a small percentage of events, non-optimal translation initiation
sequences are recognized and used by the ribosome to start
translation. The invention makes use of this principle, and allows
for decreasing and even fine-tuning of the amount of translation
and hence expression of the selectable marker polypeptide, which
can therefore be used to increase the stringency of the selection
system.
[0120] In a first embodiment of the invention, the ATG start codon
of the selectable marker polypeptide (in the coding strand of the
DNA, coding for the corresponding AUG start codon in the RNA
transcription product) is left intact, but the positions at -3 to
-1 and +4 are mutated such that they do not fulfill the optimal
Kozak sequence any more, e.g., by providing the sequence TTTATGT as
the translation start site (ATG start codon underlined). It will be
clear that other mutations around the start codon at positions -3
to -1 and/or +4 could be used with similar results using the
teaching of the invention, as can be routinely and easily tested by
the person skilled in the art. The idea of this first embodiment is
that the ATG start codon is placed in a "non-optimal" context for
translation initiation.
[0121] In a second and preferred embodiment, the ATG start codon
itself of the selectable marker polypeptide is mutated. This will
in general lead to even lower levels of translation initiation than
the first embodiment. The ATG start codon in the second embodiment
is mutated into another codon, which has been reported to provide
some translation initiation, for instance to GTG, TTG, CTG, ATT, or
ACG (collectively referred to herein as "non-optimal start
codons"). In certain embodiments, the ATG start codon is mutated
into a GTG start codon. This provides still lower expression levels
(lower translation) than with the ATG start codon intact but in a
non-optimal context. More preferably, the ATG start codon is
mutated to a TTG start codon, which provides even lower expression
levels of the selectable marker polypeptide than with the GTG start
codon (Kozak M, 1986, 1987, 1989, 1990, 1997, 2002; see also
Examples 2-6 herein). The use of non-ATG start codons in the coding
sequence for a selectable marker polypeptide in a multicistronic
transcription unit according to the invention was not disclosed nor
suggested in the prior art and, preferably in combination with
chromatin control elements, leads to very high levels of expression
of the polypeptide of interest, as also shown in the incorporated
'525 application.
[0122] For the second embodiment, i.e., where a non-ATG start codon
is used, it is strongly preferred to provide an optimal context for
such a start codon, i.e., the non-optimal start codons are
preferably directly preceded by nucleotides RCC in positions -3 to
-1 and directly followed by a G nucleotide (position +4). However,
it has been reported that using the sequence TTTGTGG (start codon
underlined), some initiation is observed at least in vitro, so
although strongly preferred it may not be absolutely required to
provide an optimal context for the non-optimal start codons.
[0123] ATG sequences within the coding sequence for a polypeptide,
but excluding the ATG start codon, are referred to as "internal
ATGs," and if these are in frame with the ORF and therefore code
for methionine, the resulting methionine in the polypeptide is
referred to as an "internal methionine." It is strongly preferred
according to certain embodiments (those of the incorporated '525
application, i.e., those where the sequence encoding the selectable
marker polypeptide is upstream of the sequence encoding the
polypeptide of interest) that the coding region (following the
start codon, not necessarily including the start codon) coding for
the selectable marker polypeptide is devoid of any ATG sequence in
the coding strand of the DNA, up to (but not including) the start
codon of the polypeptide of interest (obviously, the start codon of
the polypeptide of interest may be, and in fact preferably is, an
ATG start codon). This can be established by mutating any such ATG
sequence within the coding sequence of the selectable marker
polypeptide, following the start codon thereof (as is clear from
the teaching above, the start codon of the selectable marker
polypeptide itself may be an ATG sequence, but not necessarily so).
To this purpose preferably, the degeneracy of the genetic code is
used to avoid mutating amino acids in the selectable marker
polypeptide wherever possible. Hence, wherever an ATG is present in
the coding strand of the DNA sequence encoding the selectable
marker polypeptide, which ATG is not in frame with the selectable
marker polypeptide ORF, and therefore does not code for an internal
methionine in the selectable marker polypeptide, the ATG can be
mutated such that the resulting polypeptide has no mutations in its
internal amino acid sequence. Where the ATG is an in-frame codon
coding for an internal methionine, the codon can be mutated, and
the resulting mutated polypeptide can be routinely checked for
activity of the selectable marker polypeptide. In this way a
mutation can be chosen which leads to a mutated selectable marker
polypeptide that is still active as such (quantitative differences
may exist, but those are less relevant, and in fact it could even
be beneficial to have less active variants for the purpose of the
invention; the minimum requirement is that the selectable marker
polypeptide can still be selected for in eukaryotic cells). Amino
acids valine, threonine, isoleucine and leucine are structurally
similar to methionine, and therefore codons that code for one of
these amino acids are good starting candidates to be tested in
place of methione within the coding sequence after the start codon.
Of course, using the teachings of the invention, the skilled person
may test other amino acids as well in place of internal
methionines, using routine molecular biology techniques for
mutating the coding DNA, and routine testing for functionality of
the selectable marker polypeptide. Besides routine molecular
biology techniques for mutating DNA, it is at present also possible
to synthesise at will (if required using subcloning steps) DNA
sequences that have sufficient length for an ORF of a selectable
marker polypeptide, and such synthetic DNA sequences can nowadays
be ordered commercially from various companies. Hence, using the
teachings of the invention, the person skilled in the art may
design appropriate sequences according to the invention encoding a
selectable marker polypeptide (with a mutation decreasing
translation initiation, and preferably having no internal ATGs),
have this sequence synthesized, and test the DNA molecule for
functionality of the encoded selectable marker by introducing the
DNA molecule in eukaryotic host cells and test for expression of
functional selectable marker polypeptide. The commercial
availability of such sequences also makes feasible to provide
without undue burden for selection marker coding sequences lacking
internal ATG sequences, where the wild-type coding sequence of the
selection marker polypeptide comprises several such internal
ATGs.
[0124] By providing a coding sequence for a selectable marker
polypeptide lacking any internal ATG sequence, the chances of
inadvertent translation initiation by ribosomes that passed the
(first, non-optimal) translation start sequence of the selectable
marker polypeptide at a subsequent internal ATG trinucleotide is
diminished, so that the ribosomes will continue to scan for the
first optimal translation start sequence, i.e., that of the
polypeptide of interest.
[0125] For alternative embodiments, i.e., those where the sequence
encoding the polypeptide of interest is upstream of the sequence
encoding the selectable marker polypeptide and the latter is
operably linked to an IRES (disclosed in the incorporated '953
application), internal ATGs in the sequence encoding the selectable
marker polypeptide can remain intact.
[0126] The translation start sequence of the polypeptide of
interest may comprise an optimal translation start sequence, i.e.,
having the consensus sequence RCCATGG (start codon underlined).
This will result in a very efficient translation of the polypeptide
of interest.
[0127] By providing the coding sequence of the marker with
different mutations leading to several levels of decreased
translation efficiency, the stringency of selection can be
increased. Fine-tuning of the selection system is thus possible
using the multicistronic transcription units according to the
invention: for instance using a GTG start codon for the selection
marker polypeptide, only few ribosomes will translate from this
start codon, resulting in low levels of selectable marker protein,
and hence a high stringency of selection; using a TTG start codon
even further increases the stringency of selection because even
less ribosomes will translate the selectable marker polypeptide
from this start codon.
[0128] It is demonstrated in the incorporated '525 application that
the multicistronic expression units disclosed therein can be used
in a very robust selection system, leading to a very large
percentage of clones that express the polypeptide of interest at
high levels, as desired. In addition, the expression levels
obtained for the polypeptide of interest appear to be significantly
higher than those obtained when an even larger number of colonies
are screened using selection systems hitherto known.
[0129] In addition to a decreased translation initiation
efficiency, it could be beneficial to also provide for decreased
translation elongation efficiency of the selectable marker
polypeptide, e.g., by mutating the coding sequence thereof so that
it comprises several non-preferred codons of the host cell, in
order to further decrease the translation levels of the marker
polypeptide and allow still more stringent selection conditions, if
desired. In certain embodiments, besides the mutation(s) that
decrease the translation efficiency according to the invention, the
selectable marker polypeptide further comprises a mutation that
reduces the activity of the selectable marker polypeptide compared
to its wild-type counterpart. This may be used to increase the
stringency of selection even further. As non-limiting examples,
proline at position 9 in the ZEOCIN.TM. antibiotic-resistance
polypeptide may be mutated, e.g., to Thr or Phe, and for the
neomycin resistance polypeptide, amino acid residue 182 or 261 or
both may further be mutated (see, e.g., WO 01/32901).
[0130] In certain embodiments, for the neomycin resistance
polypeptide encoded by the sequences provided herein, amino acid
residue 185 (glutamic acid) is mutated to aspartic acid and/or
amino acid residue 201 (valine) is mutated into glycine (Sautter et
al., 2005).
[0131] In some embodiments, a so-called spacer sequence is placed
downstream of the sequence encoding the start codon of the
selectable marker polypeptide, which spacer sequence preferably is
a sequence in frame with the start codon and encoding a few amino
acids, and that does not contain a secondary structure (Kozak,
1990), and does not contain the sequence ATG. Such a spacer
sequence can be used to further decrease the translation initiation
frequency if a secondary structure is present in the RNA (Kozak,
1990) of the selectable marker polypeptide (e.g., for zeocin,
possibly for blasticidin), and hence increase the stringency of the
selection system according to the invention.
[0132] The invention also provides a DNA molecule comprising the
sequence encoding a selectable marker protein according to the
invention, which DNA molecule has been provided with a mutation
that decreases the translation efficiency of the functional
selectable marker polypeptide in a eukarytic host cell. In certain
embodiments hereof, the DNA molecule in the coding strand has been
mutated compared to the wild-type sequence encoding the selectable
marker polypeptide, such that the sequence ATG of the start codon
is mutated into GTG (encoding Valine) or into TTG (encoding
Leucine), and wherein the selectable marker polypeptide is still
functional in a eukaryotic host cell. Such DNA molecules encompass
a useful intermediate product according to the invention. These
molecules can be prepared first, introduced into eukaryotic host
cells and tested for functionality (for some markers this is even
possible in prokaryotic host cells), if desired in a (semi-)
quantitative manner, of the selectable marker polypeptide. They may
then be further used to prepare a DNA molecule according to the
invention, comprising the multicistronic transcription unit.
[0133] In one embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein that confers
resistance to zeocin, the DNA sequence comprising SEQ ID NO:92,
with the proviso that the first ATG (the start codon, encoding
Methionine) is replaced by either a GTG (encoding Valine) or a TTG
(encoding Leucine) start codon.
[0134] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein that confers
resistance to blasticidin, the DNA sequence comprising SEQ ID
NO:94, with the proviso that the first ATG (the start codon,
encoding Methionine) is replaced by either a GTG (encoding Valine)
or a TTG (encoding Leucine) start codon.
[0135] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein that confers
resistance to neomycin, the DNA sequence comprising SEQ ID NO:102,
with the proviso that the first ATG (the start codon, encoding
Methionine) is replaced by either a GTG (encoding Valine) or a TTG
(encoding Leucine) start codon.
[0136] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein that confers
resistance to puromycin, the DNA sequence comprising SEQ ID NO:96,
with the proviso that the first ATG (the start codon, encoding
Methionine) is replaced by either a GTG (encoding Valine) or a TTG
(encoding Leucine) start codon.
[0137] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein that confers
resistance to hygromycin, the DNA sequence comprising SEQ ID
NO:100, with the proviso that the first ATG (the start codon,
encoding Methionine) is replaced by either a GTG (encoding Valine)
or a TTG (encoding Leucine) start codon.
[0138] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein with dihydrofolate
reductase (dhfr) activity (conferring resistance to methotrexate),
the DNA sequence comprising SEQ ID NO:98, with the proviso that the
first ATG (the start codon, encoding Methionine) is replaced by
either a GTG (encoding Valine) or a TTG (encoding Leucine) start
codon.
[0139] In another embodiment thereof, provided is a DNA molecule
comprising a DNA sequence encoding a protein with glutamine
synthetase (GS) activity, the DNA sequence comprising SEQ ID
NO:104, with the proviso that the first ATG (the start codon,
encoding Methionine) is replaced by either a GTG (encoding Valine)
or a TTG (encoding Leucine) start codon.
[0140] It will be clear that for these embodiments, any DNA
molecules as described but having mutations in the sequence
downstream of the first ATG (start codon) coding for the selectable
marker protein are also encompassed in the invention, as long as
the respective encoded selectable marker protein still has
activity. For instance any silent mutations that do not alter the
encoded protein because of the redundancy of the genetic code are
also encompassed. Further mutations that lead to conservative amino
acid mutations or to other mutations are also encompassed, as long
as the encoded protein still has activity, which may or may not be
lower than that of the wild-type protein as encoded by the
indicated sequences. In particular, it is preferred that the
encoded protein is at least 70%, preferably at least 80%, more
preferably at least 90%, still more preferably at least 95%
identical to the proteins encoded by the respective indicated
sequences. Testing for activity of the selectable marker proteins
can be done by routine methods.
[0141] Also provided is a selectable marker proteins encoded by
these embodiments.
[0142] In one aspect, provided is an expression cassette comprising
the DNA molecule hereof, having the multicistronic transcription
unit. Such an expression cassette is useful to express sequences of
interest, for instance, in host cells. An "expression cassette" as
used herein is a nucleic acid sequence comprising at least a
promoter functionally linked to a sequence of which expression is
desired. Preferably, an expression cassette further contains
transcription termination and polyadenylation sequences. Other
regulatory sequences such as enhancers may also be included. Hence,
provided is an expression cassette comprising in the following
order: 5'-promoter-multicistronic transcription unit according to
the invention, coding for either (i) {a polypeptide of interest and
downstream thereof a selectable marker polypeptide} or (ii) {a
selectable marker polypeptide and downstream thereof a polypeptide
of interest}-transcription termination sequence-3'. The promoter is
capable of functioning in a eukaryotic host cell, i.e., it is
capable of driving transcription of the multicistronic
transcription unit. The promoter is thus operably linked to the
multicistronic transcription unit. The expression cassette may
optionally further contain other elements known in the art, e.g.,
splice sites to comprise introns, and the like. In some
embodiments, an intron is present behind the promoter and before
the sequence encoding the polypeptide of interest. In the
embodiments where the selectable marker polypeptide is encoded
downstream of the polypeptide of interest, an IRES is operably
linked to the cistron that contains the selectable marker
polypeptide coding sequence. In the embodiments where the
selectable marker polypeptide is encoded upstream of the
polypeptide of interest, the sequence encoding the selectable
marker polypeptide is devoid of ATG sequences in the coding
strand.
[0143] To obtain expression of nucleic acid sequences encoding
protein, it is well known to those skilled in the art that
sequences capable of driving such expression, can be functionally
linked to the nucleic acid sequences encoding the protein,
resulting in recombinant nucleic acid molecules encoding a protein
in expressible format. In the invention, the expression cassette
comprises a multicistronic transcription unit. In general, the
promoter sequence is placed upstream of the sequences that should
be expressed. Much used expression vectors are available in the
art, e.g., the pcDNA and pEF vector series of Invitrogen, pMSCV and
pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which
can be used to obtain suitable promoters and/or transcription
terminator sequences, polyA sequences, and the like.
[0144] Where the sequence encoding the polypeptide of interest is
properly inserted with reference to sequences governing the
transcription and translation of the encoded polypeptide, the
resulting expression cassette is useful to produce the polypeptide
of interest, referred to as expression. Sequences driving
expression may include promoters, enhancers and the like, and
combinations thereof. These should be capable of functioning in the
host cell, thereby driving expression of the nucleic acid sequences
that are functionally linked to them. The person skilled in the art
is aware that various promoters can be used to obtain expression of
a gene in host cells. Promoters can be constitutive or regulated,
and can be obtained from various sources, including viruses,
prokaryotic, or eukaryotic sources, or artificially designed.
Expression of nucleic acids of interest may be from the natural
promoter or derivative thereof or from an entirely heterologous
promoter (Kaufman, 2000). Some well-known and much used promoters
for expression in eukaryotic cells comprise promoters derived from
viruses, such as adenovirus, e.g., the E1A promoter, promoters
derived from cytomegalovirus (CMV), such as the CMV immediate early
(IE) promoter (referred to herein as the CMV promoter) (obtainable
for instance from pcDNA, Invitrogen), promoters derived from Simian
Virus 40 (SV40) (Das et al., 1985), and the like. Suitable
promoters can also be derived from eukaryotic cells, such as
methallothionein (MT) promoters, elongation factor 1.alpha.
(EF-1.alpha.) promoter (Gill et al., 2001), ubiquitin C or UB6
promoter (Gill et al., 2001; Schorpp et al., 1996), actin promoter,
an immunoglobulin promoter, heat shock promoters, and the like.
Some preferred promoters for obtaining expression in eukaryotic
cells, which are suitable promoters in the invention, are the
CMV-promoter, a mammalian EF1-alpha promoter, a mammalian ubiquitin
promoter such as a ubiquitin C promoter, or a SV40 promoter (e.g.,
obtainable from pIRES, cat.no. 631605, BD Sciences). Testing for
promoter function and strength of a promoter is a matter of routine
for a person skilled in the art, and in general may for instance
encompass cloning a test gene such as lacZ, luciferase, GFP, etc.,
behind the promoter sequence, and test for expression of the test
gene. Of course, promoters may be altered by deletion, addition,
mutation of sequences therein, and tested for functionality, to
find new, attenuated, or improved promoter sequences. According to
the invention, strong promoters that give high transcription levels
in the eukaryotic cells of choice are preferred.
[0145] In certain embodiments, a DNA molecule hereof is part of a
vector, e.g., a plasmid. Such vectors can easily be manipulated by
methods well known to the person skilled in the art, and can for
instance be designed for being capable of replication in
prokaryotic and/or eukaryotic cells. In addition, many vectors can
directly or in the form of isolated desired fragment therefrom be
used for transformation of eukaryotic cells and will integrate in
whole or in part into the genome of such cells, resulting in stable
host cells comprising the desired nucleic acid in their genome.
[0146] Conventional expression systems are DNA molecules in the
form of a recombinant plasmid or a recombinant viral genome. The
plasmid or the viral genome is introduced into (eukaryotic host)
cells and preferably integrated into their genomes by methods known
in the art. In certain embodiments, the invention also uses these
types of DNA molecules to deliver its improved transgene expression
system. A preferred embodiment of the invention is the use of
plasmid DNA for delivery of the expression system. A plasmid
contains a number of components: conventional components, known in
the art, are an origin of replication and a selectable marker for
propagation of the plasmid in bacterial cells; a selectable marker
that functions in eukaryotic cells to identify and isolate host
cells that carry an integrated transgene expression system; the
protein of interest, whose high-level transcription is brought
about by a promoter that is functional in eukaryotic cells (e.g.,
the human cytomegalovirus major immediate early promoter/enhancer,
pCMV (Boshart et al., 1985); and viral transcriptional terminators
(e.g., the SV40 polyadenylation site (Kaufman & Sharp, 1982)
for the transgene of interest and the selectable marker.
[0147] The vector used can be any vector that is suitable for
cloning DNA and that can be used for transcription of a nucleic
acid of interest. When host cells are used it is preferred that the
vector is an integrating vector. Alternatively, the vector may be
an episomally replicating vector.
[0148] It is widely appreciated that chromatin structure and other
epigenetic control mechanisms may influence the expression of
transgenes in eukaryotic cells (e.g., Whitelaw et al., 2001). The
multicistronic expression units according to the invention form
part of a selection system with a rather rigourous selection
regime. This generally requires high transcription levels in the
host cells of choice. To increase the chance of finding clones of
host cells that survive the rigorous selection regime, and possibly
to increase the stability of expression in obtained clones, it will
generally be preferable to increase the predictability of
transcription. Therefore, in certain embodiments, an expression
cassette according to the invention further comprises at least one
chromatin control element. A "chromatin control element" as used
herein is a collective term for DNA sequences that may somehow have
an effect on the chromatin structure and therewith on the
expression level and/or stability of expression of transgenes in
their vicinity (they function "in cis," and hence are placed
preferably within 5 kb, more preferably within 2 kb, still more
preferably within 1 kb from the transgene) within eukaryotic cells.
Such elements have sometimes been used to increase the number of
clones having desired levels of transgene expression. The
mechanisms by which these elements work may differ for and even
within different classes of such elements, and are not completely
known for all types of such elements. However, such elements have
been described, and for the purpose of the invention chromatin
control elements are chosen from the group consisting of matrix or
scaffold attachment regions (MARs/SARs) (e.g., Phi-Van et al.,
1990; WO 02/074969, WO 2005/040377), insulators (West et al., 2002)
such as the beta-globin insulator element (5' HS4 of the chicken
beta-globin locus), scs, scs', and the like (e.g., Chung et al.,
1993, 1997; Kellum and Schedl, 1991; WO 94/23046, WO 96/04390, WO
01/02553, WO 2004/027072), a ubiquitous chromatin opening element
(UCOE) (WO 00/05393, WO 02/24930, WO 02/099089, WO 02/099070), and
anti-repressor sequences (also referred to as "STAR" sequences)
(Kwaks et al., 2003; WO 03/004704). Non-limiting examples of
MAR/SAR sequences that could be used in the current invention are
the chicken lysosyme 5' MAR (Phi-Van et al., 1990) or fragments
thereof, e.g., the B, K and F regions as described in WO
02/074969); DNA sequences comprising at least one bent DNA element
and at least one binding site for a DNA binding protein, preferably
containing at least 10% of dinucleotide TA, and/or at least 12% of
dinucleotide AT on a stretch of 100 contiguous base pairs, such as
a sequence selected from the group of comprising the sequences SEQ
ID Nos 1 to 27 in WO 2005/040377, fragments of any one of SEQ ID
Nos 1 to 27 in WO 2005/040377 being at least 100 nucleotides in
length and having MAR activity, sequences that are at least 70%
identical in nucleotide sequence to any one of SEQ ID Nos 1 to 27
in WO 2005/040377 or fragments thereof and having MAR activity,
wherein MAR activity is defined as being capable of binding to
nuclear matrices/scaffolds in vitro and/or of altering the
expression of coding sequences operably linked to a promoter;
sequences chosen from any one of SEQ ID NO: 1 to 5 in WO 02/074969,
fragments of any one of any one of SEQ ID NO: 1 to 5 in WO
02/074969 and having MAR activity, sequences that are at least 70%
identical in nucleotide sequence to any one of SEQ ID NO: 1 to 5 in
WO 02/074969 or fragments thereof and having MAR activity;
sequences chosen from SEQ ID NO: 1 and SEQ ID NO: 2 in WO
2004/027072, functional fragments thereof and sequences being at
least 70% identical thereto. A non-limiting example of insulator
sequences that could be used in the invention is a sequence that
comprises SEQ ID NO:1 of WO 01/02553. Non-limiting examples of
UCOEs that could be used in the invention are sequences depicted in
FIGS. 2 and 7 of WO 02/24930, functional fragments thereof and
sequences being at least 70% identical thereto while still
retaining activity; sequences comprising SEQ ID NO: 28 of US
2005/181428, functional fragments thereof and sequences being at
least 70% identical thereto while still retaining activity.
[0149] Preferably, the chromatin control element is an
anti-repressor sequence, preferably chosen from the group
consisting of: a) any one SEQ ID NO:1 through SEQ ID NO:66; b)
fragments of any one of SEQ ID NO:1 through SEQ ID NO:66, wherein
the fragments have anti-repressor activity ("functional
fragments"); c) sequences that are at least 70% identical in
nucleotide sequence to a) or b) wherein the sequences have
anti-repressor activity ("functional derivatives"); and d) the
complement to any one of a) to c). Preferably, the chromatin
control element is chosen from the group consisting of STAR67 (SEQ
ID NO:66), STAR7 (SEQ ID NO:7), STAR9 (SEQ ID NO:9), STAR17 (SEQ ID
NO:17), STAR27 (SEQ ID NO:27), STAR29 (SEQ ID NO:29), STAR43 (SEQ
ID NO:43), STAR44 (SEQ ID NO:44), STAR45 (SEQ ID NO:45), STAR47
(SEQ ID NO:47), STAR61 (SEQ ID NO:61), or a functional fragment or
derivative of the STAR sequences. In a particularly preferred
embodiment, the STAR sequence is STAR 67 (SEQ ID NO:66) or a
functional fragment or derivative thereof. In certain certain
embodiments, STAR 67 or a functional fragment or derivative thereof
is positioned upstream of a promoter driving expression of the
multicistronic transcription unit. In other certain embodiments,
the expression cassettes according to the invention are flanked on
both sides by at least one anti-repressor sequence.
[0150] Sequences having anti-repressor activity as used herein are
sequences that are capable of at least in part counteracting the
repressive effect of HP1 or HPC2 proteins when these proteins are
tethered to DNA. Sequences having anti-repressor activity
(sometimes also referred to as anti-repressor sequences or
anti-repressor elements herein) suitable for the invention, have
been disclosed in WO 03/004704, incorporated herein by reference,
and were coined "STAR" sequences therein (wherever a sequence is
referred to as a STAR sequence herein, this sequence has
anti-repressor activity according to the invention). As a
non-limiting example, the sequences of 66 anti-repressor elements,
named STAR1-65 (see WO 03/004704) and STAR67 (see WO 2006/005718),
are presented herein as SEQ ID NOS:1-65 and 66, respectively.
[0151] A functional fragment or derivative of a given
anti-repressor element is considered equivalent to the
anti-repressor element, when it still has anti-repressor activity.
The presence of such anti-repressor activity can easily be checked
by the person skilled in the art, for instance by the assay
described below. Functional fragments or derivatives can easily be
obtained by a person skilled in the art of molecular biology, by
starting with a given anti-repressor sequence: and making
deletions, additions, substitutions, inversions and the like (see,
e.g., WO 03/004704). A functional fragment or derivative also
comprises orthologs from other species, which can be found using
the known anti-repressor sequences by methods known by the person
skilled in the art (see, e.g., WO 03/004704). Hence, the invention
encompasses fragments of the anti-repressor sequences, wherein the
fragments still have anti-repressor activity. The invention also
encompasses sequences that are at least 70% identical in nucleotide
sequence to the sequences having anti-repressor activity or to
functional fragments thereof having anti-repressor activity, as
long as these sequences that are at least 70% identical still have
the anti-repressor activity according to the invention. Preferably,
the sequences are at least 80% identical, more preferably at least
90% identical and still more preferably at least 95% identical to
the reference native sequence or functional fragment thereof. For
fragments of a given sequence, percent identity refers to that
portion of the reference native sequence that is found in the
fragment.
[0152] Sequences having anti-repressor activity according to the
invention can be obtained by various methods, including but not
limited to the cloning from the human genome or from the genome of
another organism, or by for instance amplifying known
anti-repressor sequences directly from such a genome by using the
knowledge of the sequences, e.g., by PCR, or can in part or wholly
be chemically synthesized.
[0153] Sequences having anti-repressor activity, and functional
fragments or derivatives thereof, are structurally defined herein
by their sequence and in addition are functionally defined as
sequences having anti-repressor activity, which can be determined
with the assay described below.
[0154] Any sequence having anti-repressor activity according to the
invention should at least be capable of surviving the following
functional assay (see WO 03/004704, example 1, incorporated herein
by reference).
[0155] Human U-2 OS cells (ATCC HTB-96) are stably transfected with
the pTet-Off plasmid (Clontech K1620-A) and with nucleic acid
encoding a LexA-repressor fusion protein containing the LexA DNA
binding domain and the coding region of either HP1 or HPC2
(Drosophila Polycomb group proteins that repress gene expression
when tethered to DNA; the assay works with either fusion protein)
under control of the Tet-Off transcriptional regulatory system
(Gossen and Bujard, 1992). These cells are referred to below as the
reporter cells for the anti-repressor activity assay. A reporter
plasmid, which provides hygromycin resistance, contains a
polylinker sequence positioned between four LexA operator sites and
the SV40 promoter that controls the zeocin-resistance gene. The
sequence to be tested for anti-repressor activity can be cloned in
the polylinker. Construction of a suitable reporter plasmid, such
as pSelect, is described in Example 1 and FIG. 1 of WO 00/004704.
The reporter plasmid is transfected into the reporter cells, and
the cells are cultured under hygromycin selection (25 .mu.g/ml;
selection for presence of the reporter plasmid) and tetracycline
repression (doxycycline, 10 ng/ml; prevents expression of the
LexA-repressor fusion protein). After 1 week of growth under these
conditions, the doxycycline concentration is reduced to 0.1 ng/ml
to induce the LexA-repressor gene, and after 2 days ZEOCIN.RTM. is
added to 250 .mu.g/ml. The cells are cultured for 5 weeks, until
the control cultures (transfected with empty reporter plasmid,
i.e., lacking a cloned anti-repressor sequence in the polylinker)
are killed by the ZEOCIN.RTM. (in this control plasmid, the SV40
promoter is repressed by the LexA-repressor fusion protein that is
tethered to the LexA operating sites, resulting in insufficient
ZEOCIN.RTM. expression in such cells to survive ZEOCIN.RTM.
selection). A sequence has anti-repressor activity according to the
invention if, when the sequence is cloned in the polylinker of the
reporter plasmid, the reporter cells survive the 5 weeks selection
under zeocin. Cells from such colonies can still be propagated onto
new medium containing ZEOCIN.RTM. after the 5 weeks ZEOCIN.RTM.
selection, whereas cells transfected with reporter plasmids lacking
anti-repressor sequences cannot be propagated onto new medium
containing zeocin. Any sequence not capable of conferring such
growth after 5 weeks on ZEOCIN.TM. in this assay, does not qualify
as a sequence having anti-repressor activity, or functional
fragment or functional derivative thereof according to the
invention. As an example, other known chromatin control elements
such as those tested by Van der Vlag et al. (2000), including
Drosophila scs (Kellum and Schedl, 1991), 5'-HS4 of the chicken
.beta.-globin locus (Chung et al., 1993, 1997) or Matrix Attachment
Regions (MARs) (Phi-Van et al., 1990), do not survive this
assay.
[0156] In addition, it is preferred that the anti-repressor
sequence or functional fragment or derivative thereof confers a
higher proportion of reporter over-expressing clones when flanking
a reporter gene (e.g., luciferase, GFP) which is integrated into
the genome of U-2 OS or CHO cells, compared to when the reporter
gene is not flanked by anti-repressor sequences, or flanked by
weaker repression blocking sequences such as Drosophila scs. This
can be verified using for instance the pSDH vector, or similar
vectors, as described in Example 1 and FIG. 2 of WO 03/004704.
[0157] Anti-repressor elements can have at least one of three
consequences for production of protein: (1) they increase the
predictability of identifying host cell lines that express a
protein at industrially acceptable levels (they impair the ability
of adjacent heterochromatin to silence the transgene, so that the
position of integration has a less pronounced effect on
expression); (2) they result in host cell lines with increased
protein yields; and/or (3) they result in host cell lines that
exhibit more stable protein production during prolonged
cultivation.
[0158] Any STAR sequence can be used in the expression cassettes
according to the invention, but the following STAR sequences are
particularly useful: STAR67 (SEQ ID NO:66), STAR7 (SEQ ID NO:7),
STAR9 (SEQ ID NO:9), STAR17 (SEQ ID NO:17), STAR27 (SEQ ID NO:27),
STAR29 (SEQ ID NO:29), STAR43 (SEQ ID NO:43), STAR44 (SEQ ID
NO:44), STAR45 (SEQ ID NO:45), STAR47 (SEQ ID NO:47), STAR61 (SEQ
ID NO:61), or functional fragments or derivatives of these STAR
sequences.
[0159] In certain embodiments, the anti-repressor sequence,
preferably STAR67, is placed upstream of the promoter, preferably
such that less than 2 kb are present between the 3' end of the
anti-repressor sequence and the start of the promoter sequence. In
certain embodiments, less than 1 kb, more preferably less than 500
nucleotides (nt), still more preferably less than about 200, less
than about 100, less than about 50, or less than about 30 nt are
present between the 3' end of the anti-repressor sequence and the
start of the promoter sequence. In certain certain embodiments, the
anti-repressor sequence is cloned directly upstream of the
promoter, resulting in only about 0-20 nt between the 3' end of the
anti-repressor sequence and the start of the promoter sequence.
[0160] For the production of multimeric proteins, two or more
expression cassettes can be used. Preferably, both expression
cassettes are multicistronic expression cassettes according to the
invention, each coding for a different selectable marker protein,
so that selection for both expression cassettes is possible. This
embodiment has proven to give good results, e.g., for the
expression of the heavy and light chain of antibodies. It will be
clear that both expression cassettes may be placed on one nucleic
acid molecule or both may be present on a separate nucleic acid
molecule, before they are introduced into host cells. An advantage
of placing them on one nucleic acid molecule is that the two
expression cassettes are present in a single predetermined ratio
(e.g., 1:1) when introduced into host cells. On the other hand,
when present on two different nucleic acid molecules, this allows
the possibility to vary the molar ratio of the two expression
cassettes when introducing them into host cells, which may be an
advantage if the preferred molar ratio is different from 1:1 or
when it is unknown beforehand what is the preferred molar ratio, so
that variation thereof and empirically finding the optimum can
easily be performed by the skilled person. According to the
invention, preferably at least one of the expression cassettes, but
more preferably each of them, comprises a chromatin control
element, more preferably an anti-repressor sequence.
[0161] In another embodiment, the different subunits or parts of a
multimeric protein are present on a single expression cassette.
[0162] Instead of or in addition to the presence of a STAR sequence
placed upstream of a promoter in an expression cassette, it has
proven highly beneficial to provide a STAR sequence on both sides
of an expression cassette, such that expression cassette comprising
the transgene is flanked by two STAR sequences, which in certain
embodiments are essentially identical to each other.
[0163] It is shown herein that the combination of a first
anti-repressor element upstream of a promoter and flanking the
expression cassette by two other anti-repressor sequences provides
superior results.
[0164] As at least some anti-repressor sequences can be directional
(WO 00/004704), the anti-repressor sequences flanking the
expression cassette (anti-repressor A and B) may beneficially
placed in opposite direction with respect to each other, such that
the 3' end of each of these anti-repressor sequences is facing
inwards to the expression cassette (and to each other). Hence, in
certain embodiments, the 5' side of an anti-repressor element faces
the DNA/chromatin of which the influence on the transgene is to be
diminished by the anti-repressor element. For an anti-repressor
sequence upstream of a promoter in an expression cassette, the 3'
end faces the promoter. The sequences of the anti-repressor
elements in the sequence listing (SEQ ID NOS:1-66) are given in 5'
to 3' direction, unless otherwise indicated.
[0165] In certain embodiments, transcription units or expression
cassettes according to the invention are provided, further
comprising: a) a transcription pause (TRAP) sequence upstream of
the promoter that drives transcription of the multicistronic
transcription unit, the TRAP being in a 5' to 3' direction; or b) a
TRAP sequence downstream of the open reading frame of the
polypeptide of interest and preferably downstream of the
transcription termination sequence of the multicistronic
transcription unit, the TRAP being in a 3' to 5' orientation; or c)
both a) and b); wherein a TRAP sequence is functionally defined as
a sequence which when placed into a transcription unit, results in
a reduced level of transcription in the nucleic acid present on the
3' side of the TRAP when compared to the level of transcription
observed in the nucleic acid on the 5' side of the TRAP.
Non-limiting examples of TRAP sequences are transcription
termination and/or polyadenylation signals. One non-limiting
example of a TRAP sequence is given in SEQ ID NO:126. Examples of
other TRAP sequences, methods to find these, and uses thereof have
been described in WO 2004/055215.
[0166] DNA molecules comprising multicistronic transcription units
and/or expression cassettes according to the invention can be used
for improving expression of nucleic acid, preferably in host cells.
The terms "cell"/"host cell" and "cell line"/"host cell line" are
respectively typically defined as a cell and homogeneous
populations thereof that can be maintained in cell culture by
methods known in the art, and that have the ability to express
heterologous or homologous proteins.
[0167] Prokaryotic host cells can be used to propagate and/or
perform genetic engineering with the DNA molecules of the
invention, especially when present on plasmids capable of
replicating in prokaryotic host cells such as bacteria.
[0168] A host cell according to the invention preferably is a
eukaryotic cell, more preferably a mammalian cell, such as a rodent
cell or a human cell or fusion between different cells. In certain
non-limiting embodiments, the host cell is a U-2 OS osteosarcoma,
CHO (Chinese hamster ovary), HEK 293, HuNS-1 myeloma, WERI-Rb-1
retinoblastoma, BHK, Vero, non-secreting mouse myeloma Sp2/0-Ag 14,
non-secreting mouse myeloma NS0, NCI-H295R adrenal gland carcinomal
or a PER.C6.RTM. cell.
[0169] In certain embodiments, a host cell is a cell expressing at
least E1A, and preferably also E1B, of an adenovirus. As
non-limiting examples, such a cell can be derived from for instance
human cells, for instance from a kidney (example: HEK 293 cells,
see Graham et al., 1977), lung (e.g., A549, see, e.g., WO 98/39411)
or retina (example: HER cells marketed under the trade mark
PER.C6.RTM., see U.S. Pat. No. 5,994,128), or from amniocytes
(e.g., N52.E6, described in U.S. Pat. No. 6,558,948), and similarly
from other cells. Methods for obtaining such cells are described
for instance in U.S. Pat. Nos. 5,994,128 and 6,558,948. PER.C6.RTM.
cells for the purpose of the invention means cells from an upstream
or downstream passage or a descendent of an upstream or downstream
passage of cells as deposited under ECACC no. 96022940, i.e.,
having the characteristics of those cells. It has been previously
shown that such cells are capable of expression of proteins at high
levels (e.g., WO 00/63403, and Jones et al., 2003). In other
certain embodiments, the host cells are CHO cells, for instance
CHO-K1, CHO-S, CHO-DG44, CHO-DUKXB11, and the like. In certain
embodiments, the CHO cells have a dhfr.sup.- phenotype.
[0170] Such eukaryotic host cells can express desired polypeptides,
and are often used for that purpose. They can be obtained by
introduction of a DNA molecule of the invention, preferably in the
form of an expression cassette, into the cells. Preferably, the
expression cassette is integrated in the genome of the host cells,
which can be in different positions in various host cells, and
selection will provide for a clone where the transgene is
integrated in a suitable position, leading to a host cell clone
with desired properties in terms of expression levels, stability,
growth characteristics, and the like. Alternatively the
multicistronic transcription unit may be targeted or randomly
selected for integration into a chromosomal region that is
transcriptionally active, e.g., behind a promoter present in the
genome. Selection for cells containing the DNA described herein can
be performed by selecting for the selectable marker polypeptide,
using routine methods known by the person skilled in the art. When
such a multicistronic transcription unit is integrated behind a
promoter in the genome, an expression cassette according to the
invention can be generated in situ, i.e., within the genome of the
host cells.
[0171] The host cells may be from a stable clone that can be
selected and propagated according to standard procedures known to
the person skilled in the art. A culture of such a clone is capable
of producing polypeptide of interest, if the cells comprise the
multicistronic transcription unit of the invention. Cells according
to the invention preferably are able to grow in suspension culture
in serum-free medium.
[0172] In certain embodiments, the DNA molecule comprising the
multicistronic transcription unit of the invention, preferably in
the form of an expression cassette, is integrated into the genome
of the eukaryotic host cell according to the invention. This will
provide for stable inheritance of the multicistronic transcription
unit.
[0173] Selection for the presence of the selectable marker
polypeptide, and hence for expression, can be performed during the
initial obtaining of the cells, and could be lowered or stopped
altogether after stable clones have been obtained. It is however
also possible to apply the selection agent during later stages
continuously, or only occasionally, possibly at lower levels than
during initial selection of the host cells.
[0174] A polypeptide of interest according to the invention can be
any protein, and may be a monomeric protein or a (part of a)
multimeric protein. A multimeric protein comprises at least two
polypeptide chains. Non-limiting examples of a protein of interest
according to the invention are enzymes, hormones, immunoglobulin
chains, therapeutic proteins like anti-cancer proteins, blood
coagulation proteins such as Factor VIII, multi-functional
proteins, such as erythropoietin, diagnostic proteins, or proteins
or fragments thereof useful for vaccination purposes, all known to
the person skilled in the art.
[0175] In certain embodiments, an expression cassette hereof
encodes an immunoglobulin heavy or light chain or an antigen
binding part, derivative and/or analogue thereof. In one
embodiment, a protein expression unit is provided, wherein the
protein of interest is an immunoglobulin heavy chain. In yet
another preferred embodiment, a protein expression unit is
provided, wherein the protein of interest is an immunoglobulin
light chain. When these two protein expression units are present
within the same (host) cell a multimeric protein and more
specifically an immunoglobulin, is assembled. Hence, in certain
embodiments, the protein of interest is an immunoglobulin, such as
an antibody, which is a multimeric protein. Preferably, such an
antibody is a human or humanized antibody. In certain embodiments
thereof, it is an IgG, IgA, or IgM antibody. An immunoglobulin may
be encoded by the heavy and light chains on different expression
cassettes, or on a single expression cassette. Preferably, the
heavy and light chain are each present on a separate expression
cassette, each having its own promoter (which may be the same or
different for the two expression cassettes), each comprising a
multicistronic transcription unit according to the invention, the
heavy and light chain being the polypeptide of interest, and
preferably each coding for a different selectable marker protein,
so that selection for both heavy and light chain expression
cassette can be performed when the expression cassettes are
introduced and/or present in a eukaryotic host cell.
[0176] The polypeptide of interest may be from any source, and in
certain embodiments is a mammalian protein, an artificial protein
(e.g., a fusion protein or mutated protein), and preferably is a
human protein.
[0177] The configurations of the expression cassettes hereof may
also be used when the ultimate goal is not the production of a
polypeptide of interest, but the RNA itself, for instance for
producing increased quantities of RNA from an expression cassette,
which may be used for purposes of regulating other genes (e.g.,
RNAi, antisense RNA), gene therapy, in vitro protein production,
etc.
[0178] In one aspect, provided is a method for generating a host
cell expressing a polypeptide of interest, the method comprising
the steps of: a) introducing into a plurality of precursor cells an
expression cassette according to the invention, and b) culturing
the generated cells under conditions selecting for expression of
the selectable marker polypeptide, and c) selecting at least one
host cell producing the polypeptide of interest. This novel method
provides a very good result in terms of the ratio of obtained
clones versus clones with high expression of the desired
polypeptide. Using the most stringent conditions, i.e., the weakest
translation efficiency for the selectable marker polypeptide (using
the weakest translation start sequence), far fewer colonies are
obtained using the same concentration of selection agent than with
known selection systems, and a relatively high percentage of the
obtained clones produces the polypeptide of interest at high
levels. In addition, the obtained levels of expression appear
higher than those obtained when an even larger number of clones
using the known selection systems are used.
[0179] The selection system is swift because it does not require
copy number amplification of the transgene. Hence, cells with low
copy numbers of the multicistronic transcription units already
provide high expression levels. High transgene copy numbers of the
transgene may be prone to genetic instability and repeat-induced
silencing (e.g., Kim et al., 1998; McBurney et al., 2002).
Therefore, an additional advantage of the embodiments with
relatively low transgene copy numbers is that lower copy numbers
are anticipated to be less prone to recombination and to
repeat-induced silencing, and therefore less problems in this
respect are anticipated when using host cells with a limited number
of copies of the transgene compared to host cells obtained using an
amplification system where hundreds or even thousands of copies of
the selectable marker and protein of interest coding sequences may
be present in the genome of the cell. Also provided are examples of
high expression levels, using the multicistronic transcription unit
selection system, while the copy number of the transgene is
relatively low, i.e., less than 30 copies per cell, or even less
than 20 copies per cell. Hence, the invention allows the generation
of host cells according to the invention, comprising less than 30
copies of the multicistronic transcription unit in the genome of
the host cells, preferably less than 25, more preferably less than
20 copies, while at the same time providing sufficient expression
levels of the polypeptide of interest for commercial purposes,
e.g., more than 15, preferably more than 20 pg/cell/day of an
antibody.
[0180] While clones having relatively low copy numbers of the
multicistronic transcription units and high expression levels can
be obtained, the selection system of the invention nevertheless can
be combined with amplification methods to even further improve
expression levels. This can, for instance, be accomplished by
amplification of a co-integrated dhfr gene using methotrexate, for
instance by placing dhfr on the same nucleic acid molecule as the
multicistronic transcription unit of the invention, or by
cotransfection when dhfr is on a separate DNA molecule.
[0181] In one aspect, provided is a method for producing a
polypeptide of interest, the method comprising culturing a host
cell, the host cell comprising a DNA molecule comprising a
multicistronic expression unit or an expression cassette according
to the invention, and expressing the polypeptide of interest from
the coding sequence for the polypeptide of interest.
[0182] The host cell for this aspect is a eukaryotic host cell,
preferably a mammalian cell, such as a CHO cell, further as
described above.
[0183] Introduction of nucleic acid that is to be expressed in a
cell, can be done by one of several methods, which as such are
known to the person skilled in the art, also dependent on the
format of the nucleic acid to be introduced. The methods include
but are not limited to transfection, infection, injection,
transformation, and the like. Suitable host cells that express the
polypeptide of interest can be obtained by selection as described
above.
[0184] In certain embodiments, selection agent is present in the
culture medium at least part of the time during the culturing,
either in sufficient concentrations to select for cells expressing
the selectable marker polypeptide or in lower concentrations. In
certain embodiments, selection agent is no longer present in the
culture medium during the production phase when the polypeptide is
expressed. In certain embodiments metabolic selection marker
proteins such as trp, his, or dhfr, are used, and selection can be
easily continued during the production phase by culturing in the
suitable culture medium described supra.
[0185] Culturing a cell is done to enable it to metabolize, and/or
grow and/or divide and/or produce recombinant proteins of interest.
This can be accomplished by methods well known to persons skilled
in the art, and includes but is not limited to providing nutrients
for the cell. The methods comprise growth adhering to surfaces,
growth in suspension, or combinations thereof. Culturing can be
done for instance in dishes, roller bottles or in bioreactors,
using batch, fed-batch, continuous systems such as perfusion
systems, and the like. In order to achieve large scale (continuous)
production of recombinant proteins through cell culture it is
preferred in the art to have cells capable of growing in
suspension, and it is preferred to have cells capable of being
cultured in the absence of animal- or human-derived serum or
animal- or human-derived serum components.
[0186] The conditions for growing or multiplying cells (see, e.g.,
Tissue Culture, Academic Press, Kruse and Paterson, editors (1973))
and the conditions for expression of the recombinant product are
known to the person skilled in the art. In general, principles,
protocols, and practical techniques for maximizing the productivity
of mammalian cell cultures can be found in Mammalian Cell
Biotechnology: a Practical Approach (M. Butler, ed., IRL Press,
1991).
[0187] In a preferred embodiment, the expressed protein is
collected (isolated), either from the cells or from the culture
medium or from both. It may then be further purified using known
methods, e.g., filtration, column chromatography, etc, by methods
generally known to the person skilled in the art.
[0188] The selection method according to the invention works in the
absence of chromatin control elements, but improved results are
obtained when the multicistronic expression units are provided with
such elements. The selection method according to the invention
works particularly well when an expression cassette according to
the invention, comprising at least one anti-repressor sequence is
used. Depending on the selection agent and conditions, the
selection can in certain cases be made so stringent, that only very
few or even no host cells survive the selection, unless
anti-repressor sequences are present. Hence, the combination of the
novel selection method and anti-repressor sequences provides a very
attractive method to obtain only limited numbers of colonies with a
greatly improved chance of high expression of the polypeptide of
interest therein, while at the same time the obtained clones
comprising the expression cassettes with anti-repressor sequences
provide for stable expression of the polypeptide of interest, i.e.,
they are less prone to silencing or other mechanisms of lowering
expression than conventional expression cassettes.
[0189] In certain embodiments, almost no clones are obtained when
no anti-repressor sequence is present in the expression cassette
according to the invention, providing for very stringent selection.
The novel selection system disclosed herein therefore also provides
the possibility to test parts of anti-repressor elements for
functionality, by analyzing the effects of such sequences when
present in expression cassettes of the invention under selection
conditions. This easy screen, which provides an almost or even
complete black and white difference in many cases, therefore can
contribute to identifying functional parts or derivatives from
anti-repressor sequences. When known anti-repressor sequences are
tested, this assay can be used to characterize them further. When
fragments of known anti-repressor sequences are tested, the assay
will provide functional fragments of such known anti-repressor
sequences.
[0190] The invention disclosed in the incorporated '953 application
provides a multicistronic transcription unit having an alternative
configuration compared to the configuration disclosed in the
incorporated '525 application: in the alternative configuration of
the '953 invention, the sequence coding for the polypeptide of
interest is upstream of the sequence coding for the selectable
marker polypeptide, and the selectable marker polypeptide is
operably linked to a cap-independent translation initiation
sequence, preferably an internal ribosome entry site (IRES). Such
multicistronic transcription units as such were known (e.g., Rees
et al., 1996, WO 03/106684), but had not been combined with a
non-optimal start codon. According to the alternative of the '953
invention, the start codon (or the context thereof) of the
selectable marker polypeptide is changed into a non-optimal start
codon, to further decrease the translation initiation rate for the
selectable marker. This therefore leads to a desired decreased
level of expression of the selectable marker polypeptide, and can
result in highly effective selection host cells expressing high
levels of the polypeptide of interest, as with the embodiments
disclosed in the incorporated '525 application. One potential
advantage of this alternative aspect of the '953 invention,
compared to the embodiments outlined in the '525 application, is
that the coding sequence of the selectable marker polypeptide needs
no further modification of internal ATG sequences, because any
internal ATG sequences therein can remain intact since they are no
longer relevant for translation of further downstream polypeptides.
This may be especially advantageous if the coding sequence for the
selectable marker polypeptide contains several internal ATG
sequences, because the task of changing these and testing the
resulting construct for functionality does not have to be performed
for the invention: only mutation of the ATG start codon (or its
context) suffices in this case. As will be understood by the person
skilled in the art after reading the description, this aspect can
further be advantageously combined with the embodiments outlined
above for the multicistronic transcription units. For instance
expression cassettes comprising the multicistronic transcription
unit can further in certain embodiments comprise at least one
chromatin control element. It is shown hereinbelow (example 19)
that this alternative provided by the invention of the '953
application also leads to very good results.
[0191] In this alternative embodiment (disclosed first in the '953
application), the coding sequence for the polypeptide of interest
comprises a stop codon, so that translation of the first cistron
(encoding the polypeptide of interest) ends upstream of the IRES,
which IRES is operably linked to the second cistron (encoding the
selectable marker polypeptide). In these embodiments, the IRES is
required for the translation of the selectable marker
polypeptide.
[0192] As used herein, an "internal ribosome entry site" or "IRES"
refers to an element that promotes direct internal ribosome entry
to the initiation codon, such as normally an ATG, but in this
invention preferably GTG or TTG, of a cistron (a protein encoding
region), thereby leading to the cap-independent translation of the
gene. See, e.g., Jackson R J, Howell M T, Kaminski A (1990) Trends
Biochem Sci 15 (12): 477-83) and Jackson R J and Kaminski, A.
(1995) RNA 1 (10): 985-1000. The invention encompasses the use of
any IRES element, which is able to promote direct internal ribosome
entry to the initiation codon of a cistron. "Under translational
control of an IRES" (also referred to as "operably linked to an
IRES") as used herein means that translation is associated with the
IRES and proceeds in a cap-independent manner. As used herein, the
term "IRES" encompasses functional variations of IRES sequences as
long as the variation is able to promote direct internal ribosome
entry to the initiation codon of a cistron. As used herein,
"cistron" refers to a polynucleotide sequence, or gene, of a
protein, polypeptide, or peptide of interest. "Operably linked"
refers to a situation where the components described are in a
relationship permitting them to function in their intended manner.
Thus, for example, a promoter "operably linked" to a cistron is
ligated in such a manner that expression of the cistron is achieved
under conditions compatible with the promoter. Similarly, a
nucleotide sequence of an IRES operably linked to a cistron is
ligated in such a manner that translation of the cistron is
achieved under conditions compatible with the IRES.
[0193] Internal ribosome binding site (IRES) elements are known
from viral and mammalian genes (Martinez-Salas, 1999), and have
also been identified in screens of small synthetic oligonucleotides
(Venkatesan & Dasgupta, 2001). The IRES from the
encephalomyocarditis virus has been analyzed in detail (Mizuguchi
et al., 2000). An IRES is an element encoded in DNA that results in
a structure in the transcribed RNA at which eukaryotic ribosomes
can bind and initiate translation. An IRES permits two or more
proteins to be produced from a single RNA molecule (the first
protein is translated by ribosomes that bind the RNA at the cap
structure of its 5' terminus, (Martinez-Salas, 1999)). Translation
of proteins from IRES elements is less efficient than cap-dependent
translation: the amount of protein from IRES-dependent open reading
frames (ORFs) ranges from less than 20% to 50% of the amount from
the first ORF (Mizuguchi et al., 2000). The reduced efficiency of
IRES-dependent translation provides an advantage that is exploited
by this embodiment of the current invention. Furthermore, mutation
of IRES elements can attenuate their activity, and lower the
expression from the IRES-dependent ORFs to below 10% of the first
ORF (Lopez de Quinto & Martinez-Salas, 1998, Rees et al.,
1996). The advantage exploited by the invention is as follows: when
the IRES-dependent ORF encodes a selectable marker protein, its low
relative level of translation means that high absolute levels of
transcription must occur in order for the recombinant host cell to
be selected. Therefore, selected recombinant host cell isolates
will by necessity express high amounts of the transgene mRNA. Since
the recombinant protein is translated from the cap-dependent ORF,
it can be produced in abundance resulting in high product yields.
On top of this, the non-optimal (i.e., non-ATG) start codon for the
selectable marker polypeptide according to the invention, further
improves the chances of obtaining a preferred host cell, i.e., a
host cell expressing high levels of recombinant protein of
interest.
[0194] It is clear to a person skilled in the art that changes to
the IRES can be made without altering the essence of the function
of the IRES (hence, providing a protein translation initiation site
with a reduced translation efficiency), resulting in a modified
IRES. Use of a modified IRES which is still capable of providing a
small percentage of translation (compared to a 5' cap translation)
is therefore also included in this invention.
[0195] The practice of this invention will employ, unless otherwise
indicated, conventional techniques of immunology, molecular
biology, microbiology, cell biology, and recombinant DNA, which are
within the skill of the art. See, e.g., Sambrook, Fritsch and
Maniatis, Molecular Cloning: A Laboratory Manual, 2.sup.nd edition,
1989; Current Protocols in Molecular Biology, Ausubel F M, et al.,
eds, 1987; the series Methods in Enzymology (Academic Press, Inc.);
PCR2: A Practical Approach, MacPherson M J, Hams B D, Taylor G R,
eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds,
1988.
[0196] The invention is further described with the aid of the
following illustrative Examples.
EXAMPLES
[0197] Examples 1-18 describe details of several embodiments of the
incorporated '525 application. Example 19 describes the selection
system with the multicistronic transcription unit of the invention,
and it will be clear that the variations described in Examples 1-18
can also be applied and tested for the multicistronic transcription
units of the present application.
Example 1
Construction and Testing of a Zeocin-Resistance Gene Product with
No Internal Methionine
[0198] The basic idea behind the development of the novel selection
system of the incorporated '525 application is to place the gene
encoding the resistance gene upstream of a gene of interest, and
one promoter drives the expression of this bicistronic mRNA. The
translation of the bicistronic mRNA is such that only in a small
percentage of translation events the resistance gene will be
translated into protein and that most of the time the downstream
gene of interest will be translated into protein. Hence the
translation efficiency of the upstream resistance gene is severely
hampered in comparison to the translation efficiency of the
downstream gene of interest. To achieve this, three steps can be
taken according to the invention of the '525 application: [0199] 1)
within the resistance gene on the mRNA, the searching ribosome
preferably should not meet another AUG, since any downstream AUG
may serve as translation start codon, resulting in a lower
translation efficiency of the second, downstream gene of interest.
Hence, preferably any AUG in the resistance gene mRNA will have to
be replaced. In case this AUG is a functional codon that encodes a
methionine, this amino acid will have to be replaced by a different
amino acid, for instance by a leucine (FIGS. 1A and B); [0200] 2)
the start codon of the resistance gene must have a bad context (be
part of a non-optimal translation start sequence); i.e., the
ribosomes must start translation at this start codon only in a
limited number of events, and hence in most events continue to
search for a better, more optimal start codon (FIG. 1C-E). Three
different stringencies can be distinguished: a) the normal ATG
start codon, but placed in a bad context (TTTATGT) (called ATGmut)
(FIG. 1C), b) preferably when placed in an optimal context, GTG can
serve as start codon (ACCGTGG) (FIG. 1D) and c) preferably when
placed in an optimal context, TTG can serve as start codon
(ACCGTGG) (FIG. 1E). The most stringent translation condition is
the TTG codon, followed by the GTG codon (FIG. 1). The Zeo mRNA
with a TTG as start codon is expected to produce the least
Zeocin-resistance protein and will hence convey the lowest
functional ZEOCIN.RTM. resistance to cells (FIGS. 1, 2). [0201] 3)
preferably, the normal start codon (ATG) of the downstream gene of
interest should have an optimal translation context (e.g., ACCATGG)
(FIG. 2A-D). This warrants that, after steps 1 and 2 have been
taken, in most events the start codon of the gene of interest will
function as start codon of the bicistronic mRNA.
[0202] In this example, step 1 is performed, that is, in the
Zeocin-resistance gene one existing internal methionine is replaced
by another amino acid (FIG. 1B-E). It is important that after such
a change the Zeo protein still confers ZEOCIN.RTM. resistance to
the transfected cells. Since it is not known beforehand which amino
acid will fulfill this criterium, three different amino acids have
been tried: leucine, threonine and valine. The different constructs
with distinct amino acids have than been tested for their ability
to still confer ZEOCIN.RTM. resistance to the transfected
cells.
Materials and Methods
Construction of the Plasmids
[0203] The original Zeo open reading frame has the following
sequence around the start codon: AAACCGCC (start codon in bold; SEQ
ID NO:67). This is a start codon with an optimal translational
context (FIG. 1A). First the optimal context of the start codon of
the Zeo open reading frame was changed through amplification from
plasmid pCMV-zeo [Invitrogen V50120], with primer pair
ZEOforwardMUT (SEQ ID NO:68):
GATCTCGCGATACAGGATTTTTGGCCAAGTTGACCAGTGCCGTTCCG and ZEO-WTreverse
(WT=Wild type; SEQ ID NO:69): AGGCGAATTCAGTCCTGCTCCTCGGC, using
pCMV-ZEO (Invitrogen; V50120) as a template. The amplified product
was cut with Nrul-EcoRI, and ligated into pcDNA3, resulting in
pZEOATGmut.
[0204] The original Zeo open reading frame contains an in frame
ATG, encoding methionine at amino acid position 94 (out of 124).
This internal ATG, encoding the methionine at position 94 was
changed in such a way that the methionine was changed into leucine,
threonine or valine respectively:
[0205] 1) To replace the internal codon for methionine in the Zeo
open reading frame with the codon for leucine (FIG. 1B), part of
the Zeo open reading frame was amplified using primer pair
ZEOforwardMUT (SEQ ID NO:68) and ZEO-LEUreverse (SEQ ID NO:70):
AGGCCCCGCCCCCACGGCTGCTCGCCGATCTCGGTCAAGGCCGGC. The PCR product was
cut with BamHI-Bgll and ligated into pZEOATGmut. This resulted in
pZEO(leu). To replace the internal codon for methionine in the Zeo
open reading frame with the codon for threonine (not shown, but as
in FIG. 1B), part of the Zeo open reading frame was amplified using
primer pair ZEOforwardMUT (SEQ ID NO:68) and ZEO-THRreverse (SEQ ID
NO:71): AGGCCCCGCCCCCACGGCTGCTCGCCGATCTCGGTGGTGGCCGGC. The PCR
product was cut with BamHI-BglI and ligated into pZEOATGmut. This
resulted in pZEO(thr). To replace the internal codon for methionine
in the Zeo open reading frame with the codon for valine (not shown,
but as in FIG. 1B) (GTG), part of the Zeo open reading frame was
amplified using primer pair ZEOforwardMUT (SEQ ID NO:68) and
ZEO-VALreverse (SEQ ID NO:72):
AGGCCCCGCCCCCACGGCTGCTCGCCGATCTCGGTCCACGCCGG. The PCR product was
cut with BamHI-BglI and ligated into pZEOATGmut. This resulted in
pZEO(val).
Transfection and Culturing of Cells
[0206] The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was
cultured in HAMS-F12 medium+10% Fetal Calf Serum containing 2 mM
glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin
at 37.degree. C./5% CO.sub.2. Cells were transfected with the
plasmids using Lipofectamine 2000 (Invitrogen) as described by the
manufacturer. Briefly, cells were seeded to culture vessels and
grown overnight to 70-90% confluence. Lipofectamine reagent was
combined with plasmid DNA at a ratio of 6 microliters per microgram
(e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120
microliters Lipofectamine) and added to the cells. After overnight
incubation the transfection mixture was replaced with fresh medium,
and the transfected cells were incubated further. After overnight
cultivation, cells were trypsinized and seeded into fresh culture
vessels with fresh medium containing ZEOCIN.RTM. (100 .mu.g/ml).
When individual colonies became visible (approximately ten days
after transfection) colonies were counted.
Results
[0207] Four plasmids were transfected to CHO-K1 cells, 1) pZEO(WT),
2) pZEO(leu), 3) pZEO(thr), and 4) pZEO(val). The cells were
selected on 100 .mu.g/ml zeocine. Transfection of pZEO(leu)
resulted in an equal number of zeocin-resistant colonies in
comparison with the control pZEO (WT). pZEO(thr) and pZEO(val) gave
less colonies, but the differences were not in the order of a
magnitude. Hence it was concluded that changes of the internal
methionine into leucine, threonine or valine all resulted in a
Zeocin-resistance protein that is still able to confer ZEOCIN.RTM.
resistance to the transfected cells. Rather arbitrarily, pZEO(leu)
was chosen as starting point for creating different start codons on
the Zeo open reading frame. Hence in the examples below the start
as well as internal methionines are always replaced by leucine, for
zeocin, but also for other selectable marker genes, as will be
clear from further examples.
Example 2
Creation and testing of Zeocin-d2EGFP Bicistronic Constructs with
Differential Translation Efficiencies
[0208] To create a bicistronic mRNA encompassing a mutated
Zeocin-resistance mRNA with less translational efficiency, and the
d2EGFP gene as downstream gene of interest, the start codon of the
d2EGFP gene was first optimized (step 3 in Example 1). After that,
the different versions of the Zeocin-resistance gene were created.
The differences between these versions are that they have different
start codons, with distinct translational efficiency (step 2 in
Example 1, FIG. 1C-E). These different ZEOCIN.RTM.-resistance gene
versions were cloned upstream of the modified d2EGFP gene (FIG.
2).
Materials and Methods
Creation of Plasmids
[0209] The d2EGFP reporter ORF was introduced into pcDNA3. The
sequence around the start codon of this d2EGFP cDNA is GAATTCGG
(start codon in bold; SEQ ID NO:73), which is not optimal. As a
first step, d2EGFP was amplified from pd2EGFP (Clontech 6010-1)
with primers d2EGFPforwardBamHI (SEQ ID NO:74):
GATCGGATCCTATGAGGAATTCGCCACCGTGAGCAAGGGCGAGGAG and
d2EGFPreverseNotI (SEQ ID NO:75):
AAGGAAAAAAGCGGCCGCCTACACATTGATCCTAGCAGAAG. This product contains
now a start codon with an optimal translational context (ACCG).
This created pd2EGFP and subsequently, the Zeo open reading frame
was ligated into pd2EGFP, resulting in pZEO-d2EGFP. It is pointed
out here that the optimization of the translational start sequence
of the gene of interest (here: EGFP as a model gene) is not
essential but preferred in order to skew the translation initiation
frequency towards the gene of interest still further.
[0210] Now three classes of constructs were made: [0211] 1) ATG as
a start codon in the Zeo resistance gene, but in a bad context
(TTTT) (not shown, but as in FIG. 2B) and followed by spacer
sequence, instead of the optimal ATG (FIG. 2A). The spacer sequence
is placed downstream of the ATG sequence. In the ZEOCIN.RTM. (and
possibly in the blasticidin) RNA, a secondary structure is present,
causing the ribosome to be temporarily delayed. Because of this, a
poor start codon can in some cases be used by the ribosome, despite
being a bad start codon or being in a non-optimal context for
translation initiation. This causes the chance of translation to
increase, and in case of the current invention therefore renders
the stringency for selection lower. To decrease this effect, and
hence to further decrease the translation initation efficiency, a
spacer sequence is introduced that does not contain a secondary
structure (Kozak, 1990). Hence, the term "space" is introduced, and
used in the plasmid and primer names to indicate the presence of
such a spacer sequence. The spacer removes the "ribosome delaying
sequence" from the neighbourhoud of the initiation codon, therewith
causing the ribosome to start translating less frequently, and
hence increasing the stringency of the selection according to the
invention. The spacer introduces some extra amino acids in the
coding sequence. This has been done in some cases for both
ZEOCIN.RTM. and for blasticidin, as will be apparent from the
examples. The nomenclature of the plasmids and primers in general
in the following is along these lines: the name of the selectable
marker polypeptide is referred to by abbreviation (e.g., Zeo, Blas,
etc); the start codon is mentioned (e.g., ATG, GTG, TTG); when this
start codon is placed in a non-optimal context for translation
initiation, the addition "mut" is used (this is usually only done
for ATG start codons, as combining a non-optimal context with a
non-ATG start codon usually does not result in sufficient
translation initiation to allow for selection); when a spacer
sequence is used behind the start codon, the addition "space" is
used (this is done usually for "ATGmut" start codons for Zeo or
Blas selectable markers). The Zeo open reading frame was amplified
with primer pair ZEOforwardBamHI-ATGmut/space (SEQ ID NO:77):
GATCGGATCCTTGGTTTTCGATCCAAAGACTGCCAAATCTAGATCCGAGATT
TTCAGGAGCTAAGGAAGCTAAAGCCAAGTTGACCAGTGAAGTTC (wherein the sequence
following the underlined sequence comprises the spacer sequence),
and ZEOWTreverse (SEQ ID NO:69), the PCR product was cut with
EcoRl-BamHI, and ligated into pd2EGF, cut with EcoRI-BamHI,
creating pZEO-ATGmut/space-d2EGFP. [0212] 2) GTG as a start codon
in the Zeo resistance gene, instead of ATG (FIG. 2C). The Zeo open
reading frame was amplified with primer pair ZEOforwardBamHI-GTG
(SEQ ID NO:78): GATCGGATCCACCGCCAAGTTGACCAGTGCCGTTC and
ZEOWTreverse (SEQ ID NO:69), the PCR product was cut with
EcoRI-BamHI, and ligated into pd2EGFP, cut with EcoRI-BamHI,
creating pZEO-GTG-d2EGFP.
[0213] 3) TTG as a start codon in the Zeo resistance gene, instead
of ATG (FIG. 2D). The Zeo open reading frame was amplified with
primer pair ZEOforwardBamHI-TTG:
GATCGGATCCACCGCCAAGTTGACCAGTGCCGTTC (SEQ ID NO:79) and ZEOWTreverse
(SEQ ID NO:69), the PCR product was cut with EcoRI-BamHI, and
ligated into pd2EGFP, cut with EcoRI-BamHI, creating
pZEO-TTG-d2EGFP.
Transfection, Culturing and Analysis of CHO Cells
[0214] The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was
cultured in HAMS-F12 medium+10% Fetal Calf Serum containing 2 mM
glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin
at 37.degree. C./5% CO.sub.2. Cells were transfected with the
plasmids using Lipofectamine 2000 (Invitrogen) as described by the
manufacturer. Briefly, cells were seeded to culture vessels and
grown overnight to 70-90% confluence. Lipofectamine reagent was
combined with plasmid DNA at a ratio of 15 microliters per 3
microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120
microliters Lipofectamine) and added after 30 minutes incubation at
25.degree. C. to the cells. After overnight incubation the
transfection mixture was replaced with fresh medium, and the
transfected cells were incubated further. After overnight
cultivation, cells were trypsinized and seeded into fresh culture
vessels with fresh medium. After another overnight incubation,
ZEOCIN.RTM. was added to a concentration of 50 .mu.g/ml and the
cells were cultured further. After another three days the medium
was replaced by fresh medium containing ZEOCIN.RTM. (100 .mu.g/ml)
and cultured further. When individual colonies became visible
(approximately ten days after transfection) medium was removed and
replaced with fresh medium without zeocin. Individual clones were
isolated and transferred to 24-well plates in medium without
zeocin. One day after isolation of the colonies, ZEOCIN.RTM. was
added to the medium. Expression of the d2EGFP reporter gene was
assessed approximately 3 weeks after transfection. d2EGFP
expression levels in the colonies were measured after periods of
two weeks.
Results
[0215] CHO-K1 cells were transfected with constructs that contain
the ATGmut/space Zeo (FIG. 2B), GTG Zeo (FIG. 2C) and TTG Zeo (FIG.
2D) genes as selection gene, all being cloned upstream of the
d2EGFP reporter gene. These three constructs were without STAR
elements (Control) or with STAR elements 7 and 67 upstream of the
CMV promoter and STAR 7 downstream from the d2EGFP gene (FIG. 3).
FIG. 3 shows that both the control (without STAR elements)
constructs with ATGmut/space Zeo (A) and GTG Zeo (B) gave colonies
that expressed d2EGFP protein. The average d2EGFP expression level
of 24 ATGmut/space Zeo colonies was 46 and of GTG Zeo colonies was
75. This higher average expression level in GTG Zeo colonies may
reflect the higher stringency of GTG, in comparison with
ATGmut/space (Example 1). Addition of STAR elements 7 and 67 to the
constructs resulted in colonies that had higher average d2EGFP
expression levels. Transfection of the ATGmut/space Zeo STAR 7/67/7
construct resulted in colonies with an average d2EGFP expression
level of 118, which is a factor 2.6 higher than the average in the
control cells (46). Addition of STAR elements to the GTG Zeo
construct resulted in an average d2EGFP expression level of 99,
which is a factor 1.3 higher than the average in the control cells
(75).
[0216] Importantly, no colonies were established when the TTG Zeo
construct was transfected. However, the construct with TTG Zeo,
flanked with STARs 7 and 67 resulted in the establishment of 6
colonies, with an average d2EGFP expression level of 576 (FIG. 3C).
Thus the highest translation stringency, brought about by the TTG
start codon (FIG. 1) yields to the highest d2EGFP expression
levels, as predicted in FIG. 2. The results also indicate that the
stringency of the TTG Zeo alone (without STAR elements) is at least
in some experiments too high for colonies to survive. However, in
later independent experiments (see below), some colonies were found
with this construct without STAR elements, indicating that the
stringency of the selection system with the TTG start codon in the
ZEOCIN.RTM. selection marker not necessarily precludes the finding
of colonies when no STAR elements are present, and that the number
of colonies obtained may vary between experiments.
[0217] It is concluded that the use of STAR elements in combination
with the stringent selection system according to the invention
allows to readily identify high producers of the gene of
interest.
Example 3
Establishment of a Higher Number of TTG Zeo STAR Colonies and
Comparison with an IRES-Zeo Construct
[0218] The results in example 2 indicate that the TTG Zeo has
extremely stringent translation efficiency, which might be to high
to convey ZEOCIN.RTM. resistance to the cells. The transfection was
scaled up to test whether there would be some colonies that have
such high expression levels that they survive. Scaling up the
experiment could also address the question whether the high average
of TTG Zeo STAR 7/67/7 would become higher when more colonies were
analyzed.
Materials and Methods
[0219] CHO-K1 cells were transfected with the constructs that have
the TTG Zeo gene as selection marker, with and without STAR
elements 7 and 67 (FIG. 4). Transfections, selection, culturing etc
were as in example 2, except that 6 times more cells, DNA and
Lipofectamine 2000 were used. Transfections and selection were done
in Petri dishes.
Results
[0220] FIG. 4A shows that transfection with the TTG Zeo STAR 7/67/7
construct resulted in the generation of many colonies with an
average d2EGFP signal of 560. This is as high as in example 2,
except that now 58 colonies were analyzed. When compared to a
construct with the Zeocin-resistance gene placed behind an IRES
sequence (FIG. 4B), the average d2EGFP expression level was 61, and
when STAR elements 7 and 67 were added to such a construct, the
average d2EGFP expression level was 125, a factor 2 above the
control (FIG. 4B). The average of the TTG Zeo STAR 7/67/7 colonies
was therefore a factor 9.2 higher than the STAR-less IRES-Zeo
colonies and a factor 4.5 higher than the STAR7/67/7 IRES Zeo
colonies.
[0221] An observation is that the form of the curve of all
expressing colonies differs between the TTG Zeo STAR7/67/7 and
IRES-Zeo STAR 7/67/7. In the first case (TTG Zeo) the curve levels
off, whereas in the second case (IRES-Zeo) the curve has a more
"exponential" shape. The plateau in the TTG Zeo curve could
indicate that the cells have reached a maximum d2EGFP expression
level, above which the d2EGFP expression levels become toxic and
the cells die. However, it later appeared that the high values were
close to the maximum value that could be detected with the settings
of the detector of the FACS analyser. In later experiments, the
settings of the FACS analyser were changed to allow for detection
of higher values, and indeed in some instances higher values than
obtained here were measured in later independent experiments (see
below).
[0222] Due to up-scaling of the transfections three colonies with
the STAR-less TTG Zeo construct could be picked. The d2EGFP
expression levels of these colonies were 475, 158 and 43. The last
colony died soon after the first measurement. This result indicates
that the TTG Zeo construct can convey ZEOCIN.RTM. resistance,
resulting in colonies that also can give high expression levels in
some instances. Hence, the novel selection method according to the
invention can be applied with expression cassettes that do not
contain chromatin control elements, although it is clearly
preferred to use expression cassettes comprising at least one such
element, preferably a STAR element.
[0223] The results indicate that STAR elements allow a more
stringent selection system according to the invention, such as
exemplified in this example, resulting in the picking of colonies
that have a very high average protein expression level.
Example 4
Creation and Testing of Blasticidin-d2EGFP Bicistronic Constructs
with Differential Translation Efficiencies
[0224] There are four internal ATGs in the blasticidine resistance
gene, none of which codes for a methionine (FIG. 14A). These ATGs
have to be eliminated though (FIG. 14B), since they will serve as
start codon when the ATG start codon (or the context thereof) has
been modified, and this will result in peptides that do not
resemble blasticidine resistance protein. More importantly, these
ATGs will prevent efficient translation of the gene of interest, as
represented by d2EGFP in this example for purposes of illustration.
To eliminate the internal ATGs, the blasticidine resistance protein
open reading frame was first amplified with 4 primer pairs,
generating 4 blasticidine resistance protein fragments. The primer
pairs were:
TABLE-US-00001 A) BSDBamHIforward (SEQ ID NO: 80):
GATCGGATCCACCATGGCCAAGCCTTTGTCTCAAG BSD150reverse (SEQ ID NO: 81):
GTAAAATGATATACGTTGACACCAG B) BSD150forward (SEQ ID NO: 82):
CTGGTGTCAACGTATATCATTTTAC BSD250reverse (SEQ ID NO: 83):
GCCCTGTTCTCGTTTCCGATCGCG C) BSD250forward (SEQ ID NO: 84):
CGCGATCGGAAACGAGAACAGGGC BSD350reverse (SEQ ID NO: 85):
GCCGTCGGCTGTCCGTCACTGTCC D) BSD350forward (SEQ ID NO: 86):
GGACAGTGACGGACAGCCGACGGC BSD399reverse (SEQ ID NO: 87):
GATCGAATTCTTAGCCCTCCCACACGTAACCAGAGGGC
[0225] Fragments A to D were isolated from an agarose gel and mixed
together. Next, only primers BSDBamHIforward and BSD399reverse were
used to create the full length blasticidine resistance protein
cDNA, but with all internal ATGs replaced. The reconstituted
blasticidine was then cut with EcoRI-BamHI, and cloned into
pZEO-GTG-d2EGFP, cut with EcoRI-BamHI (which releases Zeo),
resulting in pBSDmut-d2EGFP. The entire blasticidine resistance
protein open reading frame was sequenced to verify that all ATGs
were replaced.
[0226] With this mutated gene encoding blasticidine resistance
protein (Blas), three classes of constructs are made (FIG. 14C-E):
[0227] 1) ATG as a start codon, but in a bad context and followed
by spacer sequence. The mutated blasticidine resistance protein
open reading frame in pBSD-d2EGFP was amplified using primers
BSDforwardBamHIAvrII-ATGmut/space (SEQ ID NO:88):
GATCGGATCCTAGGTTGGTTTTC
GATCCAAAGACTGCCAAATCTAGATCCGAGATTTTCAGGAGCTAAGGAAGCTAAAGCCAAGCCT
TTGTCTCAAGAAG, [0228] and BSD399reverseEcoRIAvrII (SEQ ID NO:89):
GATCGAATTCCCTAGGTTAGCCCTCCCAC ACGTAACCAGAGGGC, the PCR product is
cut with BamHI-EcoRI, and ligated into pZEO-GTG-d2EGFP, cut with
EcoRI-BamHI. This results in pBSD-ATGmut/space-d2EGFP. [0229] 2)
GTG as a start codon instead of ATG. The mutated blasticidine
resistance protein open reading frame in pBSD-d2EGFP was amplified
using primers BSDforwardBamHIAvrII-GTG (SEQ ID NO:90):
GATCGGATCCTAGGACCGCCAAGCCTTTGTCTCAAGAAG and BSD399reverseEcoRIAvrII
(SEQ ID NO:89), the PCR product was cut with BamHI-EcoRI, and
ligated into pZEO-GTG-d2EGFP, cut with EcoRI-BamHI. This results in
pBSD-GTG-d2EGFP. [0230] 3) TTG as a start codon instead of ATG. The
mutated blasticidine open reading frame in pBSD-d2EGFP was
amplified using primers BSDforwardBamHIAvrII-TTG (SEQ ID NO:91):
GATCGGATCCTAGGACCGCCAAGCCTTTGTCTCAAGAAG and BSD399reverseEcoRIAvrII
(SEQ ID NO:89), the PCR product was cut with BamHI-EcoRI, and
ligated into pZEO-GTG-d2EGFP, cut with EcoRI-BamHI. This results in
pBSD-TTG-d2EGFP.
Results
[0231] CHO-K1 cells were transfected with constructs that contain
the GTG Blas (FIG. 5A) and TTG Blas (FIG. 5B) genes as selection
gene, all being cloned upstream of the d2EGFP reporter gene.
Selection took place in the presence of 20 .mu.g/ml Blasticidine.
The two constructs were without STAR elements (Control) or with
STAR elements 7 and 67 upstream of the CMV promoter and STAR7
downstream from the d2EGFP gene (FIG. 5). FIG. 5 shows that both
the control (without STAR elements) constructs with GTG Blas (A)
and TTG Blas (B) gave colonies that expressed d2EGFP protein. The
average d2EGFP signal of 24 GTG Blas colonies was 14.0 (FIG. 5A)
and of TTG Blas colonies was 81 (FIG. 5B). This higher average
expression level in TTG Blas colonies may reflect the higher
stringency of TTG, in comparison with GTG (see also Example 2).
However, only 8 colonies survived under the more stringent TTG
conditions.
[0232] Addition of STAR elements 7 and 67 to the constructs
resulted in colonies that had higher average d2EGFP expression
levels. Transfection of the GTG Blas STAR 7/67/7 construct resulted
in colonies with an average d2EGFP expression level of 97.2 (FIG.
5A), which is a factor 6.9 higher than the average in the control
cells (14.0). Addition of STAR elements to the TTG Blas construct
resulted in an average d2EGFP signal of 234.2 (FIG. 5B), which is a
factor 2.9 higher than the average in the control cells (81).
However, note again that only 8 colonies survived the harsh
selection conditions of TTG Blas, whereas 48 colonies survived with
TTG Blas STAR 7/67/7. When only the five highest values are
compared, the average of the five highest TTG Blas was 109.1 and
the average of the five highest TTG Blas STAR 7/67/7 was 561.2,
which is a factor 5.1 higher.
[0233] The results indicate that STAR elements allow a more
stringent selection system, resulting in the picking of colonies
that have a very high average protein expression level. They also
show that this selection is not restricted to the Zeocin-resistance
protein alone, but that also other selection marker polypeptides,
in this case the blasticidine resistance protein, can be used.
Example 5
Stability of d2EGFP Expression in the Novel Selection System
[0234] Colonies described in Example 3 were further cultured under
several conditions to assess the stability of d2EGFP expression
over an extended time period.
Results
[0235] The TTG Zeo STAR 7/67/7 containing colonies in FIG. 4A were
cultured for an additional 70 days in the presence of 100 .mu.g/ml
Zeocin. As shown in FIG. 6, the average d2EGFP signal rose from
560.2 after 35 days to 677.2 after 105 days. Except for some rare
colonies all colonies had a higher d2EGFP expression level.
[0236] When the level of ZEOCIN.RTM. was lowered to 20 .mu.g/ml
ZEOCIN.RTM., there was still an increase in the average d2EGFP
expression level, from 560.2 after 35 days to 604.5 after 105 days
(FIG. 7).
[0237] When no selection pressure was present at all due to removal
of the ZEOCIN.RTM. from the culture medium, approximately 50% of
the colonies became mosaic, that is, within one colony non-d2EGFP
expressing cells became apparent. This resulted in lowering of
d2EGFP expression levels to less than 50% of the original levels.
If the signal became less than 67% (decrease of at least one-third)
from the original signal, the colony was considered to be unstable
in respect to d2EGFP expression. Of the 57 original colonies 27
colonies remained stable according to this criterion; the average
d2EGFP signal of these colonies after 35 days (while still under
selection pressure) was 425.6, whereas the average d2EGFP signal
without selection pressure after 65 days was 290.0. When measured
after 105 days, the average signal in the 27 colonies was 300.9.
Hence, after an initial decrease, the expression levels in the 27
colonies remained stable according to this criterion (FIG. 8).
[0238] Six of the colonies were subjected to one round of
sub-cloning. Cells were sown in 96-wells plates as such that each
well contained approximately 0.3 cells. No ZEOCIN.RTM. was present
in the medium so that from the start the sub clones grew without
selection pressure. Of each original colony six sub clones were
randomly isolated and grown in 6-wells plates till analysis. In
FIG. 12 we compared the original values of the original clones, as
already shown in FIG. 4A, with one of the sub clones. In one of the
six clones (clone 25), no sub clone was present with d2EGFP signal
in the range of the original clone. However, in five out of six
cases at least one the sub clones had equal d2EGFP expression
levels as the parent clone. These expression levels were determined
after 50 days without selection pressure. We conclude that one
round of sub cloning is sufficient to obtain a high number of
colonies that remain stable for high expression in the absence of
selection pressure. This has been confirmed in a similar experiment
(not shown).
[0239] We compared the number of copies that integrated in the TTG
Zeo STAR 7/67/7 colonies. DNA was isolated when colonies were 105
days under ZEOCIN.RTM. selection pressure (see FIG. 6). As shown in
FIG. 13 two populations could be distinguished. In FIG. 13 the cut
off was made at 20 copies and the R.sup.2 value is calculated and
shown. Also the R.sup.2 value from data with higher than 20 copies
is shown. In the range from 100 to 800 d2EGFP signal there was a
high degree of copy number dependency, as signified by a relatively
high R.sup.2 of 0.5685 (FIG. 13). However, in the population of
colonies that fluctuate around a d2EGFP signal of 800 a high
variation in copy number was observed (FIG. 13), as signified with
a low R.sup.2 of 0.0328. Together the data show that in the novel
selection system, in colonies that contain TTG Zeo STAR 7/67/7
constructs there is copy number dependent d2EGFP expression up to
.about.20 copies. Also, although copy number dependency is lost
when >20 copies are present, still a substantial proportion of
the colonies with high (>800) d2EGFP signal have no more than 30
copies (FIG. 13). This combination between high d2EGFP expression
and a relatively low copy number (between 10 and 30) may be
important for identifying colonies that remain relatively stable
without selection pressure. It is an advantage to have clones with
relatively low copy numbers (less than about 30, more preferably
less than about 20) that give high expression levels, because such
clones are believed to be less amenable to genetic instability. The
present selection system allows to generate such clones, including
from CHO cells.
Example 6
Creation and Testing of Zeocin-Blasticidin-EpCAM Bicistronic
Constructs with Differential Translation Efficiencies
[0240] To test the selection system on the production of an
antibody, the anti-EpCAM antibody (see also Example 5 of the
incorporated '525 application and of WO2006/005718) was taken as
example.
Results
[0241] A plasmid was created on which both the heavy chain (HC) and
light chain (LC) were placed, each in a separate transcription unit
(FIG. 9-11). Expression of both chains was driven by the CMV
promoter. Upstream of the EpCAM heavy chain the Zeocin-resistance
gene was placed, either with the ATGmut/space (FIG. 9), GTG (FIG.
10) or TTG (FIG. 11) as start codon (See, Example 2). Upstream of
the EpCAM light chain the Blasticidine resistance gene was placed,
either with the ATGmut/space (FIG. 9), GTG (FIG. 10) or TTG (FIG.
11) as start codon (See, Example 4). Two types of constructs were
made, one construct without STAR elements (Control) and one
construct with a combination of STAR 7 and 67 elements. The STAR
elements were placed as follows: upstream of each CMV promoter
(i.e., one for the transcription unit comprising HC and one for the
transcription unit comprising LC) STAR 67 was placed and the
resulting construct was flanked with a 5' and 3' STAR 7 element
(FIGS. 9-11). All constructs were transfected to CHO-K1 cells and
selected on 100 .mu.g/ml ZEOCIN.RTM. and 20 .mu.g/ml Blasticidin
(at the same tim selection independent colonies were isolated and
propagated under continuous selection pressure (using 100 .mu.g/ml
ZEOCIN.RTM. and 20 .mu.g/ml blasticidin). FIG. 9 shows that the
STAR 7/67/7 combination had a beneficial effect on EpCAM
production. The ATGmut/space Zeo and ATGmut/space Blas had no
effect on the number of colonies that were formed with plasmids
containing STAR elements or not. However, the average EpCAM
expression levels of either 24 control versus STAR 7/67/7 colonies
ranged from 0.61 pg/cell/day in the control to 3.44 pg/cell/day in
the STAR7/67/7 construct (FIG. 9). This is a factor 5.6 increase.
Since there were many colonies in the ATGmut/space control with 0
pg/cell/day, also the average EpCAM production in the highest five
colonies was compared. In the control ATGmut/space this was 3.0
pg/cell/day, versus 7.8 pg/cell/day with the ATGmut/space STAR
7/67/7 construct, an increase of a factor 2.6.
[0242] FIG. 10 also shows that the STAR 7/67/7 combination had a
beneficial effect on EpCAM production, using the GTG start codon
for the markers. With the GTG Zeo and GTG Blas STAR 7/67/7
construct approximately 2 times more colonies were formed. Also,
the average EpCAM expression levels of either 24 control versus
STAR 7/67/7 colonies ranged from 2.44 pg/cell/day in the control to
6.51 pg/cell/day in the STAR7/67/7 construct (FIG. 10). This is a
factor 2.7 increase. Also the average EpCAM production in the
highest five colonies was compared. In the control GTG this was 5.7
pg/cell/day, versus 13.0 pg/cell/day with the GTG STAR 7/67/7
construct, an increase of a factor 2.3. Also note that the average
EpCAM production mediated by the GTG start codon for the selection
markers was significantly higher than with the ATGmut/space start
codon.
[0243] FIG. 11 shows that with the TTG Zeo and TTG Blas control
construct no colonies were formed, similar as in example 2. With
the STAR 7/67/7 TTG construct colonies were formed. The average
EpCAM expression levels of the STAR 7/67/7 TTG colonies was 10.4
pg/cell/day (FIG. 11). This is again higher than with the
ATGmut/space and GTG as start codon (see FIGS. 9, 10 for
comparison). The average EpCAM production in the highest five TTG
STAR 7/67/7 colonies was 22.5 pg/cell/day.
[0244] The results show that the selection system can also be
applied to two simultaneously produced polypeptides, in this case
two polypeptides of a multimeric protein, casu quo an antibody. The
EpCAM production closely follows the results obtained with d2EGFP.
The TTG as start codon is more stringent than the GTG start codon,
which in turn is more stringent than the ATGmut/space (FIGS. 1 and
2). Higher stringency results in a decreasing number of colonies,
with no colonies in the case of the TTG control that has no STAR
elements, and higher stringency of the selection marker is coupled
to higher expression of the protein of interest.
Example 7
Creation and Testing of Additional GTG Zeocin-d2EGFP Bicistronic
Constructs with Differential Translation Efficiencies
[0245] Different versions of the Zeocin-resistance gene with
mutated start codons were described in Example 1. Besides the
described GTG codons (Example 1, FIG. 22A), additional modified
start codons with distinct translational efficiency are possible.
These different Zeocin-resistance gene versions were created (FIG.
22) and cloned upstream of the modified d2EGFP gene, as in Example
2.
Materials and Methods
Creation of Plasmids
[0246] Four additional GIG constructs were made: [0247] 1) GTG as a
start codon in the Zeo resistance gene (FIG. 22A), but followed by
a spacer sequence (FIG. 22B). The mutspace-Zeo open reading frame
was amplified with primer pair GTGspaceBamHIF (SEQ ID NO:106):
GAATTCGGATCCACCGTGGCGATCCAAAGACTGCCAAA TCTAG and (wherein the
sequence following the underlined sequence comprises the spacer
sequence), and ZEOWTreverse (SEQ ID NO:69), the PCR product was cut
with EcoRI-BamHI, and ligated into pd2EGFP, cut with EcoRI-BamHI,
creating pZEO-GTGspace-d2EGFP. [0248] 2) GTG as a start codon in
the Zeo resistance gene, but in a bad context () (FIG. 22C). The
Zeo open reading frame was amplified with primer pair
ZEOTTTGTGBamHIF (SEQ ID NO:107):
GAATTCGGATCCTTTGTGGCCAAGTTGACCAGTGCCGTTCCG and. ZEOWTreverse (SEQ
ID NO:69), the PCR product was cut with EcoRI-BamHI, and ligated
into pd2EGFP, cut with EcoRI-BamHI, creating
pZEO(leu)-TTTGTG-d2EGFP. [0249] 3) GTG as a start codon in the Zeo
resistance gene, instead of ATG (FIG. 22A), but with an additional
mutation in the Zeo open reading frame at Pro9, which was replaced
with threonine (Thr) (FIG. 22D). The Thr9 mutation was introduced
by amplifying the Zeo open reading with primer pair
ZEOForwardGTG-Thr9 (SEQ ID NO:108):
AATTGGATCCACCGTGGCCAAGTTGACCAGTGCC GTTGTGCTC and ZEOWTreverse (SEQ
ID NO:69), the PCR product was cut with EcoRI-BamHI, and ligated
into pd2EGFP, cut with EcoRI-BamHI, creating pZEO-GTG-Thr9-d2EGFP.
[0250] 4) GTG as a start codon in the Zeo resistance gene, instead
of ATG (FIG. 22A), but with an additional mutation in the Zeo open
reading frame at Pro9, with was replaced with Phenylalanine (Phe)
(FIG. 22E). The Phe9 mutation was introduced by amplifying the Zeo
open reading with primer pair ZEOForward GTG-Phe9 (SEQ ID NO:109):
AATTGGATCCACCGTGGCCAAGTTGACCAGTG CCGTTGTGCTC and ZEOWTreverse (SEQ
ID NO:69), the PCR product was cut with EcoRI-BamHI, and ligated
into pd2EGFP, cut with EcoRI-BamHI, creating
pZEO-GTG-Phe9-d2EGFP.
Transfection, Culturing and Analysis of CHO Cells
[0251] Transfection, culturing and analysis of CHO-K1 cells was
performed as in Example 1.
Results
[0252] CHO-K1 cells were transfected with constructs that contain
the GTG Zeo (FIG. 22A), GTGspace Zeo (FIG. 22B), TTT GTG Zeo (also
called: GTGmut Zeo) (FIG. 22C), GTG Thr9 Zeo(leu) (FIG. 22D) and
GTG Phe9 Zeo(leu) (FIG. 22D) genes as selection gene, all being
cloned upstream of the d2EGFP reporter gene. These five constructs
were without STAR elements (Control) or with STAR elements 7 and 67
upstream of the CMV promoter and STAR 7 downstream from the d2EGFP
gene (FIG. 22). FIG. 23 shows that of the control constructs
without STAR elements only the GTG Zeo construct without STAR
elements gave colonies that expressed d2EGFP protein. In contrast,
all constructs containing STAR elements gave colonies that
expressed d2EGFP protein. The mean d2EGFP fluorescence signal of 11
GTG Zeo Control colonies was 20.3, of 13 GTG Zeo colonies with
STARs 7/67/7 104.9, of 24 GTG space Zeo 7/67/7 colonies 201.5, of 6
TTT GTG Zeo 7/67/7 colonies 310.5, of 22 GTG Thr9 Zeo 7/67/7
colonies 423, and of 16 GTG Phe9 Zeo colonies 550.2 (FIG. 23).
[0253] The higher stringencies of the novel GTG mutations correlate
with higher mean fluorescence signals (FIG. 23). The TTT GTG Zeo
7/67/7, however, gave only two high expressing colonies and a few
low expressing colonies. This may indicate that this mutation is at
the brink of the stringency that these cells can bear with a fixed
concentration of ZEOCIN.RTM. added to the culture medium.
[0254] The Thr9 and Phe9 mutations do not influence the translation
efficiency of the Zeo mutants. Instead they reduce the
functionality of the Zeocin-resistance protein, by preventing an
optimal interaction between the two halves of the
ZEOCIN.RTM.-resistance protein (Dumas et al., 1994). This implies
that more of the protein has to be produced to achieve resistance
against the ZEOCIN.RTM. in the culture medium. As a consequence,
the entire cassette has to be transcribed at a higher level,
eventually resulting in a higher d2EGFP expression level.
[0255] It is concluded that the use of the described translation
efficiencies of the Zeocin-resistance mRNA result in higher
expression levels of the d2EGFP protein, this in combination with
STAR elements.
[0256] This example further demonstrates the possibility to provide
for fine-tuning of the stringency of the selection system of the
invention, to achieve optimal expression levels of a protein of
interest. Clearly, the person skilled in the art will be capable of
combining these and other possibilities within the concepts
disclosed herein (e.g., mutate the ZEOCIN.RTM. at position 9 to
other amino acids, or mutate it in other positions; use a GTG or
other start codon in a non-optimal translation initition context
for ZEOCIN.RTM. or other selection markers; or mutate other
selection markers to reduce their functionality, for instance use a
sequence coding for a neomycin resistance gene having a mutation at
amino acid residue 182 or 261 or both, see, e.g., WO 01/32901), and
the like, to provide for such fine-tuning, and by simply testing
determine a suitable combination of features for the selection
marker, leading to enhanced expression of the polypeptide of
interest.
Example 8
Creation and Testing of Additional TTG Zeocin-d2EGFP Bicistronic
Constructs with Differential Translation Efficiencies
[0257] Different versions of the Zeocin-resistance gene with
mutated start codons were described in Example 1. Besides the
described TTG codons (FIG. 24A) additional modified start codons
with distinct translational efficiency are possible. These
different Zeocin-resistance gene versions were created and cloned
upstream of the modified d2EGFP gene (FIG. 24).
Materials and Methods
Creation of Plasmids
[0258] Three additional TTG constructs were made: [0259] 1) TTG as
a start codon in the Zeo resistance gene (FIG. 24A), but followed
by a spacer sequence (FIG. 24B). The Zeo open reading frame (with
the spacer sequence) was amplified with primer pair TTGspaceBamHIF
(SEQ ID NO:110): GAATTCGGATCCACCTTGGCGATCCAAAGACTGCCAA ATCTAG and
ZEOWTreverse(SEQ ID NO:69), the PCR product was cut with
EcoRI-BamHI, and ligated into pd2EGFP, cut with EcoRI-BamHI,
creating pZEO-TTGspace-d2EGFP. [0260] 2) TTG as a start codon in
the Zeo resistance gene, instead of ATG (FIG. 24A), but with an
additional mutation in the Zeo open reading frame at Pro9, with was
replaced with threonine (Thr) (FIG. 24C). The Thr9 mutation was
introduced by amplifying the Zeo open reading with primer pair
ZEOForwardTTG-Thr9 (SEQ ID NO:111):
AATTGGATCCACCTTGGCCAAGTTGACCAGTGCCGT TGTGCTC and ZEOWTreverse (SEQ
ID NO:69), the PCR product was cut with EcoRI-BamHI, and ligated
into pd2EGFP, cut with EcoRI-BamHI, creating pZEO-TTG-Thr9-d2EGFP.
[0261] 3) TTG as a start codon in the Zeo resistance gene, instead
of ATG (FIG. 24A), but with an additional mutation in the Zeo open
reading frame at Pro9, with was replaced with Phenylalanine (Phe)
(FIG. 24D). The Phe9 mutation was introduced by amplifying the Zeo
open reading with primer pair ZEOForwardTTG-Phe9 (SEQ ID NO:112):
AATTGGATCCACCTTGGCCAAGTTGACCAGTGCC GTTGTGCTC and ZEOWTreverse (SEQ
ID NO:69), the PCR product was cut with EcoRI-BamHI, and ligated
into pd2EGFP, cut with EcoRI-BamHI, creating
pZEO-TTG-Phe9-d2EGFP.
Results
[0262] CHO-K1 cells were transfected with constructs that contain
the TTG Zeo (FIG. 24A), TTGspace Zeo (FIG. 24B), TTG Thr9 Zeo (FIG.
24C) and TTG Phe9 Zeo (FIG. 24D) genes as selection gene, all being
cloned upstream of the d2EGFP reporter gene. These four constructs
were without STAR elements (Control) or with STAR elements 7 and 67
upstream of the CMV promoter and STAR 7 downstream from the d2EGFP
gene (FIG. 24). FIG. 25 shows that of the control constructs
without STAR elements only the TTG Zeo construct without STAR
elements gave colonies that expressed d2EGFP protein. In contrast,
all constructs containing STAR elements gave colonies that
expressed d2EGFP protein. The mean d2EGFP fluorescence signal of 3
TTG Zeo Control colonies was 26.8, of 24 TTG Zeo colonies with
STARs 7/67/7 426.8, of 24 TTGspace Zeo 7/67/7 colonies 595.7, of 2
TTG Thr9 Zeo 7/67/7 colonies 712.1, and of 3 TTG Phe9 Zeo colonies
677.1 (FIG. 25).
[0263] The higher stringencies of the novel TTG mutations correlate
with higher mean fluorescence signals (FIG. 25). The TTG Thr9 Zeo
7/67/7 and TTG Phe9 Zeo 7/67/7 constructs, however, gave only two
high expressing colonies each and a few low expressing colonies.
This may indicate that these mutations are at the brink of the
stringency that the cells can bear with a fixed concentration of
ZEOCIN.RTM. added to the culture medium.
[0264] It is concluded that the use of the described translation
efficiencies of the Zeocin-resistance mRNA result in higher
expression levels of the d2EGFP protein, this in combination with
STAR elements.
Example 9
Creation and Testing of Puromycin-d2EGFP Bicistronic Constructs
with Differential Translation Efficiencies
[0265] There are three internal ATGs in the puromycin resistance
gene, each of which codes for a methionine (FIG. 17, FIG. 26A).
These ATGs have to be eliminated (FIG. 26B,C), since they will
serve as start codon when the ATG start codon (or the context
thereof) has been modified, and this will result in peptides that
do not resemble puromycin resistance protein. More importantly,
these ATGs will prevent efficient translation of the gene of
interest, as represented by d2EGFP in this example for purposes of
illustration. The methionines were changed into leucine, like in
the zeocin-resistance protein (Example 1). However, instead of
using the TTG codon for leucine (for instance in ZEOCIN.RTM. in
Example 1), now the CTG codon for leucine was chosen (in humans,
for leucine the CTG codon is used more often than the TTG codon).
To eliminate the internal ATGs, the puromycin resistance protein
open reading frame was first amplified with 4 primer pairs,
generating 4 puromycin resistance protein fragments. The primer
pairs were: PURO BamHI F (SEQ ID NO:113):
GATCGGATCCATGGTTACCGAGTACAAGCCCACGGT, PURO300 R LEU (SEQ ID
NO:114): CAGCCGGGAACCGCTCAACTCGGCCAGGCGCGGGC; and PURO300FLEU (SEQ
ID NO:115): CGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGCTGGAAGGCCTC,
PURO600RLEU (SEQ ID NO:116):
AAGCTTGAATTCAGGCACCGGGCTTGCGGGTCAGGCACCAGGTC.
[0266] This generates two PCR products, corresponding to the 5' and
3' part of the puromycin resistance gene. The two products were
added together and amplified with PURO BamHI F (SEQ ID
NO:113)-PURO600RLEU (SEQ ID NO:116). The resulting PCR product was
cut with BamHI-EcoRI and ligated, creating pCMV-ATGPURO (leu).
Sequencing of this clone verified that all three internal ATGs had
been converted. The entire puromycin open reading frame was then
amplified with PUROBamHI TTG1F (SEQ ID NO:117):
GAATTCGGATCCACCTTGGTTACCGAGTACAAGCCCACGGTG and PURO600RLEU (SEQ ID
NO:116). This primer introduces an extra codon (GTT) directly after
the TTG start codon, because the "G" at nucleotide +4 is introduced
for an optimal context, and hence two more nucleotides are
introduced to preserve the reading frame.
Results
[0267] CHO-K1 cells were transfected with the construct that
contains the TTG Puro (FIG. 27) gene as selection gene, cloned
upstream of the d2EGFP reporter gene. Selection was under 10
.mu.g/ml puromycin. The construct was without STAR elements
(Control) or with STAR elements 7 and 67 upstream of the CMV
promoter and STAR 7 downstream from the d2EGFP gene (FIG. 27). FIG.
27 shows that the average d2EGFP fluorescence signal of 24 TTG Puro
Control colonies was 37.9, of 24 TTG Puro colonies with STARs
7/67/7 75.5. Moreover, when the average of the five highest values
is taken, the d2EGFP fluorescence signal of TTG Puro Control
colonies was 69.5, and of TTG Puro colonies with STARs 7/67/7
186.1, an almost three-fold increase in d2EGFP fluorescence signal.
This shows that the described, modified translation efficiency of
the Puromycin resistance mRNA result in higher expression levels of
the d2EGFP protein, this in combination with STAR elements.
[0268] This experiment demonstrates that the puromycin resistance
gene can be mutated to remove the ATG sequences therefrom, while
remaining functional. Moreover it is concluded that the selection
method of the invention also works with yet another selection
marker, puromycin.
Example 10
Creation and Testing of Neomycin Constructs with Differential
Translation Efficiencies
[0269] There are sixteen internal ATGs in the neomycin resistance
gene, five of which code for a methionine in the neomycin open
reading frame (FIG. 20, FIG. 28A). All these sixteen ATGs have to
be eliminated (FIG. 28B,C), since they will serve as start codon
when the ATG start codon (or the context thereof) has been
modified, and this will result in peptides that do not resemble
neomycin resistance protein, and this will decrease the translation
from the downstream open reading frame coding for the polypeptide
of interest in the transcription units of the invention. To
eliminate the internal ATGs, the neomycin resistance protein open
reading frame was entirely synthesized by a commercial provider
(GeneArt, Germany), wherein all internal coding ATGs (for Met)
where replaced by CTGs (coding for Leu), and non-coding ATGs were
replaced such that a degenerated codon was used and hence no
mutations in the protein sequence resulted; the synthesised
sequence of the neomycin is given in SEQ ID NO:118. In order to
replace the ATG start codon with GTG (FIG. 28B) or TTG FIG. 28C),
the synthesized neomycin gene was amplified with primer pairs
NEO-F-HindIII (SEQ ID NO:120): GATCAAGCTTTTGGATCGGCCATTGAA
ACAAGACGGATTG and NEO EcoRI 800R (SEQ ID NO:121):
AAGCTTGAATTCTCAGAAGAACTCGT CAAGAAGGCG.
Results
[0270] E. coli bacteria were used to test the functionality of the
neomycin resistance protein from which all ATGs were removed. E.
coli bacteria were transformed with the constructs that contain the
GTG Neo (FIG. 28B) or TTG Neo (FIG. 28C) gene as selection gene.
Selection took place by growing the bacteria on kanamycin. Only a
functional neomycin resistance gene can give resistance against
kanamycin. Transformation with either modified Neo gene resulted in
the formation of E. coli colonies, from which the plasmid
containing the gene could be isolated. This shows that the
described, modified translation efficiencies of the Neomycin
resistance mRNAs, as well as the removal of all ATGs from the Neo
open reading frame result in the production of functional neomycin
resistance protein.
[0271] The mutated neomycin resistance genes are incorporated in a
multicistronic transcription unit of the invention, and used for
selection with G418 or neomycin in eukaryotic host cells.
Example 11
Creation and Testing of dhfr Constructs with Differential
Translation Efficiencies
[0272] There are eight internal ATGs in the dhfr gene, six of which
code for a methionine in the dhfr open reading frame (FIG. 18, FIG.
29A). All these ATGs have to be eliminated (FIGS. 29B,C), since
they will serve as start codon when the ATG start codon (or the
context thereof) has been modified, and this will result in
peptides that do not resemble dhfr protein, and will decrease the
translation from the downstream open reading frame coding for the
polypeptide of interest in the transcription units of the
invention. To eliminate the internal ATGs, the dhfr protein open
reading frame was entirely synthesized (SEQ ID NO:122), as
described above for neomycin. In order to replace the ATG start
codon with GTG (FIG. 29B) or TTG (FIG. 29C), the synthesized DHFR
gene was amplified with primers DHFR-F-HindIII (SEQ ID NO:124):
GATCAAGCTTTTGTTCGACCATTGAACTGCATCGTC and DHFR-EcoRI-600-R (SEQ ID
NO:125): AGCTTGAATTCTTAGTCTTTCTTCTCGTAGACTTC.
Results
[0273] E. coli bacteria were used to test the functionality of the
dhfr protein from which all ATGs were removed. E. coli was
transformed with the constructs that contain the GTG dhfr (FIG.
29B) or TTG dhfr (FIG. 29C) gene. Selection took place by growing
the bateria on trimethoprim (Sigma T7883-56). Only a functional
dhfr gene can give resistance against trimethoprim. Transformation
with either modified dhfr gene resulted in the formation of E. coli
colonies, from which the plasmid containing the gene could be
isolated. This shows that the described, modified translation
efficiencies of the dhfr mRNAs, as well as the removal of all ATGs
from the dhfr open reading frame result in the production of
functional dhfr protein.
[0274] The mutated dhfr genes are incorporated in a multicistronic
transcription unit of the invention, and used for selection with
methotrexate in eukaryotic host cells.
Example 12
Testing of Zeocin- and Blasticidin Constructs with Differential
Translation Efficiencies in PER.C6.RTM. Cells
[0275] Various ZEOCIN.RTM. and blasticidin genes with mutated start
codons, all cloned upstream of the d2EGFP gene were tested in the
PER.C6.RTM. cell line.
Results
[0276] The GTG ZEOCIN.RTM. and GTGspace Zeocin-resistance gene
modifications (see also Example 7; FIG. 30) and the GTG blasticidin
and TTG blasticidin resistance gene modifications (see also Example
4; FIG. 31), all cloned upstream of the d2EGFP gene were
transfected to PER.C6.RTM. cells. As shown in FIG. 30, transfection
with both the GTG ZEOCIN.RTM. and GTGspace ZEOCIN.RTM. gene
resulted in colonies that expressed d2EGFP. The average d2EGFP
fluorescence signal of 20 GTG Zeo colonies was 63.8, while the
average d2EGFP signal of 20 GTGspace Zeo colonies was 185,
demonstrating that also in PER.C6.RTM. cells the GTGspace Zeo has a
higher translation stringency than the GTG Zeo mRNA.
[0277] As shown in FIG. 31, transfection with both the GTG
Blasticidin and TTG Blasticidin gene resulted in colonies that
expressed d2EGFP. The average d2EGFP fluorescence signal of 20 GTG
Blasticidin colonies was 71.4, while the average d2EGFP
fluorescence signal of 20 TTG Blasticidin colonies was 135,
demonstrating that also in PER.C6.RTM. cells the TTG Blasticidin
has a higher translation stringency than the GTG Blasticidin
mRNA.
[0278] This example demonstrates that the selection system of the
invention can also be used in other cells than CHO cells.
Example 13
Testing of the Addition of a Transcriptional Pause Signal to a TTG
Zeocin-d2EGFP Construct
[0279] A TRAnscription Pause (TRAP) sequence is thought to, at
least in part, prevent formation of antisense RNA or, to at least
in part, prevent transcription to enter the protein expression unit
(see WO 2004/055215). A TRAP sequence is functionally defined as a
sequence which when placed into a transcription unit, results in a
reduced level of transcription in the nucleic acid present on the
3' side of the TRAP when compared to the level of transcription
observed in the nucleic acid on the 5' side of the TRAP, and
non-limiting examples of TRAP sequences are transcription
termination signals. In order to function to prevent or decrease
transcription to enter the transcription unit, the TRAP is to be
placed upstream of a promoter driving expression of the
transcription unit and the TRAP should be in a 5' to 3' direction.
In order to prevent at least in part formation of antisense RNA,
the TRAP should be located downstream of the open reading frame in
a transcription unit and present in a 3' to 5' direction (that is,
in an opposite orientation as the normal orientation of a
transcriptional termination sequence that is usually present behind
the open reading frame in a transcription unit). A combination of a
TRAP upstream of the promoter in a 5' to 3' orientation and a TRAP
downstream of the open reading frame in a 3' to 5' oreintation is
preferred. Adding a TRAP sequence to a STAR element improves the
effects of STAR elements on transgene expression (see WO
2004/055215). Here we test the effects of the TRAP sequence in the
context of the TTG Zeo resistance gene.
Results
[0280] The TTG Zeocin-d2EGFP cassette that was flanked with STAR7
elements (FIG. 32) was modified by the addition of the SPA/pause
TRAP sequence (see WO 2004/055215); SEQ ID NO:126), both upstream
of the 5' STAR7 (in 5' to 3' direction) and downstream of the 3'
STAR7 (in 3' to 5' direction) (FIG. 32). Both STAR 7/7 and
TRAP-STAR 7/7-TRAP containing vectors were transfected to CHO-K1.
Stable colonies were isolated and the d2EGFP fluorescence
intensities were measured. As shown in FIG. 43 the average d2EGFP
fluorescence signal of 23 TTG Zeo STAR 7/7 colonies was 455.1,
while the average d2EGFP fluorescence signal of 23 TTG Zeo
TRAP-STAR 7/7-TRAP colonies was 642.3. The average d2EGFP
fluorescence signal in highest 5 TTG Zeo STAR 7/7 colonies was
705.1, while the average d2EGFP fluorescence signal of 5 TTG Zeo
TRAP-STAR 7/7-TRAP colonies was 784.7.
[0281] This result indicates that the addition of TRAPs does not
enhance the d2EGFP fluorescence signal in the highest colonies, but
that there is a significant raise in the number of high expressing
colonies. Whereas only 5 TTG Zeo STAR 7/7 colonies had d2EGFP
signal above 600, 17 TTG Zeo TRAP-STAR 7/7-TRAP colonies had a
d2EGFP fluorescence signal above 600.
[0282] In the experiment 3 .mu.g DNA of each plasmid was
transfected. However, whereas the transfection efficiency was
similar, the total number of colonies with the TTG Zeo STAR 7/7
plasmid was 62, while the total number of colonies with the TTG Zeo
TRAP-STAR 7/7-TRAP plasmid was 116, almost a doubling.
[0283] We conclude that addition of TRAP elements to the STAR
containing plasmids with modified Zeocin-resistance gene
translation codons results in a significantly higher overall number
of colonies and that more colonies are present with the highest
expression levels.
Example 14
Copy-Number Dependency of Expression
[0284] We analyzed the EpCAM antibody expression levels in relation
to the number of integrated EpCAM DNA copies.
Results
[0285] The construct that was tested was TTG-Zeo-Light Chain
(LC)-TTG-Blas-Heavy Chain (HC), both expression units being under
the control of the CMV promoter (see FIG. 33). This construct
contained STAR 7 and 67 (see FIG. 33). Selection conditions were
such that with 200 .mu.g/ml ZEOCIN.RTM. and 20 .mu.g/ml Blasticidin
in the culture medium no control colonies (no STARs) survived and
only STAR 7/67/7 colonies survived.
[0286] DNA was isolated when colonies were 60 days under
ZEOCIN.RTM. and Blasticidin selection pressure (see FIG. 33). The
R.sup.2 value is calculated and shown. In the entire range from 5
to 40 pg/cell/day EpCAM there was a high degree of copy number
dependency, as signified by a relatively high R.sup.2 of 0.5978
(FIG. 33). The data show that in the novel selection system, in
colonies that contain TTG Zeo-TTG Blas EpCAM STAR 7/67/7 constructs
there is copy number dependent EpCAM expression.
Example 15
Methotrexate Induction of Higher EpCAM Expression
[0287] We analyzed EpCAM antibody expression levels after
incubation of clones with methotrexate (MTX). The purpose of this
experiment was to determine whether amplification of a
STAR-containing construct would result in higher EpCAM expression.
MTX acts through inhibition of the dhfr gene product. While some
CHO strains that are dhfr-deficient have been described, CHO-K1 is
dhfr.sup.+. Therefore relatively high concentrations of MTX in the
culture medium have to be present to select for amplification by
increased MTX concentrations in CHO-K1 cells.
Results
[0288] The construct that was tested was TTG-Zeo-Heavy Chain
(HC)-TTG-Blas-Light Chain (LC), both expression units being under
the control of the CMV promoter. Upstream of each CMV promoter
STAR67 was positioned and STAR7 was used to flank the entire
cassette (see also Example 6, FIG. 11 for such a construct). This
construct was further modified by placing an SV40-dhfr cassette (a
mouse dhfr gene under control of an SV40 promoter) between the HC
and LC cassettes, upstream of the second STAR67 (FIG. 34). CHO-K1
cells were transfected. Selection was done with 100 .mu.g/ml
ZEOCIN.RTM. and 10 .mu.g/ml Blasticidin in the culture medium. No
control colonies (without STAR elements) survived and only colonies
with constructs containing the STAR elements survived. Colonies
were isolated and propagated before measuring EpCAM expression
levels. Six colonies that produced between 20 and 35 pg/cell/day
were transferred to medium containing 100 nM MTX. This
concentration was raised to 500 nM, 1000 nM and finally to 2000 nM
with two weeks periods in between each step. After two weeks on
2000 nM MTX, EpCAM concentrations were measured. As shown in FIG.
34, four colonies showed enhanced EpCAM production. Colony 13: from
22 to 30; colony 14: from 28 to 42; colony 17: from 20 to 67 and
colony 19: from 37 to 67 pg/cell/day. Colonies 4 and 16 showed no
enhanced EpCAM expression. We conclude that addition of
methotrexate to the culture medium of CHO-K1 colonies created with
the selection system of the invention can result in enhanced
protein expression. Hence, STAR elements and the selection method
of the invention can be combined with and are compatible with
MTX-induced enhancement of protein expression levels.
Example 16
TTG-Zeo Selection Operates in the Context of Different
Promoters
[0289] We analyzed d2EGFP expression levels in the context of the
TTG Zeo selection marker and different promoters. We compared the
action of STAR elements in the context of the CMV
enhancer/promoter, the SV40 enhancer/promoter and the CMV
enhancer/fl-actin promoter.
Results
[0290] In FIG. 35 we indicate the promoters we tested in the
context of the TTG Zeo selection marker. The tested plasmids
consisted of the indicated control constructs with three different
promoters and STAR constructs which were flanked with STAR 7 and
STAR 67 at the 5' end and STAR 7 at the 3' end. The constructs were
transfected to CHO-K1 cells and selection was performed with 200
.mu.g/ml ZEOCIN.RTM. in the culture medium. Up to 23 independent
colonies were isolated and propagated before analysis of d2EGFP
expression levels. As shown in FIG. 35, incorporation of STAR
elements in constructs with the CMV enhancer/promoter, the SV40
enhancer/promoter or the CMV enhancer/.beta.-actin promoter all
resulted in the formation of colonies with higher d2EGFP expression
levels than with the corresponding control constructs. This shows
that the selection system of the invention, in combination with
STAR elements, operates well in the context of different promoters.
Further analysis showed that the mean of CMV-driven d2EGFP values
was significantly higher than the mean of SV40-driven d2EGFP values
(p<0.05). In contrast, the mean of CMV-driven d2EGFP values did
not significantly differ from CMV/.beta. actin-driven d2EGFP values
(p=0.2).
Example 17
Comparison of Different STAR Elements in the TTG-Zeo Selection
System
[0291] We analyzed d2EGFP expression levels in the context of the
CMV promoter-TTG Zeo selection marker and 53 different STAR
elements, to obtain more insight in which STAR elements give the
best results in this context.
Results
[0292] We cloned 53 STAR elements up-and downstream of the CMV
promoter-TTG Zeo-d2EGFP cassette. The following STAR elements were
tested in such constructs: STAR2-12, 14, 15, 17-20, 26-34, 36, 37,
39, 40, 42-49, 51, 52, 54, 55, 57-62, 64, 65, 67. The constructs
were transfected to CHO-K1 cells and selection was performed with
200 .mu.g/ml ZEOCIN.RTM. in the culture medium. Up to 24
independent colonies were isolated and propagated before analysis
of d2EGFP expression levels. Incorporation of STAR elements in the
constructs resulted in different degrees of enhanced d2EGFP
expression, as compared to the control. Incorporation of STAR
elements 14, 18 and 55 in this experiment did not result in an
increase of average d2EGFP expression over the control (no STAR
element). Although some constructs (with STAR elements 2, 3, 10,
42, 48 and 49) in this experiment gave rise to only a few colonies,
all tested STAR elements except 14, 18 and 55 resulted in average
d2EGFP expression levels higher than for the control. It should be
noted that some STAR elements may act in a more cell type specific
manner and that it is well possible that STAR 14, 18 and 55 work
better in other cell types, with other promoters, other selection
markers, or in different context or configuration than in the
particular set of conditions tested here. Addition of 10 STAR
elements, namely STAR elements 7, 9, 17, 27, 29, 43, 44, 45, 47 and
61, induced average d2EGFP expression levels higher than 5 times
the average d2EGFP expression level of the control. We
retransformed the control and 7 constructs with STAR elements and
repeated the experiment. The results are shown in FIG. 36.
Incorporation of STAR elements in the constructs resulted in
different degrees of enhanced d2EGFP expression, as compared to the
control (FIG. 47). The average d2EGFP expression level in colonies
transfected with the control construct was 29. The averages from
d2EGFP expression levels in colonies with the 7 different STAR
constructs ranged between 151 (STAR 67) and 297 (STAR 29). This is
a factor of 5 to 10-fold higher than the average in the control
colonies.
[0293] We conclude that a) the vast majority of STAR elements have
a positive effect on gene expression levels, b) there is variation
in the degree of positive effects induced by the different STAR
elements, and c) 10 out of 53 tested STAR elements induce more than
5-fold average d2EGFP expression levels, as compared to the
control, and that STAR elements can induce a 10-fold higher average
d2EGFP expression level, as compared to the control.
Example 18
Other Chromatin Control Elements in the Context of a Selection
System of the Invention
[0294] DNA elements such as the HS4 hypersensitive site in the
locus control region of the chicken .beta.-globin locus (Chung et
al., 1997), matrix attachment regions (MAR) (Stief et al., 1989)
and a ubiquitous chromatin opening element (UCOE) (Williams et al.,
2005) have been reported to have beneficial effects on gene
expression when these DNA elements are incorporated in a vector. We
combined these DNA elements with the selection system of the
invention.
Results
[0295] The 1.25 kb HS4 element was cloned into the cassette
encompassing the CMV promoter, TTG Zeo and d2EGFP by a three way
ligation step to obtain a construct with a tandem of 2 HS4 elements
(Chung et al., 1997). This step was done both for the 5' and 3' of
the cassette encompassing the CMV promoter, TTG Zeo and d2EGFP. The
2959 by long chicken lysozyme MAR (Stief et al., 1989) was cloned
5' and 3' of the cassette encompassing the CMV promoter, TTG Zeo
and d2EGFP. The 2614 by long UCOE (Williams et al., 2005) was a
NotI-KpnI fragment, excised from a human BAC clone (RP11-93D5),
corresponding to nucleotide 29449 to 32063. This fragment was
cloned 5' of the CMV promoter. The STAR construct contained STAR7
and STAR67 5' of the CMV promoter and STAR7 3' of the cassette.
These four constructs, as well as the control construct without
flanking chromatin control DNA elements, were transfected to CHO-K1
cells. Selection was performed by 200 .mu.g/ml ZEOCIN.RTM. in the
culture medium. Colonies were isolated, propagated and d2EGFP
expression levels were measured. As shown in FIG. 37, constructs
with all DNA elements resulted in the formation of d2EGFP
expressing colonies. However, incorporation of 2.times. HS4
elements and the UCOE did not result in the formation of colonies
that displayed higher d2EGFP expression levels, in comparison with
the control colonies. In contrast, incorporation of the lysozyme
MAR resulted in the formation of colonies that expressed d2EGFP
significantly higher. The mean expression level induced by MAR
containing constructs was four-fold higher than in the control
colonies. Best results were obtained, however, by incorporating
STAR 7 and 67 in the construct. An almost ten-fold increase in the
mean d2EGFP expression level was observed, as compared to the
control colonies. We conclude that other chromatin control DNA
elements such as MARs can be used in the context of the selection
system of the invention. However, the best results were obtained
when STAR elements were used as chromatin control elements.
Example 19
Stringent Selection by Placing a Modified ZEOCIN.degree. Resistance
Gene Behind an IRES Sequence
[0296] The previous examples (all from the incorporated '525
application) have shown a selection system where a sequence
encoding a selectable marker protein is upstream of a sequence
encoding a protein of interest in a multicistonic transcription
unit, and wherein the translation initiation sequence of the
selectable marker is non-optimal, and wherein further internal ATGs
have been removed from the selectable marker coding sequence. This
system results in a high stringency selection system. For instance
the Zeo selection marker wherein the translation initiation codon
is changed into TTG was shown to give very high selection
stringency, and very high levels of expression of the protein of
interest encoded downstream.
[0297] In another possible selection system the selection marker,
e.g., Zeo, is placed downstream from an IRES sequence. This creates
a multicistronic mRNA from which the Zeo gene product is translated
by IRES-dependent initiation. In the usual d2EGFP-IRES-Zeo
construct, the Zeo start codon is the optimal ATG. It is therefore
possible that changing the Zeo ATG start codon into for instance
TTG (referred to as IRES-TTG Zeo) may result in increased selection
stringencies compared to the usual IRES-ATG Zeo.
Results
[0298] The used constructs are schematically shown in FIG. 38. The
control construct consisted of a CMV promoter, the d2EGFP gene, an
IRES sequence (the sequence of the used IRES (Rees et al., 1996) in
this example was:
GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTC
TATATGTGATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTG
ACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAG
CAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCC
ACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACC
CCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAG
GGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA
CATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAA
AACACGATGATAAGCTTGCCACAACCCCGGGATA; SEQ ID NO:127), and a TTG Zeo
selection marker, i.e., the zeocin-resistance gene with a TTG start
codon ("d2EGFP-IRES-TTG Zeo"). The other construct was the same,
but with a combination of STAR 7 and STAR 67 placed upstream of the
expression cassette and STAR 7 downstream of the cassette
("STAR7/67 d2EGFP-IRES-TTG Zeo STAR7"). Both constructs were
transfected to CHO-K1 cells and selection was performed with 100
.mu.g/ml ZEOCIN.RTM. in the culture medium. Four colonies emerged
after transfection with the control construct and six with the STAR
containing construct. These independent colonies were isolated
propagated before analysis of d2EGFP expression levels. As shown in
FIG. 38, incorporation of STAR elements in the construct resulted
in the formation of colonies with high d2EGFP expression levels. Of
the control colonies without STAR elements ("d2EGFP-IRES-TTG Zeo")
only one colony displayed some d2EGFP expression. The expression
levels are also much higher than those obtained with other control
constructs, containing the IRES with a normal Zeo with standard ATG
start codon, either with or without STAR elements ("d2EGFP-IRES-ATG
Zeo" and "STAR 7/67 d2EGFP-IRES-ATG Zeo STAR7"; also in these ATG
Zeo constructs there was an enhancing effect of the STAR elements,
but these are modest as compared to the novel TTG Zeo variant).
[0299] These results show that placing a Zeo selection marker with
a TTG start codon downstream of an IRES sequence, in combination
with STAR elements, operates well and establishes a stringent
selection system.
[0300] From these data and the previous examples it will be clear
that the marker can be varied along the same lines of the previous
examples. For instance, instead of a TTG start codon, a GTG start
codon can be used, and the marker can be changed from Zeo into a
different marker, e.g., Neo, Blas, dhfr, puro, etc, all with either
GTG or TTG as start codon. The STAR elements can be varied by using
different STAR sequences or different placement thereof, or by
substituting them for other chromatin control elements, e.g., MAR
sequences. This leads to improvements over the prior art selection
systems having an IRES with a marker with a normal ATG start
codon.
[0301] As a non-limiting example, instead of the modified Zeo
resistance gene (TTG Zeo) a modified Neomycin resistance gene is
placed downstream of an IRES sequence. The modification consists of
a replacement of the ATG translation initiation codon of the Neo
coding sequence by a TTG translation initiation codon, creating TTG
Neo. The CMV-d2EGF-IRES-TTG Neo construct, either surrounded by
STAR elements or not, is transfected to CHO-K1 cells. Colonies are
picked, cells are propagated and d2EGFP values are measured. This
("IRES-TTG Neo") leads to improvement over the known selection
system having Neo with an ATG start codon downstream of an IRES
("IRES-ATG Neo"). The improvement is especially apparent when the
TTG Neo construct comprises STAR elements.
Example 20
Increased Expression of Erythropoietin Using a Selection System of
the Invention
[0302] The previous examples (of the incorporated '525 and '953
applications) have shown selection systems based on altered
translation initiation codons for the selectable marker gene, and
have employed d2EGFP as test protein of interest, and antibodies as
protein of interest. This example shows the applicability of a
selection system of the invention for improving protein expression
levels of a secreted single chain protein that has therapeutic
significance, viz. erythropoietin (EPO). EPO expression levels were
analysed in the context of the TTG Zeo selection marker, using the
CMV promoter and STAR elements in CHO cells.
[0303] STAR 7 and 67 were cloned to shield a TTG Zeo EPO cassette.
Human EPO cDNA was derived from the plasmid pORF-hEPO (Invivogen).
As control construct the TTG Zeo EPO cassette was not flanked with
STAR elements (FIG. 39). In another control construct we used the
IRES Zeo (with normal ATG start codon) configuration as selection
system, considered the method giving the best results prior to the
invention, which control contruct was either flanked with STARs 7
and 67 or not (FIG. 39). The constructs were transfected to CHO-K1
cells with Lipofectamine 2000 (Invitrogen) and selection was
performed with 150 .mu.g/ml ZEOCIN.RTM. in the culture medium. The
culture medium consisted of HAMF 12: DMEM=1:1, +10% foetal bovine
serum. Up to 24 independent colonies were isolated and propagated
before analysis of EPO expression levels. Per independent colony,
10.sup.5 cells were seeded and cultured in 6-well dishes for two
days before cells were counted and the medium was collected. The
amount of secreted human recombinant erythropoietin was determined
using an ELISA-kit (R&D systems).
[0304] We found that the TTG Zeo-EPO control construct (A in FIG.
39) generated much less clones (5), as compared to the STAR
containing TTG Zeo EPO construct (B in FIG. 39) (41 clones). Mean
EPO expression levels increased from 3.3 pg/cell/day with the TTG
Zeo-EPO control construct, to 17.7 pg/cell/day with the STAR
containing TTG Zeo-EPO construct. The peak EPO expression level
increased from respectively 5.5 to 28.3 pg/cell/day (FIG. 39). Also
in comparison with the STAR containing EPO-IRES-Zeo construct (D in
FIG. 39; 300 clones) and with the IRES contruct without STARs (C in
FIG. 39; 164 clones) we again found that much less clones were
formed with the STAR containing TTG Zeo-EPO construct of the
invention (B in FIG. 39; 41 clones). Also, mean EPO expression
levels increased from 9.0 pg/cell/day with the STAR containing
EPO-IRES-Zeo control construct (D), to 17.7 pg/cell/day in the STAR
containing TTG Zeo-EPO construct of the invention (B; see FIG.
39).
[0305] The obtained EPO expression levels with the construct of the
invention are high in comparison to reported values of 12
pg/cell/day, which was achieved after gene amplification (Yoon et
al., 2003, 2005). This result shows that the selection system of
the invention can readily be applied for the production of
important therapeutic proteins, such as EPO. As shown in FIG. 39
incorporation of STAR elements gave significantly higher EPO
expression levels. The results further demonstrate that STAR
elements are able to increase EPO expression levels.
[0306] In an alternative embodiment, the EPO sequence is cloned
upstream of an IRES, which IRES is operably linked to a sequence
encoding ZEOCIN.RTM. resistance having a TTG start codon,
analogously to example 19, and STAR sequences are included in the
expression construct as described above. It is expected that also
this embodiment will improve expression of EPO compared to the
situation where the sequence encoding ZEOCIN.RTM. resistance has a
normal ATG start codon (such as in situation D in FIG. 39).
Example 21
STAR Sequences Operate Well in the Context of the Selection System
of the Invention in CHO-DG44 Cells
[0307] Several previous examples show the selection system of the
invention, with an impaired start codon for the selectable marker
sequence, and preferably with the use of STAR sequences. In most
cases in the examples above, CHO-K1 cells were used. CHO-DG44 is a
different CHO cell line, which is dhfr.sup.-, and is a good
suspension grower in contrast to CHO-K1, and hence has advantages
for recombinant protein production on an industrial scale. Here it
is shown that the selection system of the invention works well with
several tested STAR sequences also in the CHO-DG44 cell line.
[0308] Seven different STAR elements were tested in a construct
that encompasses the CMV promoter, upstream of the TTG Zeo
selection marker and the d2EGFP gene. In all constructs STAR 67 was
included, cloned immediately upstream of the CMV promoter (FIG.
40). As a control, a construct without STAR elements was included.
The following STAR elements were tested in such constructs: STAR
7/67-7, 9/67-9, 17/67-17, 27/67-27, 43/67-43, 44/67-44 and
45/67-45. The constructs were transfected to CHO-DG44 cells with
Lipofectamine 2000 (Invitrogen) and selection was performed with
150 .mu.g/ml ZEOCIN.RTM. in the culture medium. The culture medium
consisted of HAMF12:DMEM=1:1, +10% foetal bovine serum. Up to 24
independent colonies were isolated and propagated before analysis
of d2EGFP expression levels. As expected and as shown in FIG. 40,
incorporation of the seven different STAR elements gave
significantly higher d2EGFP expression levels, compared to the
control without STAR elements. From the results it is clear that
STAR elements are able to increase d2EGFP expression levels also in
the CHO-DG44 cell line.
Example 22
Removing CpG Dinucleotides from the Selectable Marker Coding
Sequence Improves Expression Using a Selection Method of the
Invention
[0309] The selection methods of the invention, using different
translation initiation codons for the selectable marker, such as
GTG or TTG, can result in very stringent selection, and in very
high levels of production for the polypeptide of interest, as shown
in several examples above. In this example, the coding region of
the selectable marker polypeptide gene itself was modified by
removing CpG dinucleotides. The rationale is that the C nucleotide
in the CpG nucleotide may be prone to methylation, which might
result in gene silencing of the selectable marker, and thus
removing CpG dinucleotides might improve the results. The
zeocin-resistance gene with a TTG start codon was taken as the
marker, and as many CpG dinucleotides were removed as was possible,
without changing the amino acid sequence of the zeocin-resistance
protein, and further without introducing ATG sequences in the
coding strand, to prevent undesired translation initiation within
the coding region of the zeocin-resistance protein (as explained,
e.g., in Examples 1 and 2). Hence, some CpGs were not removed. The
CpG content of the native sequence (here: containing a TTG start
codon, and a mutation to remove the internal ATG sequence, see,
e.g., Examples 1 and 2) is 13.3%, whereas after mutating the CpGs,
the CpG content was reduced to 1.8% [referred to as "TTG Zeo (CpG
poor)"]. The zeocin-resitance gene with decreased CpG content was
cloned upstream of the d2EGFP coding sequence to result in a
multicistronic expression construct of the invention (see, e.g.,
Example 2). Expression levels of d2EGFP were measured.
[0310] Constructs were prepared containing STARs 7 and 67 upstream
of the CMV promoter, followed by the TTG Zeo (CpG poor) selection
marker (synthesized by GeneArt GmbH, Regensburg, Germany; see SEQ
ID NO:132; see SEQ ID NO:92 for the ZEOCIN.TM.
antibiotic-resistance coding sequence with its natural CpG
content), the d2EGFP gene and STAR 7 (FIG. 41). The constructs were
transfected to CHO-K1 cells. DNA was transfected using
Lipofectamine 2000 (Invitrogen) and cells were grown in the
presence of 150 .mu.g/ml ZEOCIN.TM. antibiotic in HAM-F12 medium
(Invitrogen)+10% FBS (Invitrogen).
[0311] Eight colonies emerged after transfection with the control
"CpG-rich" TTG Zeo construct (A in FIG. 41) and none with the
"CpG-poor" TTG Zeo containing construct (C in FIG. 41). In
contrast, with both "CpG-rich" TTG Zeo (B in FIG. 41) and
"CpG-poor" TTG Zeo (D in FIG. 41) selection markers more than 24
colonies emerged when STARs 7/67-7 was included in the construct.
With the "CpG-rich" TTG ZEOCIN.TM. antibiotic selection marker (A
in FIG. 41), the average d2EGFP expression with the STAR-less
control construct was 140, and with the STAR containing construct
1332 (B in FIG. 41). This is an increase due to the presence of the
STAR elements. The average d2EGFP expression with the STAR
containing construct and the "CpG-poor" Zeo was 2453 (D in FIG.
41), an almost two-fold increase in comparison with the "CpG-rich"
TTG Zeo (B in FIG. 41). Furthermore, the highest d2EGFP value
achieved with the "CpG-rich" TTG Zeo construct (B) was 2481 and
with the "CpG-poor" TTG Zeo (D) 4308.
[0312] We conclude that lowering the CpG content of the ZEOCIN.TM.
antibiotic marker gene raises the stringency of the selection
system. This results in higher d2EGFP expression values when STAR
elements are included in the construct and no colonies with the
control construct.
[0313] The same constructs were also transfected to CHO-DG44 cells,
under the same conditions as in Example 21. With the "CpG-rich" TTG
ZEOCIN.RTM. selection marker, the average d2EGFP expression with
the STAR-less control construct was 43 (A in FIG. 42), and the
average d2EGFP expression with the STAR containing constructs was
586 (B in FIG. 42). This is an increase due to the presence of the
STAR elements. The average d2EGFP expression with the STAR
constructs and the "CpG-poor" Zeo was 1152 (D in FIG. 42), an
almost two-fold increase in comparison with the "CpG-rich" TTG Zeo
(B in FIG. 42). Furthermore, the highest d2EGFP value achieved with
the "CpG-rich" TTG Zeo construct was 1296 (B in FIG. 42) and with
the "CpG-poor" TTG Zeo 2416 (D in FIG. 42). In contrast with
CHO-K1, where no control colonies emerged with the "CpG-poor" TTG
Zeo construct (C in FIG. 41), control colonies emerged with
CHO-DG44, but the average d2EGFP value was 52 and the highest value
in a colony was 115 (C in FIG. 42).
[0314] We conclude that also in CHO-DG44 addition of the "CpG-poor"
TTG Zeo selection marker to the construct results in higher protein
expression when STAR elements are employed.
[0315] It will be clear that the configuration where a
zeocin-resistance gene with decreased CpG content and with a GTG or
TTG start codon could also be placed downstream from the coding
sequence for the polypeptide of interest (here d2EGFP as a model)
when the zeocin-resistance protein coding sequences are placed
under control of an IRES (see, e.g., Example 19). In that case, no
care needs to be taken that mutation of CpG dinucleotides would
introduce ATG sequences (as explained in the incorporated '953
application). It is expected that also in such embodiments, similar
results can be obtained, i.e., that reduction of the CpG content of
the selectable marker protein coding sequence will improve
expression levels.
Example 23
Modifications in the Neomycin Resistance Coding Sequence in the
Selection System of the Invention
[0316] The selection system of the invention, in which a modified
start codon is employed for the sequence encoding the selectable
marker polypeptide, is used here for the neomycin resistance gene.
As described in examples above (from the incorporated '525 and '953
applications), different stringencies for selection can be designed
by using different translation initiation codons for the selectable
marker coding sequence, such as GTG or TTG. In this example, also
the coding region of the neomycin resistance gene itself was
modified, by removing as many CpG dinucleotides of the (ATG-less,
so already devoid of ATG sequences in the coding strand) neomycin
resistance gene as possible, while not changing the amino acid
sequence of the neomycin resistance protein (except for the
Met>Leu mutations where the internal ATG sequences were in-frame
and replaced by CTG as compared to the wild-type sequence:
obviously this was done for reasons of removing ATG sequences from
the coding strand and independent from the effort of reducing the
CpG content), and without introducing new ATG sequences in the
coding strand, analogously to what was done in example 22 for the
zeocin-resistance gene. The CpG content of the "wild type" neomycin
selection marker gene is 10.4% (SEQ ID NO:128), while after the
changes the CpG content was reduced to 2.3% (SEQ ID NO:130).
Constructs containing the sequences for the neomycin resistance
gene in this example were ordered from GeneArt GmbH, Regensburg,
Germany. As a start codon, TTG was used in this example. The
sequences used therefore consisted of SEQ ID NO:130, with the
proviso that the start codon (first three nucleotides, ATG) was
replaced by a TTG start codon, and further in certain cases
contained one of the mutations indicated below.
[0317] In the "CpG poor" neomycin resistance gene, some mutations
were made to change amino acids in the neomycin resistance protein,
to test whether these have influence on the expression levels of
the polypeptide of interest when used in the multicistronic
transcription units of the invention. The mutations (Sautter et
al., 2005; it is noted that the neo sequence used in the present
application encodes three additional amino acids immediately after
the start codon as compared to the sequence used by (Sautter et
al., 2005), and hence the amino acid numbering in the present
application is three higher as compared to the numbering in
(Sautter et al., 2005)) consisted of a change from amino acid
valine 201 (198 in Sautter et al., 2005) to glycine 201 (TTG Neo
201V>G), glutamic acid 185 (182 in Sautter et al., 2005) to
aspartic acid 185 (TTG Neo 185E>D) and a double mutation in
which both amino acid valine 201 and glutamic acid 185 were changed
to glycine 201 and aspartic acid 185, respectively (TTG Neo
185E>D/201V>G) (FIG. 43). These modifications were compared
with the control Neomycin (CpG poor TTG Neo 185E/201V). In all
cases constructs were prepared with and without STAR elements (FIG.
43).
[0318] The modified TTG Neo selection marker was incorporated in a
construct containing STARs 7 and 67 upstream of the CMV promoter,
followed by the TTG Neo selection marker, the d2EGFP gene and STAR
7 (FIG. 43). The constructs were transfected to CHO-K1 cells. DNA
was transfected using Lipofectamine 2000 (Invitrogen) and cells
were grown in the presence of 500 .mu.g/ml G418 geneticin in
HAM-F12 medium (Invitrogen)+10% FBS (Invitrogen).
[0319] With the control Neo construct (185E/201V) only a very
limited effect of STAR elements was observed. This may at least in
part be due to the numerous colonies that were generated under 500
.mu.g/ml G418 geneticin, indicating that the stringency of the TTG
neomycin modification is low. However, the neomycin with
modifications of the invention is operational: in the TTG Neo 185E
201V construct all ATGs were removed from the coding strand of the
neomycin resistance gene, and although d2EGFP values were low, it
is clear that the removal of ATGs still allowed proper selection
under Geneticin selection pressure. When the Neomycin resistance
gene was further modified, a distinctive effect of the addition of
STAR elements was observed. The mean of 21 TTG Neo 201V>G
control colonies was 65 (A2 in FIG. 43), whereas the mean d2EGFP
signal of the 24 TTG Neo 201V>G colonies with STAR elements was
150 (B2 in FIG. 43). The selection stringency with the TTG Neo
185E>D mutation was further increased, since no control colonies
survived without STAR elements (A3 in FIG. 43), whereas the mean
d2EGFP signal of 17 surviving TTG Neo 185E>D STAR colonies was
204 (B3 in FIG. 43). This mean GFP fluorescence is higher than with
the TTG Neo 201V>G colonies (B2 in FIG. 43). Also the highest
d2EGFP value in TTG Neo 185E>D colonies was 715, as compared to
433 in the TTG Neo 201V>G colonies (compare B3 and B2 in FIG.
43). The highest stringency was observed in the double Neo mutant,
TTG Neo 185E>D 201V>G. No control colonies survived (A4 in
FIG. 43) and the mean d2EGFP value of 7 surviving STAR TTG Neo
185E>D 201V>G colonies was 513, with as highest d2EGFP value
923 (B4 in FIG. 43).
[0320] It is concluded that the introduction of specific mutations
raises the stringency of selection of the Neomycin resistance gene
when used according to the invention. Some of these modifications
convey such selection stringency to the Neomycin resistance gene
that only after incorporation with STAR elements colonies are able
to survive, due to higher expression values. This concomitantly
results in higher d2EGFP expression values. Clearly, the
advantageous embodiments described herein of the neomycin
resistance gene further improve the suitability of this gene for
use according to the invention.
[0321] It will be clear that the configuration where a neomycin
resistance gene with decreased CpG content and with a GTG or TTG
start codon, and with the indicated mutations (185E>D and/or
201V>G) could also be placed downstream from the coding sequence
for the polypeptide of interest (here d2EGFP as a model) when the
neomycin resistance protein coding sequences are placed under
control of an IRES (see, e.g., Example 19). In that case, no care
needs to be taken that mutation of CpG dinucleotides would
introduce ATG sequences (as explained in the incorporated '953
application). It is expected that also in such embodiments, good
results can be obtained, i.e., that reduction of the CpG content
and specific mutation at the indicated positions of the selectable
marker protein coding sequence will improve expression levels.
Example 24
Use of Tryptophan Synthesizing Enzyme as Selection Marker in the
Selection System of the Invention
[0322] Enzymes that are part of metabolic pathways can be
effectively used as a selection marker. For instance, mammalian
cells lack enzymes that are part of the metabolic pathway to create
the amino acids tryptophan or histidine. Hence these amino acids
need to be present in our food or, in case of cell lines, in the
culture medium. These amino acids are therefore called essential.
When the amino acids are omitted from the culture medium, the cells
will die, unless a plasmid is transfected to the cells that
encompass the (bacterial derived) enzymes that are lacking from the
mammalian cell and that are essential for the synthesis of the
respective amino acid. In this and the following two examples we
describe the use of three enzymes that can be used as selection
marker. Specifically, these markers with a GTG or TTG start codon
are used in the context of constructs containing STAR elements, and
are incorporated in the selection systems of the invention.
[0323] In this example, the tryptophan synthesizing enzyme (trp) is
used as a selectable marker polypeptide. The trp protein
specifically converts indole and L-serine into L-tryptophan. For
use of trp as a selectable marker, a culture medium that is
essentially devoid of tryptophan and which contains the non-toxic
substance indol is used (Hartman and Mulligan, 1988). Indol is used
as substrate for the synthesis of tryptophan. Constructs are
designed to contain the CMV promoter, the d2EGFP gene and the
tryptophan synthesizing enzyme coding sequence (trp) in several
configurations (FIG. 44).
[0324] The synthesized constructs are flanked by STAR elements 7
and 67. trp (the trpB gene) can be derived from E. coli by PCR.
More conveniently, the desired trp gene is synthesized using
standard DNA synthesis methods (e.g., by GeneArt GmbH, Regensburg,
Germany).
[0325] In a first embodiment the trp gene is modified such that all
ATGs are removed. These include 14 ATGs that encode methionine (SEQ
ID NO:136). The translation initiation codon is either GTG or TTG.
These modified trp genes are placed upstream of d2EGFP (FIG.
44A).
[0326] Alternatively the wild type trp gene (containing all
internal ATGs; SEQ ID NO:134) is placed downstream of the d2EGFP
gene, but separated by an IRES sequence (See, Example 19) (FIG.
44B). Translation initiation of the trp mRNA will start at the
translation initiation codon of trp. The first ATG (start codon) is
replaced by GTG or TTG as a start codon. As a control in this
configuration, a construct is also prepared with the normal ATG
start codon for trp.
[0327] The constructs are transfected to CHO-K1 cells that are
cultured in HAMF12 medium that is devoid of the amino acid
tryptophan (obtained from Invitrogen). The medium contains 0.3 mM
of the tryptophan precursor indole.
Example 25
Use of Histidine Synthesizing enzyme as Selection Marker in the
Selection System of the Invention
[0328] In this example, the enzyme that is involved in the
synthesis of the essential amino acid histidine, named histidinol
dehydrogenase (hisD, herein referred to as his), is used as a
selectable marker. The hisD protein specifically converts
l-histidinol into l-histidine. For use of his as a selectable
marker, a culture medium that is essentially devoid of histidine
and which contains the substance histidinol is used (Hartman and
Mulligan, 1988). Histidinol is used as substrate for the synthesis
of histidine. Constructs are designed to contain the CMV promoter,
the d2EGFP gene and the hsitidine syntesizing enzyme coding
sequence (his) in several configurations (FIG. 45).
[0329] The synthesized constructs are flanked by STAR elements 7
and 67. his can be derived from Salmonella typhimurium by PCR. More
conveniently, the desired his gene is synthesized using standard
DNA synthesis methods (e.g., by GeneArt GmbH, Regensburg,
Germany).
[0330] In a first embodiment the his gene is modified such that all
ATGs are removed. These include 4 ATGs that encode methionine (SEQ
ID NO:140). The translation initiation codon is either GTG or TTG.
These modified his genes are placed upstream of d2EGFP (FIG.
45A).
[0331] Alternatively the wild type his gene (containing all
internal ATGs; SEQ ID NO:138) is placed downstream of the d2EGFP
gene, but separated by an IRES sequence (See, Example 19) (FIG.
45B). Translation initiation of the his mRNA will start at the
translation initiation codon of his. The first ATG (start codon) is
replaced by GTG or TTG as a start codon. As a control in this
configuration, a construct is also prepared with the normal ATG
start codon for his.
[0332] The constructs are transfected to CHO-K1 cells, that are
cultured in HAMF12 medium that is devoid of the amino acid
histidine (obtained from Invitrogen). The medium contains 0.125 mM
of the histidine precursor histidinol.
Example 26
Use of dhfr Enzyme as Selection Marker in the Selection System of
the Invention
[0333] In this example, the 5,6,7,8 tetrahydrofolate synthesizing
enzyme dihydrofolate reductase (dhfr) is used as a selectable
marker. The dhfr protein specifically converts folate into 5,6,7,8
tetrahydrofolate. For use of dhfr as a selectable marker according
to this aspect of the invention, the non-toxic substance folate has
to be present in the culture medium (Simonsen et al., 1988).
Furthermore, the medium is essentially devoid of glycine,
hypoxanthine and thymidine, since when these are available for the
cell, the need for the dhfr enzyme is bypassed. Constructs are
designed to contain the CMV promoter, the d2EGFP gene and the dhfr
coding sequence in several configurations (FIG. 46).
[0334] The synthesized constructs are flanked by STAR elements 7
and 67. dhfr can be derived from mouse by PCR. More conveniently,
the desired dhfr gene is synthesized using standard DNA synthesis
methods (e.g., by GeneArt GmbH, Regensburg, Germany).
[0335] In a first embodiment the dhfr gene is modified such that
all ATGs are removed. These include 6 ATGs that encode methionine,
which are changed for codons that encode leucine (SEQ ID NO:122).
The translation initiation codon is either GTG or TTG. These
modified dhfr genes are placed upstream of d2EGFP (FIG. 46A).
[0336] Alternatively the wild type dhfr gene (containing all
internal ATGs; SEQ ID NO:98) is placed downstream of the d2EGFP
gene, but separated by an IRES sequence (See, Example 19) (FIG.
46B). Translation initiation of the dhfr mRNA will start at the
translation initiation codon of dhfr. The first ATG (start codon)
is replaced by GTG or TTG as a start codon. As a control in this
configuration, a construct is also prepared with the normal ATG
start codon for dhfr.
[0337] The constructs are transfected to CHO-DG44 cells, that are
cultured in DMEM:HAMF12 (1:1) medium (Gibco, cat no. 11320-074),
supplemented with 2 mM L-glutamine (Gibco, 25030-024), which medium
is essentially devoid of glycine, hypoxanthine and thymidine, and
which medium contains 6 .mu.M folic acid.
Example 27
Use of the trp and dhfr Enzymes as Additional Selection Markers
Combined with the Selection System of the Invention
[0338] In certain embodiments, it may be beneficial to maintain
(some) selection pressure during culturing of host cells for
expression of polypeptides of interest from expression cassettes in
the host cell. Although it is possible to do this using selectable
marker polypeptides that confer resistance to antibiotics, it is
more advantageous in view of costs and/or regulatory/safety issues
to use for instance metabolic enzymes such as trp and/or dhfr, as
described in Examples 24 and 26, respectively. The present example
describes the use of trp and dhfr as an additional selectable
marker in combination with the selection system of the invention,
to be able to continuously select for the expression and of the
expression unit that also expresses the polypeptide of interest.
This selection pressure during the stage of expression of the
polypeptide of interest may increase the expression levels in this
stage as compared to a situation wherein only initially (for the
establishment of selected clones) selection pressure is
applied.
[0339] Constructs are designed to encompass the light (LC) and
heavy chain (HC) of a monoclonal antibody, each under the control
of the CMV promoter (FIG. 47A). The constructs are flanked by STAR
elements 7 and 67. Also, between the expression cassettes for the
LC and HC, STAR67 is placed. The cassette with the LC is placed
upstream of the cassette with the HC, but of course the reverse
order would also be possible, or alternatively the HC and LC
expression cassettes could be on separate DNA molecules. The
cassette with the LC is constructed as follows: the CMV promoter,
the TTG Zeo selection marker (e.g., SEQ ID NO:132), the LC and an
IRES sequence, followed by the trp gene (See, Example 24; SEQ ID
NO:134). The trp gene is tested with an ATG, GTG or TTG translation
initiation codon. The cassette with the HC is constructed as
follows: the CMV promoter, the TTG Neo selection marker (See,
Example 23; SEQ ID NO:130, but with a TTG start codon), the HC and
an IRES sequence (see, e.g., Example 19), followed by the dhfr gene
(See, Example 26; SEQ ID NO:98). The dhfr gene is tested with an
ATG, GTG or TTG translation initiation codon (FIG. 47A).
[0340] Alternatively, a cassette can be constructed wherein the HC
and/or LC are upstream of the two selectable marker sequences,
wherein the selectable marker sequences each are preceded by an
IRES (FIG. 47B).
[0341] It is clear that the same principle can be used for a single
expression cassette, i.e., for expression of only one polypeptide
of interest, for instance if that is not part of a multimeric
protein. In that case only one of the two expression cassettes
needs to be constructed (e.g., the one for HC, but with HC replaced
by a sequence encoding another polypeptide of interest).
[0342] The constructs are transfected to CHO-DG44 cells that are
cultured in DMEM:HAMF12 (1:1) medium. Selection takes place by 150
.mu.g/ml ZEOCIN.RTM. and 500 .mu.g/ml geneticin G418. Colonies are
isolatated and cells are propagated. After first measurements of
secreted monoclonal antibody in the culture medium, the cells are
changed to DMEM:HAMF12 (1:1) medium (without ZEOCIN.RTM. and
geneticin G418) (Gibco, cat no. 11320-074), supplemented with 2 mM
L-glutamine (Gibco, 25030-024), which medium is essentially devoid
of glycine, hypoxanthine and thymidine, and which medium contains 6
.mu.M folic acid, and/or to medium devoid of tryptophan, while
containing 0.3 mM indole.
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Sequence CWU 1
1
1411749DNAHomo sapiensmisc_featuresequence of STAR1 1atgcggtggg
ggcgcgccag agactcgtgg gatccttggc ttggatgttt ggatctttct 60gagttgcctg
tgccgcgaaa gacaggtaca tttctgatta ggcctgtgaa gcctcctgga
120ggaccatctc attaagacga tggtattgga gggagagtca cagaaagaac
tgtggcccct 180ccctcactgc aaaacggaag tgattttatt ttaatgggag
ttggaatatg tgagggctgc 240aggaaccagt ctccctcctt cttggttgga
aaagctgggg ctggcctcag agacaggttt 300tttggccccg ctgggctggg
cagtctagtc gaccctttgt agactgtgca cacccctaga 360agagcaacta
cccctataca ccaggctggc tcaagtgaaa ggggctctgg gctccagtct
420ggaaaatctg gtgtcctggg gacctctggt cttgcttctc tcctcccctg
cactggctct 480gggtgcttat ctctgcagaa gcttctcgct agcaaaccca
cattcagcgc cctgtagctg 540aacacagcac aaaaagccct agagatcaaa
agcattagta tgggcagttg agcgggaggt 600gaatatttaa cgcttttgtt
catcaataac tcgttggctt tgacctgtct gaacaagtcg 660agcaataagg
tgaaatgcag gtcacagcgt ctaacaaata tgaaaatgtg tatattcacc
720ccggtctcca gccggcgcgc caggctccc 7492883DNAHomo
sapiensmisc_featuresequence of STAR2 2gggtgcttcc tgaattcttc
cctgagaagg atggtggccg gtaaggtccg tgtaggtggg 60gtgcggctcc ccaggccccg
gcccgtggtg gtggccgctg cccagcggcc cggcaccccc 120atagtccatg
gcgcccgagg cagcgtgggg gaggtgagtt agaccaaaga gggctggccc
180ggagttgctc atgggctcca catagctgcc ccccacgaag acggggcttc
cctgtatgtg 240tggggtccca tagctgccgt tgccctgcag gccatgagcg
tgcgggtcat agtcgggggt 300gccccctgcg cccgcccctg ccgccgtgta
gcgcttctgt gggggtggcg ggggtgcgca 360gctgggcagg gacgcagggt
aggaggcggg gggcagcccg taggtaccct gggggggctt 420ggagaagggc
gggggcgact ggggctcata cgggacgctg ttgaccagcg aatgcataga
480gttcagatag ccaccggctc cggggggcac ggggctgcga cttggagact
ggccccccga 540tgacgttagc atgcccttgc ccttctgatc ctttttgtac
ttcatgcggc gattctggaa 600ccagatcttg atctggcgct cagtgaggtt
cagcagattg gccatctcca cccggcgcgg 660ccggcacagg tagcggttga
agtggaactc tttctccagc tccaccagct gcgcgctcgt 720gtaggccgtg
cgcgcgcgct tggacgaagc ctgccccggc gggctcttgt cgccagcgca
780gctttcgcct gcgaggacag agagaggaag agcggcgtca ggggctgccg
cggccccgcc 840cagcccctga cccagcccgg cccctccttc caccaggccc caa
88332126DNAHomo sapiensmisc_featuresequence of STAR3 3atctcgagta
ctgaaatagg agtaaatctg aagagcaaat aagatgagcc agaaaaccat 60gaaaagaaca
gggactacca gttgattcca caaggacatt cccaaggtga gaaggccata
120tacctccact acctgaacca attctctgta tgcagattta gcaaggttat
aaggtagcaa 180aagattagac ccaagaaaat agagaacttc caatccagta
aaaatcatag caaatttatt 240gatgataaca attgtctcca aaggaacaag
gcagagtcgt gctagcagag gaagcacgtg 300agctgaaaac agccaaatct
gctttgtttt catgacacag gagcataaag tacacaccac 360caactgacct
attaaggctg tggtaaaccg attcatagag agaggttcta aatacattgg
420tccctcacag gcaaactgca gttcgctccg aacgtagtcc ctggaaattt
gatgtccagt 480atagaaaagc agagcagtca aaaaatatag ataaagctga
accagatgtt gcctgggcaa 540tgttagcagc accacactta agatataacc
tcaggctgtg gactccctcc ctggggagcg 600gtgctgccgg cggcgggcgg
gctccgcaac tccccggctc tctcgcccgc cctcccgttc 660tcctcgggcg
gcggcggggg ccgggactgc gccgctcaca gcggcggctc ttctgcgccc
720ggcctcggag gcagtggcgg tggcggccat ggcctcctgc gttcgccgat
gtcagcattt 780cgaactgagg gtcatctcct tgggactggt tagacagtgg
gtgcagccca cggagggcga 840gttgaagcag ggtggggtgt cacctccccc
aggaagtcca gtgggtcagg gaactccctc 900ccctagccaa gggaggccgt
gagggactgt gcccggtgag agactgtgcc ctgaggaaag 960gtgcactctg
gcccagatac tacacttttc ccacggtctt caaaacccgc agaccaggag
1020attccctcgg gttcctacac caccaggacc ctgggtttca accacaaaac
cgggccattt 1080gggcagacac ccagctagct gcaagagttg tttttttttt
tatactcctg tggcacctgg 1140aacgccagcg agagagcacc tttcactccc
ctggaaaggg ggctgaaggc agggaccttt 1200agctgcgggc tagggggttt
ggggttgagt gggggagggg agagggaaaa ggcctcgtca 1260ttggcgtcgt
ctgcagccaa taaggctacg ctcctctgct gcgagtagac ccaatccttt
1320cctagaggtg gagggggcgg gtaggtggaa gtagaggtgg cgcggtatct
aggagagaga 1380aaaagggctg gaccaatagg tgcccggaag aggcggaccc
agcggtctgt tgattggtat 1440tggcagtgga ccctcccccg gggtggtgcc
ggaggggggg atgatgggtc gaggggtgtg 1500tttatgtgga agcgagatga
ccggcaggaa cctgccccaa tgggctgcag agtggttagt 1560gagtgggtga
cagacagacc cgtaggccaa cgggtggcct taagtgtctt tggtctcctc
1620caatggagca gcggcggggc gggaccgcga ctcgggttta atgagactcc
attgggctgt 1680aatcagtgtc atgtcggatt catgtcaacg acaacaacag
ggggacacaa aatggcggcg 1740gcttagtcct acccctggcg gcggcggcag
cggtggcgga ggcgacggca ctcctccagg 1800cggcagccgc agtttctcag
gcagcggcag cgcccccggc aggcgcggtg gcggtggcgc 1860gcagccaggt
ctgtcaccca ccccgcgcgt tcccaggggg aggagactgg gcgggagggg
1920ggaacagacg gggggggatt caggggcttg cgacgcccct cccacaggcc
tctgcgcgag 1980ggtcaccgcg gggccgctcg gggtcaggct gcccctgagc
gtgacggtag ggggcggggg 2040aaaggggagg agggacaggc cccgcccctc
ggcagggcct ctagggcaag ggggcggggc 2100tcgaggagcg gaggggggcg gggcgg
212641625DNAHomo sapiensmisc_featuresequence of STAR4 4gatctgagtc
atgttttaag gggaggattc ttttggctgc tgagttgaga ttaggttgag 60ggtagtgaag
gtaaaggcag tgagaccacg taggggtcat tgcagtaatc caggctggag
120atgatggtgg ttcagttgga atagcagtgc atgtgctgta acaacctcag
ctgggaagca 180gtatatgtgg cgttatgacc tcagctggaa cagcaatgca
tgtggtggtg taatgacccc 240agctgggtag ggtgcatgtg gtgtaacgac
ctcagctggg tagcagtgtg tgtgatgtaa 300caacctcagc tgggtagcag
tgtacttgat aaaatgttgg catactctag atttgttatg 360agggtagtgc
cattaaattt ctccacaaat tggttgtcac gtatgagtga aaagaggaag
420tgatggaaga cttcagtgct tttggcctga ataaatagaa gacgtcattt
ccagttaatg 480gagacaggga agactaaagg tagggtggga ttcagtagag
caggtgttca gttttgaata 540tgatgaactc tgagagagga aaaacttttt
ctacctctta gtttttgtga ctggacttaa 600gaattaaagt gacataagac
agagtaacaa gacaaaaata tgcgaggtta tttaatattt 660ttacttgcag
aggggaatct tcaaaagaaa aatgaagacc caaagaagcc attagggtca
720aaagctcata tgccttttta agtagaaaat gataaatttt aacaatgtga
gaagacaaag 780gtgtttgagc tgagggcaat aaattgtggg acagtgatta
agaaatatat gggggaaatg 840aaatgataag ttattttagt agatttattc
ttcatatcta ttttggcttc aacttccagt 900ctctagtgat aagaatgttc
ttctcttcct ggtacagaga gagcaccttt ctcatgggaa 960attttatgac
cttgctgtaa gtagaaaggg gaagatcgat ctcctgtttc ccagcatcag
1020gatgcaaaca tttccctcca ttccagttct caaccccatg gctgggcctc
atggcattcc 1080agcatcgcta tgagtgcacc tttcctgcag gctgcctcgg
gtagctggtg cactgctagg 1140tcagtctatg tgaccaggag ctgggcctct
gggcaatgcc agttggcagc ccccatccct 1200ccactgctgg gggcctccta
tccagaaggg cttggtgtgc agaacgatgg tgcaccatca 1260tcattcccca
cttgccatct ttcaggggac agccagctgc tttgggcgcg gcaaaaaaca
1320cccaactcac tcctcttcag gggcctctgg tctgatgcca ccacaggaca
tccttgagtg 1380ctgggcagtc tgaggacagg gaaggagtga tgaccacaaa
acaggaatgg cagcagcagt 1440gacaggagga agtcaaaggc ttgtgtgtcc
tggccctgct gagggctggc gagggccctg 1500ggatggcgct cagtgcctgg
tcggctgcaa gaggccagcc ctctgcccat gaggggagct 1560ggcagtgacc
aagctgcact gccctggtgg tgcatttcct gccccactct ttccttctaa 1620gatcc
162551571DNAHomo sapiensmisc_featuresequence of STAR5 5cacctgattt
aaatgatctg tctggtgagc tcactgggtc tttactcgca tgctgggtcc 60acagctccac
tgtcctgcag ggtccgtgag tgtgggcccc ttatctattt catcatcata
120accctgcgtg tcctcaactc ctggcacata ttgggtggcc ccatccacac
acggttgttg 180agtgaatcca tgagatgaca aaggctatga tgtagactat
atcatgagcc agaaccaggc 240tttcctacct ccagacaatc aagggccttg
atttgggatt gagggagaaa ggagtagaag 300ccaggaagga gaagagattg
aggtttacca agggtgcaaa gtcctggccc ctgactgtag 360gctgaaaact
atagaaatga tagaacaatt ttgcaatgaa atgcagaaga ccctgcatca
420actttaggtg ggacttcggg tatttttatg gccacagaac atcctcccat
ttacctgcat 480ggcccagaca cagacttcaa aacagttgag gccagcaggc
tccaggtaag tggtaggatt 540ccagaatgcc ctcagagtgt tgtgggaggc
agcaggcgat tttcctggac ttctgagttt 600atgagaaccc caaaccccaa
ttggcattaa cattgaggtc tcaatgtatc atggcaggaa 660gcttccgagt
ggtgaaaagg aaagtgaaca tcaaagctcg gaagacaaga gggtggagtg
720atggcaacca agagcaagac ccttccctct cctgtgatgg ggtggctcta
tgtgaagccc 780ccaaactgga cacaggtctg gcagaatgag gaacccactg
agatttagcg ccaacatcca 840gcataaaagg gagactgaca tagaatttga
gttagttaaa aataaggcac aatgcttttc 900atgtattcct gagttttgtg
gactggtgtt caatttgcag cattcttagt tgattaaatc 960tgagatgaag
aaagagtgtc caacactttc accttggaaa gctctggaaa agcaaaaggg
1020agagacaatt agcttcatcc attaactcac ttagtcatta tgcattcatt
catgtaacta 1080ccaaacacgt actgagtgcc taacactcct gagacactga
gaagtttctt gggaatacaa 1140agatgaataa aaaccacgcc aggcaggagt
tggaggaagg ttctggatgc caccacgctc 1200tacctcctgg ctggacacca
ggcaatgttg gtaaccttct gcctccaatt tctgcaaata 1260cataattaat
aaacacaagg ttatcttcta aacagttctt aaaatgagtc aactttgttt
1320aaacttgttc tttttagaga aaaatgtatt tttgaaagag ttggttagtg
ctaggggaaa 1380tgtctgggca cagctcagtc tggtgtgaga gcaggaagca
gctctgtgtg tctggggtgg 1440gtacgtatgt aggacctgtg ggagaccagg
ttgggggaag gcccctcctc atcaagggct 1500cctttgcttt ggtttgcttt
ggcgtgggag gtgctgtgcc acaagggaat acgggaaata 1560agatctctgc t
157161173DNAHomo sapiensmisc_featuresequence of STAR6 6tgacccacca
cagacatccc ctctggcctc ctgagtggtt tcttcagcac agcttccaga 60gccaaattaa
acgttcactc tatgtctata gacaaaaagg gttttgacta aactctgtgt
120tttagagagg gagttaaatg ctgttaactt tttaggggtg ggcgagaggg
atgacaaata 180acaacttgtc tgaatgtttt acatttctcc ccactgcctc
aagaaggttc acaacgaggt 240catccatgat aaggagtaag acctcccagc
cggactgtcc ctcggccccc agaggacact 300ccacagagat atgctaactg
gacttggaga ctggctcaca ctccagagaa aagcatggag 360cacgagcgca
cagagcaggg ccaaggtccc agggacagaa tgtctaggag ggagattggg
420gtgagggtaa tctgatgcaa ttactgtggc agctcaacat tcaagggagg
gggaagaaag 480aaacagtccc tgtcaagtaa gttgtgcagc agagatggta
agctccaaaa tttgaaactt 540tggctgctgg aaagttttag ggggcagaga
taagaagaca taagagactt tgagggttta 600ctacacacta gacgctctat
gcatttattt atttattatc tcttatttat tactttgtat 660aactcttata
ataatcttat gaaaacggaa accctcatat acccatttta cagatgagaa
720aagtgacaat tttgagagca tagctaagaa tagctagtaa gtaaaggagc
tgggacctaa 780accaaaccct atctcaccag agtacacact cttttttttt
ttccagtgta atttttttta 840atttttattt tactttaagt tctgggatac
atgtgcagaa ggtatggttt gttacatagg 900tatatgtgtg ccatagtgga
ttgctgcacc tatcaacccg tcatctaggt ttaagcccca 960catgcattag
ctatttgtcc tgatgctctc cctcccctcc ccacaccaga caggccttgg
1020tgtgtgatgt tcccctccct gtgtccatgt gttctcactg ttcagctccc
acttatgagt 1080gagaacgtgt ggtatttggt tttctgttcc tgtgttagtt
tgctgaggat gatggcttcc 1140agcttcatcc atgtccctgc aaaggacacg atc
117372101DNAHomo sapiensmisc_featuresequence of STAR7 7aggtgggtgg
atcacccgag gtcaggagtt caagaccagc ctggccaaca tggtaaaacc 60tcgtctctac
taaaaaatac gaaaaattag ctggttgtgg tggtgcgtgc ttgtaatccc
120agctactcgg gaggctgagg caggagaatc acttgaatct gggaggcaga
ggttgcagtg 180agctgagata gtgccattgc actccagcct gggcaacaga
cggagactct gtctccaaaa 240aaaaaaaaaa aaatcttaga ggacaagaat
ggctctctca aacttttgaa gaaagaataa 300ataaattatg cagttctaga
agaagtaatg gggatatagg tgcagctcat gatgaggaag 360acttagctta
actttcataa tgcatctgtc tggcctaaga cgtggtgagc tttttatgtc
420tgaaaacatt ccaatataga atgataataa taatcacttc tgacccccct
tttttttcct 480ctccctagac tgtgaagcag aaaccccata tttttcttag
ggaagtggct acgcactttg 540tatttatatt aacaactacc ttatcaggaa
attcatattg ttgccctttt atggatgggg 600aaactggaca agtgacagag
caaaatccaa acacagctgg ggatttccct cttttagatg 660atgattttaa
aagaatgctg ccagagagat tcttgcagtg ttggaggaca tatatgacct
720ttaagatatt ttccagctca gagatgctat gaatgtatcc tgagtgcatg
gatggacctc 780agttttgcag attctgtagc ttatacaatt tggtggtttt
ctttagaaga aaataacaca 840tttataaata ttaaaatagg cccaagacct
tacaagggca ttcatacaaa tgagaggctc 900tgaagtttga gtttgttcac
tttctagtta attatctcct gcctgtttgt cataaatgcg 960tttagtaggg
agctgctaat gacaggttcc tccaacagag tgtggaagaa ggagatgaca
1020gctggcttcc cctctgggac agcctcagag ctagtgggga aactatgtta
gcagagtgat 1080gcagtgacca agaaaatagc actaggagaa agctggtcca
tgagcagctg gtgagaaaag 1140gggtggtaat catgtatgcc ctttcctgtt
ttatttttta ttgggtttcc ttttgcctct 1200caattccttc tgacaataca
aaatgttggt tggaacatgg agcacctgga agtctggttc 1260attttctctc
agtctcttga tgttctctcg ggttcactgc ctattgttct cagttctaca
1320cttgagcaat ctcctcaata gctaaagctt ccacaatgca gattttgtga
tgacaaattc 1380agcatcaccc agcagaactt aggttttttt ctgtcctccg
tttcctgacc tttttcttct 1440gagtgcttta tgtcacctcg tgaaccatcc
tttccttagt catctaccta gcagtcctga 1500ttcttttgac ttgtctccct
acaccacaat aaatcactaa ttactatgga ttcaatccct 1560aaaatttgca
caaacttgca aatagattac gggttgaaac ttagagattt caaacttgag
1620aaaaaagttt aaatcaagaa aaatgacctt taccttgaga gtagaggcaa
tgtcatttcc 1680aggaataatt ataataatat tgtgtttaat atttgtatgt
aacatttgaa taccttcaat 1740gttcttattt gtgttatttt aatctcttga
tgttactaac tcatttggta gggaagaaaa 1800catgctaaaa taggcatgag
tgtcttatta aatgtgacaa gtgaatagat ggcagaaggt 1860ggattcatat
tcagttttcc atcaccctgg aaatcatgcg gagatgattt ctgcttgcaa
1920ataaaactaa cccaatgagg ggaacagctg ttcttaggtg aaaacaaaac
aaacacgcca 1980aaaaccttta ttctctttat tatgaatcaa atttttcctc
tcagataatt gttttattta 2040tttattttta ttattattgt tattatgtcc
agtctcactc tgtcgcctaa gctggcatga 2100t 210181821DNAHomo
sapiensmisc_featuresequence of STAR8 8gagatcacct cgaagagagt
ctaacgtccg taggaacgct ctcgggttca caaggattga 60ccgaacccca ggatacgtcg
ctctccatct gaggcttgct ccaaatggcc ctccactatt 120ccaggcacgt
gggtgtctcc cctaactctc cctgctctcc tgagcccatg ctgcctatca
180cccatcggtg caggtccttt ctgaagagct cgggtggatt ctctccatcc
cacttccttt 240cccaagaaag aagccaccgt tccaagacac ccaatgggac
attccccttc cacctccttc 300tccaaagttg cccaggtgtt catcacaggt
tagggagaga agcccccagg tttcagttac 360aaggcatagg acgctggcat
gaacacacac acacacacac acacacacac acacacacac 420acacgactcg
aagaggtagc cacaagggtc attaaacact tgacgactgt tttccaaaaa
480cgtggatgca gttcatccac gccaaagcca agggtgcaaa gcaaacacgg
aatggtggag 540agattccaga ggctcaccaa accctctcag gaatattttc
ctgaccctgg gggcagaggt 600tggaaacatt gaggacattt cttgggacac
acggagaagc tgaccgacca ggcattttcc 660tttccactgc aaatgaccta
tggcgggggc atttcacttt cccctgcaaa tcacctatgg 720cgaggtacct
ccccaagccc ccacccccac ttccgcgaat cggcatggct cggcctctat
780ccgggtgtca ctccaggtag gcttctcaac gctctcggct caaagaagga
caatcacagg 840tccaagccca aagcccacac ctcttccttt tgttataccc
acagaagtta gagaaaacgc 900cacactttga gacaaattaa gagtccttta
tttaagccgg cggccaaaga gatggctaac 960gctcaaaatt ctctgggccc
cgaggaaggg gcttgactaa cttctatacc ttggtttagg 1020aaggggaggg
gaactcaaat gcggtaattc tacagaagta aaaacatgca ggaatcaaaa
1080gaagcaaatg gttatagaga gataaacagt tttaaaaggc aaatggttac
aaaaggcaac 1140ggtaccaggt gcggggctct aaatccttca tgacacttag
atataggtgc tatgctggac 1200acgaactcaa ggctttatgt tgttatctct
tcgagaaaaa tcctgggaac ttcatgcact 1260gtttgtgcca gtatcttatc
agttgattgg gctcccttga aatgctgagt atctgcttac 1320acaggtcaac
tccttgcgga agggggttgg gtaaggagcc cttcgtgtct cgtaaattaa
1380ggggtcgatt ggagtttgtc cagcattccc agctacagag agccttattt
acatgagaag 1440caaggctagg tgattaaaga gaccaacagg gaagattcaa
agtagcgact tagagtaaaa 1500acaaggttag gcatttcact ttcccagaga
acgcgcaaac attcaatggg agagaggtcc 1560cgagtcgtca aagtcccaga
tgtggcgagc ccccgggagg aaaaaccgtg tcttccttag 1620gatgcccgga
acaagagcta ggcttccgga gctaggcagc catctatgtc cgtgagccgg
1680cgggagggag accgccggga ggcgaagtgg ggcggggcca tccttctttc
tgctctgctg 1740ctgccgggga gctcctggct ggcgtccaag cggcaggagg
ccgccgtcct gcagggcgcc 1800gtagagtttg cggtgcagag t 182191929DNAHomo
sapiensmisc_featuresequence of STAR9 9cacttcctgg gagtggagca
gaggctctgc gtggagcatc catgtgcagt actcttaggt 60acggaaggga ttgggctaaa
ccatggatgg gagctgggaa gggaagggac caacttcagg 120ccccactggg
acactggagc tgccaccctt tagagccctc ctaaccctac accagaggct
180gagggggacc tcagacatca cacacatgct ttcccatgtt ttcagaaatc
tggaaacgta 240gaacttcagg ggtgagagtg cctagatatt gaatacaagg
ctagattggg cttctgtaat 300atcccaaagg accctccagc tttttcacca
gcacctaatg cccatcagat accaaagaca 360cagcttagga gaggttcacc
ctgaagctga ggaggaggca gccggattag agttgactga 420gcaaggatga
ctgccttctc cacctgacga tttcagctgc tgcccttttc ttttcctggg
480aatgcctgtc gccatggcct tctgtgtcca caggagagtt tgacccagat
actcatggac 540caggcaaagg tgctgttcct cccagcccag ggcccaccat
gaagcatgcc tgggagcctg 600gtaaggaccc agccactcct gggctgttga
cattggcttc tcttgcccag cattgtagcc 660acgccactgc attgtactgt
gagataagtc aaggtgggct caccaggacc tgcactaaat 720tgtgaaattc
agctccaaag aactttggaa attacccatg catttaagca aaatgaatga
780tacctgagca aaccctttca cattggcaca agttacaatc ctgtctcatc
ctcttgatta 840caaattccat ccaggcaaga gctgtatcac cctgaggtct
ccccattcat gttttggtca 900ataatattta gtttcctttt gaaaatagat
ttttgtgtta ctccattatg atgggcagag 960gccagatgct tatattctat
ttaaatgact atgtttttct atctgtaact gggtttgtgt 1020tcaggtggta
aatgcttttt ttttgcagtc agaagattcc tggaaggcga ccagaaatta
1080gctggccgct gtcagacctg aagttacttc taaagggcct ttagaaatga
attctttttt 1140atgccttctc tgaattctga gaagtaggct tgacttcccc
taagtgtgga gttgggagtc 1200aactcttctg aaaagaaagt ttcagagcat
tttccaaagc catggtcagc tgtgggaagg 1260gaagacgatg gatagtacag
ttgccggaaa acactgatgg aggcggatgc tccagctcag 1320ccaaagacct
ttgttctgcc caccccagaa atgccccttc ctcaatcgca gaaacgttgc
1380cccatggctc ctgatactca gaatgcagcc tctgaccagg accatctgca
tcctccagga 1440gctcgtaaga aatgcagcat cgtgggacct gctggcacct
ggtgaaccca aacctgcagg 1500gctcctgggt gtgcttgggg cggctgcagg
ggaagaggga gtcagcagcc tcctcctgac 1560cttcccgggg gctgcttttc
tgaggggcca gaatgcaccg gttgaccttg ttgcatcact 1620ggcccatgac
tggctgcttt ggtcaggtgt aaaaaggtgt ttccagaggg tctgctcctc
1680tcactatcgg accaggtttc catggagagc tcagcctccc agcaaggata
gagaacttca 1740aatggctcaa agaactgaga ggccacacat gtgtgacctg
aatagtctct gctgcaaaac 1800aaagggtttc ttaatgtaaa acgttctctt
cctcacagag gggttcccag ctgctagtgg 1860gcatgttgca ggcatttcct
gggctgcatc aggttgtcat aagccagagg atcatttttg 1920ggggctcat
1929101167DNAHomo sapiensmisc_featuresequence of STAR10
10aggtcaggag ttcaagacca gcctggccaa catggtgaaa ccctgtccct acaaaaaata
60caaaaattag ccgggcgtgg tggggggcgc ctataatccc agctactcag gatgctgaga
120caggagaatt gtttgaaccc gggaggtgga
ggttgcagtg aactgagatc gcgccactgc 180actccagcct ggtgacagag
agagactccg tctcaacaac agacaaacaa acaaacaaac 240aacaacaaaa
atgtttactg acagctttat tgagataaaa ttcacatgcc ataaaggtca
300ccttctacag tatacaattc agtggattta gtatgttcac aaagttgtac
gttgttcacc 360atctactcca gaacatttac atcaccccta aaagaagctc
tttagcagtc acttctcatt 420ctccccagcc cctgccaacc acgaatctac
tntctgtctc tattctgaat atttcatata 480aaggagtcct atcatatggg
ccttttacgt ctaccttctt tcacttagca tcatgttttt 540aagattcatc
cacagtgtag cacgtgtcag ttaattcatt tcatcttatg gctggataat
600gctctattgt atgcatatcc ctcactttgc ttatccattc atcaactgat
tgacatttgg 660gttatttcta ctttttgact attatgagta atgctgctat
gaacattcct gtaccaatcg 720ttacgtggac atatgctttc aattctcctg
agtatgtaac tagggttgga gttgctgggt 780catatgttaa ctcagtgttt
catttttttg aagaactacc aaatggtttt ccaaagtgga 840tgcaacactt
tacattccca ccagcaagat atgaaggttc caatgtctct acatttttgc
900caacacttgt gattttcttt tatttattta tttatttatt tatttttgag
atggagtctc 960actctgtcac ccaggctgga gtgcagtggc acaatttcag
ctcactgcaa tctccacctc 1020tcgggctcaa gcgatactcc tgcctcaacc
tcccgagtaa ctgggattac aggcgcccac 1080caccacacca agctaatttt
ttgtattttt agtagagacg gggtttcatc atgtcggcca 1140ggntgtactc
gaactctgac ctcaagt 1167111377DNAHomo sapiensmisc_featuresequence of
STAR11 11aggatcactt gagcccagga gttcaagacc agcctgggca acatagcgag
aacatgtctc 60aaaaaggaaa aaaatggggg aaaaaaccct cccagggaca gatatccaca
gccagtcttg 120ataagctcca tcattttaaa gtgcaaggcg gtgcctccca
tgtggatgat tatttaatcc 180tcttgtactt tgtttagtcc tttgtggaaa
tgcccatctt ataaattaat agaattctag 240aatctaatta aaatggttca
actctacatt ttactttagg ataatatcag gaccatcaca 300gaatgtctga
gatgtggatt taccctatct gtagctcact tcttcaacca ttcttttagc
360aaggctagtt atcttcagtg acaacccctt gctgccctct actatctcct
ccctcagatg 420gactactctg attaagcttg agctagaata agcatgttat
cccgggattt catatggaat 480attttataca tgagtgagcc attatgagtt
gtttgaaaat ttattatgtt gagggagggt 540aaccgctgta acaaccatca
ccaaatctaa tcgactgaat acatttgacg tttatttctt 600gttcacctga
cagttcagtg ttacctaaat ttacatgaag acccagaggc ccacgctcct
660tcattttggg ctccaccgac ctccaaggtt tcagggccct ctgccccgcc
ttctgcaccc 720acaggggaag agagtggagg atgcacacgc ccaggcctgg
aagtgacgca tgtggcttcc 780ccgtccacag acttcaccca cagtccattg
gccttcttaa gtcatggact cctgctgagc 840tgccagggtg catgggaaat
ccatgtgact gtgtgccctg gaggaagggg agcgtttcgg 900tgagcacaca
ggagtctttg ccactagacg ctgatgagga ttccccacag gcgatgaagc
960atggagactc atcttgtaac aaacagatga gttgttgaca tctcttaagt
ttactttgtg 1020tgcagttttt attcagatag gaaaggctgt taaaatctta
acacctaact ggaagaaggg 1080ttttagagaa gtgtggtttt cagtaagcca
gttctttcca caatccaaga aacgaaataa 1140atttccagca tggagcagtt
ggcaggtaag gtttttgttg tggtctcgcc caggcttgag 1200tgtaaccggt
gtggtcatag ctcactacat tctcaaactc ctggccttaa gtcatcctcc
1260tgcctcagcc tcccaaaggc aagtaaggtt aagaataggg gaaaggtgaa
gtttcacagc 1320ttttctagaa ttctttttat tcaagggact ctcagatcat
caaacccacc cagaatc 1377121051DNAHomo sapiensmisc_featuresequence of
STAR12 12atcctgcttc tgggaagaga gtggcctccc ttgtgcaggt gactttggca
ggaccagcag 60aaacccaggt ttcctgtcag gaggaagtgc tcagcttatc tctgtgaagg
gtcgtgataa 120ggcacgagga ggcaggggct tgccaggatg ttgcctttct
gtgccatatg ggacatctca 180gcttacgttg ttaagaaata tttggcaaga
agatgcacac agaatttctg taacgaatag 240gatggagttt taagggttac
tacgaaaaaa agaaaactac tggagaagag ggaagccaaa 300caccaccaag
tttgaaatcg attttattgg acgaatgtct cactttaaat ttaaatggag
360tccaacttcc ttttctcacc cagacgtcga gaaggtggca ttcaaaatgt
ttacacttgt 420ttcatctgcc tttttgctaa gtcctggtcc cctacctcct
ttccctcact tcacatttgt 480cgtttcatcg cacacatatg ctcatcttta
tatttacata tatataattt ttatatatgg 540cttgtgaaat atgccagacg
agggatgaaa tagtcctgaa aacagctgga aaattatgca 600acagtgggga
gattgggcac atgtacattc tgtactgcaa agttgcacaa cagaccaagt
660ttgttataag tgaggctggg tggtttttat tttttctcta ggacaacagc
ttgcctggtg 720gagtaggcct cctgcagaag gcattttctt aggagcctca
acttccccaa gaagaggaga 780gggcgagact ggagttgtgc tggcagcaca
gagacaaggg ggcacggcag gactgcagcc 840tgcagagggg ctggagaagc
ggaggctggc acccagtggc cagcgaggcc caggtccaag 900tccagcgagg
tcgaggtcta gagtacagca aggccaaggt ccaaggtcag tgagtctaag
960gtccatggtc agtgaggctg agacccaggg tccaatgagg ccaaggtcca
gagtccagta 1020aggccgagat ccagggtcca gggaggtcaa g 1051131291DNAHomo
sapiensmisc_featuresequence of STAR13 13agccactgag gtcctaactg
cagccaaggg gccgttctgc acatgtcgct caccctctgt 60gctctgttcc ccacagagca
aacgcacatg gcaacgttgg tccgctcagc cactggttct 120gtggtggaac
ggtggatgtc tgcactgtga catcagctga gtaagtaaca acgactgagg
180atgccgctga cccagggctg gggaagggga ctcccagctc agacaggctt
ggctgtggtt 240tgctttggga ggagagtgaa catcacaggg aatggctcat
gtcagcccca ggagggtggg 300ctggcccctg gtccccgggc tccttctggc
cctgcaggcg atagagagcc tcaacctgct 360gccgcttctc cttggcccgg
gtgatggccg tctggaagag cctgcagtag aggtgcacag 420ccagcggaga
gtcgtcattg ccgggtacag ggtaggtgat gaggcagggg ttgcagttgg
480tgtccacgat gcccactgtg gggatgttca tcttggctgc gtctctcacg
gccacgtgtg 540gctcaaagat gttgttgagc gtgtgcagga agatgatgag
gtccggcagg cggaccgtgg 600ggccaaagag gaggcgcgcg ttggtcagca
tgccgcccct gaagtagcga gtgtgggcgt 660actcgccaca gtcacgggcc
atgttctcaa tcaggtacga gaactgccgg ttgcggctta 720taaacaagat
gatgcccttg cggtaggcca tgtgggcggt gaagttcaag gccagctgga
780ggtgcgtggc tgtctgttcc aggtcgatga tgtcgtggtc caggcggctc
ccaaagatgt 840acggctccat aaacctgcca gagaccccac caaggcaagg
gggatgagag ttcacggggc 900catctccact ggctccttgc aggaacacag
acgcccacca gggactcccg ggctcctctg 960tgggggcact atgggctggg
aagcacaatt tgcaacgctc cccgtgtgca tggacagcag 1020tgcagaccca
tccaggccac ccctctgcat gcctcgtctc gtggcttaac ccctcctacc
1080ctctacctct tcccgaagga atcctaatag aactgacccc atatggatgt
gtggacatcc 1140aacatgacgc caaaaggaca ttctgccccg tgcagctcac
agggcagccg cctccgtcac 1200tgtcctcttc ccgaggcttt gcggatgagg
cccctctggg gttggactta gcggggtgct 1260ctgggccaaa agcattaagg
gatcagggca g 129114711DNAHomo sapiensmisc_featuresequence of STAR14
14ccctggacca gggtccgtgg tcttggtggg cactggcttc ttcttgctgg gtgttttcct
60gtgggtctct ggcaaggcac tttttgtggc gctgcttgtg ctgtgtgcgg gaggggcagg
120tgctctttcc tcttggagct ggaccctctg gggcgggtcc ccgtcggcct
ccttgtgtgt 180tttctgcacc tggtacagct ggatggcctc ctcaatgccg
tcgtcgctgc tggagtcgga 240cgcctcgggc gcctgtacgg cgctcgtgac
tcgctttccc ctccttgcgg tgctggcgtt 300ccttttaatc ccacttttat
tctgtactgc ttctgaaggg cggtgggggt tgctggcttt 360gtgctgccct
ccttctcctg cgtggtcgtg gtcgtgacct tggacctgag gcttctgggc
420tgcacgtttg tctttgctaa ccgggggagg tctgcagaag gcgaactcct
tctggacgcc 480catcaggccc tgccggtgca ccacctttgt agccggctct
tggtgggatt tcgagagtga 540cttcgccgaa ttttcatgtg tgtctggttt
cttctccact gacccatcac atttttgggt 600ctcatgctgt cttttctcat
tcagaaactg ttctatttct gccctgatgc tctgctcaaa 660ggagtctgct
ctgctcatgc tgactgggga ggcagagccc tggtccttgc t 711151876DNAHomo
sapiensmisc_featuresequence of STAR15 15gagtccaaga tcaaggtgcc
agcatcttgt gagggccttc ttgttacgtc actccctagc 60gaaagggcaa agagagggtg
agcaagagaa aggggggctg aactcgtcct tgtagaagag 120gcccattccc
gagacaatgg cattcatcca ttcactccac cctcatggcc tcaccacctc
180tcatgaggct ccacctccca gccctggttt gttggggatt aaatttccaa
cacatgcctt 240ttgggggaca tgttaaaatt atagcacccc aaatgttaca
ctatcttttg atgagcggta 300gttctgattt taagtctagc tggcctactt
tttcttgcac gtgggatgct ttctgcctgt 360tccagggcag gcagctcttc
tctgtccctc tgctggcccc acctcatcct ctgttgtcct 420cttccctcct
tctgtgccct ggggtcctgg tgggggtgtg actgtcaact gcgttgggct
480aacttttttc cctgctggtg gcccgtaatg aaagaaagct tcttgctccc
aagttcctta 540aatccaagct catagacaac gcggtctcac agcaggcctg
gggccagcct cacgtgagcc 600ccttccctgg tgtagtcact ggcatggggg
aatgggattt cctgttgccc tactgtgtgg 660ctgaggtggg ggttgcttcc
tggagccagg ccttgtggaa gggcagtgcc cactgcagtg 720gatgctgggc
cctgaatctg accccagtgt tcattggctc tgtgagaccc agtgagggca
780gggagggaag tggagctggg gtgagaagta gaggccctgc agggcccacg
tgccagccac 840caggcctcag actaggctca gatgacggag agctgcacac
ctgcccaacc caggccctgc 900agtgcccaca tgccagccgc tggggcccag
acttgctcca gagggcggag agctttacac 960cggcccaacc caggccatgg
ctccaaatgc gtgacagttt tgctgttgct tcttttagtc 1020attgtcaagt
tgatgcttgt tttgcagagg accaaggctt tatgaaccta ttaccctgtg
1080tgaagagttt caccaggtta tggaaatttc tttaaaacca taccacagtt
ttttcattat 1140tcatgtatat ttttaaaaat aattactgca ctcagtagaa
taacatgaaa atgttgcctg 1200ttagcccttt tccagtttgc cccgagaata
ctgggggcac ttgtggctgc aatgtttatc 1260ctgcggcagc tttgccatga
agtatctcac ttttattatt atttttgcat tgctcgagta 1320tattgacttt
ggaaacaaaa gacatcattc tatttatagc attatgtttt tagtagtggt
1380atttccatat acaagataca gtaattttcc gtcaatgaaa atgtcaaatt
ctagaaaatg 1440taacattcct atgcgtggtg ttaacatcgt tctctaacag
ttgttggccg aagattcgtt 1500tgatgaatcc gatttttcca aaatagccga
ttctgatgat tcagacgatt ctgatgttct 1560gtttagaaat aattccaaga
acagttttta cattttattt tcacattgaa aatcagtcag 1620atttgcttca
gcctcaaaga gcacgtttat gtaaaattaa atgagtgctg gcagccagct
1680gcgctttgtt tttctaaatg ggaaaagggt taaatttcac tcagctttta
aatgacagcg 1740cacagcctgt gtcatagagg gttggaggag atgactttaa
ctgcctgtgg ttaggatccc 1800tttcccccag gaatgtctgg gagcccactg
ccgggtttgc tgtccgtctc gtttggactc 1860agttctgcat gtactg
1876161282DNAHomo sapiensmisc_featuresequence of STAR16
16cgcccacctc ggctttccaa agtgctggga ttacaggcat gagtcactgc gcccatcctg
60attccaagtc tttagataat aacttaactt tttcgaccaa ttgccaatca ggcaatcttt
120gaatctgcct atgacctagg acatccctct ccctacaagt tgccccgcgt
ttccagacca 180aaccaatgta catcttacat gtattgattg aagttttaca
tctccctaaa acatataaaa 240ccaagctata gtctgaccac ctcaggcacg
tgttctcagg acctccctgg ggctatggca 300tgggtcctgg tcctcagatt
tggctcagaa taaatctctt caaatatttt ccagaatttt 360actcttttca
tcaccattac ctatcaccca taagtcagag ttttccacaa ccccttcctc
420agattcagta atttgctaga atggccacca aactcaggaa agtattttac
ttacaattac 480caatttatta tgaagaactc aaatcaggaa tagccaaatg
gaagaggcat agggaaaggt 540atggaggaag gggcacaaag cttccatgcc
ctgtgtgcac accaccctct cagcatcttc 600atgtgttcac caactcagaa
gctcttcaaa ctttgtcatt taggggtttt tatggcagtt 660ccactatgta
ggcatggttg ataaatcact ggtcatcggt gatagaactc tgtctccagc
720tcctctctct ctcctcccca gaagtcctga ggtggggctg aaagtttcac
aaggttagtt 780gctctgacaa ccagccccta tcctgaagct attgaggggt
cccccaaaag ttaccttagt 840atggttggaa gaggcttatt atgaataaca
aaagatgctc ctatttttac cactagggag 900catatccaag tcttgcggga
acaaagcatg ttactggtag caaattcata caggtagata 960gcaatctcaa
ttcttgcctt ctcagaagaa agaatttgac caagggggca taaggcagag
1020tgagggacca agataagttt tagagcagga gtgaaagttt attaaaaagt
tttaggcagg 1080aatgaaagaa agtaaagtac atttggaaga gggccaagtg
ggcgacatga gagagtcaaa 1140caccatgccc tgtttgatgt ttggcttggg
gtcttatatg atgacatgct tctgagggtt 1200gcatccttct cccctgattc
ttcccttggg gtgggctgtc cgcatgcaca atggcctgcc 1260agcagtaggg
aggggccgca tg 128217793DNAHomo sapiensmisc_featuresequence of
STAR17 17atccgagggg aggaggagaa gaggaaggcg agcagggcgc cggagcccga
ggtgtctgcg 60agaactgttt taaatggttg gcttgaaaat gtcactagtg ctaagtggct
tttcggattg 120tcttatttat tactttgtca ggtttcctta aggagagggt
gtgttggggg tgggggagga 180ggtggactgg ggaaacctct gcgtttctcc
tcctcggctg cacagggtga gtaggaaacg 240cctcgctgcc acttaacaat
ccctctatta gtaaatctac gcggagactc tatgggaagc 300cgagaaccag
tgtcttcttc cagggcagaa gtcacctgtt gggaacggcc cccgggtccc
360cctgctgggc tttccggctc ttctaggcgg cctgatttct cctcagccct
ccacccagcg 420tccctcaggg acttttcaca cctccccacc cccatttcca
ctacagtctc ccagggcaca 480gcacttcatt gacagccaca cgagccttct
cgttctcttc tcctctgttc cttctctttc 540tcttctcctc tgttccttct
ctttctctgt cataatttcc ttggtgcttt cgccacctta 600aacaaaaaag
agaaaaaaat aaaataaaaa aaacccattc tgagccaaag tattttaaga
660tgaatccaag aaagcgaccc acatagccct ccccacccac ggagtgcgcc
aagacgcacc 720caggctccat cacagggccg agagcagcgc cactctggtc
gtacttttgg gtcaagagat 780cttgcaaaag agg 79318492DNAHomo
sapiensmisc_featuresequence of STAR18 18atctttttgc tctctaaatg
tattgatggg ttgtgttttt tttcccacct gctaataaat 60attacattgc aacattcttc
cctcaacttc aaaactgctg aactgaaaca atatgcataa 120aagaaaatcc
tttgcagaag aaaaaaagct attttctccc actgattttg aatggcactt
180gcggatgcag ttcgcaaatc ctattgccta ttccctcatg aacattgtga
aatgaaacct 240ttggacagtc tgccgcattg cgcatgagac tgcctgcgca
aggcaagggt atggttccca 300aagcacccag tggtaaatcc taacttatta
ttcccttaaa attccaatgt aacaacgtgg 360gccataaaag agtttctgaa
caaaacatgt catctttgtg gaaaggtgtt tttcgtaatt 420aatgatggaa
tcatgctcat ttcaaaatgg aggtccacga tttgtggcca gctgatgcct
480gcaaattatc ct 492191840DNAHomo sapiensmisc_featuresequence of
STAR19 19tcacttcctg atattttaca ttcaaggcta gctttatgca tatgcaacct
gtgcagttgc 60acagggcttt gtgttcagaa agactagctc ttggtttaat actctgttgt
tgccatcttg 120agattcatta taatataatt tttgaatttg tgttttgaac
gtgatgtcca atgggacaat 180ggaacattca cataacagag gagacaggtc
aggtggcagc ctcaattcct tgccaccctt 240ttcacataca gcattggcaa
tgccccatga gcacaaaatt tgggggaacc atgatgctaa 300gactcaaagc
acatataaac atgttacctc tgtgactaaa agaagtggag gtgctgacag
360cccccagagg ccacagttta tgttcaaacc aaaacttgct tagggtgcag
aaagaaggca 420atggcagggt ctaagaaaca gcccatcata tccttgttta
ttcatgttac gtccctgcat 480gaactaatca cttacactga aaatattgac
agaggaggaa atggaaagat agggcaaccc 540atagttcttt ttccttttag
tctttcctta tcagtaaacc aaagatagta ttggtaaaat 600gtgtgtgagt
taattaatga gttagtttta ggcagtgttt ccactgttgg ggtaagaaca
660aaatatatag gcttgtattg agctattaaa tgtaaattgt ggaatgtcag
tgattccaag 720tatgaattaa atatccttgt atttgcattt aaaattggca
ctgaacaaca aagattaaca 780gtaaaattaa taatgtaaaa gtttaatttt
tacttagaat gacattaaat agcaaataaa 840agcaccatga taaatcaaga
gagagactgt ggaaagaagg aaaacgtttt tattttagta 900tatttaatgg
gactttcttc ctgatgtttt gttttgtttt gagagagagg gatgtggggg
960cagggaggtc tcattttgtt gcccaggctg gacttgaact cctgggctcc
agctatcctg 1020ccttagcttc ttgagtagct gggactacag gcacacacca
cagtgtctga cattttctgg 1080attttttttt tttttttatt ttttttgtga
gacaggttct ggctctgtta ctcaggttgc 1140agtgcagtgg catgatagcg
gctcactgca gcctcaacct cctcagctta agctactctc 1200ccacttcagc
ctcctgagta gccaggacta cagttgtgtg ccaccacacc tgtggctaat
1260ttttgtagag atggggtctc tccacgttgc cgaggctggt ctccaactcc
tggtctcaag 1320cgaacctcct gacttggcct cccgaagtgc tgggattaca
ggcttgagcc actgcatcca 1380gcctgtcctc tgtgttaaac ctactccaat
ttgtctttca tctctacata aacggctctt 1440ttcaaagttc ccatagacct
cactgttgct aatctaataa taaattatct gccttttctt 1500acatggttca
tcagtagcag cattagattg ggctgctcaa ttcttcttgg tatattttct
1560tcatttggct tctggggcat cacactctct ttgagttact cattcctcat
tgatagcttc 1620ttcctagtct tctttactgg ttcttcctct tctccctgac
tccttaatat tgtttttctc 1680cccaggcttt agttcttagt cctcttctgt
tatctattta cacccaattc tttcagagtc 1740tcatccagag tcatgaactt
aaacctgttt ctgtgcagat aattcacatt attatatctc 1800cagcccagac
tctcccgcaa actgcagact gatcctactg 184020780DNAHomo
sapiensmisc_featuresequence of STAR20 20gatctcaagt ttcaatatca
tgttttggca aaacattcga tgctcccaca tccttaccta 60aagctaccag aaaggctttg
ggaactgtca acagagctac agaaaagtca gtaaagacca 120atggacccct
caaacaaaaa cagccaagct tttctgccaa aaagatgact gagaagactg
180ttaaagcaaa aaactctgtt cctgcctcag atgatggcta tccagaaata
gaaaaattat 240ttcccttcaa tcctctaggc ttcgagagtt ttgacctgcc
tgaagagcac cagattgcac 300atctcccctt gagtgaagtg cctctcatga
tacttgatga ggagagagag cttgaaaagc 360tgtttcagct gggcccccct
tcacctttga agatgccctc tccaccatgg aaatccaatc 420tgttgcagtc
tcctttaagc attctgttga ccctggatgt tgaattgcca cctgtttgct
480ctgacataga tatttaaatt tcttagtgct ttagagtttg tgtatatttc
tattaataaa 540gcattatttg tttaacagaa aaaaagatat atacttaaat
cctaaaataa aataaccatt 600aaaaggaaaa acaggagtta taactaataa
gggaacaaag gacataaaat gggataataa 660tgcttaatcc aaaataaagc
agaaaatgaa gaaaaatgaa atgaagaaca gataaataga 720aaacaaatag
caatatgaaa gacaaacttg accgggtgtg gtggctgatg cctgtaatcc
78021607DNAHomo sapiensmisc_featuresequence of STAR21 21gatcaataat
ttgtaatagt cagtgaatac aaaggggtat atactaaatg ctacagaaat 60tccattcctg
ggtataaatc ctagacatat ttatgcatat gtacaccaag atatatctgc
120aagaatgttc acagcaaatc tctttgtagt agcaaaaggc caaaaggtct
atcaacaaga 180aaattaatac attgtggcac ataatggcat ccttatgcca
ataaaaatgg atgaaattat 240agttaggttc aaaaggcaag cctccagata
atttatatca tataattcca tgtacaacat 300tcaacaacaa gcaaaactaa
acatatacaa atgtcaggga aaatgatgaa caaggttaga 360aaatgattaa
tataaaaata ctgcacagtg ataacattta atgagaaaaa aagaaggaag
420ggcttaggga gggacctaca gggaactcca aagttcatgg taagtactaa
atacataatc 480aaagcactca aaatagaaaa tattttagta atgttttagc
tagttaatat cttacttaaa 540acaaggtcta ggccaggcac ggtggctcac
acctgtaatc ccagcacttt gggaggctga 600ggcgggt 607221380DNAHomo
sapiensmisc_featuresequence of STAR22 22cccttgtgat ccacccgcct
tggcctccca aagtgctggg attacaggcg tgagtcacta 60cgcccggcca ccctccctgt
atattatttc taagtatact attatgttaa aaaaagttta 120aaaatattga
tttaatgaat tcccagaaac taggatttta catgtcacgt tttcttatta
180taaaaataaa aatcaacaat aaatatatgg taaaagtaaa aagaaaaaca
aaaacaaaaa 240gtgaaaaaaa taaacaacac tcctgtcaaa aaacaacagt
tgtgataaaa cttaagtgcc 300tgaaaattta gaaacatcct tctaaagaag
ttctgaataa aataaggaat aaaataatca 360catagttttg gtcattggtt
ctgtttatgt gatggattat gtttattgat ttgtgtatgt 420tgaacttatc
tcaatagatg cagacaaggc cttgataaaa gtttttaaca ccttttcatg
480ttgaaaactc tcaatagact aggtattgat gaaacatatc tcaaaataat
agaagctatt 540tatgataaac ccatagccaa tatcatactg agtgggcaaa
agctggaagc attccctttg 600aaaactggca caagacaagg atgccctctc
tcaccactcc tattaaatgt agtattggaa 660gttctggcca gagcaatcag
gcaggagaaa gaaaaggtat taaaatagga agagaggaag 720tcaaattgtc
tctgtttgca gtaaacatga ttgtatattt agaaaacccc attgtctcat
780cctaaaaact ccttaagctg ataaacaact tcagcaaagt ctcaggatac
aaaatcaatg 840tgcaaaaatc acaagcattc ctatacaccg
ataatagaca gcagagagcc aaatcatgag 900tgaagtccca ttcacaattg
cttcaaagaa aataaaatac ttaggaatac aactttcacg 960ggacatgaag
gacattttca aggacaacta aaaaccactg ctcaaggaaa tgagagagga
1020cacaaagaaa tggaaaaaca ttccatgctc atggaagaat caatatcatg
aaaatggcca 1080tactgcccaa agtaatttat agattcaatg ctaaccccat
caagccacca ttgactttct 1140tcacagaact agaaaaaaac tattttaaaa
ctcatatgta gtcaaaaaga gtcggtatag 1200ccaagacaat cctaagcata
aagaacaaag ctggatgcat cacgctgact tcaaaccata 1260ctacaaggct
acagtaacca aaacagcatg gtactggtac caaaacagat agatagaccg
1320atagaacaga acagaggcct cggaaataac accacacatc tacaaccctt
tgatcttcaa 1380231246DNAHomo sapiensmisc_featuresequence of STAR23
23atcccctcat ccttcagggc agctgagcag ggcctcgagc agctggggga gcctcactta
60atgctcctgg gagggcagcc agggagcatg gggtctgcag gcatggtcca gggtcctgca
120ggcggcacgc accatgtgca gccgccccca cctgttgctc tgcctccgcc
acctggccat 180gggcttcagc agccagccac aaagtctgca gctgctgtac
atggacaaga agcccacaag 240cagctagagg accttgtgtt ccacgtgccc
agggagcatg gcccacagcc caaagaccag 300tcaggagcag gcaggggctt
ctggcaggcc cagctctacc tctgtcttca cacagatggg 360agatttctgt
tgtgattttg agtgatgtgc ccctttggtg acatccaaga tagttgctga
420agcaccgctc taacaatgtg tgtgtattct gaaaacgaga acttctttat
tctgaaataa 480ttgatgcaaa ataaattagt ttggatttga aattctattc
atgtaggcat gcacacaaaa 540gtccaacatt gcatatgaca caaagaaaag
aaaaagcttg cattccttaa atacaaatat 600ctgttaacta tatttgcaaa
tatatttgaa tacacttcta ttatgttaca tataatatta 660tatgtatatg
tatatataat atacatatat atgttacata taatatactt ctattatgtt
720acatataata tttatctata agtaaataca taaatataaa gatttgagta
gctgtagaac 780attgtcttat gtgttatcag ctactactac aaaaatatct
cttccactta tgccagtttg 840ccatataaat atgatcttct cattgatggc
ccagggcaag agtgcagtgg gtacttattc 900tctgtgagga gggaggagaa
aagggaacaa ggagaaagtc acaaagggaa aactctggtg 960ttgccaaaat
gtcaagtttc acatattccg agacggaaaa tgacatgtcc cacagaagga
1020ccctgcccag ctaatgtgtc acagatatct caggaagctt aaatgatttt
tttaaaagaa 1080aagagatggc attgtcactt gtttcttgta gctgaggctg
tgggatgatg cagatttctg 1140gaaggcaaag agctcctgct ttttccacac
cgagggactt tcaggaatga ggccagggtg 1200ctgagcacta caccaggaaa
tccctggaga gtgtttttct tactta 124624939DNAHomo
sapiensmisc_featuresequence of STAR24 24acgaggtcac gagttcgaga
ccagcctggc caagatggtg aagccctgtc tctactaaaa 60atacaacaag tagccgggcg
cggtgacggg cgcctgtaat cccagctact caggaggctg 120aagcaggaga
atctctagaa cccaggaggc ggaggtgcag tgagctgaga ctgccccgct
180gcactctagc ctgggcaaca cagcaagact ctgtctcaaa taaataaata
aataaataaa 240taaataaata aataaataaa tagaaaggga gagttggaag
tagatgaaag agaagaaaag 300aaatcctaga tttcctatct gaaggcacca
tgaagatgaa ggccacctct tctgggccag 360gtcctcccgt tgcaggtgaa
ccgagttctg gcctccattg gagaccaaag gagatgactt 420tggcctggct
cctagtgagg aagccatgcc tagtcctgtt ctgtttgggc ttgatcctgt
480atcacttgat tgtctctcct ggactttcca tggattccag ggatgcaact
gagaagttta 540tttttaatgc acttacttga agtaagagtt attttaaaac
attttagcaa aggaaatgaa 600ttctgacagg ttttgcactg aagacattca
catgtgagga aaacaggaaa accactatgc 660tagaaaaagc aaatgctgtt
gagattgtct cacaaacaca aattgcgtgc cagcaggtag 720gtttgagcct
caggttgggc acattttacc ttaagcgcac tgttggtgga acttaaggtg
780actgtaggac ttatatatac atacatacat ataatatata tacatattta
tgtgtatata 840cacacacaca cacacacaca cacacagggt cttgctatct
tgcccagggt ggtctccaac 900tctgggtctc aagcgatcct ctgcctcccc ttcccaaag
939251067DNAHomo sapiensmisc_featuresequence of STAR25 25cagcccctct
tgtgtttttc tttatttctc gtacacacac gcagttttaa gggtgatgtg 60tgtataatta
aaaggaccct tggcccatac tttcctaatt ctttagggac tgggattggg
120tttgactgaa atatgttttg gtggggatgg gacggtggac ttccattctc
cctaaactgg 180agttttggtc ggtaatcaaa actaaaagaa acctctggga
gactggaaac ctgattggag 240cactgaggaa caagggaatg aaaaggcaga
ctctctgaac gtttgatgaa atggactctt 300gtgaaaatta acagtgaata
ttcactgttg cactgtacga agtctctgaa atgtaattaa 360aagtttttat
tgagcccccg agctttggct tgcgcgtatt tttccggtcg cggacatccc
420accgcgcaga gcctcgcctc cccgctgccc tcagcctccg atgacttccc
cgcccccgcc 480ctgctcggtg acagacgttc tactgcttcc aatcggaggc
acccttcgcg ggagcggcca 540atcgggagct ccggcaggcg gggaggccgg
gccagttaga tttggaggtt caacttcaac 600atggccgaag caagtagcgc
caatctaggc agcggctgtg aggaaaaaag gcatgagggg 660tcgtcttcgg
aatctgtgcc acccggcact accatttcga gggtgaagct cctcgacacc
720atggtggaca cttttcttca gaagctggtc gccgccggca ggtaaagtgg
acgcagccgc 780ggtgggagtg tttgttggca ccgaagctca aatcccgcga
ggtcaggacg gccgcaggct 840ggcgcgcggt gacgtgggtc cgcgttgggg
gcggggcagt cggacgaggc gacccagtca 900aatcctgagc cttaggagtc
agggtattca cgcactgata acctgtagcg gaccgggata 960gctagctact
ccttcctaca ggaagccccg ttttcactaa aatttcaggt ggttgggagg
1020aaagatagag cctttgcaaa ttagagcagg gttttttatt tttttat
106726540DNAHomo sapiensmisc_featuresequence of STAR26 26ccccctgaca
agccccagtg tgtgatgttc cccactctgt gtccatgcat tctcattgtt 60caactcccat
ctgtgagtga gaacatgcag tgtttggttt tctgtccttg agatagtttg
120ctgagaatga tggtttccag cttcatccat gtccttgcaa aggaagtgaa
cttatccttt 180tttatggctt catagtattc catggcacat atgtgccaca
tttttttaat ccagtctatc 240attgatggac atttgggttg gttccaagtc
tttgctattg tgaatagcac cacaattaac 300atatgtgtgc atgtatacat
ctttatagta gcatgattta taatccttcg ggtatatacc 360ctgtaatggg
atcgctgggt caaatggtat ttctagttct agatccttga ggaatcacca
420cactgctttc cacaatggtt gaactaattt acgctcccac cagcagtgta
aaagcattcc 480tatttctcca cgtcctctcc agtatctgtt gtttcctgac
tttttaatga tcatcattct 540271520DNAHomo sapiensmisc_featuresequence
of STAR27 27cttggccctc acaaagcctg tggccaggga acaattagcg agctgcttat
tttgctttgt 60atccccaatg ctgggcataa tgcctgccat tatgagtaat gccggtagaa
gtatgtgttc 120aaggaccaaa gttgataaat accaaagaat ccagagaagg
gagagaacat tgagtagagg 180atagtgacag aagagatggg aacttctgac
aagagttgtg aagatgtact aggcaggggg 240aacagcttaa ggagagtcac
acaggaccga gctcttgtca agccggctgc catggaggct 300gggtggggcc
atggtagctt tcccttcctt ctcaggttca gagtgtcagc cttgaacttc
360taattcccag aggcatttat tcaatgtttt cttctagggg catacctgcc
ctgctgtgga 420agactttctt ccctgtgggt cgccccagtc cccagatgag
acggtttggg tcagggccag 480gtgcaccgtt gggtgtgtgc ttatgtctga
tgacagttag ttactcagtc attagtcatt 540gagggaggtg tggtaaagat
ggagatgctg ggtcacatcc ctagagaggt gttccagtat 600gggcacatgg
gagggctgga aggataggtt actgctagac gtagagaagc cacatccttt
660aacaccctgg cttttcccac tgccaagatc cagaaagtcc ttgtggtttc
gctgctttct 720cctttttttt tttttttttt tttctgagat ggagtctggc
tctgtcgccc aggctggagt 780gcagtggcac gatttcggct cactgcaagt
tccgcctcct aggttcatac cattctccca 840cctcagcctc ccgagtagct
gggactacag gcgccaccac acccagctaa ttttttgtat 900ttttagtaga
gacggcgttt caccatgtta gccaggatgg tcttgatccg cctgcctcag
960cctcccaaag tgctgggatt acaggcgtga gccaccgcgc ccggcctgct
ttcttctttc 1020atgaagcatt cagctggtga aaaagctcag ccaggctggt
ctggaactct tgacctcaag 1080tgatctgcct gcctcagcct cccaaagtgc
tgagattaca ggcatgagcc agtccgaatg 1140tggctttttt tgttttgttt
tgaaacaagg tctcactgtt gcccaggctg cagtgcagtg 1200gcatacctca
gctccactgc agcctcgacc tcctgggctc aagcaatcct cccaactgag
1260cctccccagt agctggggct acaagcgcat gccaccacgc ctggctattt
tttttttttt 1320tttttttttt gagaaggagt ttcattcttg ttgcccaggc
tggagtgcaa tggcacagtc 1380tcagctcact gcagcctccg cctcctgggt
tcaagcgatt ctcctgcctc agcctcccga 1440gtagctggga ttataggcac
ctgccaccat gcctggctaa tttttttgta tttttagtag 1500ggatggggtt
tcaccatgtt 152028961DNAHomo sapiensmisc_featuresequence of STAR28
28aggaggttat tcctgagcaa atggccagcc tagtgaactg gataaatgcc catgtaagat
60ctgtttaccc tgagaagggc atttcctaac tctccctata aaatgccaag tggagcaccc
120cagatgaaat agctgatatg ctttctatac aagccatcta ggactggctt
tatcatgacc 180aggatattca cccactgaat atggctatta cccaagttat
ggtaaatgct gtagttaagg 240gggtcccttc cacatggaca ccccaggtta
taaccagaaa gggttcccaa tctagactcc 300aagagagggt tcttagacct
catgcaagaa agaacttggg gcaagtacat aaagtgaaag 360caagtttatt
aagaaagtaa agaaacaaaa aaatggctac tccataagca aagttatttc
420tcacttatat gattaataag agatggatta ttcatgagtt ttctgggaaa
ggggtgggca 480attcctggaa ctgagggttc ctcccacttt tagaccatat
agggtatctt cctgatattg 540ccatggcatt tgtaaactgt catggcactg
atgggagtgt cttttagcat tctaatgcat 600tataattagc atataatgag
cagtgaggat gaccagaggt cacttctgtt gccatattgg 660tttcagtggg
gtttggttgg cttttttttt tttttaacca caacctgttt tttatttatt
720tatttattta tttatttatt tatatttttt attttttttt agatggagtc
ttgctctgtc 780acccaggtta gagtgcagtg gcaccatctc ggctcactgc
aagctctgcc tccttggttc 840acgccattct gctgcctcag cctcccgagt
agctgggact acaggtgcct gccaccatac 900ccggctaatt ttttctattt
ttcagtagag acggggtttc accgtgttag ccaggatggt 960c 961292233DNAHomo
sapiensmisc_featuresequence of STAR29 29agcttggaca cttgctgatg
ccactttgga tgttgaaggg ccgccctctc ccacaccgct 60ggccactttt aaatatgtcc
cctctgccca gaagggcccc agaggagggg ctggtgaggg 120tgacaggagt
tgactgctct cacagcaggg ggttccggag ggaccttttc tccccattgg
180gcagcataga aggacctaga agggccccct ccaagcccag ctgggcgtgc
agggccagcg 240attcgatgcc ttcccctgac tcaggtggcg ctgtcctaaa
ggtgtgtgtg ttttctgttc 300gccagggggt ggcggataca gtggagcatc
gtgcccgaag tgtctgagcc cgtggtaagt 360ccctggaggg tgcacggtct
cctccgactg tctccatcac gtcaggcctc acagcctgta 420ggcaccgctc
ggggaagcct ctggatgagg ccatgtggtc atccccctgg agtcctggcc
480tggcctgaag aggaggggag gaggaggcca gcccctccct agccccaagg
cctgcgaggc 540tgcaagcccg gccccacatt ctagtccagg cttggctgtg
caagaagcag attgcctggc 600cctggccagg cttcccagct aggatgtggt
atggcagggg tgggggacat tgaggggctg 660ctgtagcccc cacaacctcc
ccaggtaggg tggtgaacag taggctggac aagtggacct 720gttcccatct
gagattcaag agcccacctc tcggaggttg cagtgagccg agatccctcc
780actgcactcc agcctgggca acagagcaag actctgtctc aaaaaaacag
aacaacgaca 840acaaaaaacc cacctctggc ccactgccta actttgtaaa
taaagtttta ttggcacata 900gacacaccca ttcatttaca tactgctgcg
gctgcttttg cattaccctt gagtagacga 960cagaccacgt ggccatggaa
gccaaaaata tttactgtct ggccctttac agaagtctgc 1020tctagaggga
gaccccggcc catggggcag gaccactggg cgtgggcaga agggaggcct
1080cggtgcctcc acgggcctag ttgggtatct cagtgcctgt ttcttgcatg
gagcaccagg 1140ggtcagggca agtacctgga ggaggcaggc tgttgcccgc
ccagcactgg gacccaggag 1200accttgagag gctcttaacg aatgggagac
aagcaggacc agggctccca ttggctgggc 1260ctcagtttcc ctgcctgtaa
gtgagggagg gcagctgtga aggtgaactg tgaggcagag 1320cctctgctca
gccattgcag gggcggctct gccccactcc tgttgtgcac ccagagtgag
1380gggcacgggg tgagatgtca ccatcagccc ataggggtgt cctcctggtg
ccaggtcccc 1440aagggatgtc ccatcccccc tggctgtgtg gggacagcag
agtccctggg gctgggaggg 1500ctccacactg ttttgtcagt ggtttttctg
aactgttaaa tttcagtgga aaattctctt 1560tcccctttta ctgaaggaac
ctccaaagga agacctgact gtgtctgaga agttccagct 1620ggtgctggac
gtcgcccaga aagcccaggt actgccacgg gcgccggcca ggggtgtgtc
1680tgcgccagcc atgggcacca gccaggggtg tgtctacgcc ggccaggggt
aggtctccgc 1740cggcctccgc tgctgcctgg ggagggccgt gcctgacact
gcaggcccgg tttgtccgcg 1800gtcagctgac ttgtagtcac cctgcccttg
gatggtcgtt acagcaactc tggtggttgg 1860ggaaggggcc tcctgattca
gcctctgcgg acggtgcgcg agggtggagc tcccctccct 1920ccccaccgcc
cctggccagg gttgaacgcc cctgggaagg actcaggccc gggtctgctg
1980ttgctgtgag cgtggccacc tctgccctag accagagctg ggccttcccc
ggcctaggag 2040cagccgggca ggaccacagg gctccgagtg acctcagggc
tgcccgacct ggaggccctc 2100ctggcgtcgc ggtgtgactg acagcccagg
agcgggggct gttgtaattg ctgtttctcc 2160ttcacacaga accttttcgg
gaagatggct gacatcctgg agaagatcaa gaagtaagtc 2220ccgcccccca ccc
2233301851DNAHomo sapiensmisc_featuresequence of STAR30
30gggtgcattt ccacccaggg gacacttggc aatggtggga gacattgctt gttgtcacaa
60ctgggcatgg gagtgctgct gcgtctagtg ggtagaggcc agagatgctc ctaatatcct
120acaaggcaca gaacagcccc ccacaacaga gaattatcca gcctgaaaat
gtccacagtg 180ctgaggttgg gaaaccctat tctagagcca acaggctgtg
aagcttgact catggttcca 240tcaccaatag ctgcgtgacc ttggtgagtt
ccttagctgc tctgtgcctc ggattcatgg 300taggttttcc ttgttaggtt
taaatgagtg aagttataca gagggcctga agtctcatgg 360tattttacta
gagcctcatt gtgttttagt tataattaga aattgggtaa ggtaaggaca
420cagaagaagc catctgatct gggggcttca cacttagaag tgacctcgga
gcaattgtat 480tggggtggaa agggactaac agccaggagc agagggcaca
ttggaattgg ggccagaggg 540cacagactgc cttgtccatc aggcatagca
atggacagag gaaggggaat gactagttat 600ggctgcaagg ccaagtacag
gggacttatt tctcatatct atctatctat ctacctaccg 660tctatttatc
tatcatctat ctacttattt atctatctat ttatgcatgt gtaccaaccg
720aaagttttag taaatgcaca aactgcgata taatgaaaat ggaaattttc
aaaagaagag 780aaatcacctg ccacctgact accttaacaa atgagtggtt
ttcatctctc cttccaggcc 840tgtcattttt acagtgcttt agtcataaaa
caggtcctct attctattgt tttatgtcac 900atgaaattgt accataagca
ttttccatga tgtgactcca ctgtttcatt ttccattttt 960ttccagaatg
aagataacct cattgttttt ttcctgattg taaaaatgct ctgtgctctt
1020tttttttttt tttaacaatg caggcagtac caaaaagtat gaagaagaat
gtaatagttc 1080ccatttccca tctcactctt taaggccagc attttggtga
acatccatcc gaacaaatct 1140ccacgcgttt atcaatttgt tgacttactc
cttcttttat gtaaatatga acatgattta 1200actgccagtc catttggaac
cttaaagtga aggtttttta ttgttggggt ttgctatggt 1260ctgaatatgt
gtgtcccccc aaaatttatg ttgaatccta acgcccaatg cgattaggag
1320gtggggccat taggaggtga ttaagtcatg aagtcatcag ccctaatgaa
tgggatttgt 1380ggccttgaaa agggacccca gagagctgcc ttgccccttc
tgccatgtaa ggacacagtg 1440aggagctagg aagggggcct cagcagagac
caaatgtgat ggtgcctcga tattggactt 1500cccagcctcc agaatgtgag
aaatgaattt ctgttgttta taagtcaccc agtctatagt 1560attttgttct
agcagcccaa acagactaag tcagggttgt tgttttagga agtggggaat
1620ggggccatgc atgggtgtac gccagaacaa aggaagccag caagtcctga
aagatactgg 1680aaaagggaat agtgggcacg tgcagtgtgt tagtttcctg
aggctgctat aacaaagcac 1740cacaggttgg gtggcttaaa taacagaaat
tcattctccc atcattctgg ggaccagacg 1800tctgaaatca agactcctat
gccatgctcc ttctgaaggc tccaggggag g 1851311701DNAHomo
sapiensmisc_featuresequence of STAR31 31cacccgcctt ggccccccag
agtgctggga ttacaagtgt aaaccaccat tcctggctag 60atttaatttt ttaaaaaata
aagagaagta ggaatagttc attttaggga gagcccctta 120actgggacag
gggcaggaca ggggtgaggc ttcccttant tcaagctcac ctcaaaccca
180cccaggactg tgtgtcacat tctccaataa aggaaaggtt gctgcccccg
cctgtgagtg 240ctgcagtgga gggtagaggg ccgtgggcag agtgcttcat
ggactgctca tcaagaaagg 300cttcatgaca atcggcccag ctgctgtcat
cccacattct acttccagct aggagaaggc 360ggcttgccca cagtcaccca
gccggcaagt gtcacccctg ggttggaccc agagctatga 420tcctgcccag
gggtccagct gagaatcagg cccacgttct aggcagaggg gctcacctac
480tgggactcca gtagctgtag tgcatggagg catcatggct gcagcagcct
ggacctggtc 540tcacactggc tgtccctgtg ggcaggccat cctcaatgcc
aggtcaggcc caagcatgta 600tcccagacaa tgacaatggg gtggaatcct
ctcttgtccc agaagccact cctcactgtt 660ctacctgagg aaggcagggg
catggtggaa tcctgaagcc tgctgtgagg gtctccagcg 720aacttgcaca
tggtcagccc tgccttctcc tccctgaact agattgagcg agagcaagaa
780ggacattgaa ccagcaccca aagaattttg gggaacggcc tctcatccag
gtcaggctca 840cctccttttt aaaatttaat taattaatta attaattttt
ttttagagac agagtcttac 900tgtgtggccc aggctgtagt gcagtggcac
aatcatagtt cactgcagcc tcaaactccc 960cacctcagcc tctggattag
ctgagactac aggtgcacca ccaccacacc cagctaatat 1020ttttattttt
gtagagagag ggtttcacca tcttgcccag gctggtctca aactcctggg
1080ctcaagtgat cccgcccagg tctgaaagcc cccaggctgg cctcagactg
tggggttttc 1140catgcagcca cccgagggcg cccccaagcc agttcatctc
ggagtccagg cctggccctg 1200ggagacagag tgaaaccagt ggtttttatg
aacttaactt agagtttaaa agatttctac 1260tcgatcactt gtcaagatgc
gccctctctg gggagaaggg aacgtgactg gattccctca 1320ctgttgtatc
ttgaataaac gctgctgctt catcctgtgg gggccgtggc cctgtccctg
1380tgtgggtggg gcctcttcca tttccctgac ttagaaacca cagtccacct
agaacagggt 1440ttgagaggct tagtcagcac tgggtagcgt tttgactcca
ttctcggctt tcttcttttt 1500ctttccagga tttttgtgca gaaatggttc
ttttgttgcc gtgttagtcc tccttggaag 1560gcagctcaga aggcccgtga
aatgtcgggg gacaggaccc ccagggaggg aaccccaggc 1620tacgcacttt
agggttcgtt ctccagggag ggcgacctga cccccgnatc cgtcggngcg
1680cgnngnnacn aannnnttcc c 170132771DNAHomo
sapiensmisc_featuresequence of STAR32 32gatcacacag cttgtatgtg
ggagctagga ttggaacccc agaagtctgg ccccaggttc 60atgctctcac ccactgcata
caatggcctc tcataaatca atccagtata aaacattaga 120atctgcttta
aaaccataga attagtagcg taagtaataa atgcagagac catgcagtga
180atggcattcc tggaaaaagc ccccagaagg aattttaaat cagctttcgt
ctaatcttga 240gcagctagtt agcaaatatg agaatacagt tgttcccaga
taatgcttta tgtctgacca 300tcttaaactg gcgctgtttt tcaaaaactt
aaaaacaaaa tccatgactc ttttaattat 360aaaagtgata catgtctact
tgggaggctg aggtggtggg aggatggctt gagtttgagg 420ctgcagtatg
ctactatcat gcctataaat agccgctgca ttccagcttg ggcaacatac
480ccaggcccta tctcaaaaaa ataaaaagta atacatctac attgaagaaa
attaatttta 540ttgggttttt ttgcattttt attatacaca gcacacacag
cacatatgaa aaaatgggta 600tgaactcagg cattcaactg gaagaacagt
actaaatcaa tgtccatgta gtcagcgtga 660ctgaggttgg tttgtttttt
cttttttctt ctcttctctt ctcttttctt tttttttgag 720acggagcttt
gctctttttg cccaggcttg attgcaatgg cgtgatctca g 771331368DNAHomo
sapiensmisc_featuresequence of STAR33 33gcttttatcc tccattcaca
gctagcctgg cccccagagt acccaattct ccctaaaaaa 60cggtcatgct gtatagatgt
gtgtggcttg gtagtgctaa agtggccaca tacagagctc 120tgacaccaaa
cctcaggacc atgttcatgc cttctcactg agttctggct tgttcgtgac
180acattatgac attatgatta tgatgacttg tgagagcctc agtcttctat
agcactttta 240gaatgcttta taaaaaccat ggggatgtca ttatattcta
acctgttagc acttctgttc 300gtattaccca tcacatccca acatcaattc
tcatatatgc aggtacctct tgtcacgcgc 360gtccatgtaa ggagaccaca
aaacaggctt tgtttgagca acaaggtttt tatttcacct 420gggtgcaggt
gggctgagtc tgaaaagaga gtcagtgaag ggagacaggg gtgggtccac
480tttataagat ttgggtaggt agtggaaaat tacaatcaaa gggggttgtt
ctctggctgg 540ccagggtggg ggtcacaagg tgctcagtgg gagagccttt
gagccaggat gagccagaag 600gaatttcaca aggtaatgtc atcagttaag
gcagggactg gccattttca cttcttttgt 660ggtggaatgt catcagttaa
ggcaggaacc ggccattttc acttcttttg tgattcttca 720cttgcttcag
gccatctgga cgtataggtg caggtcacag tcacagggga taagatggca
780atggcatagc ttgggctcag aggcctgaca
cctctgagaa actaaagatt ataaaaatga 840tggtcgcttc tattgcaaat
ctgtgtttat tgtcaagagg cacttatttg tcaattaaga 900acccagtggt
agaatcgaat gtccgaatgt aaaacaaaat acaaaacctc tgtgtgtgtg
960tgtgtgtgag tgtgtgtgta tgtgtgtgtg tgtgtattag agaggaaaag
cctgtatttg 1020gaggtgtgat tcttagattc taggttcttt cctgcccacc
ccatatgcac ccaccccaca 1080aaagaacaaa caacaaatcc caggacatct
tagcgcaaca tttcagtttg catattttac 1140atatttactt ttcttacata
ttaaaaaact gaaaatttta tgaacacgct aagttagatt 1200ttaaattaag
tttgttttta cactgaaaat aatttaatat ttgtgaagaa tactaataca
1260ttggtatatt tcattttctt aaaattctga acccctcttc ccttatttcc
ttttgacccg 1320attggtgtat tggtcatgtg actcatggat ttgccttaag gcaggagg
136834755DNAHomo sapiensmisc_featuresequence of STAR34 34actgggcacc
ctcctaggca ggggaatgtg agaactgccg ctgctctggg gctgggcgcc 60atgtcacagc
aggagggagg acggtgttac accacgtggg aaggactcag ggtggtcagc
120cacaaagctg ctggtgatga ccaggggctt gtgtcttcac tctgcagccc
taacacccag 180gctgggttcg ctaggctcca tcctgggggt gcagaccctg
agagtgatgc cagtgggagc 240ctcccgcccc tccccttcct cgaaggccca
ggggtcaaac agtgtagact cagaggcctg 300agggcacatg tttatttagc
agacaaggtg gggctccatc agcggggtgg cctggggagc 360agctgcatgg
gtggcactgt ggggagggtc tcccagctcc ctcaatggtg ttcgggctgg
420tgcggcagct ggcggcaccc tggacagagg tggatatgag ggtgatgggt
ggggaaatgg 480gaggcacccg agatggggac agcagaataa agacagcagc
agtgctgggg ggcaggggga 540tgagcaaagg caggcccaag acccccagcc
cactgcaccc tggcctccca caagccccct 600cgcagccgcc cagccacact
cactgtgcac tcagccgtcg atacactggt ctgttaggga 660gaaagtccgt
cagaacaggc agctgtgtgt gtgtgtgcgt gtatgagtgt gtgtgtgtga
720tccctgactg ccaggtcctc tgcactgccc ctggg 755351193DNAHomo
sapiensmisc_featuresequence of STAR35 35cgacttggtg atgcgggctc
ttttttggtt ccatatgaac tttaaagtag tcttttccaa 60ttctgtgaag aaagtcattg
gtaggttgat ggggatggca ttgaatctgt aaattacctt 120gggcagtatg
gccattttca caatgttgat tcttcctatc catgatgatg gaatgttctt
180ccattagttt gtatcctctt ttatttcctt gagcagtggt ttgtagttct
ccttgaagag 240gtccttcaca tcccttgtaa gttggattcc taggtatttt
attctctttg aagcaaattg 300tgaatgggag tncactcacg atttggctct
ctgtttgtct gctgggtgta taaanaatgt 360ngtgatnttn gtacattgat
ttngtatccn tgagacttng ctgaatttgc ttnatcngct 420tnngggaacc
ttttgggctg aaacnatggg attttctaaa tatacaatca tgtcgtctgc
480aaacagggaa caatttgact tcctcttttc ctaattgaat acactttatc
tccttctcct 540gcctaattgc cctgggcaaa acttccaaca ctatgntngn
aataggagnt ggtgagagag 600ggcatccctg ttcttgttgc cagnttttca
aagggaatgc ttccagtttt ggcccattca 660gtatgatatg ggctgtgggt
ngtgtcataa atagctctta tnattttgaa atgtgtccca 720tcaataccta
atttattgaa agtttttagc atgaangcat ngttgaattt ggtcaaaggc
780tttttctgca tctatggaaa taatcatgtg gtttttgtct ttggctcntg
tttatatgct 840ggatnacatt tattgatttg tgtatatnga acccagcctn
ncatcccagg gatgaagccc 900acttgatcca agcttggcgc gcngnctagc
tcgaggcagg caaaagtatg caaagcatgc 960atctcaatta gtcagcaccc
atagtccgcc cctacctccg cccatccgcc cctaactcng 1020nccgttcgcc
cattctcgcc catggctgac taatnttttt annatccaag cggngccgcc
1080ctgcttganc attcagagtn nagagnnttg gaggccnagc cttgcaaaac
tccggacngn 1140ttctnnggat tgaccccnnt taaatatttg gttttttgtn
ttttcanngg nga 1193361712DNAHomo sapiensmisc_featuresequence of
STAR36 36gatcccatcc ttagcctcat cgatacctcc tgctcacctg tcagtgcctc
tggagtgtgt 60gtctagccca ggcccatccc ctggaactca ggggactcag gactagtggg
catgtacact 120tggcctcagg ggactcagga ttagtgagcc ccacatgtac
acttggcctc agtggactca 180ggactagtga gccccacatg tacacttggc
ctcaggggac tcaggattag tgagccccca 240catgtacact tggcctcagg
ggactcagga ttagtgagcc ccacatgtac acttggcctc 300aggggactca
ggactagtga gccccacatg tacacttggc ctcaggggac tcagaactag
360tgagccccac atgtacactt ggcttcaggg gactcaggat tagtgagccc
cacatgtaca 420cttggacacg tgaaccacat cgatgtgctg cagagctcag
ccctctgcag atgaaatgtg 480gtcatggcat tccttcacag tggcacccct
cgttccctcc ccacctcatc tcccattctt 540gtctgtcttc agcacctgcc
atgtccagcc ggcagattcc accgcagcat cttctgcagc 600acccccgacc
acacacctcc ccagcgcctg cttggccctc cagcccagct cccgcctttc
660ttccttgggg aagctccctg gacagacacc ccctcctccc agccatggct
ttttcctgct 720ctgccccacg cgggaccctg ccctggatgt gctacaatag
acacatcaga tacagtcctt 780cctcagcagc cggcagaccc agggtggact
gctcggggcc tgcctgtgag gtcacacagg 840tgtcgttaac ttgccatctc
agcaactagt gaatatgggc agatgctacc ttccttccgg 900ttccctggtg
agaggtactg gtggatgtcc tgtgttgccg gccacctttt gtccctggat
960gccatttatt tttttccaca aatatttccc aggtctcttc tgtgtgcaag
gtattagggc 1020tgcagcgggg gccaggccac agatctctgt cctgagaaga
cttggattct agtgcaggag 1080actgaagtgt atcacaccaa tcagtgtaaa
ttgttaactg ccacaaggag aaaggccagg 1140aaggagtggg gcatggtggt
gttctagtgt tacaagaaga agccagggag ggcttcctgg 1200atgaagtggc
atctgacctg ggatctggag gaggagaaaa atgtcccaaa agagcagaga
1260gcccacccta ggctctgcac caggaggcaa cttgctgggc ttatggaatt
cagagggcaa 1320gtgataagca gaaagtcctt gggggccaca attaggattt
ctgtcttcta aagggcctct 1380gccctctgct gtgtgacctt gggcaagtta
cttcacctct agtgctttgg ttgcctcatc 1440tgtaaagtgg tgaggataat
gctatcacac tggttgagaa ttgaagtaat tattgctgca 1500aagggcttat
aagggtgtct aatactagta ctagtaggta cttcatgtgt cttgacaatt
1560ttaatcatta ttattttgtc atcaccgtca ctcttccagg ggactaatgt
ccctgctgtt 1620ctgtccaaat taaacattgt ttatccctgt gggcatctgg
cgaggtggct aggaaagcct 1680ggagctgttt cctgttgacg tgccagacta gt
1712371321DNAHomo sapiensmisc_featuresequence of STAR37
37aggatcacat ttaaggaagt gtgtggggtc cctggatgac accagcaccc agtgcggctc
60tgtctggcaa ccgctcccaa ggtggcagga gtgggtgtcc cctgtgtgtc agtgggcagc
120tcctgctgag cctacagctc actggggagc ctgacagcgg ggccatgtgc
ctgacactcc 180tctctgcttg tggacctggc aaggcaggga gcagaaaaca
gagccacttg aaggctttct 240gtctgcgtct gtgtgcagtg tggatttagt
tgtgcttttt tcttgctggg agagcacagc 300caccatttac aagcagtgtc
accctcatgg gtggcgagga cagaacagga gcctctgctc 360tctgtaccta
tctgggcccg gtgggctccc ttgtcctggc ttccatctct gtctcagcga
420ccattcagcc ctgcgcagga acacatgttg cttagaaaag ccaaattcag
cccttgtctc 480tgcctcctct ggtctcatga tgtgcatctg ttaccttgaa
actggaaacc agtctatcaa 540tgtctgtgcc aattttttat tccctcccca
acctccttcc ccatacgact ttttatttat 600gtaggatgtg tgctgtctaa
tgatgggatg accacatttt tccatgttct aaaagtgctc 660ctctcccgca
gggtcccagg gctggtggtt gctttgggtc tacagctacg tcttacccgc
720ctcctgcctc aacagcctgt gtggtggcaa agccggtgtg gggctgggga
acgcagcgtt 780ctccaggagg gggacccggc tctccttctg cagtgcaggc
gaaggcctag atgccagtgt 840gacctcccac aaggcgtggc ttccagactc
cccggctgga agtgatgctt ttttgcctcc 900ggccctgggt ttgaagcagc
ctggctttct cttggtaagt ggctggtgtc ttagcagctg 960caatctgagc
tcagccacct acacaccacc gtggccgaca ctttcattaa aaagtttcct
1020gagacgactt gcgtgcatgt tgacttcatg atcagcgccg ctgggaagaa
cccctgagcc 1080ggtggggtgg ggctggaagc agcaggtgca gtgatggggc
tgggtgccca ggaggcctca 1140gtgctcaatc aggccaaggt ggccaagccc
aggctgcagg gaaggccggc ctgggggttg 1200tgggtgagca caggcaggca
ccagctgggc agtgttagga tgctggagca gcatccgtaa 1260ccccactgag
tggggtagtc tggttggggc agggaccgct gttgctttgg cagagagaga 1320t
1321381445DNAHomo sapiensmisc_featuresequence of STAR38
38gatctatggg agtagcttcc ttagtgagct ttcccttcaa atactttgca accaggtaga
60gaattttgga gtgaaggttt tgttcttcgt ttcttcacaa tatggatatg catcttcttt
120tgaaaatgtt aaagtaaatt acctctcttt tcagatactg tcttcatgcg
aacttggtat 180cctgtttcca tcccagcctt ctataaccca gtaacatctt
ttttgaaacc agtgggtgag 240aaagacacct ggtcaggaac gcggaccaca
ggacaactca ggctcaccca cggcatcaga 300ctaaaggcaa acaaggactc
tgtataaagt accggtggca tgtgtatnag tggagatgca 360gcctgtgctc
tgcagacagg gagtcacaca gacacttttc tataatttct taagtgcttt
420gaatgttcaa gtagaaagtc taacattaaa tttgattgaa caattgtata
ttcatggaat 480attttggaac ggaataccaa aaaatggcaa tagtggttct
ttctggatgg aagacaaact 540tttcttgttt aaaataaatt ttattttata
tatttgaggt tgaccacatg accttaagga 600tacatataga cagtaaactg
gttactacag tgaagcaaat taacatatct accatcgtac 660atagttacat
ttttttgtgt gacaggaaca gctaaaatct acgtatttaa caaaaatcct
720aaagacaata catttttatt aactatagcc ctcatgatgt acattagatc
gtgtggttgt 780ttcttccgtc cccgccacgc cttcctcctg ggatggggat
tcattcccta gcaggtgtcg 840gagaactggc gcccttgcag ggtaggtgcc
ccggagcctg aggcgggnac tttaanatca 900gacgcttggg ggccggctgg
gaaaaactgg cggaaaatat tataactgna ctctcaatgc 960cagctgttgt
agaagctcct gggacaagcc gtggaagtcc cctcaggagg cttccgcgat
1020gtcctaggtg gctgctccgc ccgccacggt catttccatt gactcacacg
cgccgcctgg 1080aggaggaggc tgcgctggac acgccggtgg cgcctttgcc
tgggggagcg cagcctggag 1140ctctggcggc agcgctggga gcggggcctc
ggaggctggg cctggggacc caaggttggg 1200cggggcgcag gaggtgggct
cagggttctc cagagaatcc ccatgagctg acccgcaggg 1260cggccgggcc
agtaggcacc gggcccccgc ggtgacctgc ggacccgaag ctggagcagc
1320cactgcaaat gctgcgctga ccccaaatgc tgtgtccttt aaatgtttta
attaagaata 1380attaataggt ccgggtgtgg aggctcaagc cttaatcccc
agcacctggc gaggccgagg 1440aggga 1445392331DNAHomo
sapiensmisc_featuresequence of STAR39 39gtgaaataga tcactaaagc
tgattcctct tgtctaaatg aaactttcta ccctttgatg 60gacagctatg ctttccccat
cctctcccgt cccccagccc ttggtaacca tcatcctact 120ctctacttgt
aggagttcaa cttgtttaga ttttgtgagt gagaacatgt ggtatttgcc
180tttagagtcc tctaggttta tccatattgt gttaaatgac aggattccct
gcctttttaa 240ggctgaatag tatttcattg taatatatat acatacacac
acacatatac acacacatat 300atatacatat atacatatat gtacatagat
acatatatat gtacatatat acacacacat 360atacacacat atatacacat
atatacatat acatatatac acatatatgt acatatatat 420aacttttttt
catttatcca ttcacttaat acatatgatg gagggcttta tatatgccag
480gctctgtgat gaatgctgga aattcaatag tgagaaagac tcagtctctg
cctccaaaga 540gcatcatggg ctaggtgctg caacgaggaa ttgccaactg
ttgtcatgag agcacagaga 600agggactcaa ccagccttga agaatcaggg
gaggcttcta agctaatggt gtgtgcctgg 660ggatcacatt gtttcaagca
gcagtaacag gatgtgctca ggtccagatg tgagagagag 720agagagcata
tgtcttcaag aaactaacag tagctcccta tagctgaagc aggagtacaa
780aatagtgagt ttaagtgatg aggcaagaga tatgaagaag cttgaccatg
cagctacacc 840gggcagcatg ccctctgaga catctcatgg aagccggaaa
tgggagtgcc ttgataccaa 900gccagagaaa ttataatact aagtagatag
actgagcagc actcctcctg ggaagaatga 960gacaagccct gaatttggag
gtaagttgtg gattggtgat tagaggagag gtaacaggca 1020ccaaagcaag
aaatagtatt gatgcaaagc tgaggttaat tggatgacaa aatgaagagc
1080ataaggggct cagacacaga ctgagcagaa aacgagtagc atctgaacct
agattgagtt 1140actaatggat gagaaagagt tcttaaagtt gatgaccacg
ggatccatat ataagaatgt 1200ccaatctccc caaattgatc cacgagttca
gtgcaatgcc aatcaaaatc ccactaacaa 1260gtttatttta aaatgtaaat
gaaaatacaa aatttttaaa aagcaaagca atattgaaaa 1320cccaggaaaa
attaggagga cttacacaac ctgatctcaa aacttaccat tatcaagaca
1380gagtgttatt gacacaagga gagacaaata gataaacgga atgtggtagt
ctggagatgc 1440acccacatgt atgtggtcaa ttgatttttg gccaaggcac
caagtcaatt caaaggagca 1500aggaaagtag tacagaaaca accaaatatt
gttttggaaa ataatgacaa agggcttata 1560accagaatat aagcatataa
atataattct ttcaaatcaa taataagaag gcaaatatct 1620aataaaaatg
agcaaagact tgaaaagtca cttaaaaagg cttattaatt agaaatatgc
1680aaatgttatt agtcttcagt ggaatttaca ttaaaccaca agggatacta
ttatatctta 1740tgcccactag aataaccaaa ggaaaaaaga cagacaaaac
aaaatgctgg tgaggatgtg 1800aagcaactgg aactctcata cattattggt
ggtaatgtaa aatttataca accattatga 1860ataaaggttt ggcagtttct
tacaaagttg aatgcacttc tccacgatga ctaggctttt 1920cactcatagg
cgtctggctc cctagaactg aaaacatatg ttcacaagaa gacttgcaaa
1980tatatattct cccacgtcag gagatatttg ctatgcattt aactgacata
agattagtgc 2040tagagtttat aatgaggttc ttcaaatcta aaagaaaatg
caaagcatat aatagtaagg 2100ggtgcaggcc aggcgcagtg gctcactctg
taatcccagc actttgggag gccgaggtgg 2160gcggatcaca aggtcaggag
ttcgagacca acctggccaa catagtgaaa ccctgtctct 2220actaaaaata
caaaaactag ccaggtgcgg tgtcatgcac ctgtagtccc agctactcgg
2280gaggccgagg caggagaatc acttgaacct gggaggtgga ggttgcagtg a
2331401071DNAHomo sapiensmisc_featuresequence of STAR40
40gctgtgattc aaactgtcag cgagataagg cagcagatca agaaagcact ccgggctcca
60gaaggagcct tccaggccag ctttgagcat aagctgctga tgagcagtga gtgtcttgag
120tagtgttcag ggcagcatgt taccattcat gcttgacttc tagccagtgt
gacgagaggc 180tggagtcagg tctctagaga gttgagcagc tccagcctta
gatctcccag tcttatgcgg 240tgtgcccatt cgctttgtgt ctgcagtccc
ctggccacac ccagtaacag ttctgggatc 300tatgggagta gcttccttag
tgagctttcc cttcaaatac tttgcaacca ggtagagaat 360tttggagtga
aggttttgtt cttcgtttct tcacaatatg gatatgcatc ttcttttgaa
420aatgttaaag taaattacct ctcttttcag atactgtctt catgcgaact
tggtatcctg 480tttccatccc agccttctat aacccagtaa catctttttt
gaaaccagtg ggtgagaaag 540acacctggtc aggaacgcgg accacaggac
aactcaggct cacccacggc atcagactaa 600aggcaaacaa ggactctgta
taaagtaccg gtggcatgtg tattagtgga gatgcagcct 660gtgctctgca
gacagggagt cacacagaca cttttctata atttcttaag tgctttgaat
720gttcaagtag aaagtctaac attaaatttg attgaacaat tgtatattca
tggaatattt 780tggaacggaa taccaaaaaa tggcaatagt ggttctttct
ggatggaaga caaacttttc 840ttgtttaaaa taaattttat tttatatatt
tgaggttgac cacatgacct taaggataca 900tatagacagt aaactggtta
ctacagtgaa gcaaattaac atatctacca tcgtacatag 960ttacattttt
ttgtgtgaca ggaacagcta aaatctacgt atttaacaaa aatcctaaag
1020acaatacatt tttattaact atagccctca tgatgtacat tagatctcta a
1071411135DNAHomo sapiensmisc_featuresequence of STAR41
41cgtgtgcagt ccacggagag tgtgttctcc tcatcctcgt tccggtggtt gtggcgggaa
60acgtggcgct gcaggacacc aacatcagtc acgtatttca ttctggaaaa aaaagtagca
120caagcctcgg ctggttccct ccagctctta ccaggcagcc taagcctagg
ctccattccc 180gctcaaggcc ttcctcaggg gcctgctcac cacaggagct
gttcccatgc agggactaag 240gacatgcagc ctgcatagaa accaagcacc
caggaaaaca tgattggatg gagcgggggg 300gtgtggtctc tagccttgtc
cacctccggt cctcatgggt ctcacacctc ctgagaatgg 360gcaccgcaga
ggccacagcc catacagcca agatgacaga ctccgtaagt gacagggatc
420cacagcagag tgggtgaaat gttccctata aactttacaa aattaatgag
ggcaggggga 480ggggagaaat gaaaatgaac ccagctcgca gcacatcagc
atcagtcact aggtcggcgt 540gctctctgac tgcttcctcg tagctgcttg
gtgtctcatt gcctcagaag catgtagacc 600ctgtcacaag attgtagttc
ccctaactgc tccgtagatc acaacttgaa ccttaggaaa 660tgctgttttc
cctttgagat attcctttgg gtcctgtata ctgatggagc tactgactga
720gctgctccga aggaccccac gaggagctga ctaaaccaag agtgcagttt
gtacaccctg 780atgattacat cccccttgcc ccaccaatca actctcccaa
ttttccagcc cctcaccctc 840cagtcccctt aaaagcccca gcccaggccg
ggcacagtgg ctcatgcctg taatcccagc 900actttgggag gccaaggtgg
gcagatcacc tgagggcagg aatttgagac cagcctgacc 960aacatgaaga
aaccccgtct ctattacaaa tacaaaatta gccgggcgtg ttgctgcata
1020ctggtaatcc cagctacttg ggagggtgag gcaggagaat cacttgaatc
tgggaggcgg 1080aggttgcgat gagccgagac agcgccattg cactgcagcc
tgggcaacaa gagca 113542735DNAHomo sapiensmisc_featuresequence of
STAR42 42aagggtgaga tcactaggga gggaggaagg agctataaaa gaaagaggtc
actcatcaca 60tcttacacac tttttaaaac cttggttttt taatgtccgt gttcctcatt
agcagtaagc 120cctgtggaag caggagtctt tctcattgac caccatgaca
agaccctatt tatgaaacat 180aatagacaca caaatgttta tcggatattt
attgaaatat aggaattttt cccctcacac 240ctcatgacca cattctggta
cattgtatga atgaatatac cataatttta cctatggctg 300tatatttagg
tcttttcgtg caggctataa aaatatgtat gggccggtca cagtgactta
360cgcccgtagt cccagaactt tgggaggccg aggcgggtgg atcacctgag
gtcgggagtt 420caaaaccagc ctgaccaaca tggagaaacc ccgtctctgc
taaaaataca aaaattaact 480ggacacggtg gcgtatgcct gtaatcccag
ctactcggga agctgaggca ggagaactgc 540ttgaacccag gaggcggagg
ttgtggtgag tcgagattgc gccattgcac tccagcctgg 600gcaacaagag
cgaaattcca tctcaaaaaa aagaaaaaag tatgactgta tttagagtag
660tatgtggatt tgaaaaatta ataagtgttg ccaacttacc ttagggttta
taccatttat 720gagggtgtcg gtttc 735431227DNAHomo
sapiensmisc_featuresequence of STAR43 43caaatagatc tacacaaaac
aagataatgt ctgcccattt ttccaaagat aatgtggtga 60agtgggtaga gagaaatgca
tccattctcc ccacccaacc tctgctaaat tgtccatgtc 120acagtactga
gaccaggggg cttattccca gcgggcagaa tgtgcaccaa gcacctcttg
180tctcaatttg cagtctaggc cctgctattt gatggtgtga aggcttgcac
ctggcatgga 240aggtccgttt tgtacttctt gctttagcag ttcaaagagc
agggagagct gcgagggcct 300ctgcagcttc agatggatgt ggtcagcttg
ttggaggcgc cttctgtggt ccattatctc 360cagcccccct gcggtgttgc
tgtttgcttg gcttgtctgg ctctccatgc cttgttggct 420ccaaaatgtc
atcatgctgc accccaggaa gaatgtgcag gcccatctct tttatgtgct
480ttgggctatt ttgattcccc gttgggtata ttccctaggt aagacccaga
agacacagga 540ggtagttgct ttgggagagt ttggacctat gggtatgagg
taatagacac agtatcttct 600ctttcatttg gtgagactgt tagctctggc
cgcggactga attccacaca gctcacttgg 660gaaaacttta ttccaaaaca
tagtcacatt gaacattgtg gagaatgagg gacagagaag 720aggccctaga
tttgtacatc tgggtgttat gtctataaat agaatgcttt ggtggtcaac
780tagacttgtt catgttgaca tttagtcttg ccttttcggt ggtgatttaa
aaattatgta 840tatcttgttt ggaatatagt ggagctatgg tgtggcattt
tcatctggct ttttgtttag 900ctcagcccgt cctgttatgg gcagccttga
agctcagtag ctaatgaaga ggtatcctca 960ctccctccag agagcggtcc
cctcacggct cattgagagt ttgtcagcac cttgaaatga 1020gtttaaactt
gtttattttt aaaacattct tggttatgaa tgtgcctata ttgaattact
1080gaacaacctt atggttgtga agaattgatt tggtgctaag gtgtataaat
ttcaggacca 1140gtgtctctga agagttcatt tagcatgaag tcagcctgtg
gcaggttggg tggagccagg 1200gaacaatgga gaagctttca tgggtgg
1227441586DNAHomo sapiensmisc_featuresequence of STAR44
44cacctgcctc agcctcccaa agtgctgaga ttcaaagaaa ttttcatgga gaggggacag
60atggagtcaa ttcttgtggg gtgaacatga gtaccacagt tagactgagg ttgggaaaga
120ttttccagac aattggaaga gcatgtgaaa gacacagatt ttgagaaatg
ttaagtctag 180ggaactgcaa ggcttttggc acaagaaagc cactgtagac
tatagaggca ggatgcctag 240attcaaatcc caactgctac acttctaagc
tttgtaattt tggcaagttt ttaccctcta 300ttttcttatc tataaaatat
agattttata tatatagata tagatatata gatagataat 360aattgtgcat
gcctaataaa gttgtcaaag attaaatgtt atatgtgaag tattttgtac
420ggtgatagga acccaggaag ggctctatga atattatgta ttattattat
tctaaagtag 480ctggaataca atgttcaaag gagatagtgg caggagataa
gtttgaattg aaagattgag 540gccagaacat aaagtgcctc ctatattata
ttttacataa ttggaacatc attgaaaaat 600ttaagtatta tttatgtgtg
tatgtgtgtt
ttatataatt aattctagtt catcatttta 660aaatatcttt ctgatgtcac
tgtgaacaac agatgagaag aagtgaatcc tgagttaagg 720agaccagctc
tctgattact gccataatcc agggagggta ccataaggat ttcaactgga
780agtgaatcca tcatgatgga gaggaaggac agggctgaaa aatacttagg
aagtagtatc 840agtaggactg gttaagagag agcagaggca ggctacaggg
gttggaggtg tcaatcacag 900agatagggaa aatgggagga gaagcaggct
ttgaaaaagt ggcttgtctt gtaaaattat 960gtgctgttaa aacagtacaa
gaaattaata tattcaatcc caaaatacag ggacaattct 1020ttttgaaaga
gttacccaga tagtcttcct tgaagttttc agttaaagaa atttcttgtt
1080aacaaataat gtagtcatag aagaaaacac ttaaaacttt attgaataaa
gctaataaat 1140catttaatat aatttatagg aaattgttac ataacacaca
cattcaatac tttttgctaa 1200agtataaatt aatggaagga gagcacgcac
acagaggttg aattatgttt atgactttat 1260tagtcaagaa tacaaaattg
agtagctaca tcaagcagaa gcacatgctt tacaatccag 1320cacagaatcc
cttgacatcc aaactcccga aacagacatg taaatacaga tgacattgtc
1380agaacaaaat agggtctcac ccgacctata atgttctttt cttgatataa
atatgcacat 1440gaattgcata cggtcatatg gttccaatta ccattatttc
ctctgggctt agctatccat 1500ctaaggggaa tttacaccaa cactgtactt
ctacttgcaa gaatatatga aagcatagtt 1560aacttctggc ttaggacccc aactca
1586451981DNAHomo sapiensmisc_featuresequence of STAR45
45atggatcata gggtaaataa atttataatt tcttgagaaa gcttcgtact gttttccaag
60atggctgtac taatttccat tcctaccaac agtgtacagg gtttcttttt ctccacatcc
120tcaccaacac ttatcttcca tcttttttta taatagccct agtaaaatgt
gtgaggtgat 180atctcattgt ggcattgatt tgcacttctc tgataattag
gaatgtttat gattttttca 240tgtacctggt tggccttttg tatgatgtag
gaaatgtcta ttctgattct ttgcttattt 300tttaataagc atagtttttt
tcttattttt gagtaggttg agttgcttat atattattat 360atgagcccct
tacctgatgt atggtttaaa aatattatcc catttgtggg ttctcttaat
420tctatcattg cttcttttcc tgtggaaaag ttttaagttt tatgcagtct
catttgtgtg 480ttttgctttt gttgcctttt ggaataatct acagaaaatc
atagctcagg ccaatgtcat 540acagtctcct tctatatttc cttgtagtag
ttttacattt aaactttaat tttgatttga 600tgcttgtata aagagcaaaa
taaaagtcaa attttattct tctgtatgtg gatagtcagt 660tttgtctaca
ccatttattg aaaataattt tctttcttca ctgtgtattt ttagttattt
720tatcaaaaaa tcaattgacc acagacacac ggatttattt acaggttcta
tatccctttg 780tactgtttta catgtctgtt tttatgccat tgctatgctg
ttttaattcc tatagctttg 840taatagagtt tggagtcagg tagtctgatg
cctccagctt tgttcttttt gttcaagatt 900gctttggttg gtccaggtct
tttgtggttc catacaaatt ttagcagtaa tttttctatt 960tctgtgaaga
atgacattgg aatttgatag tggttgcatt taatctgtag attgctttgg
1020gtagcattga cacttttaca atactaattt ttgaatccat caatgaagga
tgtttctcca 1080tttatttatg ccattttaat ttttttcatc aatgtgctat
agttttcagt atgtaaatct 1140tttatggttt tgattaaatt tactcctgtc
ttttatatat ttatatatct gttttgattc 1200tattataaat tgaattgcct
ttatttttca ggtaatagtt tgtcattagt taatagaaac 1260aataatgata
tttgtatgtt gattttgtaa ctattaactt tattgaattt cttcatcagc
1320tataaccatt tattttggtg gaatctttaa gattttctct atcttaagat
tatattttca 1380aaaaacagaa acaatcttac ctcttccttc cctatgtgga
tttcttttac gtctttgtct 1440tgtgtaactg ttctggctag gcaattacac
ataatgtttt catcatttat aattttacat 1500cacatccatc tattgtggca
cattgattgc tacttttcaa gttgtaaacc tggacattta 1560tcactactct
tcctccaata caggagtcca tggcgtggtg tgggccctac tgtgccacag
1620tccagggcac ggctgggctg aggttctctt gtgcaagagt ccgtggctct
gcggagcaag 1680agttctccag tgccttagtc cagggttagg caggggtggg
gctccttcag tagcttagtc 1740cagtgcgccg ccctgcgagg gtcctcctga
gcaggagtac acgatgaggc agggtcctac 1800tgtgccttag cccaggaagc
ggggggctgg gtcctctggt gccatagtcc aggctgccgg 1860gagctgggtc
ctctggtgcc atagctcagg ccggcgggag ctgggtcctc tggtgccgta
1920gtccagggtg cagcagaaca ggagtcctgc ggagcagtag tccagggcac
gctggggcgt 1980g 1981461859DNAHomo sapiensmisc_featuresequence of
STAR46 46attgtttttc tcgcccttct gcattttctg caaattctgt tgaatcattg
cagttactta 60ggtttgcttc gtctccccca ttacaaacta cttactgggt ttttcaaccc
tagttccctc 120atttttatga tttatgctca tttctttgta cacttcgtct
tgctccatct cccaactcat 180ggcccctggc tttggattat tgttttggtc
ttttattttt tgtcttcttc tacctcaaca 240cttatcttcc tctcccagtc
tccggtaccc tatcaccaag gttgtcatta acctttcata 300ttattcctca
ttatccatgt attcatttgc aaataagcgt atattaacaa aatcacaggt
360ttatggagat ataattcaca taccttaaaa ttcaggcttt taaagtgtac
ctttcatgtg 420gtttttggta tattcacaaa gttatgcatt gatcaccacc
atctgattcc ataacatgtt 480caatacctca aaaagaagtc tgtactcatt
agtagtcatt tcacattcac cactccctct 540ggctctgggc agtcactgat
ctttgtgtct ctatggattt gcctagtcta ggtattttta 600tgtaaatggc
atcatacaac atgtgacctt ttgtttggct tttttcattt agcaaaatgt
660tatcaaggtc tgtccctgtt gtagcatgta ttagcacttc atttcttata
tgctgaatga 720tatactttat ttgtccatca gttgttcatg ctttatttgt
ccatcagttg atgaacattt 780gcgtttttgc cactttgggc tattaagaat
aatgctactg tgaacaagtg tgtacaagtt 840cctctacaaa tttttgtgtg
gacatatcct ttcagttctc tcaggtgtat atctgggaat 900tgaattgctg
ggtcgtgtag tagctatgtt aaacactttg agaaactgct ataatgttct
960ccagagctgt accattttaa attctgtgta tgaggattcc acgttctcca
cttcctcacc 1020agtgtatgga tttgggggta tactttttaa aaagtgggat
taggctgggc acagtggctc 1080acacctgtaa tcccaacact tcaggaagct
gaggtgggag gatcacttga gcctagtagt 1140ttgagaccag cctgggcaac
atagggagac cctgtctcta caaaaaataa tttaaaataa 1200attagctggg
cgttgtggca cacacctgta gtcccagcta catgggaggc tgaggtggaa
1260ggattccctg agcccagaag tttgaggttg cagtgagcca tgatggcagc
actatactgt 1320agcctgggtg tcagagcaag actccgtttc agggaagaaa
aaaaaaagtg ggatgatatt 1380tttgacactt ttcttcttgt tttcttaatt
tcatacttct ggaaattcca ttaaattagc 1440tggtaccact ctaactcatt
gtgtttcatg gctgcatagt aatattgcat aatataaata 1500taccattcat
tcatcaaagt tagcagatat tgactgttag gtgccaggca ctgctctaag
1560cgttaaagaa aaacacacaa aaacttttgc attcttagag tttattttcc
aatggagggg 1620gtggagggag gtaagaattt aggaaataaa ttaattacat
atatagcata gggtttcacc 1680agtgagtgca gcttgaatcg ttggcagctt
tcttagtagt ataaatacag tactaaagat 1740gaaattactc taaatggtgt
tacttaaatt actggaatag gtattactat tagtcacttt 1800gcaggtgaaa
gtggaaacac catcgtaaaa tgtaaaatag gaaacagctg gttaatgtt
1859471082DNAHomo sapiensmisc_featuresequence of STAR47
47atcattagtc attagggaaa tgcaaatgaa aaacacaagc agccaccaat atacacctac
60taggatgatt taaaggaaaa taagtgtgaa gaaggacgta aagaaattgt aaccctgata
120cattgatggt agaaatggat aaagttgcag ccactgtgaa aaacagtctg
cagtggctca 180gaaggttaaa tatagaaccc ctgttggacc caggaactct
actcttaggc accccaaaga 240atagagaaca gaaatcaaac agatgtttgt
atactaatgt ttgtagcatc acttttcaca 300ggagccaaaa ggtggaaata
atccaaccat cagtgaacaa atgaatgtaa taaaagcaag 360gtggtctgca
tgcaatgcta catcatccat ctgtaaaaaa cgaacatcat tttgatagat
420gatacaacat gggtggacat tgagaacatt atgcttagtg aaataagcca
gacacaaaag 480gaatatattg tataattgta attacatgaa gtgcctagaa
tagtcaaatt catacaagag 540aaagtgggat aggaatcacc atgggctgga
aataggggga aggtgctata ctgcttattg 600tggacaaggt ttcgtaagaa
atcatcaaaa ttgtgggtgt agatagtggt gttggttatg 660caaccctgtg
aatatattga atgccatgga gtgcacactt tggttaaaag gttcaaatga
720taaatattgt gttatatata tttccccacg atagaaaaca cgcacagcca
agcccacatg 780ccagtcttgt tagctgcctt cctttacctt caagagtggg
ctgaagcttg tccaatcttt 840caaggttgct gaagactgta tgatggaagt
catctgcatt gggaaagaaa ttaatggaga 900gaggagaaaa cttgagaatc
cacactactc accctgcagg gccaagaact ctgtctccca 960tgctttgctg
tcctgtctca gtatttcctg tgaccacctc ctttttcaac tgaagacttt
1020gtacctgaag gggttcccag gtttttcacc tcggcccttg tcaggactga
tcctctcaac 1080ta 1082481242DNAHomo sapiensmisc_featuresequence of
STAR48 48atcatgtatt tgttttctga attaattctt agatacatta atgttttatg
ttaccatgaa 60tgtgatatta taatataata tttttaattg gttgctactg tttataagaa
tttcattttc 120tgtttacttt gccttcatat ctgaaaacct tgctgatttg
attagtgcat ccacaaattt 180tcttggattt tctatgggta attacaaatc
tccacacaat gaggttgcag tgagccaaga 240tcacaccact gtactccagc
ctgggcgaca gagtgagaca ccatctcaca aaaacacata 300aacaaacaaa
cagaaactcc acacaatgac aacgtatgtg ctttcttttt ttcttcctct
360ttctataata tttctttgtc ctatcttaac tgaactggcc agaaacccca
ggacaatgat 420aaatacgagc agtgtcaaca gacatctcat tccctttcct
agcttttata aaaataacga 480ttatgcttca acattacata tggtggtgtc
gatggttttg ttatagataa gcttatcagg 540ttaagaaatt tgtctgcgtt
tcctagtttg gtataaagat tttaatataa atgaatgttg 600tattttatca
tcttattttt ttcctacatc tgctaaggta atcctgtgtt ttcccctttt
660caatctccta atgtggtgaa tgacattaaa ataccttcta ttgttaaaat
attcttgcaa 720cgctgtatag aaccaatgcc tttattctgt attgctgatg
gatttttgaa aaatatgtag 780gtggacttag ttttctaagg ggaatagaat
ttctaatata tttaaaatat tttgcatgta 840tgttctgaag gacattggtg
tgtcatttct ataccatctg gctactagag gagccgactg 900aaagtcacac
tgccggagga ggggagaggt gctcttccgt ttctggtgtc tgtagccatc
960tccagtggta gctgcagtga taataatgct gcagtgccga cagttctgga
aggagcaaca 1020acagtgattt cagcagcagc agtattgcgg gatccccacg
atggagcaag ggaaataatt 1080ctggaagcaa tgacaatatc agctgtggct
atagcagctg agatgtgagt tctcacggtg 1140gcagcttcaa ggacagtagt
gatggtccaa tggcgcccag acctagaaat gcacatttcc 1200tcagcaccgg
ctccagatgc tgagcttgga cagctgacgc ct 1242491015DNAHomo
sapiensmisc_featuresequence of STAR49 49aaaccagaaa cccaaaacaa
tgggagtgac atgctaaaac cagaaaccca aaacaatggg 60agggtcctgc taaaccagaa
acccaaaaca atgggagtga agtgctaaaa ccagaaaccc 120aaaacaatgg
gagtgtcctg ctacaccaga aacccaaaac gatgggagtg acgtgataaa
180accagacacc caaaacaatg ggagtgacgt gctaaaccag aaacccaaaa
caatgggagt 240gacgtgctaa aacctggaaa cctaaaacaa tgcgagtgag
gtgctaacac cagaatccat 300aacaatgtga gtgacgtgct aaaccagaac
ccaaaacaat gggagtgacg tgctaaaaca 360ggaacccaaa acaatgagag
tgacgtgcta aaccagaaac ccaaaacaat gggaatgacg 420tgctaaaacc
ggaacccaaa acaatgggag tgatgtgcta aaccagaaac ccaaaacaat
480gggaatgaca tgctaaaact ggaacccaaa acaatggtaa ctaagagtga
tgctaaggcc 540ctacattttg gtcacactct caactaagtg agaacttgac
tgaaaaggag gatttttttt 600tctaagacag agttttggtc tgtcccccag
agtggagtgc agtggcatga tctcggctca 660ctgcaagctc tgcctcccgg
gttcaggcca ttctcctgcc tcagcctcct gagtagctgg 720gaatacaggc
acccgccacc acacttggct aattttttgt atttttagta gagatggggt
780ttcaccatat tagcaaggat ggtctcaatc tcctgacctc gtgatctgcc
cacctcaggc 840tcccaaagtg ctgggattac aggtgtgagc caccacaccc
agcaaaaagg aggaattttt 900aaagcaaaat tatgggaggc cattgttttg
aactaagctc atgcaatagg tcccaacaga 960ccaaaccaaa ccaaaccaaa
atggagtcac tcatgctaaa tgtagcataa tcaaa 1015502355DNAHomo
sapiensmisc_featuresequence of STAR50 50caaccatcgt tccgcaagag
cggcttgttt attaaacatg aaatgaggga aaagcctagt 60agctccattg gattgggaag
aatggcaaag agagacaggc gtcattttct agaaagcaat 120cttcacacct
gttggtcctc acccattgaa tgtcctcacc caatctccaa cacagaaatg
180agtgactgtg tgtgcacatg cgtgtgcatg tgtgaaagta tgagtgtgaa
tgtgtctata 240tgggaacata tatgtgattg tatgtgtgta actatgtgtg
actggcagcg tggggagtgc 300tggttggagt gtggtgtgat gtgagtatgc
atgagtggct gtgtgtatga ctgtggcggg 360aggcggaagg ggagaagcag
caggctcagg tgtcgccaga gaggctggga ggaaactata 420aacctgggca
atttcctcct catcagcgag cctttcttgg gcaatagggg cagagctcaa
480agttcacaga gatagtgcct gggaggcatg aggcaaggcg gaagtactgc
gaggaggggc 540agagggtctg acacttgagg ggttctaatg ggaaaggaaa
gacccacact gaattccact 600tagccccaga ccctgggccc agcggtgccg
gcttccaacc ataccaacca tttccaagtg 660ttgccggcag aagttaacct
ctcttagcct cagtttcccc acctgtaaaa tggcagaagt 720aaccaagctt
accttcccgg cagtgtgtga ggatgaaaag agctatgtac gtgatgcact
780tagaagaagg tctagggtgt gagtggtact cgtctggtgg gtgtggagaa
gacattctag 840gcaatgagga ctggggagag cctggcccat ggcttccact
cagcaaggtc agtctcttgt 900cctctgcact cccagccttc cagagaggac
cttcccaacc agcactcccc acgctgccag 960tcacacatag ttacacacat
acaatcacat atatgttccc atatagacac attcacactc 1020ataccttcac
acatgcacac gcatgtgcac acacagtcac tcatttctgt gttggagatt
1080gggtgaggac attcaatggg tgaggaccaa caggtgtgaa gattgctttc
tagaaaatga 1140ctcctgtctc tctttgccat tcttcccaat ccgatggagc
tactaggctt ttccctcatt 1200tcatgtttaa taaaccttcc caatggcgaa
atgggctttc tcaagaagtg gtgagtgtcc 1260catccctgcg gtggggacag
gggtggcagc ggacaagcct gcctggaggg aactgtcagg 1320ctgattccca
gtccaactcc agcttccaac acctcatcct ccaggcagtc ttcattcttg
1380gctctaattt cgctcttgtt ttctttttta tttttatcga gaactgggtg
gagagctttt 1440ggtgtcattg gggattgctt tgaaaccctt ctctgcctca
cactgggagc tggcttgagt 1500caactggtct ccatggaatt tcttttttta
gtgtgtaaac agctaagttt taggcagctg 1560ttgtgccgtc cagggtggaa
agcagcctgt tgatgtggaa ctgcttggct cagatttctt 1620gggcaaacag
atgccgtgtc tctcaactca ccaattaaga agcccagaaa atgtggcttg
1680gagaccacat gtctggttat gtctagtaat tcagatggct tcacctggga
agccctttct 1740gaatgtcaaa gccatgagat aaaggacata tatatagtag
ctagggtggt ccacttctta 1800ggggccatct ccggaggtgg tgagcactaa
gtgccaggaa gagaggaaac tctgttttgg 1860agccaaagca taaaaaaacc
ttagccacaa accactgaac atttgttttg tgcaggttct 1920gagtccaggg
agggcttctg aggagagggg cagctggagc tggtaggagt tatgtgagat
1980ggagcaaggg ccctttaaga ggtgggagca gcatgagcaa aggcagagag
gtggtaatgt 2040ataaggtatg tcatgggaaa gagtttggct ggaacagagt
ttacagaata gaaaaattca 2100acactattaa ttgagcctct actacgtgct
cgacattgtt ctagtcactg agataggttt 2160ggtatacaaa acaaaatcca
tcctctatgg acattttagt gactaacaac aatataaata 2220ataaaagtga
acaaaagctc aaaacatgcc aggcactatt atttatttat ttatttattt
2280atttatttat tttttgaaac agagtctcgc tctgttgccc aggctggagt
gtagtggtgc 2340gatctcggct cactg 2355512289DNAHomo
sapiensmisc_featuresequence of STAR51 51tcacaggtga caccaatccc
ctgaccacgc tttgagaagc actgtactag attgactttc 60taatgtcagt cttcattttc
tagctctgtt acagccatgg tctccatatt atctagtaca 120acacacatac
aaatatgtgt gatacagtat gaatataata taaaaatatg tgttataata
180taaatataat attaaaatat gtctttatac tagataataa tacttaataa
cgttgagtgt 240ttaactgctc taagcacttt acctgcagga aacagttttt
tttttatttt ggtgaaatac 300aactaacata aatttattta caattttaag
catttttaag tgtatagttt agtggagtta 360atatattcaa aatgttgtgc
agccgtcacc atcatcagtc ttcataactc ttttcatatt 420gtaaaattaa
aagtttatgc tcatttaaaa atgactccca atttcccccc tcctcaacct
480ctggaaacta ccattctatt ttctgcctcc gtagttttgc ccactctaag
tacctcacat 540aagtggaatt tgtcttattt gcctgtttgt gaccggctga
tttcatttag tataatgtcc 600tcaagtttta ttcacgttat atagcatatg
tcataatttt cttcactttt aagcttgagt 660aatatttcat cgtatgtatc
tcacattttg cttatccatt catctctcag tggacacttg 720agttgcttct
acattttagc tgttgtgaat actgctgcta tgaacatggg tgtataaata
780tctcaagacc tttttatcag ttttttaaaa tatatactca gtagtagttt
agctggatta 840tatggtaatt ttatttttaa tttttgagga actgtcctac
ccttttattc aatagtagct 900ataccaattg acaattggca ttcctaccaa
cagggcataa gggttctcaa ttctccacat 960attccctgat acttgttatt
ttcaggtgtt tttttttttt tttttttttt atgggagcca 1020tgttaatggg
tgtaaggtga tatttcatta tagttttgat ttgcatttcc ctaatgatta
1080gtgatgttaa gcatctcttc atgtgcctat tggccatttg tatatcttct
ttaaaaatat 1140atatatactc attcctttgc ccatttttga attatgttta
ttttttgtta ttgagtttca 1200atacttttct atataaccta ggtattaatc
ctttatcaga cttaagattt gcaaatattc 1260tctttcattc cacaggttgc
taattctctc tgttggtaat atcttttgat gctgttgtgt 1320ccagaattga
ttcattcctg tgggttcttg gtctcactga cttcaagaat aaagctgcgg
1380accctagtgg tgagtgttac acttcttata gatggtgttt ccggagtttg
ttccttcaga 1440tgtgtccaga gtttcttcct tccaatgggt tcatggtctt
gctgacttca ggaatgaagc 1500cgcagacctt cgcagtgagg tttacagctc
ttaaaggtgg cgtgtccaga gttgtttgtt 1560ccccctggtg ggttcgtggt
cttgctgact tcaggaatga agccgcagac cctcgcagtg 1620agtgttacag
ctcataaagg tagtgcggac acagagtgag ctgcagcaag atttactgtg
1680aagagcaaaa gaacaaagct tccacagcat agaaggacac cccagcgggt
tcctgctgct 1740ggctcaggtg gccagttatt attcccttat ttgccctgcc
cacatcctgc tgattggtcc 1800attttacaga gtactgattg gtccatttta
cagagtgctg attggtgcat ttacaatcct 1860ttagctagac acagagtgct
gattgctgca ttcttacaga gtgctgattg gtgcatttac 1920agtcctttag
ctagatacag aacgctgatt gctgcgtttt ttacagagtg ctgattggtg
1980catttacaat cctttagcta gacacagtgc tgattggtgg gtttttacag
agtgctgatt 2040ggtgcgtctt tacagagtgc tgattggtgc atttacaatc
ctttagctag acacagagtg 2100ctgattggtg cgtttataat cctctagcta
gacagaaaag ttttccaagt ccccacctga 2160ccgagaagcc ccactggctt
cacctctcac tgttatactt tggacatttg tccccccaaa 2220atctcatgtt
gaaatgtaac ccctaatgtt ggaactgagg ccagactgga tgtggctggg
2280ccatgggga 2289521184DNAHomo sapiensmisc_featuresequence of
STAR52 52ctcttctttg tttttttatt ttggggtgtg tgggtacgtg taagatgaga
aatgtacaaa 60cacaagtatt tcagaaactc caagtaatat tctgtctgtg agttcacggt
aaataaataa 120aaagggcaaa gtgacagaaa tacaggatta ttaaaagcaa
aataatgttc tttgaaatcc 180cccccttggt gtatttttta tcttaggatg
cagcactttc agcatgccca agtattgaaa 240gcagtgtttt tacgctacca
cggtaatttt atttagaaac cccatgttca cttttagttt 300taaaatggtc
tttatgacat aaaattatca gcattcatat ttttgtgttt taatattcct
360ttggctactt attgaaacag taaacattac gaaaattagt aaacaaatct
ttgatagttg 420cttatttttg tttaattgaa tgtttatttt attaggtaaa
tatacaatca aatttattta 480aaaataatga ggaaaagaat acttttcttt
cgctttgcga aagcaaagtg atttttcatt 540cttctccgtc cgattccttc
tcttccagct gccacagccg actgacaggc tcccggcggc 600ctgaggagta
gtatgcaaat tttggatgat tgacacctac agtagaagcc aatcacgtca
660aagtaggatg ctgattggtt gacaacaata ggcgtaaacc ttgacgtttt
aaaaacctga 720cacccaatcc aggcgattca tgcaaataaa ggaagggagt
cacattacca ggggccagag 780agacttgagt acgacctcac gtgttcagtg
gtggatattg cacagacgtc tgcaaggtct 840atataaacgc tacataatgt
tcaactcaat tgcttgcctt ggcctttccc aaacttgtca 900ctggaatata
aattatccct tttttaaaaa taaaaaaata agaattatgt agtgcacata
960tatgatggtt catgtagaaa tctaaatgga cttccaacgc atggaatttt
cctatttccc 1020cctttcttta aattaatcct cagtgaagga ggctgttttc
ccctagattt caaaaggacg 1080agatttacag agcctttcct tggagaaacc
cgctctaggc acagatggtc agtaaattta 1140gcttcttcag cgaagttcca
catggcaccg ccagatggca taag 1184531431DNAHomo
sapiensmisc_featuresequence of STAR53 53ccctgaggaa gatgacgagt
aactccgtaa gagaaccttc cactcatccc ccacatccct 60gcagacgtgc tattctgtta
tgatactggt atcccatctg tcacttgctc cccaaatcat 120tcccttctta
caattttcta ctgtacagca ttgaggctga acgatgagag atttcccatg
180ctctttctac tccctgccct gtatatatcc ggggatcctc cctacccagg
atgctgtggg 240gtcccaaacc ccaagtaagc cctgatatgc
gggccacacc tttctctagc ctaggaattg 300ataacccagg cgaggaagtc
actgtggcat gaacagatgg ttcacttcga ggaaccgtgg 360aaggcgtgtg
caggtcctga gatagggcag aatcggagtg tgcagggtct gcaggtcagg
420aggagttgag attgcgttgc cacgtggtgg gaactcactg ccacttattt
ccttctctct 480tcttgcctca gcctcaggga tacgacacat gcccatgatg
agaagcagaa cgtggtgacc 540tttcacgaac atgggcatgg ctgcggaccc
ctcgtcatca ggtgcatagc aagtgaaagc 600aagtgttcac aacagtgaaa
agttgagcgt catttttctt agtgtgccaa gagttcgatg 660ttagcgttta
cgttgtattt tcttacactg tgtcattctg ttagatacta acattttcat
720tgatgagcaa gacatactta atgcatattt tggtttgtgt atccatgcac
ctaccttaga 780aaacaagtat tgtcggttac ctctgcatgg aacagcatta
ccctcctctc tccccagatg 840tgactactga gggcagttct gagtgtttaa
tttcagattt tttcctctgc atttacacac 900acacgcacac aaaccacacc
acacacacac acacacacac acacacacac acacacacac 960acacaccaag
taccagtata agcatctgcc atctgctttt cccattgcca tgcgtcctgg
1020tcaagctccc ctcactctgt ttcctggtca gcatgtactc ccctcatccg
attcccctgt 1080agcagtcact gacagttaat aaacctttgc aaacgttccc
cagttgtttg ctcgtgccat 1140tattgtgcac acagctctgt gcacgtgtgt
gcatatttct ttaggaaaga ttcttagaag 1200tggaattgct gtgtcaaagg
agtcatttat tcaacaaaac actaatgagt gcgtcctcgt 1260gctgagcgct
gttctaggtg ctggagcgac gtcagggaac aaggcagaca ggagttcctg
1320acccccgttc tagaggagga tgtttccagt tgttgggttt tgtttgtttg
tttcttctag 1380agatggtggt cttgctctgt ccaggctaga gtgcagtggc
atgatcatag c 143154975DNAHomo sapiensmisc_featuresequence of STAR54
54ccataaaagt gtttctaaac tgcagaaaaa tccccctaca gtcttacagt tcaagaattt
60tcagcatgaa atgcctggta gattacctga ctttttttgc caaaaataag gcacagcagc
120tctctcctga ctctgacttt ctatagtcct tactgaatta tagtccttac
tgaattcatt 180cttcagtgtt gcagtctgaa ggacacccac attttctctt
tgtctttgtc aattctttgt 240gttgtaaggg caggatgttt aaaagttgaa
gtcattgact tgcaaaatga gaaatttcag 300agggcatttt gttctctaga
ccatgtagct tagagcagtg ttcacactga ggttgctgct 360aatgtttctg
cagttcttac caatagtatc atttacccag caacaggata tgatagagga
420cttcgaaaac cccagaaaat gttttgccat atatccaaag ccctttggga
aatggaaagg 480aattgcgggc tcccattttt atatatggat agatagagac
caagaaagac caaggcaact 540ccatgtgctt tacattaata aagtacaaaa
tgttaacatg taggaagtct aggcgaagtt 600tatgtgagaa ttctttacac
taattttgca acattttaat gcaagtctga aattatgtca 660aaataagtaa
aaatttttac aagttaagca gagaataaca atgattagtc agagaaataa
720gtagcaaaat cttcttctca gtattgactt ggttgctttt caatctctga
ggacacagca 780gtcttcgctt ccaaatccac aagtcacatc agtgaggaga
ctcagctgag actttggcta 840atgttggggg gtccctcctg tgtctcccca
ggcgcagtga gcctgcaggc cgacctcact 900cgtggcacac aactaaatct
ggggagaagc aacccgatgc cagcatgatg cagatatctc 960agggtatgat cggcc
97555501DNAHomo sapiensmisc_featuresequence of STAR55 55cctgaactca
tgatccgccc acctcagcct cctgaagtgc tgggattaca ggtgtgagcc 60accacaccca
gccgcaacac actcttgagc aaccaatgtg tcataaaaga aataaaatgg
120aaatcagaaa gtatcttgag acagacaaaa atggaaacac aacataccaa
aatttatggg 180acacagcaaa agcagtttta ggagggaagt ttatagtgat
gaatacctac ctcaaaatca 240ttagcctgat tggatgacac tacagtgtat
aaatgaattg aaaaccacat tgtgccccat 300acatatatac aatttttatt
tgttaattaa aaataaaata aaactttaaa aaagaagaaa 360gagctcaaat
aaacaaccta actttatacc tcaaggaaat agaagagcca gctaagccca
420aagttgacag aaggaaaaaa atattggcag aaagaaatga aacagagact
agaaagacaa 480ttgaagagat cagcaaaact a 50156741DNAHomo
sapiensmisc_featuresequence of STAR56 56acacaggaaa agatcgcaat
tgttcagcag agctttgaac cggggatgac ggtctccctc 60gttgcccggc aacatggtgt
agcagccagc cagttatttc tctggcgtaa gcaataccag 120gaaggaagtc
ttactgctgt cgccgccgga gaacaggttg ttcctgcctc tgaacttgct
180gccgccatga agcagattaa agaactccag cgcctgctcg gcaagaaaac
gatggaaaat 240gaactcctca aagaagccgt tgaatatgga cgggcaaaaa
agtggatagc gcacgcgccc 300ttattgcccg gggatgggga gtaagcttag
tcagccgttg tctccgggtg tcgcgtgcgc 360agttgcacgt cattctcaga
cgaaccgatg actggatgga tggccgccgc agtcgtcaca 420ctgatgatac
ggatgtgctt ctccgtatac accatgttat cggagagctg ccaacgtatg
480gttatcgtcg ggtatgggcg ctgcttcgca gacaggcaga acttgatggt
atgcctgcga 540tcaatgccaa acgtgtttac cggatcatgc gccagaatgc
gctgttgctt gagcgaaaac 600ctgctgtacc gccatcgaaa cgggcacata
caggcagagt ggccgtgaaa gaaagcaatc 660agcgatggtg ctctgacggg
ttcgagttct gctgtgataa cggagagaga ctgcgtgtca 720cgttcgcgct
ggactgctgt g 741571365DNAHomo sapiensmisc_featuresequence of STAR57
57tccttctgta aataggcaaa atgtatttta gtttccacca cacatgttct tttctgtagg
60gcttgtatgt tggaaatttt atccaattat tcaattaaca ctataccaac aatctgctaa
120ttctggagat gtggcagtga ataaaaaagt tatagtttct gattttgtgg
agcttggact 180ttaatgatgg acaaaacaac acattcttaa atatatattt
catcaaaatt atagtgggtg 240aattatttat atgtgcattt acatgtgtat
gtatacataa atgggcggtt actggctgca 300ctgagaatgt acacgtggcg
cgaacgaggc tgggcggtca gagaaggcct cccaaggagg 360tggctttgaa
gctgagtggt gcttccacgt gaaaaggctg gaaagggcat tccaagaaaa
420ggctgaggcc agcgggaaag aggttccagt gcgctctggg aacggaaagc
gcacctgcct 480gaaacgaaaa tgagtgtgct gaaataggac gctagaaagg
gaggcagagg ctggcaaaag 540cgaccgagga ggagctcaaa ggagcgagcg
gggaaggccg ctgtggagcc tggaggaagc 600acttcggaag cgcttctgag
cgggtaaggc cgctgggagc atgaactgct gagcaggtgt 660gtccagaatt
cgtgggttct tggtctcact gacttcaaga atgaagaggg accgcggacc
720ctcgcggtga gtgttacagc tcttaaggtg gcgcgtctgg agtttgttcc
ttctgatgtt 780cggatgtgtt cagagtttct tccttctggt gggttcgtgg
tctcgctggc tcaggagtga 840agctgcagac cttcgcggtg agtgttacag
ctcataaaag cagggtggac tcaaagagtg 900agcagcagca agatttattg
caaagaatga aagaacaaag cttccacact gtggaagggg 960accccagcgg
gttgccactg ctggctccgc agcctgcttt tattctctta tctggcccca
1020cccacatcct gctgattggt agagccgaat ggtctgtttt gacggcgctg
attggtgcgt 1080ttacaatccc tgcgctagat acaaaggttc tccacgtccc
caccagatta gctagataga 1140gtctccacac aaaggttctc caaggcccca
ccagagtagc tagatacaga gtgttgattg 1200gtgcattcac aaaccctgag
ctagacacag ggtgatgact ggtgtgttta caaaccttgc 1260ggtagataca
gagtatcaat tggcgtattt acaatcactg agctaggcat aaaggttctc
1320caggtcccca ccagactcag gagcccagct ggcttcaccc agtgg
1365581401DNAHomo sapiensmisc_featuresequence of STAR58
58aagtttacct tagccctaaa ttatttcatt gtgattggca ttttaggaaa tatgtattaa
60ggaatgtctc ttaggagata aggataacat atgtctaaga aaattatatt gaaatattat
120tacatgaact aaaatgttag aactgaaaaa aaattattgt aactccttcc
agcgtaggca 180ggagtatcta gataccaact ttaacaactc aactttaaca
acttcgaacc aaccagatgg 240ctaggagatt cacctattta gcatgatatc
ttttattgat aaaaaaatat aaaacttcca 300ttaaattttt aagctactac
aatcctatta aattttaact taccagtgtt ctcaatgcta 360cataatttaa
aatcattgaa atcttctgat tttaactcct cagtcttgaa atctacttat
420ttttagttac atatatatcc aatctactgc cgctagtaga agaagcttgg
aatttgagaa 480aaaaatcaga cgttttgtat attctcatat tcactaattt
attttttaaa tgagtttctg 540caatgcatca agcagtggca aaacaggaga
aaaattaaaa ttggttgaaa agatatgtgt 600gccaaacaat cccttgaaat
ttgatgaagt gactaatcct gagttattgt ttcaaatgtg 660tacctgttta
tacaagggta tcacctttga aatctcaaca ttaaatgaaa ttttataagc
720aatttgttgt aacatgatta ttataaaatt ctgatataac attttttatt
acctgtttag 780agtttaaaga gagaaaagga gttaagaata attacatttt
cattagcatt gtccgggtgc 840aaaaacttct aacactatct tcaaatcttt
ttctccattg ccttctgaac atacccactt 900gggtatctca ttagcactgc
aaattcaaca ttttcgattg ctaatttttc tccctaaata 960tttatttgtt
ttctcagctt tagccaatgt ttcactattg accatttgct caagtatagt
1020gacgcttcaa tgaccttcag agagctgttt cagtccttcc tggactactt
gcatgcttcc 1080aacaaaatga agcactcttg atgtcagtca ctcaaataaa
tggaaatggg cccatttact 1140aggaatgtta acagaataaa aagatagacg
tgacaccagt tgcttcagtc catctccatt 1200tacttgctta aggcctggcc
atatttctca cagttgatat ggcgcagggc acatgtttaa 1260atggctgttc
ttgtaggatg gtttgactgt tggattcctc atcttccctc tccttaggaa
1320ggaaggttac agtagtactg ttggctcctg gaatatagat tcataaagaa
ctaatggagt 1380atcatctccc actgctcttg t 140159866DNAHomo
sapiensmisc_featuresequence of STAR59 59gagatcacgc cactgcactc
cagcctgggg gacagagcaa gactccatct cagaaacaaa 60caaacacaca aagccagtca
aggtgtttaa ttcgacggtg tcaggctcag gtctcttgac 120aggatacatc
cagcacccgg gggaaacgtc gatgggtggg gtggaatcta ttttgtggcc
180tcaagggagg gtttgagagg tagtcccgca agcggtgatg gcctaaggaa
gcccctccgc 240ccaagaagcg atattcattt ctagcctgta gccacccaag
agggagaatc gggctcgcca 300cagaccccac aacccccaac ccaccccacc
cccacccctc ccacctcgtg aaatgggctc 360tcgctccgtc aggctctagt
cacaccgtgt ggttttggaa cctccagcgt gtgtgcgtgg 420gttgcgtggt
ggggtggggc cggctgtgga cagaggaggg gataaagcgg cggtgtcccg
480cgggtgcccg ggacgtgggg cgtggggcgt gggtggggtg gccagagcct
tgggaactcg 540tcgcctgtcg ggacgtctcc cctcctggtc ccctctctga
cctacgctcc acatcttcgc 600cgttcagtgg ggaccttgtg ggtggaagtc
accatccctt tggactttag ccgacgaagg 660ccgggctccc aagagtctcc
ccggaggcgg ggccttgggc aggctcacaa ggatgctgac 720ggtgacggtt
ggtgacggtg atgtacttcg gaggcctcgg gccaatgcag aggtatccat
780ttgacctcgg tgggacaggt cagctttgcg gagtcccgtg cgtccttcca
gagactcatc 840cagcgctagc aagcatggtc ccgagg 866602067DNAHomo
sapiensmisc_featuresequence of STAR60 60agcagtgcag aactggggaa
gaagaagagt ccctacacca cttaatactc aaaagtactc 60gcaaaaaata acacccctca
ccaggtggca tnattactct ccttcattga gaaaattagg 120aaactggact
tcgtagaagc taattgcttt atccagagcc acctgcatac aaacctgcag
180cgccacctgc atacaaacct gtcagccgac cccaaagccc tcagtcgcac
caagcctctg 240ctgcacaccc tcgtgccttc acactggccg ttccccaagc
ctggggcata ctncccagct 300ctgagaaatg tattcatcct tcaaagccct
gctcatgtgt cctnntcaac aggaaaatct 360cccatgagat gctctgctat
ccccatctct cctgccccat agcttaggca nacttctgtg 420gtggtgagtc
ctgggctgtg ctgtgatgtg ttcgcctgcn atgtntgttc ttccccacaa
480tgatgggccc ctgaattctc tatctctagc acctgtgctc agtaaaggct
tgggaaacca 540ggctcaaagc ctggcccaga tgccaccttt tccagggtgc
ttccgggggc caccaaccag 600agtgcagcct tctcctccac caggaactct
tgcagcccca cccctgagca cctgcacccc 660attacccatc tttgtttctc
cgtgtgatcg tattattaca gaattatata ctgtattctt 720aatacagtat
ataattgtat aattattctt aatacagtat ataattatac aaatacaaaa
780tatgtgttaa tggaccgttt atgttactgg taaagcttta agtcaacagt
gggacattag 840ttaggttttt ggcgaagtca aaagttatat gtgcattttc
aacttcttga ggggtcggta 900cntctnaccc ccatgttgtt caanggtcaa
ctgtctacac atatcatagc taattcacta 960cagaaatgtt agcttgtgtc
actagtatct ccccttctca taagcttaat acacatacct 1020tgagagagct
cttggccatc tctactaatg actgaagttt ttatttatta tagatgtcat
1080aataggcata aaactacatt acatcattcg agtgccaatt ttgccacctt
gaccctcttt 1140tgcaaaacac caacgtcagt acacatatga agaggaaact
gcccgagaac tgaagttcct 1200gagaccagga gctgcaggcg ttagatagaa
tatggtgacg agagttacga ggatgacgag 1260agtaaatact tcatactcag
tacgtgccaa gcactgctat aagcgctctg tatgtgtgaa 1320gtcatttaat
cctcacagca tcccacggtg taattatttt cattatcccc atgagggaac
1380agaaactcag aacggttcaa cacatatgcg agaagtcgca gccggtcagt
gagagagcag 1440gttcccgtcc aagcagtcag accccgagtg cacactctcg
acccctgtcc agcagactca 1500ctcgtcataa ggcggggagt gntctgtttc
agccagatgc tttatgcatc tcagagtacc 1560caaaccatga aagaatgagg
cagtattcan gagcagatgg ngctgggcag taaggctggg 1620cttcagaata
gctggaaagc tcaagtnatg ggacctgcaa gaaaaatcca ttgtttngat
1680aaatagccaa agtccctagg ctgtaagggg aaggtgtgcc aggtgcaagt
ggagctctaa 1740tgtaaaatcg cacctgagtc tcctggtctt atgagtnctg
ggtgtacccc agtgaaaggt 1800cctgctgcca ccaagtgggc catggttcag
ctgtgtaagt gctgagcggc agccggaccg 1860cttcctctaa cttcacctcc
aaaggcacag tgcacctggt tcctccagca ctcagctgcg 1920aggcccctag
ccagggtccc ggcccccggc ccccggcagc tgctccagct tccttcccca
1980cagcattcag gatggtctgc gttcatgtag acctttgttt tcagtctgtg
ctccgaggtc 2040actggcagca ctagccccgg ctcctgt 2067611470DNAHomo
sapiensmisc_featuresequence of STAR61 61cagcccccac atgcccagcc
ctgtgctcag ctctgcagcg gggcatggtg ggcagagaca 60cagaggccaa ggccctgctt
cggggacggt gggcctggga tgagcatggc cttggccttc 120gccgagagtn
ctcttgtgaa ggaggggtca ggaggggctg ctgcagctgg ggaggagggc
180gatggcactg tggcangaag tgaantagtg tgggtgcctn gcaccccagg
cacggccagc 240ctggggtatg gacccggggc cntctgttct agagcaggaa
ggtatggtga ggacctcaaa 300aggacagcca ctggagagct ccaggcagag
gnacttgaga ggccctgggg ccatcctgtc 360tcttttctgg gtctgtgtgc
tctgggcctg ggcccttcct ctgctccccc gggcttggag 420agggctggcc
ttgcctcgtg caaaggacca ctctagactg gtaccaagtc tggcccatgg
480cctcctgtgg gtgcaggcct gtgcgggtga cctgagagcc agggctggca
ggtcagagtc 540aggagaggga tggcagtgga tgccctgtgc aggatctgcc
taatcatggt gaggctggag 600gaatccaaag tgggcatgca ctctgcactc
atttctttat tcatgtgtgc ccatcccaac 660aagcagggag cctggccagg
agggcccctg ggagaaggca ctgatgggct gtgttccatt 720taggaaggat
ggacggttgt gagacgggta agtcagaacg ggctgcccac ctcggccgag
780agggccccgt ggtgggttgg caccatctgg gcctggagag ctgctcagga
ggctctctag 840ggctgggtga ccaggnctgg ggtacagtag ccatgggagc
aggtgcttac ctggggctgt 900ccctgagcag gggctgcatt gggtgctctg
tgagcacaca cttctctatt cacctgagtc 960ccnctgagtg atgagnacac
ccttgttttg cagatgaatc tgagcatgga gatgttaagt 1020ggcttgcctg
agccacacag cagatggatg gtgtagctgg gacctgaggg caggcagtcc
1080cagcccgagg acttcccaag gttgtggcaa actctgacag catgacccca
gggaacaccc 1140atctcagctc tggtcagaca ctgcggagtt gtgttgtaac
ccacacagct ggagacagcc 1200accctagccc cacccttatc ctctcccaaa
ggaacctgcc ctttcccttc attttcctct 1260tactgcattg agggaccaca
cagtgtggca gaaggaacat gggttcagga cccagatgga 1320cttgcttcac
agtgcagccc tcctgtcctc ttgcagagtg cgtcttccac tgtgaagttg
1380ggacagtcac accaactcaa tactgctggg cccgtcacac ggtgggcagg
caacggatgg 1440cagtcactgg ctgtgggtct gcagaggtgg 1470621011DNAHomo
sapiensmisc_featuresequence of STAR62 62agtgtcaaat agatctacac
aaaacaagat aatgtctgcc catttttcca aagataatgt 60ggtgaagtgg gtagagagaa
atgcatccat tctccccacc caacctctgc taaattgtcc 120atgtcacagt
actgagacca gggggcttat tcccagcggg cagaatgtgc accaagcacc
180tcttgtctca atttgcagtc taggccctgc tatttgatgg tgtgaaggct
tgcacctggc 240atggaaggtc cgttttgtac ttcttgcttt agcagttcaa
agagcaggga gagctgcgag 300ggcctctgca gcttcagatg gatgtggtca
gcttgttgga ggcgccttct gtggtccatt 360atctccagcc cccctgcggt
gttgctgttt gcttggcttg tctggctctc catgccttgt 420tggctccaaa
atgtcatcat gctgcacccc aggaagaatg tgcaggccca tctcttttat
480gtgctttggg ctattttgat tccccgttgg gtatattccc taggtaagac
ccagaagaca 540caggaggtag ttgctttggg agagtttgga cctatgggta
tgaggtaata gacacagtat 600cttctctttc atttggtgag actgttagct
ctggccgcgg actgaattcc acacagctca 660cttgggaaaa ctttattcca
aaacatagtc acattgaaca ttgtggagaa tgagggacag 720agaagaggcc
ctagatttgt acatctgggt gttatgtcta taaatagaat gctttggtgg
780tcaactagac ttgttcatgt tgacatttag tcttgccttt tcggtggtga
tttaaaaatt 840atgtatatct tgtttggaat atagtggagc tatggtgtgg
cattttcatc tggctttttg 900tttagctcag cccgtcctgt tatgggcagc
cttgaagctc agtagctaat gaagaggtat 960cctcactccc tccagagagc
ggtcccctca cggctcattg agagtttgtc a 1011631410DNAHomo
sapiensmisc_featuresequence of STAR63 63ccacagcctg atcgtgctgt
cgatgagagg aatctgctct aagggtctga gcggagggag 60atgccgaagc tttgagcttt
ttgtttctgg cttaaccttg gtggattttc accctctggg 120cattacctct
tgtccagggg aggggctggg ggagtgcctg gagctgtagg gacagagggc
180tgagtggggg ggactgcttg ggctgaccac ataatattct gctgcgtatt
aatttttttt 240tgagacagtc tttctctgtt gcccaggctg gagtgtaatg
gcttgatagc tcactgccac 300ctccgcctcc tgggttcaag tgattctcct
gcttcagctt ccggagtagc tgggactgca 360ggtgcccgcc accatggctg
gctaattttt gtatttttat tagcaatggg gttttgctat 420gttgcccagg
ccggtcccga actcctgccc tcaagtgata cacctgcctc ggcctcccaa
480agtgctggga ttagaggctt gagccactgc gcctggccag ctgcatattg
ttaattagac 540ataaaatgca aaataagatg atataaacac aaaggtgtga
aataagatgg acacctgctg 600agcgcgcctg tcctgaagca tcgcccctct
gcaaaagcag gggtcagcat gtgttctccg 660gtccttgctc ttacagagga
gtgagctgcc tatgcgtctt ccagccactt cctgggctgc 720tcagaggcct
ctcacgggtg ttctgggttg ctgccacttg caggggtgct gaggcggggc
780tcctcccgtg cggggcatgt ccaggccgcc ctctctgaag gcttggcagg
tacaggtggg 840agtgggggtc tctgggctgc tgtggggact gggcaggctc
ctggaagacc tccctgtgtt 900tgggctgaaa gcgcagcccg aggggaggtc
cccagggagg ccgctgtcgg gggtgggggc 960ttggaggagg gaggggccga
ggagccggcg acactccgtg acggcccagg aacgtcccta 1020aacaaggcgc
cgcgttctcg atggggtggg gtccgctttc ttttctcaaa agctgcagtt
1080actccatgct cggaggactg gcgtccgcgc cctgttccaa tgctgccccg
gggccctggc 1140cttggggaat cggggccttg gactggaccc tgggggcttc
gcggagccgg gcctggcggg 1200gcgagcggag cagaggctgg gcagccccgg
ggaagcgctc gccaaagccg ggcgctgctc 1260ccagagcgcg aggtgcagaa
ccagaggctg gtcccgcggc gctaacgaga gaagaggaag 1320cgcgctgtgt
agagggcgcc caccccgtgg ggcgaacccc cttcctcaac tccatggacg
1380gggctcatgg gttcccagcg gctcagacgc 1410641414DNAHomo
sapiensmisc_featuresequence of STAR64 64tggatcagat ttgttttata
ccctcccttc tactgctctg agagttgtac atcacagtct 60actgtatctg tttcccatta
ttataatttt tttgcactgt gcttgcctga agggagcctc 120aagttcatga
gtctccctac cctcctccca aatgagacat ggacctttga atgctttcct
180gggaccacca ccccaccttt catgctgctg ttatccagga ttttagttca
acagtgtttt 240aaccccccaa atgagtcatt tttattgttt cgtatagtga
atgtgtattt gggtttgctt 300atatggtgac ctgtttattt gctcctcatt
gtacctcatg ctctgctctt tccttctaga 360ttcagtctct ttcctaatga
ggtgtctcgc agcaattctt tacaagacag ccaagatagg 420ccagctctca
gagcacttgt tgtctgaaaa agtcttgtct tatttaattt ctttttctta
480gagatggggt ctcattatgt tacccacact ggtctcaaac ttctggctta
aagcggtcct 540cccaccttgg cctcccaaag tgctaggatt acaggcgtga
gcgacctcgt ccagcctgtc 600tgagaaagcg tttgttttgc ccttgctctc
agatgacagt ttggggatag aattctaggt 660ggacggtttt tttccttcag
ccctttgaag agtctgtatt ttcattatct ccctgcatta 720gatgttcttt
tgcaagtaac gtgtcttttc tctctgggta ttcttaaggt tttctctttg
780cctttggtga gctgcagtgg atttgctttt ttcaagaggt caagagaaag
gaaagtgtga 840ggtttctgtt ttttactgac aatttgtttg ttgatttgtt
ttcccaccca gaggttcctt 900gccactttgc caggctggaa ggcagacttc
ttctggtgtc ctgttcacag acggggcagc 960ctgcggaagg ccctgccaca
tgcagggcct cggtcctcat tcccttgcat gtggacccgg 1020gcgtgactcc
tgttcaggct ggcacttccc agagctgagc cccagcctga ccttcctccc
1080atactgtctt cacaccccct cctttcttct
gatacctgga ggttttcctt tctttcctgt 1140cacctccact tggattttaa
atcctctgtc tgtggaattg tattcggcac aggaagatgc 1200ttgcaagggc
caggctcatc agccctgtcc ctgctgctgg aagcagcaca gcagagcctc
1260atgctcaggc tgagatggag cagaggcctg cagacgagca cccagctcag
ctggggttgg 1320cgccgatggt ggagggtcct cgaaagctct ggggacgatg
gcagagctat tggcagggga 1380gccgcagggt cttttgagcc cttaaaagat ctct
1414651310DNAHomo sapiensmisc_featuresequence of STAR65
65gtgaatgttg atggatcaaa tatctttctg tgttgtttat caaagttaaa ataaatgtgg
60tcatttaaag gacaaaagat gaggggttgg agtctgttca agcaaagggt atattaggag
120aaaagcagaa ttctctccct gtgaagggac agtgactcct attttccacc
tcatttttac 180taactctcct aactatctgc ttaggtagag atatatccat
gtacatttat aaaccacagt 240gaatcatttg attttggaat aaagatagta
taaaatgtgt cccagtgttg atatacatca 300tacattaaat atgtctggca
gtgttctaat tttacagttg tccaaagata atgttagggc 360atactggcta
tggatgaagc tccaatgttc agattgcaaa gaaacttaga attttactaa
420tgaaaccaaa tacatcccaa gaaatttttc agaagaaaaa aagagaaact
agtagcaaag 480taaagaatca ccacaatatc atcagatttt ttttatatgt
agaatattta ttcagttctt 540ttttcaagta caccttgtct tcattcattg
tactttattt tttgtgaagg tttaaattta 600tttcttctat gtgtttagtg
atatttaaaa tttttattta atcaagttta tcagaaagtt 660ctgttagaaa
atatgacgag gctttaattc cgccatctat attttccgct attatataaa
720gataattgtt ttctcttttt aaaacaactt gaattgggat tttatatcat
aattttttaa 780tgtctttttt tattatactt taagttctgg gatacatgtg
cagaacgtgc aggtgtgtta 840catagatata cacgtgccat ggtggtttgc
tgcacccact aacctgttat cgacattagg 900tatttctcct aatgctatca
ccccctattt ccccaccccc cgagaggccc cagtgtgtga 960tgttctcctc
cctgtgtcca tgtgttctca ttgttcatct cccacttatg gtatctacca
1020taaccttgaa attgtcttat gcattcactt gtttggttgt tatatagcct
ccatcaggac 1080agggatattt gctgctgctt cttttttttt tctttttgag
acagtcttgc tccgtcatcc 1140aggctggagt gcttctcggc tcaatgcaac
ctccacctcc caggtttaag cgattctcca 1200acttcagcct cccaaatggc
tgggactgca ggcatgcacc actacacctg gctaattttt 1260gtatttgtaa
tagagacaat gtttcaccat gttggccagg ctggtctcga 1310661917DNAHomo
sapiensmisc_featuresequence of STAR67 66aggatcctaa aattttgtga
ccctagagca agtactaact atgaaagtga aatagagaat 60gaaggaatta tttaattaag
tccagcaaaa cccaaccaaa tcatctgtaa aatatatttg 120ttttcaacat
ccaggtattt tctgtgtaaa aggttgagtt gtatgctgac ttattgggaa
180aaataattga gttttcccct tcactttgcc agtgagagga aatcagtact
gtaattgtta 240aaggttaccc atacctacct ctactaccgt ctagcatagg
taaagtaatg tacactgtga 300agtttcctgc ttgactgtaa tgttttcagt
ttcatcccat tgattcaaca gctatttatt 360cagcacttac tacaaccatg
ctggaaaccc aagagtaaat aggctgtgtt actcaacagg 420actgaggtac
agccgaactg tcaggcaagg ttgctgtcct ttggacttgc ctgctttctc
480tctatgtagg aagaagaaat ggacataccg tccaggaaat agatatatgt
tacatttcct 540tattccataa ttaatattaa taaccctgga cagaaactac
caagtttcta gacccttata 600gtaccacctt accctttctg gatgaatcct
tcacatgttg atacatttta tccaaatgaa 660aattttggta ctgtaggtat
aacagacaaa gagagaacag aaaactagag atgaagtttg 720ggaaaaggtc
aagaaagtaa ataatgcttc tagaagacac aaaaagaaaa atgaaatggt
780aatgttggga aagttttaat acattttgcc ctaaggaaaa aaactacttg
ttgaaattct 840acttaagact ggaccttttc tctaaaaatt gtgcttgatg
tgaattaaag caacacaggg 900aaatttatgg gctccttcta agttctaccc
aactcaccgc aaaactgttc ctagtaggtg 960tggtatactc tttcagattc
tttgtgtgta tgtatatgtg tgtgtgtgtg tgtgtttgta 1020tgtgtacagt
ctatatacat atgtgtacct acatgtgtgt atatataaat atatatttac
1080ctggatgaaa tagcatatta tagaatattc ttttttcttt aaatatatat
gtgcatacat 1140atgtatatgc acatatatac ataaatgtag atatagctag
gtaggcattc atgtgaaaca 1200aagaagccta ttacttttta atggttgcat
gatattccat cataggagta tagtacaact 1260tatgtaacac acatttggct
tgttgtaaaa ttttggtatt aataaaatag cacatatcat 1320gcaaagacac
ccttgcatag gtctattcat tctttgattt ttaccttagg acaaaattta
1380aaagtagaat ttctgggtca agcagtatgc tcatttaaaa tgtcattgca
tatttccaaa 1440ttgtcctcca gaaaagtagt aacagtaaca attgatggac
tgcgtgtttt ctaaaacttg 1500catttttttc cttattggtg aggtttggca
ttttccatat gtttattggc attttaattt 1560tttttggttc atgtctttta
ttcccttcct gcaaatttgt ggtgtgtctc aactttattt 1620atactctcat
tttcataatt ttctaaagga atttgacttt aaaaaaataa gacagccaat
1680gctttggttt aatttcattg ctgctttttg aagtgactgc tgtgttttta
tatactttta 1740tattttgttg ttttagcaaa ttcttctata ttataattgt
gtatgctgga acaaaaagtt 1800atatttctta atctagataa aatatttcaa
gatgttgtaa ttacagtccc ctctaaaatc 1860atataaatag acgcatagct
gtgtgatttg taattagtta tgtccattga tagatcc
19176711DNAArtificialsequence around startcodon of wild-type zeocin
resistance gene 67aaaccatggc c 116850DNAArtificialprimer
ZEOforwardMUT 68gatctcgcga tacaggattt atgttggcca agttgaccag
tgccgttccg 506926DNAArtificialprimer ZEO-WTreverse 69aggcgaattc
agtcctgctc ctcggc 267045DNAArtificialprimer ZEO-LEUreverse
70aggccccgcc cccacggctg ctcgccgatc tcggtcaagg ccggc
457145DNAArtificialprimer ZEO-THRreverse 71aggccccgcc cccacggctg
ctcgccgatc tcggtggtgg ccggc 457244DNAArtificialprimer
ZEO-VALreverse 72aggccccgcc cccacggctg ctcgccgatc tcggtccacg ccgg
447311DNAArtificialsequence around startcodon of wt d2EGFP
73gaattcatgg g 117449DNAArtificialprimer d2EGFPforwardBamHI
74gatcggatcc tatgaggaat tcgccaccat ggtgagcaag ggcgaggag
497541DNAArtificialprimer d2EGFPreverseNotI 75aaggaaaaaa gcggccgcct
acacattgat cctagcagaa g 417657DNAArtificialspacer sequence
76tcgatccaaa gactgccaaa tctagatccg agattttcag gagctaagga agctaaa
577799DNAArtificialprimer ZEOforwardBamHI-ATGmut/space 77gatcggatcc
ttggtttatg tcgatccaaa gactgccaaa tctagatccg agattttcag 60gagctaagga
agctaaagcc aagttgacca gtgaagttc 997838DNAArtificialprimer
ZEOforwardBamHI-GTG 78gatcggatcc accgtggcca agttgaccag tgccgttc
387938DNAArtificialprimer ZEOforwardBamHI-TTG 79gatcggatcc
accttggcca agttgaccag tgccgttc 388035DNAArtificialprimer
BSDBamHIforward 80gatcggatcc accatggcca agcctttgtc tcaag
358125DNAArtificialprimer BSD150reverse 81gtaaaatgat atacgttgac
accag 258225DNAArtificialprimer BSD150forward 82ctggtgtcaa
cgtatatcat tttac 258324DNAArtificialprimer BSD250reverse
83gccctgttct cgtttccgat cgcg 248424DNAArtificialprimer
BSD250forward 84cgcgatcgga aacgagaaca gggc
248524DNAArtificialprimer BSD350reverse 85gccgtcggct gtccgtcact
gtcc 248624DNAArtificialprimer BSD350forward 86ggacagtgac
ggacagccga cggc 248738DNAArtificialprimer BSD399reverse
87gatcgaattc ttagccctcc cacacgtaac cagagggc
3888103DNAArtificialprimer BSDforwardBamHIAvrII-ATGmut/space
88gatcggatcc taggttggtt tatgtcgatc caaagactgc caaatctaga tccgagattt
60tcaggagcta aggaagctaa agccaagcct ttgtctcaag aag
1038944DNAArtificialprimer BSD399reverseEcoRIAvrII 89gatcgaattc
cctaggttag ccctcccaca cgtaaccaga gggc 449042DNAArtificialprimer
BSDforwardBamHIAvrII-GTG 90gatcggatcc taggaccgtg gccaagcctt
tgtctcaaga ag 429142DNAArtificialprimer BSDforwardBamHIAvrII-TTG
91gatcggatcc taggaccttg gccaagcctt tgtctcaaga ag
4292375DNAArtificialwt zeocin resistance gene 92atg gcc aag ttg acc
agt gcc gtt ccg gtg ctc acc gcg cgc gac gtc 48Met Ala Lys Leu Thr
Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val1 5 10 15gcc gga gcg gtc
gag ttc tgg acc gac cgg ctc ggg ttc tcc cgg gac 96Ala Gly Ala Val
Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30ttc gtg gag
gac gac ttc gcc ggt gtg gtc cgg gac gac gtg acc ctg 144Phe Val Glu
Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45ttc atc
agc gcg gtc cag gac cag gtg gtg ccg gac aac acc ctg gcc 192Phe Ile
Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60tgg
gtg tgg gtg cgc ggc ctg gac gag ctg tac gcc gag tgg tcg gag 240Trp
Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu65 70 75
80gtc gtg tcc acg aac ttc cgg gac gcc tcc ggg ccg gcc atg acc gag
288Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu
85 90 95atc ggc gag cag ccg tgg ggg cgg gag ttc gcc ctg cgc gac ccg
gcc 336Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro
Ala 100 105 110ggc aac tgc gtg cac ttc gtg gcc gag gag cag gac tga
375Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115
12093124PRTArtificialSynthetic Construct 93Met Ala Lys Leu Thr Ser
Ala Val Pro Val Leu Thr Ala Arg Asp Val1 5 10 15Ala Gly Ala Val Glu
Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30Phe Val Glu Asp
Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45Phe Ile Ser
Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60Trp Val
Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu65 70 75
80Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu
85 90 95Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro
Ala 100 105 110Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115
12094399DNAArtificialwt blasticidin resistance gene 94atg gcc aag
cct ttg tct caa gaa gaa tcc acc ctc att gaa aga gca 48Met Ala Lys
Pro Leu Ser Gln Glu Glu Ser Thr Leu Ile Glu Arg Ala1 5 10 15acg gct
aca atc aac agc atc ccc atc tct gaa gac tac agc gtc gcc 96Thr Ala
Thr Ile Asn Ser Ile Pro Ile Ser Glu Asp Tyr Ser Val Ala 20 25 30agc
gca gct ctc tct agc gac ggc cgc atc ttc act ggt gtc aat gta 144Ser
Ala Ala Leu Ser Ser Asp Gly Arg Ile Phe Thr Gly Val Asn Val 35 40
45tat cat ttt act ggg gga cct tgt gca gaa ctc gtg gtg ctg ggc act
192Tyr His Phe Thr Gly Gly Pro Cys Ala Glu Leu Val Val Leu Gly Thr
50 55 60gct gct gct gcg gca gct ggc aac ctg act tgt atc gtc gcg atc
gga 240Ala Ala Ala Ala Ala Ala Gly Asn Leu Thr Cys Ile Val Ala Ile
Gly65 70 75 80aat gag aac agg ggc atc ttg agc ccc tgc gga cgg tgc
cga cag gtg 288Asn Glu Asn Arg Gly Ile Leu Ser Pro Cys Gly Arg Cys
Arg Gln Val 85 90 95ctt ctc gat ctg cat cct ggg atc aaa gcc ata gtg
aag gac agt gat 336Leu Leu Asp Leu His Pro Gly Ile Lys Ala Ile Val
Lys Asp Ser Asp 100 105 110gga cag ccg acg gca gtt ggg att cgt gaa
ttg ctg ccc tct ggt tat 384Gly Gln Pro Thr Ala Val Gly Ile Arg Glu
Leu Leu Pro Ser Gly Tyr 115 120 125gtg tgg gag ggc taa 399Val Trp
Glu Gly 13095132PRTArtificialSynthetic Construct 95Met Ala Lys Pro
Leu Ser Gln Glu Glu Ser Thr Leu Ile Glu Arg Ala1 5 10 15Thr Ala Thr
Ile Asn Ser Ile Pro Ile Ser Glu Asp Tyr Ser Val Ala 20 25 30Ser Ala
Ala Leu Ser Ser Asp Gly Arg Ile Phe Thr Gly Val Asn Val 35 40 45Tyr
His Phe Thr Gly Gly Pro Cys Ala Glu Leu Val Val Leu Gly Thr 50 55
60Ala Ala Ala Ala Ala Ala Gly Asn Leu Thr Cys Ile Val Ala Ile Gly65
70 75 80Asn Glu Asn Arg Gly Ile Leu Ser Pro Cys Gly Arg Cys Arg Gln
Val 85 90 95Leu Leu Asp Leu His Pro Gly Ile Lys Ala Ile Val Lys Asp
Ser Asp 100 105 110Gly Gln Pro Thr Ala Val Gly Ile Arg Glu Leu Leu
Pro Ser Gly Tyr 115 120 125Val Trp Glu Gly 13096600DNAArtificialwt
puromycin resistance gene 96atg acc gag tac aag ccc acg gtg cgc ctc
gcc acc cgc gac gac gtc 48Met Thr Glu Tyr Lys Pro Thr Val Arg Leu
Ala Thr Arg Asp Asp Val1 5 10 15ccc agg gcc gta cgc acc ctc gcc gcc
gcg ttc gcc gac tac ccc gcc 96Pro Arg Ala Val Arg Thr Leu Ala Ala
Ala Phe Ala Asp Tyr Pro Ala 20 25 30acg cgc cac acc gtc gat ccg gac
cgc cac atc gag cgg gtc acc gag 144Thr Arg His Thr Val Asp Pro Asp
Arg His Ile Glu Arg Val Thr Glu 35 40 45ctg caa gaa ctc ttc ctc acg
cgc gtc ggg ctc gac atc ggc aag gtg 192Leu Gln Glu Leu Phe Leu Thr
Arg Val Gly Leu Asp Ile Gly Lys Val 50 55 60tgg gtc gcg gac gac ggc
gcc gcg gtg gcg gtc tgg acc acg ccg gag 240Trp Val Ala Asp Asp Gly
Ala Ala Val Ala Val Trp Thr Thr Pro Glu65 70 75 80agc gtc gaa gcg
ggg gcg gtg ttc gcc gag atc ggc ccg cgc atg gcc 288Ser Val Glu Ala
Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala 85 90 95gag ttg agc
ggt tcc cgg ctg gcc gcg cag caa cag atg gaa ggc ctc 336Glu Leu Ser
Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu 100 105 110ctg
gcg ccg cac cgg ccc aag gag ccc gcg tgg ttc ctg gcc acc gtc 384Leu
Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val 115 120
125ggc gtc tcg ccc gac cac cag ggc aag ggt ctg ggc agc gcc gtc gtg
432Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val
130 135 140ctc ccc gga gtg gag gcg gcc gag cgc gcc ggg gtg ccc gcc
ttc ctg 480Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala
Phe Leu145 150 155 160gag acc tcc gcg ccc cgc aac ctc ccc ttc tac
gag cgg ctc ggc ttc 528Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr
Glu Arg Leu Gly Phe 165 170 175acc gtc acc gcc gac gtc gag tgc ccg
aag gac cgc gcg acc tgg tgc 576Thr Val Thr Ala Asp Val Glu Cys Pro
Lys Asp Arg Ala Thr Trp Cys 180 185 190atg acc cgc aag ccc ggt gcc
tga 600Met Thr Arg Lys Pro Gly Ala 19597199PRTArtificialSynthetic
Construct 97Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp
Asp Val1 5 10 15Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp
Tyr Pro Ala 20 25 30Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu
Arg Val Thr Glu 35 40 45Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu
Asp Ile Gly Lys Val 50 55 60Trp Val Ala Asp Asp Gly Ala Ala Val Ala
Val Trp Thr Thr Pro Glu65 70 75 80Ser Val Glu Ala Gly Ala Val Phe
Ala Glu Ile Gly Pro Arg Met Ala 85 90 95Glu Leu Ser Gly Ser Arg Leu
Ala Ala Gln Gln Gln Met Glu Gly Leu 100 105 110Leu Ala Pro His Arg
Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val 115 120 125Gly Val Ser
Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val 130 135 140Leu
Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu145 150
155 160Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly
Phe 165 170 175Thr Val Thr Ala Asp Val Glu Cys Pro Lys Asp Arg Ala
Thr Trp Cys 180 185 190Met Thr Arg Lys Pro Gly Ala
19598564DNAArtificialwt DHFR gene (from mouse) 98atg gtt cga cca
ttg aac tgc atc gtc gcc gtg tcc caa aat atg ggg 48Met Val Arg Pro
Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly1 5 10 15att ggc aag
aac gga gac cta ccc tgg cct ccg ctc agg aac gag ttc 96Ile Gly Lys
Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30aag tac
ttc caa aga atg acc aca acc tct tca gtg gaa ggt aaa cag 144Lys Tyr
Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45aat
ctg gtg att atg ggt agg aaa acc tgg ttc tcc att cct gag aag 192Asn
Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55
60aat cga cct tta aag gac aga att aat ata gtt ctc agt aga gaa ctc
240Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu
Leu65 70 75 80aaa gaa cca cca cga gga gct cat ttt ctt gcc aaa agt
ttg gat gat 288Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser
Leu Asp Asp 85 90 95gcc tta aga ctt att gaa caa ccg gaa ttg gca agt
aaa gta gac atg 336Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu
Ala Ser Lys Val Asp Met 100 105 110gtt tgg ata gtc gga ggc agt tct
gtt tac cag gaa gcc atg aat caa 384Val Trp Ile Val Gly Gly Ser Ser
Val Tyr Gln Glu Ala Met Asn Gln 115 120 125cca ggc cac ctc aga ctc
ttt gtg aca agg atc atg cag gaa ttt gaa 432Pro Gly His Leu Arg Leu
Phe Val Thr Arg Ile Met Gln Glu Phe Glu 130 135 140agt gac acg ttt
ttc cca gaa att gat ttg ggg aaa tat aaa ctt ctc 480Ser Asp Thr Phe
Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu145 150 155 160cca
gaa tac cca ggc gtc ctc tct gag gtc cag gag gaa aaa ggc atc 528Pro
Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ile 165 170
175aag tat aag ttt gaa gtc tac gag aag aaa gac taa 564Lys Tyr Lys
Phe Glu Val Tyr Glu Lys Lys Asp 180 18599187PRTArtificialSynthetic
Construct 99Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn
Met Gly1 5 10 15Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg
Asn Glu Phe 20 25 30Lys Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val
Glu Gly Lys Gln 35 40 45Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe
Ser Ile Pro Glu Lys 50 55 60Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile
Val Leu Ser Arg Glu Leu65 70 75 80Lys Glu Pro Pro Arg Gly Ala His
Phe Leu Ala Lys Ser Leu Asp Asp 85 90 95Ala Leu Arg Leu Ile Glu Gln
Pro Glu Leu Ala Ser Lys Val Asp Met 100 105 110Val Trp Ile Val Gly
Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Gln 115 120 125Pro Gly His
Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Glu 130 135 140Ser
Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu145 150
155 160Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly
Ile 165 170 175Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp 180
1851001143DNAArtificialwt hygromycin resistance gene 100atg aaa aag
cct gaa ctc acc gcg acg tct gtc gag aag ttt ctg atc 48Met Lys Lys
Pro Glu Leu Thr Ala Thr Ser Val Glu Lys Phe Leu Ile1 5 10 15gaa aag
ttc gac agc gtc tcc gac ctg atg cag ctc tcg gag ggc gaa 96Glu Lys
Phe Asp Ser Val Ser Asp Leu Met Gln Leu Ser Glu Gly Glu 20 25 30gaa
tct cgt gct ttc agc ttc gat gta gga ggg cgt gga tat gtc ctg 144Glu
Ser Arg Ala Phe Ser Phe Asp Val Gly Gly Arg Gly Tyr Val Leu 35 40
45cgg gta aat agc tgc gcc gat ggt ttc tac aaa gat cgt tat gtt tat
192Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr
50 55 60cgg cac ttt gca tcg gcc gcg ctc ccg att ccg gaa gtg ctt gac
att 240Arg His Phe Ala Ser Ala Ala Leu Pro Ile Pro Glu Val Leu Asp
Ile65 70 75 80ggg gaa ttc agc gag agc ctg acc tat tgc atc tcc cgc
cgt gca cag 288Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg
Arg Ala Gln 85 90 95ggt gtc acg ttg caa gac ctg cct gaa acc gaa ctg
ccc gct gtt ctg 336Gly Val Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu
Pro Ala Val Leu 100 105 110cag ccg gtc gcg gag gcc atg gat gcg atc
gct gcg gcc gat ctt agc 384Gln Pro Val Ala Glu Ala Met Asp Ala Ile
Ala Ala Ala Asp Leu Ser 115 120 125cag acg agc ggg ttc ggc cca ttc
gga ccg caa gga atc ggt caa tac 432Gln Thr Ser Gly Phe Gly Pro Phe
Gly Pro Gln Gly Ile Gly Gln Tyr 130 135 140act aca tgg cgt gat ttc
ata tgc gcg att gct gat ccc cat gtg tat 480Thr Thr Trp Arg Asp Phe
Ile Cys Ala Ile Ala Asp Pro His Val Tyr145 150 155 160cac tgg caa
act gtg atg gac gac acc gtc agt gcg tcc gtc gcg cag 528His Trp Gln
Thr Val Met Asp Asp Thr Val Ser Ala Ser Val Ala Gln 165 170 175gct
ctc gat gag ctg atg ctt tgg gcc gag gac tgc ccc gaa gtc cgg 576Ala
Leu Asp Glu Leu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg 180 185
190cac ctc gtg cac gcg gat ttc ggc tcc aac aat gtc ctg acg gac aat
624His Leu Val His Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn
195 200 205ggc cgc ata aca gcg gtc att gac tgg agc gag gcg atg ttc
ggg gat 672Gly Arg Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe
Gly Asp 210 215 220tcc caa tac gag gtc gcc aac atc ttc ttc tgg agg
ccg tgg ttg gct 720Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg
Pro Trp Leu Ala225 230 235 240tgt atg gag cag cag acg cgc tac ttc
gag cgg agg cat ccg gag ctt 768Cys Met Glu Gln Gln Thr Arg Tyr Phe
Glu Arg Arg His Pro Glu Leu 245 250 255gca gga tcg ccg cgg ctc cgg
gcg tat atg ctc cgc att ggt ctt gac 816Ala Gly Ser Pro Arg Leu Arg
Ala Tyr Met Leu Arg Ile Gly Leu Asp 260 265 270caa ctc tat cag agc
ttg gtt gac ggc aat ttc gat gat gca gct tgg 864Gln Leu Tyr Gln Ser
Leu Val Asp Gly Asn Phe Asp Asp Ala Ala Trp 275 280 285gcg cag ggt
cga tgc gac gca atc gtc cga tcc gga gcc ggg act gtc 912Ala Gln Gly
Arg Cys Asp Ala Ile Val Arg Ser Gly Ala Gly Thr Val 290 295 300ggg
cgt aca caa atc gcc cgc aga agc gcg gcc gtc tgg acc gat ggc 960Gly
Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly305 310
315 320tgt gta gaa gta ctc gcc gat agt gga aac cga cgc ccc agc act
cgt 1008Cys Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr
Arg 325 330 335ccg gag gca aag gaa ttc ggg aga tgg ggg agg cta act
gaa aca cgg 1056Pro Glu Ala Lys Glu Phe Gly Arg Trp Gly Arg Leu Thr
Glu Thr Arg 340 345 350aag gag aca ata ccg gaa gga acc cgc gct atg
acg gca ata aaa aga 1104Lys Glu Thr Ile Pro Glu Gly Thr Arg Ala Met
Thr Ala Ile Lys Arg 355 360 365cag aat aaa acg cac ggg tgt tgg gtc
gtt tgt tca taa 1143Gln Asn Lys Thr His Gly Cys Trp Val Val Cys Ser
370 375 380101380PRTArtificialSynthetic Construct 101Met Lys Lys
Pro Glu Leu Thr Ala Thr Ser Val Glu Lys Phe Leu Ile1 5 10 15Glu Lys
Phe Asp Ser Val Ser Asp Leu Met Gln Leu Ser Glu Gly Glu 20 25 30Glu
Ser Arg Ala Phe Ser Phe Asp Val Gly Gly Arg Gly Tyr Val Leu 35 40
45Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr
50 55 60Arg His Phe Ala Ser Ala Ala Leu Pro Ile Pro Glu Val Leu Asp
Ile65 70 75 80Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg
Arg Ala Gln 85 90 95Gly Val Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu
Pro Ala Val Leu 100 105 110Gln Pro Val Ala Glu Ala Met Asp Ala Ile
Ala Ala Ala Asp Leu Ser 115 120 125Gln Thr Ser Gly Phe Gly Pro Phe
Gly Pro Gln Gly Ile Gly Gln Tyr 130 135 140Thr Thr Trp Arg Asp Phe
Ile Cys Ala Ile Ala Asp Pro His Val Tyr145 150 155 160His Trp Gln
Thr Val Met Asp Asp Thr Val Ser Ala Ser Val Ala Gln 165 170 175Ala
Leu Asp Glu Leu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg 180 185
190His Leu Val His Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn
195 200 205Gly Arg Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe
Gly Asp 210 215 220Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg
Pro Trp Leu Ala225 230 235 240Cys Met Glu Gln Gln Thr Arg Tyr Phe
Glu Arg Arg His Pro Glu Leu 245 250 255Ala Gly Ser Pro Arg Leu Arg
Ala Tyr Met Leu Arg Ile Gly Leu Asp 260 265 270Gln Leu Tyr Gln Ser
Leu Val Asp Gly Asn Phe Asp Asp Ala Ala Trp 275 280 285Ala Gln Gly
Arg Cys Asp Ala Ile Val Arg Ser Gly Ala Gly Thr Val 290 295 300Gly
Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly305 310
315 320Cys Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr
Arg 325 330 335Pro Glu Ala Lys Glu Phe Gly Arg Trp Gly Arg Leu Thr
Glu Thr Arg 340 345 350Lys Glu Thr Ile Pro Glu Gly Thr Arg Ala Met
Thr Ala Ile Lys Arg 355 360 365Gln Asn Lys Thr His Gly Cys Trp Val
Val Cys Ser 370 375 380102804DNAArtificialwt neomycin resistance
gene 102atg gga tcg gcc att gaa caa gat gga ttg cac gca ggt tct ccg
gcc 48Met Gly Ser Ala Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro
Ala1 5 10 15gct tgg gtg gag agg cta ttc ggc tat gac tgg gca caa cag
aca atc 96Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln
Thr Ile 20 25 30ggc tgc tct gat gcc gcc gtg ttc cgg ctg tca gcg cag
ggg cgc ccg 144Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gln
Gly Arg Pro 35 40 45gtt ctt ttt gtc aag acc gac ctg tcc ggt gcc ctg
aat gaa ctg cag 192Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu
Asn Glu Leu Gln 50 55 60gac gag gca gcg cgg cta tcg tgg ctg gcc acg
acg ggc gtt cct tgc 240Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr
Thr Gly Val Pro Cys65 70 75 80gca gct gtg ctc gac gtt gtc act gaa
gcg gga agg gac tgg ctg cta 288Ala Ala Val Leu Asp Val Val Thr Glu
Ala Gly Arg Asp Trp Leu Leu 85 90 95ttg ggc gaa gtg ccg ggg cag gat
ctc ctg tca tct cac ctt gct cct 336Leu Gly Glu Val Pro Gly Gln Asp
Leu Leu Ser Ser His Leu Ala Pro 100 105 110gcc gag aaa gta tcc atc
atg gct gat gca atg cgg cgg ctg cat acg 384Ala Glu Lys Val Ser Ile
Met Ala Asp Ala Met Arg Arg Leu His Thr 115 120 125ctt gat ccg gct
acc tgc cca ttc gac cac caa gcg aaa cat cgc atc 432Leu Asp Pro Ala
Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile 130 135 140gag cga
gca cgt act cgg atg gaa gcc ggt ctt gtc gat cag gat gat 480Glu Arg
Ala Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp145 150 155
160ctg gac gaa gag cat cag ggg ctc gcg cca gcc gaa ctg ttc gcc agg
528Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg
165 170 175ctc aag gcg cgc atg ccc gac ggc gat gat ctc gtc gtg acc
cat ggc 576Leu Lys Ala Arg Met Pro Asp Gly Asp Asp Leu Val Val Thr
His Gly 180 185 190gat gcc tgc ttg ccg aat atc atg gtg gaa aat ggc
cgc ttt tct gga 624Asp Ala Cys Leu Pro Asn Ile Met Val Glu Asn Gly
Arg Phe Ser Gly 195 200 205ttc atc gac tgt ggc cgg ctg ggt gtg gcg
gac cgc tat cag gac ata 672Phe Ile Asp Cys Gly Arg Leu Gly Val Ala
Asp Arg Tyr Gln Asp Ile 210 215 220gcg ttg gct acc cgt gat att gct
gaa gag ctt ggc ggc gaa tgg gct 720Ala Leu Ala Thr Arg Asp Ile Ala
Glu Glu Leu Gly Gly Glu Trp Ala225 230 235 240gac cgc ttc ctc gtg
ctt tac ggt atc gcc gct ccc gat tcg cag cgc 768Asp Arg Phe Leu Val
Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg 245 250 255atc gcc ttc
tat cgc ctt ctt gac gag ttc ttc tga 804Ile Ala Phe Tyr Arg Leu Leu
Asp Glu Phe Phe 260 265103267PRTArtificialSynthetic Construct
103Met Gly Ser Ala Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala1
5 10 15Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr
Ile 20 25 30Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly
Arg Pro 35 40 45Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn
Glu Leu Gln 50 55 60Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr
Gly Val Pro Cys65 70 75 80Ala Ala Val Leu Asp Val Val Thr Glu Ala
Gly Arg Asp Trp Leu Leu 85 90 95Leu Gly Glu Val Pro Gly Gln Asp Leu
Leu Ser Ser His Leu Ala Pro 100 105 110Ala Glu Lys Val Ser Ile Met
Ala Asp Ala Met Arg Arg Leu His Thr 115 120 125Leu Asp Pro Ala Thr
Cys Pro Phe Asp His Gln Ala Lys His Arg Ile 130 135 140Glu Arg Ala
Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp145 150 155
160Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg
165 170 175Leu Lys Ala Arg Met Pro Asp Gly Asp Asp Leu Val Val Thr
His Gly 180 185 190Asp Ala Cys Leu Pro Asn Ile Met Val Glu Asn Gly
Arg Phe Ser Gly 195 200 205Phe Ile Asp Cys Gly Arg Leu Gly Val Ala
Asp Arg Tyr Gln Asp Ile 210 215 220Ala Leu Ala Thr Arg Asp Ile Ala
Glu Glu Leu Gly Gly Glu Trp Ala225 230 235 240Asp Arg Phe Leu Val
Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg 245 250 255Ile Ala Phe
Tyr Arg Leu Leu Asp Glu Phe Phe 260 2651041121DNAArtificialwt
glutamine synthase gene (human) 104atg acc acc tca gca agt tcc cac
tta aat aaa ggc atc aag cag gtg 48Met Thr Thr Ser Ala Ser Ser His
Leu Asn Lys Gly Ile Lys Gln Val1 5 10 15tac atg tcc ctg cct cag ggt
gag aaa gtc cag gcc atg tat atc tgg 96Tyr Met Ser Leu Pro Gln Gly
Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30atc gat ggt act gga gaa
gga ctg cgc tgc aag acc cgg acc ctg gac 144Ile Asp Gly Thr Gly Glu
Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45agt gag ccc aag tgt
gtg gaa gag ttg cct gag tgg aat ttc gat ggc 192Ser Glu Pro Lys Cys
Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60tcc agt act tta
cag tct gag ggt tcc aac agt gac atg tat ctc gtg 240Ser Ser Thr Leu
Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Val65 70 75 80cct gct
gcc atg ttt cgg gac ccc ttc cgt aag gac cct aac aag ctg 288Pro Ala
Ala Met Phe Arg Asp Pro Phe Arg Lys Asp Pro Asn Lys Leu 85 90 95gtg
tta tgt gaa gtt ttc aag tac aat cga agg cct gca gag acc aat 336Val
Leu Cys Glu Val Phe Lys Tyr Asn Arg Arg Pro Ala Glu Thr Asn 100 105
110ttg agg cac acc tgt aaa cgg ata atg gac atg gtg agc aac cag cac
384Leu Arg His Thr Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His
115 120 125ccc tgg ttt ggc atg gag cag gag tat acc ctc atg ggg aca
gat ggg 432Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr
Asp Gly 130 135 140cac ccc ttt ggt tgg cct tcc aac ggc ttc cca ggg
ccc cag ggt cca 480His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly
Pro Gln Gly Pro145 150 155 160tat tac tgt ggt gtg gga gca gac aga
gcc tat ggc agg gac atc gtg 528Tyr Tyr Cys Gly Val Gly Ala Asp Arg
Ala Tyr Gly Arg Asp Ile Val 165 170 175gag gcc cat tac cgg gcc tgc
ttg tat gct gga gtc aag att gcg ggg 576Glu Ala His Tyr Arg Ala Cys
Leu Tyr Ala Gly Val Lys Ile Ala Gly 180 185 190act aat gcc gag gtc
atg cct gcc cag tgg gaa ttt cag att gga cct 624Thr Asn Ala Glu Val
Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro 195 200 205tgt gaa gga
atc agc atg gga gat cat ctc tgg gtg gcc cgt ttc atc 672Cys Glu Gly
Ile Ser Met Gly Asp His Leu Trp Val Ala Arg Phe Ile 210 215 220ttg
cat cgt gtg tgt gaa gac ttt gga gtg ata gca acc ttt gat cct 720Leu
His Arg Val Cys Glu Asp Phe Gly Val Ile Ala Thr Phe Asp Pro225 230
235 240aag ccc att cct ggg aac tgg aat ggt gca ggc tgc cat acc aac
ttc 768Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn
Phe 245 250 255agc acc aag gcc atg cgg gag gag aat ggt ctg aag tac
atc gag gag 816Ser Thr Lys Ala Met
Arg Glu Glu Asn Gly Leu Lys Tyr Ile Glu Glu 260 265 270gcc att gag
aaa cta agc aag cgg cac cag tac cac atc cgt gcc tat 864Ala Ile Glu
Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280 285gat
ccc aag gga ggc ctg gac aat gcc cga cgt cta act gga ttc cat 912Asp
Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His 290 295
300gaa acc tcc aac atc aac gac ttt tct ggt ggt gta gcc aat cgt agc
960Glu Thr Ser Asn Ile Asn Asp Phe Ser Gly Gly Val Ala Asn Arg
Ser305 310 315 320gcc agc ata cgc att ccc cgg act gtt ggc cag gag
aag aag ggt tac 1008Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu
Lys Lys Gly Tyr 325 330 335ttt gaa gat cgt cgc ccc tct gcc aac tgc
gac ccc ttt tcg gtg aca 1056Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys
Asp Pro Phe Ser Val Thr 340 345 350gaa gcc ctc atc cgc acg tgt ctt
ctc aat gaa acc ggc gat gag ccc 1104Glu Ala Leu Ile Arg Thr Cys Leu
Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365ttc cag tac aaa aat ta
1121Phe Gln Tyr Lys Asn 370105373PRTArtificialSynthetic Construct
105Met Thr Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Val1
5 10 15Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile
Trp 20 25 30Ile Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr
Leu Asp 35 40 45Ser Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn
Phe Asp Gly 50 55 60Ser Ser Thr Leu Gln Ser Glu Gly Ser Asn Ser Asp
Met Tyr Leu Val65 70 75 80Pro Ala Ala Met Phe Arg Asp Pro Phe Arg
Lys Asp Pro Asn Lys Leu 85 90 95Val Leu Cys Glu Val Phe Lys Tyr Asn
Arg Arg Pro Ala Glu Thr Asn 100 105 110Leu Arg His Thr Cys Lys Arg
Ile Met Asp Met Val Ser Asn Gln His 115 120 125Pro Trp Phe Gly Met
Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140His Pro Phe
Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro145 150 155
160Tyr Tyr Cys Gly Val Gly Ala Asp Arg Ala Tyr Gly Arg Asp Ile Val
165 170 175Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile
Ala Gly 180 185 190Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe
Gln Ile Gly Pro 195 200 205Cys Glu Gly Ile Ser Met Gly Asp His Leu
Trp Val Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Glu Asp Phe
Gly Val Ile Ala Thr Phe Asp Pro225 230 235 240Lys Pro Ile Pro Gly
Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245 250 255Ser Thr Lys
Ala Met Arg Glu Glu Asn Gly Leu Lys Tyr Ile Glu Glu 260 265 270Ala
Ile Glu Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280
285Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His
290 295 300Glu Thr Ser Asn Ile Asn Asp Phe Ser Gly Gly Val Ala Asn
Arg Ser305 310 315 320Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln
Glu Lys Lys Gly Tyr 325 330 335Phe Glu Asp Arg Arg Pro Ser Ala Asn
Cys Asp Pro Phe Ser Val Thr 340 345 350Glu Ala Leu Ile Arg Thr Cys
Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365Phe Gln Tyr Lys Asn
37010643DNAArtificialprimer GTGspaceBamHIF 106gaattcggat ccaccgtggc
gatccaaaga ctgccaaatc tag 4310742DNAArtificialprimer
ZEOTTTGTGBamHIF 107gaattcggat cctttgtggc caagttgacc agtgccgttc cg
4210846DNAArtificialprimer ZEOForwardGTG-Thr9 108aattggatcc
accgtggcca agttgaccag tgccgttacc gtgctc 4610946DNAArtificialpimer
ZEOForward GTG-Phe9 109aattggatcc accgtggcca agttgaccag tgccgttttc
gtgctc 4611043DNAArtificialprimer TTGspaceBamHIF 110gaattcggat
ccaccttggc gatccaaaga ctgccaaatc tag 4311146DNAArtificialprimer
ZEOForwardTTG-Thr9 111aattggatcc accttggcca agttgaccag tgccgttacc
gtgctc 4611246DNAArtificialpimer ZEOForwardTTG-Phe9 112aattggatcc
accttggcca agttgaccag tgccgttttc gtgctc 4611337DNAArtificialprimer
PURO BamHI F 113gatcggatcc atggttaccg agtacaagcc cacggtg
3711435DNAArtificialprimer PURO300 R LEU 114cagccgggaa ccgctcaact
cggccaggcg cgggc 3511549DNAArtificialprimer PURO300FLEU
115cgagttgagc ggttcccggc tggccgcgca gcaacagctg gaaggcctc
4911644DNAArtificialprimer PURO600RLEU 116aagcttgaat tcaggcaccg
ggcttgcggg tcaggcacca ggtc 4411742DNAArtificialprimer PUROBamHI
TTG1F 117gaattcggat ccaccttggt taccgagtac aagcccacgg tg
42118804DNAArtificialmodified neomycin resistance gene lacking
internal ATG sequences 118atg gga tcg gcc att gaa caa gac gga ttg
cac gca ggt tct ccg gcc 48Met Gly Ser Ala Ile Glu Gln Asp Gly Leu
His Ala Gly Ser Pro Ala1 5 10 15gct tgg gtg gag agg cta ttc ggc tac
gac tgg gca caa cag aca atc 96Ala Trp Val Glu Arg Leu Phe Gly Tyr
Asp Trp Ala Gln Gln Thr Ile 20 25 30ggc tgc tct gac gcc gcc gtg ttc
cgg ctg tca gcg cag ggg cgc ccg 144Gly Cys Ser Asp Ala Ala Val Phe
Arg Leu Ser Ala Gln Gly Arg Pro 35 40 45gtt ctt ttt gtc aag acc gac
ctg tcc ggt gcc ctg aac gaa ctg cag 192Val Leu Phe Val Lys Thr Asp
Leu Ser Gly Ala Leu Asn Glu Leu Gln 50 55 60gac gag gca gcg cgg cta
tcg tgg ctg gcc acg acg ggc gtt cct tgc 240Asp Glu Ala Ala Arg Leu
Ser Trp Leu Ala Thr Thr Gly Val Pro Cys65 70 75 80gca gct gtg ctc
gac gtt gtc act gaa gcg gga agg gac tgg ctg cta 288Ala Ala Val Leu
Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu 85 90 95ttg ggc gaa
gtg ccg ggg cag gat ctc ctg tca tct cac ctt gct cct 336Leu Gly Glu
Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro 100 105 110gcc
gag aaa gta tcc atc ctg gct gac gca ctg cgg cgg ctg cat acg 384Ala
Glu Lys Val Ser Ile Leu Ala Asp Ala Leu Arg Arg Leu His Thr 115 120
125ctt gat ccg gct acc tgc cca ttc gac cac caa gcg aaa cat cgc atc
432Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile
130 135 140gag cga gca cgt act cgg ctg gaa gcc ggt ctt gtc gat cag
gac gat 480Glu Arg Ala Arg Thr Arg Leu Glu Ala Gly Leu Val Asp Gln
Asp Asp145 150 155 160ctg gac gaa gag cat cag ggg ctc gcg cca gcc
gaa ctg ttc gcc agg 528Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala
Glu Leu Phe Ala Arg 165 170 175ctc aag gcg cgc ctg ccc gac ggc gac
gat ctc gtc gtg acc cac ggc 576Leu Lys Ala Arg Leu Pro Asp Gly Asp
Asp Leu Val Val Thr His Gly 180 185 190gac gcc tgc ttg ccg aat atc
ctg gtg gaa aac ggc cgc ttt tct gga 624Asp Ala Cys Leu Pro Asn Ile
Leu Val Glu Asn Gly Arg Phe Ser Gly 195 200 205ttc atc gac tgt ggc
cgg ctg ggt gtg gcg gac cgc tat cag gac ata 672Phe Ile Asp Cys Gly
Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile 210 215 220gcg ttg gct
acc cgt gat att gct gaa gag ctt ggc ggc gag tgg gct 720Ala Leu Ala
Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala225 230 235
240gac cgc ttc ctc gtg ctt tac ggt atc gcc gct ccc gat tcg cag cgc
768Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg
245 250 255atc gcc ttc tat cgc ctt ctt gac gag ttc ttc tga 804Ile
Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 260
265119267PRTArtificialSynthetic Construct 119Met Gly Ser Ala Ile
Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala1 5 10 15Ala Trp Val Glu
Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile 20 25 30Gly Cys Ser
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro 35 40 45Val Leu
Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln 50 55 60Asp
Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys65 70 75
80Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu
85 90 95Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala
Pro 100 105 110Ala Glu Lys Val Ser Ile Leu Ala Asp Ala Leu Arg Arg
Leu His Thr 115 120 125Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln
Ala Lys His Arg Ile 130 135 140Glu Arg Ala Arg Thr Arg Leu Glu Ala
Gly Leu Val Asp Gln Asp Asp145 150 155 160Leu Asp Glu Glu His Gln
Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg 165 170 175Leu Lys Ala Arg
Leu Pro Asp Gly Asp Asp Leu Val Val Thr His Gly 180 185 190Asp Ala
Cys Leu Pro Asn Ile Leu Val Glu Asn Gly Arg Phe Ser Gly 195 200
205Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile
210 215 220Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu
Trp Ala225 230 235 240Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala
Pro Asp Ser Gln Arg 245 250 255Ile Ala Phe Tyr Arg Leu Leu Asp Glu
Phe Phe 260 26512040DNAArtificialprimer NEO-F-HindIII 120gatcaagctt
ttggatcggc cattgaaaca agacggattg 4012136DNAArtificialprimer NEO
EcoRI 800R 121aagcttgaat tctcagaaga actcgtcaag aaggcg
36122564DNAArtificialmodified dhfr gene lacking internal ATG
sequences 122atg gtt cga cca ttg aac tgc atc gtc gcc gtg tcc caa
aat ctg ggg 48Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln
Asn Leu Gly1 5 10 15att ggc aag aac gga gac cta ccc tgg cct ccg ctc
agg aac gag ttc 96Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu
Arg Asn Glu Phe 20 25 30aag tac ttc caa aga ctg acc aca acc tct tca
gtg gaa ggt aaa cag 144Lys Tyr Phe Gln Arg Leu Thr Thr Thr Ser Ser
Val Glu Gly Lys Gln 35 40 45aat ctg gtg att ctg ggt agg aaa acc tgg
ttc tcc att cct gag aag 192Asn Leu Val Ile Leu Gly Arg Lys Thr Trp
Phe Ser Ile Pro Glu Lys 50 55 60aat cga cct tta aag gac aga att aat
ata gtt ctc agt aga gaa ctc 240Asn Arg Pro Leu Lys Asp Arg Ile Asn
Ile Val Leu Ser Arg Glu Leu65 70 75 80aaa gaa cca cca cga gga gct
cat ttt ctt gcc aaa agt ttg gac gac 288Lys Glu Pro Pro Arg Gly Ala
His Phe Leu Ala Lys Ser Leu Asp Asp 85 90 95gcc tta aga ctt att gaa
caa ccg gaa ttg gca agt aaa gta gac ctg 336Ala Leu Arg Leu Ile Glu
Gln Pro Glu Leu Ala Ser Lys Val Asp Leu 100 105 110gtt tgg ata gtc
gga ggc agt tct gtt tac cag gaa gcc ctg aat caa 384Val Trp Ile Val
Gly Gly Ser Ser Val Tyr Gln Glu Ala Leu Asn Gln 115 120 125cca ggc
cac ctc aga ctc ttt gtg aca agg att ctg cag gaa ttt gaa 432Pro Gly
His Leu Arg Leu Phe Val Thr Arg Ile Leu Gln Glu Phe Glu 130 135
140agt gac acg ttt ttc cca gaa att gat ttg ggg aaa tat aaa ctt ctc
480Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu
Leu145 150 155 160cca gaa tac cca ggc gtc ctc tct gag gtc cag gag
gaa aaa ggc atc 528Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu
Glu Lys Gly Ile 165 170 175aag tat aag ttt gaa gtc tac gag aag aaa
gac taa 564Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp 180
185123187PRTArtificialSynthetic Construct 123Met Val Arg Pro Leu
Asn Cys Ile Val Ala Val Ser Gln Asn Leu Gly1 5 10 15Ile Gly Lys Asn
Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30Lys Tyr Phe
Gln Arg Leu Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu
Val Ile Leu Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn
Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp
85 90 95Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp
Leu 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala
Leu Asn Gln 115 120 125Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile
Leu Gln Glu Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Gly Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Glu Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Lys Asp 180 18512436DNAArtificialprimer
DHFR-F-HindIII 124gatcaagctt ttgttcgacc attgaactgc atcgtc
3612536DNAArtificialprimer DHFR-EcoRI-600-R 125aagcttgaat
tcttagtctt tcttctcgta gacttc 36126154DNAArtificialcombined
synthetic polyadenylation sequence and pausing signal from the
human alpha2 globin gene 126aataaaatat ctttattttc attacatctg
tgtgttggtt ttttgtgtga atcgatagta 60ctaacatacg ctctccatca aaacaaaacg
aaacaaaaca aactagcaaa ataggctgtc 120cccagtgcaa gtgcaggtgc
cagaacattt ctct 154127596DNAArtificialIRES sequence 127gcccctctcc
ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60gtgcgtttgt
ctatatgtga ttttccacca tattgccgtc ttttggcaat gtgagggccc
120ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
ctcgccaaag 180gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
tctggaagct tcttgaagac 240aaacaacgtc tgtagcgacc ctttgcaggc
agcggaaccc cccacctggc gacaggtgcc 300tctgcggcca aaagccacgt
gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360acgttgtgag
ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
420aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
gggcctcggt 480gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
tctaggcccc ccgaaccacg 540gggacgtggt tttcctttga aaaacacgat
gataagcttg ccacaacccc gggata 596128804DNAArtificialwild type
neomycin (Neo) resistance sequence 128atg gga tcg gcc att gaa caa
gat gga ttg cac gca ggt tct ccg gcc 48Met Gly Ser Ala Ile Glu Gln
Asp Gly Leu His Ala Gly Ser Pro Ala1 5 10 15gct tgg gtg gag agg cta
ttc ggc tat gac tgg gca caa cag aca atc 96Ala Trp Val Glu Arg Leu
Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile 20 25 30ggc tgc tct gat gcc
gcc gtg ttc cgg ctg tca gcg cag ggg cgc ccg 144Gly Cys Ser Asp Ala
Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro 35 40 45gtt ctt ttt gtc
aag acc gac ctg tcc ggt gcc ctg aat gaa ctg cag 192Val Leu Phe Val
Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln 50 55 60gac gag gca
gcg cgg cta tcg tgg ctg gcc acg acg ggc gtt cct tgc 240Asp Glu Ala
Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys65 70 75 80gca
gct gtg ctc gac gtt gtc act gaa gcg gga agg gac tgg ctg cta 288Ala
Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu 85 90
95ttg ggc gaa gtg ccg ggg cag gat ctc ctg tca tct cac ctt gct cct
336Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro
100 105 110gcc gag aaa gta tcc atc atg gct gat gca atg cgg cgg ctg
cat acg 384Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu
His Thr 115 120 125ctt gat ccg gct acc tgc cca ttc gac cac caa gcg
aaa cat cgc atc 432Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln Ala
Lys His Arg Ile 130 135 140gag cga gca cgt act cgg atg gaa
gcc ggt ctt gtc gat cag gat gat 480Glu Arg Ala Arg Thr Arg Met Glu
Ala Gly Leu Val Asp Gln Asp Asp145 150 155 160ctg gac gaa gag cat
cag ggg ctc gcg cca gcc gaa ctg ttc gcc agg 528Leu Asp Glu Glu His
Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg 165 170 175ctc aag gcg
cgc atg ccc gac ggc gag gat ctc gtc gtg acc cat ggc 576Leu Lys Ala
Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly 180 185 190gat
gcc tgc ttg ccg aat atc atg gtg gaa aat ggc cgc ttt tct gga 624Asp
Ala Cys Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly 195 200
205ttc atc gac tgt ggc cgg ctg ggt gtg gcg gac cgc tat cag gac ata
672Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile
210 215 220gcg ttg gct acc cgt gat att gct gaa gag ctt ggc ggc gaa
tgg gct 720Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu
Trp Ala225 230 235 240gac cgc ttc ctc gtg ctt tac ggt atc gcc gct
ccc gat tcg cag cgc 768Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala
Pro Asp Ser Gln Arg 245 250 255atc gcc ttc tat cgc ctt ctt gac gag
ttc ttc tga 804Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 260
265129267PRTArtificialSynthetic Construct 129Met Gly Ser Ala Ile
Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala1 5 10 15Ala Trp Val Glu
Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile 20 25 30Gly Cys Ser
Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro 35 40 45Val Leu
Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln 50 55 60Asp
Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys65 70 75
80Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu
85 90 95Leu Gly Glu Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala
Pro 100 105 110Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met Arg Arg
Leu His Thr 115 120 125Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gln
Ala Lys His Arg Ile 130 135 140Glu Arg Ala Arg Thr Arg Met Glu Ala
Gly Leu Val Asp Gln Asp Asp145 150 155 160Leu Asp Glu Glu His Gln
Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg 165 170 175Leu Lys Ala Arg
Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly 180 185 190Asp Ala
Cys Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly 195 200
205Phe Ile Asp Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile
210 215 220Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu
Trp Ala225 230 235 240Asp Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala
Pro Asp Ser Gln Arg 245 250 255Ile Ala Phe Tyr Arg Leu Leu Asp Glu
Phe Phe 260 265130804DNAArtificialCpG poor Neo resistance sequence
130atg gga agt gcc att gaa caa gac gga ttg cac gca ggt tct cct gca
48Met Gly Ser Ala Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala1
5 10 15gct tgg gtg gag agg cta ttt ggc tac gac tgg gca caa cag aca
ata 96Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr
Ile 20 25 30ggc tgc tct gac gca gca gtg ttc aga ctg tca gca cag ggg
aga cca 144Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly
Arg Pro 35 40 45gtt ctt ttt gtc aag act gac ctg tca ggt gcc ctg aac
gaa ctg cag 192Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn
Glu Leu Gln 50 55 60gac gag gca gca aga cta agt tgg ctg gcc act act
ggt gtt cct tgt 240Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr
Gly Val Pro Cys65 70 75 80gca gct gtg ttg gac gtt gtc act gaa gca
gga agg gac tgg ctg cta 288Ala Ala Val Leu Asp Val Val Thr Glu Ala
Gly Arg Asp Trp Leu Leu 85 90 95ttg ggt gaa gtg cct ggg cag gat ctc
ctg tca tct cac ctt gct cct 336Leu Gly Glu Val Pro Gly Gln Asp Leu
Leu Ser Ser His Leu Ala Pro 100 105 110gca gag aaa gta tcc atc ctg
gct gac gca ctg aga aga ctg cat act 384Ala Glu Lys Val Ser Ile Leu
Ala Asp Ala Leu Arg Arg Leu His Thr 115 120 125ctt gat cca gct acc
tgc cca ttt gac cac caa gca aaa cat aga att 432Leu Asp Pro Ala Thr
Cys Pro Phe Asp His Gln Ala Lys His Arg Ile 130 135 140gag aga gca
cga act aga ctg gaa gca ggt ctt gta gat cag gac gat 480Glu Arg Ala
Arg Thr Arg Leu Glu Ala Gly Leu Val Asp Gln Asp Asp145 150 155
160ctg gac gaa gag cat cag ggg ttg gca cca gca gaa ctg ttt gcc agg
528Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg
165 170 175ctc aag gca aga ctg cct gac ggt gaa gat ttg gtt gtg acc
cac ggt 576Leu Lys Ala Arg Leu Pro Asp Gly Glu Asp Leu Val Val Thr
His Gly 180 185 190gac gcc tgc ttg cct aat atc ctg gtg gaa aac ggc
aga ttt tct gga 624Asp Ala Cys Leu Pro Asn Ile Leu Val Glu Asn Gly
Arg Phe Ser Gly 195 200 205ttc att gac tgt ggc aga ctg ggt gtg gca
gac aga tat cag gac ata 672Phe Ile Asp Cys Gly Arg Leu Gly Val Ala
Asp Arg Tyr Gln Asp Ile 210 215 220gca ttg gct acc aga gat att gct
gaa gag ctt ggt ggt gag tgg gct 720Ala Leu Ala Thr Arg Asp Ile Ala
Glu Glu Leu Gly Gly Glu Trp Ala225 230 235 240gac aga ttc ttg gtg
ctt tac ggt ata gcc gct cct gat tca cag aga 768Asp Arg Phe Leu Val
Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg 245 250 255ata gcc ttc
tat aga ctt ctt gac gag ttc ttc tga 804Ile Ala Phe Tyr Arg Leu Leu
Asp Glu Phe Phe 260 265131267PRTArtificialSynthetic Construct
131Met Gly Ser Ala Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala1
5 10 15Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr
Ile 20 25 30Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly
Arg Pro 35 40 45Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn
Glu Leu Gln 50 55 60Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr
Gly Val Pro Cys65 70 75 80Ala Ala Val Leu Asp Val Val Thr Glu Ala
Gly Arg Asp Trp Leu Leu 85 90 95Leu Gly Glu Val Pro Gly Gln Asp Leu
Leu Ser Ser His Leu Ala Pro 100 105 110Ala Glu Lys Val Ser Ile Leu
Ala Asp Ala Leu Arg Arg Leu His Thr 115 120 125Leu Asp Pro Ala Thr
Cys Pro Phe Asp His Gln Ala Lys His Arg Ile 130 135 140Glu Arg Ala
Arg Thr Arg Leu Glu Ala Gly Leu Val Asp Gln Asp Asp145 150 155
160Leu Asp Glu Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg
165 170 175Leu Lys Ala Arg Leu Pro Asp Gly Glu Asp Leu Val Val Thr
His Gly 180 185 190Asp Ala Cys Leu Pro Asn Ile Leu Val Glu Asn Gly
Arg Phe Ser Gly 195 200 205Phe Ile Asp Cys Gly Arg Leu Gly Val Ala
Asp Arg Tyr Gln Asp Ile 210 215 220Ala Leu Ala Thr Arg Asp Ile Ala
Glu Glu Leu Gly Gly Glu Trp Ala225 230 235 240Asp Arg Phe Leu Val
Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg 245 250 255Ile Ala Phe
Tyr Arg Leu Leu Asp Glu Phe Phe 260 265132375DNAArtificialCpG poor
and ATG-less zeocin (Zeo) resistance sequence 132ttg gcc aag ttg
acc agt gct gtc cca gtg ctc aca gcc agg gac gtg 48Leu Ala Lys Leu
Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val1 5 10 15gct gga gct
gtt gag ttc tgg act gac agg ttg ggg ttc tcc aga gat 96Ala Gly Ala
Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30ttt gtg
gag gac gac ttt gca ggt gtg gtc aga gac gac gtc acc ctg 144Phe Val
Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45ttc
atc tca gca gtc cag gac cag gtg gtg cct gac aac acc ctg gct 192Phe
Ile Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55
60tgg gtg tgg gtg aga gga ctg gac gag ctg tac gct gag tgg agt gag
240Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser
Glu65 70 75 80gtg gtc tcc acc aac ttc agg gac gcc agt ggc cct gcc
ttg aca gag 288Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala
Leu Thr Glu 85 90 95att gga gag cag ccc tgg ggg aga gag ttt gcc ctg
aga gac cca gca 336Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu
Arg Asp Pro Ala 100 105 110ggc aac tgt gtg cac ttt gtg gca gag gag
cag gac tga 375Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115
120133124PRTArtificialSynthetic Construct 133Leu Ala Lys Leu Thr
Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val1 5 10 15Ala Gly Ala Val
Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30Phe Val Glu
Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45Phe Ile
Ser Ala Val Gln Asp Gln Val Val Pro Asp Asn Thr Leu Ala 50 55 60Trp
Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu65 70 75
80Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Leu Thr Glu
85 90 95Ile Gly Glu Gln Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro
Ala 100 105 110Gly Asn Cys Val His Phe Val Ala Glu Glu Gln Asp 115
1201341194DNAEscherichia coliwt trp
sequence(1)..(1194)CDS(1)..(1194) 134atg aca aca tta ctt aac ccc
tat ttt ggt gag ttt ggc ggc atg tac 48Met Thr Thr Leu Leu Asn Pro
Tyr Phe Gly Glu Phe Gly Gly Met Tyr1 5 10 15gtg cca caa atc ctg atg
cct gct ctg cgc cag ctg gaa gaa gct ttt 96Val Pro Gln Ile Leu Met
Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe 20 25 30gtc agt gcg caa aaa
gat cct gaa ttt cag gct cag ttc aac gac ctg 144Val Ser Ala Gln Lys
Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp Leu 35 40 45ctg aaa aac tat
gcc ggg cgt cca acc gcg ctg acc aaa tgc cag aac 192Leu Lys Asn Tyr
Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys Gln Asn 50 55 60att aca gcc
ggg acg aac acc acg ctg tat ctc aag cgt gaa gat ttg 240Ile Thr Ala
Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg Glu Asp Leu65 70 75 80ctg
cac ggc ggc gcg cat aaa act aac cag gtg ctg ggg cag gcg ttg 288Leu
His Gly Gly Ala His Lys Thr Asn Gln Val Leu Gly Gln Ala Leu 85 90
95ctg gcg aag cgg atg ggt aaa acc gaa atc atc gcc gaa acc ggt gcc
336Leu Ala Lys Arg Met Gly Lys Thr Glu Ile Ile Ala Glu Thr Gly Ala
100 105 110ggt cag cat ggc gtg gcg tcg gcc ctg gcc agc gcc ctg ctc
ggc ctg 384Gly Gln His Gly Val Ala Ser Ala Leu Ala Ser Ala Leu Leu
Gly Leu 115 120 125aaa tgc cgt att tat atg ggt gcc aaa gac gtt gaa
cgc cag tcg cct 432Lys Cys Arg Ile Tyr Met Gly Ala Lys Asp Val Glu
Arg Gln Ser Pro 130 135 140aac gtt ttt cgt atg cgc tta atg ggt gcg
gaa gtg atc ccg gtg cat 480Asn Val Phe Arg Met Arg Leu Met Gly Ala
Glu Val Ile Pro Val His145 150 155 160agc ggt tcc gcg acg ctg aaa
gat gcc tgt aac gag gcg ctg cgc gac 528Ser Gly Ser Ala Thr Leu Lys
Asp Ala Cys Asn Glu Ala Leu Arg Asp 165 170 175tgg tcc ggt agt tac
gaa acc gcg cac tat atg ctg ggc acc gca gct 576Trp Ser Gly Ser Tyr
Glu Thr Ala His Tyr Met Leu Gly Thr Ala Ala 180 185 190ggc ccg cat
cct tat ccg acc att gtg cgt gag ttt cag cgg atg att 624Gly Pro His
Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg Met Ile 195 200 205ggc
gaa gaa acc aaa gcg cag att ctg gaa aga gaa ggt cgc ctg ccg 672Gly
Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu Gly Arg Leu Pro 210 215
220gat gcc gtt atc gcc tgt gtt ggc ggc ggt tcg aat gcc atc ggc atg
720Asp Ala Val Ile Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly
Met225 230 235 240ttt gct gat ttc atc aat gaa acc aac gtc ggc ctg
att ggt gtg gag 768Phe Ala Asp Phe Ile Asn Glu Thr Asn Val Gly Leu
Ile Gly Val Glu 245 250 255cca ggt ggt cac ggt atc gaa act ggc gag
cac ggc gca ccg cta aaa 816Pro Gly Gly His Gly Ile Glu Thr Gly Glu
His Gly Ala Pro Leu Lys 260 265 270cat ggt cgc gtg ggt atc tat ttc
ggt atg aaa gcg ccg atg atg caa 864His Gly Arg Val Gly Ile Tyr Phe
Gly Met Lys Ala Pro Met Met Gln 275 280 285acc gaa gac ggg cag att
gaa gaa tct tac tcc atc tcc gcc gga ctg 912Thr Glu Asp Gly Gln Ile
Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu 290 295 300gat ttc ccg tct
gtc ggc cca caa cac gcg tat ctt aac agc act gga 960Asp Phe Pro Ser
Val Gly Pro Gln His Ala Tyr Leu Asn Ser Thr Gly305 310 315 320cgc
gct gat tac gtg tct att acc gat gat gaa gcc ctt gaa gcc ttc 1008Arg
Ala Asp Tyr Val Ser Ile Thr Asp Asp Glu Ala Leu Glu Ala Phe 325 330
335aaa acg ctg tgc ctg cac gaa ggg atc atc ccg gcg ctg gaa tcc tcc
1056Lys Thr Leu Cys Leu His Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser
340 345 350cac gcc ttg gcc cat gcg ttg aaa atg atg cgc gaa aac ccg
gat aaa 1104His Ala Leu Ala His Ala Leu Lys Met Met Arg Glu Asn Pro
Asp Lys 355 360 365gag cag cta ctg gtg gtt aac ctt tcc ggt cgc ggc
gat aaa gac atc 1152Glu Gln Leu Leu Val Val Asn Leu Ser Gly Arg Gly
Asp Lys Asp Ile 370 375 380ttc acc gtt cac gat att ttg aaa gca cga
ggg gaa atc tga 1194Phe Thr Val His Asp Ile Leu Lys Ala Arg Gly Glu
Ile385 390 395135397PRTEscherichia coli 135Met Thr Thr Leu Leu Asn
Pro Tyr Phe Gly Glu Phe Gly Gly Met Tyr1 5 10 15Val Pro Gln Ile Leu
Met Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe 20 25 30Val Ser Ala Gln
Lys Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp Leu 35 40 45Leu Lys Asn
Tyr Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys Gln Asn 50 55 60Ile Thr
Ala Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg Glu Asp Leu65 70 75
80Leu His Gly Gly Ala His Lys Thr Asn Gln Val Leu Gly Gln Ala Leu
85 90 95Leu Ala Lys Arg Met Gly Lys Thr Glu Ile Ile Ala Glu Thr Gly
Ala 100 105 110Gly Gln His Gly Val Ala Ser Ala Leu Ala Ser Ala Leu
Leu Gly Leu 115 120 125Lys Cys Arg Ile Tyr Met Gly Ala Lys Asp Val
Glu Arg Gln Ser Pro 130 135 140Asn Val Phe Arg Met Arg Leu Met Gly
Ala Glu Val Ile Pro Val His145 150 155 160Ser Gly Ser Ala Thr Leu
Lys Asp Ala Cys Asn Glu Ala Leu Arg Asp 165 170 175Trp Ser Gly Ser
Tyr Glu Thr Ala His Tyr Met Leu Gly Thr Ala Ala 180 185 190Gly Pro
His Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg Met Ile 195 200
205Gly Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu Gly Arg Leu Pro
210 215 220Asp Ala Val Ile Ala Cys Val Gly Gly Gly Ser Asn Ala Ile
Gly Met225 230 235 240Phe Ala Asp Phe Ile Asn Glu Thr Asn Val Gly
Leu Ile Gly Val Glu 245 250 255Pro Gly Gly His Gly Ile Glu Thr Gly
Glu His Gly Ala Pro Leu Lys 260 265 270His Gly Arg Val Gly Ile Tyr
Phe Gly Met Lys Ala Pro Met Met Gln 275 280 285Thr Glu Asp Gly Gln
Ile Glu Glu Ser Tyr Ser Ile
Ser Ala Gly Leu 290 295 300Asp Phe Pro Ser Val Gly Pro Gln His Ala
Tyr Leu Asn Ser Thr Gly305 310 315 320Arg Ala Asp Tyr Val Ser Ile
Thr Asp Asp Glu Ala Leu Glu Ala Phe 325 330 335Lys Thr Leu Cys Leu
His Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser 340 345 350His Ala Leu
Ala His Ala Leu Lys Met Met Arg Glu Asn Pro Asp Lys 355 360 365Glu
Gln Leu Leu Val Val Asn Leu Ser Gly Arg Gly Asp Lys Asp Ile 370 375
380Phe Thr Val His Asp Ile Leu Lys Ala Arg Gly Glu Ile385 390
3951361194DNAArtificialATG-less trp sequence 136atg aca aca tta ctt
aac ccc tat ttt ggt gag ttt ggc ggc cag tac 48Met Thr Thr Leu Leu
Asn Pro Tyr Phe Gly Glu Phe Gly Gly Gln Tyr1 5 10 15gtg cca caa atc
ctg gtc cct gct ctg cgc cag ctg gaa gag gct ttt 96Val Pro Gln Ile
Leu Val Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe 20 25 30gtc agt gcc
caa aaa gat cct gaa ttt caa gct cag ttc aac gac ctg 144Val Ser Ala
Gln Lys Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp Leu 35 40 45ctg aaa
aac tac gcc ggg cgt cca acc gcg ctg acc aag tgc cag aac 192Leu Lys
Asn Tyr Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys Gln Asn 50 55 60att
acc gcc ggg acg aac acc acg ctg tat ctc aag cgt gaa gat ttg 240Ile
Thr Ala Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg Glu Asp Leu65 70 75
80ctg cac ggc ggc gcg cat aaa act aac cag gtg ctg ggg cag gcg ttg
288Leu His Gly Gly Ala His Lys Thr Asn Gln Val Leu Gly Gln Ala Leu
85 90 95ctg gcg aag cgg ctg ggt aaa acc gaa atc atc gcc gaa act ggt
gcc 336Leu Ala Lys Arg Leu Gly Lys Thr Glu Ile Ile Ala Glu Thr Gly
Ala 100 105 110ggt cag cac ggc gtg gcg tcg gcc ctt gcc agc gcc ctg
ctc ggc ctg 384Gly Gln His Gly Val Ala Ser Ala Leu Ala Ser Ala Leu
Leu Gly Leu 115 120 125aag tgc cgt att tat ctg ggt gcc aaa gac gtt
gaa cgc cag tcg cct 432Lys Cys Arg Ile Tyr Leu Gly Ala Lys Asp Val
Glu Arg Gln Ser Pro 130 135 140aac gtt ttt cgt ctg cgc tta ctg ggt
gcg gaa gtg atc ccg gtg cat 480Asn Val Phe Arg Leu Arg Leu Leu Gly
Ala Glu Val Ile Pro Val His145 150 155 160agc ggt tcc gcg acg ctg
aaa gac gcc tgt aac gag gcg ctg cgc gac 528Ser Gly Ser Ala Thr Leu
Lys Asp Ala Cys Asn Glu Ala Leu Arg Asp 165 170 175tgg tcc ggt agt
tac gaa acc gcg cac tat ctg ctg ggc acc gca gct 576Trp Ser Gly Ser
Tyr Glu Thr Ala His Tyr Leu Leu Gly Thr Ala Ala 180 185 190ggc ccg
cat cct tat ccg acc att gtg cgt gag ttt caa cgg atc att 624Gly Pro
His Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg Ile Ile 195 200
205ggc gaa gaa acc aaa gcg cag att ctg gaa aga gaa ggt cgc ctg ccg
672Gly Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu Gly Arg Leu Pro
210 215 220gac gcc gtt atc gcc tgt gtt ggc ggc ggt tct aac gcc atc
ggc atc 720Asp Ala Val Ile Ala Cys Val Gly Gly Gly Ser Asn Ala Ile
Gly Ile225 230 235 240ttt gct gat ttc atc aac gaa acc aac gtc ggc
ctg att ggt gtg gag 768Phe Ala Asp Phe Ile Asn Glu Thr Asn Val Gly
Leu Ile Gly Val Glu 245 250 255cca ggt ggt cac ggt atc gaa act ggc
gag cac ggc gca ccg cta aaa 816Pro Gly Gly His Gly Ile Glu Thr Gly
Glu His Gly Ala Pro Leu Lys 260 265 270cac ggt cgc gtg ggt atc tat
ttc ggt ctg aaa gcg ccg atc ctg caa 864His Gly Arg Val Gly Ile Tyr
Phe Gly Leu Lys Ala Pro Ile Leu Gln 275 280 285acc gaa gac ggg cag
att gaa gaa tct tac tcc atc tcc gcc gga ctg 912Thr Glu Asp Gly Gln
Ile Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu 290 295 300gat ttc ccg
tct gtc ggc cca caa cac gcc tat ctt aac agc act gga 960Asp Phe Pro
Ser Val Gly Pro Gln His Ala Tyr Leu Asn Ser Thr Gly305 310 315
320cgc gct gat tac gtg tct att acc gac gac gaa gcc ctt gaa gcc ttc
1008Arg Ala Asp Tyr Val Ser Ile Thr Asp Asp Glu Ala Leu Glu Ala Phe
325 330 335aaa acg ctg tgc ctg cac gaa ggg atc atc ccg gcg ctg gaa
tcc tcc 1056Lys Thr Leu Cys Leu His Glu Gly Ile Ile Pro Ala Leu Glu
Ser Ser 340 345 350cac gcc ctg gcc cac gcc ttg aaa ctg gct cgc gaa
aac ccg gat aaa 1104His Ala Leu Ala His Ala Leu Lys Leu Ala Arg Glu
Asn Pro Asp Lys 355 360 365gag cag cta ctg gtg gtc aac ctt tcc ggt
cgc ggc gat aaa gac atc 1152Glu Gln Leu Leu Val Val Asn Leu Ser Gly
Arg Gly Asp Lys Asp Ile 370 375 380ttc acc gtt cac gat att ttg aaa
gca cga ggg gaa atc tga 1194Phe Thr Val His Asp Ile Leu Lys Ala Arg
Gly Glu Ile385 390 395137397PRTArtificialSynthetic Construct 137Met
Thr Thr Leu Leu Asn Pro Tyr Phe Gly Glu Phe Gly Gly Gln Tyr1 5 10
15Val Pro Gln Ile Leu Val Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe
20 25 30Val Ser Ala Gln Lys Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp
Leu 35 40 45Leu Lys Asn Tyr Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys
Gln Asn 50 55 60Ile Thr Ala Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg
Glu Asp Leu65 70 75 80Leu His Gly Gly Ala His Lys Thr Asn Gln Val
Leu Gly Gln Ala Leu 85 90 95Leu Ala Lys Arg Leu Gly Lys Thr Glu Ile
Ile Ala Glu Thr Gly Ala 100 105 110Gly Gln His Gly Val Ala Ser Ala
Leu Ala Ser Ala Leu Leu Gly Leu 115 120 125Lys Cys Arg Ile Tyr Leu
Gly Ala Lys Asp Val Glu Arg Gln Ser Pro 130 135 140Asn Val Phe Arg
Leu Arg Leu Leu Gly Ala Glu Val Ile Pro Val His145 150 155 160Ser
Gly Ser Ala Thr Leu Lys Asp Ala Cys Asn Glu Ala Leu Arg Asp 165 170
175Trp Ser Gly Ser Tyr Glu Thr Ala His Tyr Leu Leu Gly Thr Ala Ala
180 185 190Gly Pro His Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg
Ile Ile 195 200 205Gly Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu
Gly Arg Leu Pro 210 215 220Asp Ala Val Ile Ala Cys Val Gly Gly Gly
Ser Asn Ala Ile Gly Ile225 230 235 240Phe Ala Asp Phe Ile Asn Glu
Thr Asn Val Gly Leu Ile Gly Val Glu 245 250 255Pro Gly Gly His Gly
Ile Glu Thr Gly Glu His Gly Ala Pro Leu Lys 260 265 270His Gly Arg
Val Gly Ile Tyr Phe Gly Leu Lys Ala Pro Ile Leu Gln 275 280 285Thr
Glu Asp Gly Gln Ile Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu 290 295
300Asp Phe Pro Ser Val Gly Pro Gln His Ala Tyr Leu Asn Ser Thr
Gly305 310 315 320Arg Ala Asp Tyr Val Ser Ile Thr Asp Asp Glu Ala
Leu Glu Ala Phe 325 330 335Lys Thr Leu Cys Leu His Glu Gly Ile Ile
Pro Ala Leu Glu Ser Ser 340 345 350His Ala Leu Ala His Ala Leu Lys
Leu Ala Arg Glu Asn Pro Asp Lys 355 360 365Glu Gln Leu Leu Val Val
Asn Leu Ser Gly Arg Gly Asp Lys Asp Ile 370 375 380Phe Thr Val His
Asp Ile Leu Lys Ala Arg Gly Glu Ile385 390 3951381305DNASalmonella
typhimuriumwt his sequence(1)..(1305)CDS(1)..(1305) 138atg agc ttc
aat acc ctg att gac tgg aac agc tgt agc cct gaa cag 48Met Ser Phe
Asn Thr Leu Ile Asp Trp Asn Ser Cys Ser Pro Glu Gln1 5 10 15cag cgt
gcg ctg ctg acg cgt ccg gcg att tcc gcc tct gac agt att 96Gln Arg
Ala Leu Leu Thr Arg Pro Ala Ile Ser Ala Ser Asp Ser Ile 20 25 30acc
cgg acg gtc agc gat att ctg gat aat gta aaa acg cgc ggt gac 144Thr
Arg Thr Val Ser Asp Ile Leu Asp Asn Val Lys Thr Arg Gly Asp 35 40
45gat gcc ctg cgt gaa tac agc gct aaa ttt gat aaa aca gaa gtg aca
192Asp Ala Leu Arg Glu Tyr Ser Ala Lys Phe Asp Lys Thr Glu Val Thr
50 55 60gcg cta cgc gtc acc cct gaa gag atc gcc gcc gcc ggc gcg cgt
ctg 240Ala Leu Arg Val Thr Pro Glu Glu Ile Ala Ala Ala Gly Ala Arg
Leu65 70 75 80agc gac gaa tta aaa cag gcg atg acc gct gcc gtc aaa
aat att gaa 288Ser Asp Glu Leu Lys Gln Ala Met Thr Ala Ala Val Lys
Asn Ile Glu 85 90 95acg ttc cat tcc gcg cag acg cta ccg cct gta gat
gtg gaa acc cag 336Thr Phe His Ser Ala Gln Thr Leu Pro Pro Val Asp
Val Glu Thr Gln 100 105 110cca ggc gtg cgt tgc cag cag gtt acg cgt
ccc gtc tcg tct gtc ggt 384Pro Gly Val Arg Cys Gln Gln Val Thr Arg
Pro Val Ser Ser Val Gly 115 120 125ctg tat att ccc ggc ggc tcg gct
ccg ctc ttc tca acg gtg ctg atg 432Leu Tyr Ile Pro Gly Gly Ser Ala
Pro Leu Phe Ser Thr Val Leu Met 130 135 140ctg gcg acg ccg gcg cgc
att gcg gga tgc cag aag gtg gtt ctg tgc 480Leu Ala Thr Pro Ala Arg
Ile Ala Gly Cys Gln Lys Val Val Leu Cys145 150 155 160tcg ccg ccg
ccc atc gct gat gaa atc ctc tat gcg gcg caa ctg tgt 528Ser Pro Pro
Pro Ile Ala Asp Glu Ile Leu Tyr Ala Ala Gln Leu Cys 165 170 175ggc
gtg cag gaa atc ttt aac gtc ggc ggc gcg cag gcg att gcc gct 576Gly
Val Gln Glu Ile Phe Asn Val Gly Gly Ala Gln Ala Ile Ala Ala 180 185
190ctg gcc ttc ggc agc gag tcc gta ccg aaa gtg gat aaa att ttt ggc
624Leu Ala Phe Gly Ser Glu Ser Val Pro Lys Val Asp Lys Ile Phe Gly
195 200 205ccc ggc aac gcc ttt gta acc gaa gcc aaa cgt cag gtc agc
cag cgt 672Pro Gly Asn Ala Phe Val Thr Glu Ala Lys Arg Gln Val Ser
Gln Arg 210 215 220ctc gac ggc gcg gct atc gat atg cca gcc ggg ccg
tct gaa gta ctg 720Leu Asp Gly Ala Ala Ile Asp Met Pro Ala Gly Pro
Ser Glu Val Leu225 230 235 240gtg atc gca gac agc ggc gca aca ccg
gat ttc gtc gct tct gac ctg 768Val Ile Ala Asp Ser Gly Ala Thr Pro
Asp Phe Val Ala Ser Asp Leu 245 250 255ctc tcc cag gct gag cac ggc
ccg gat tcc cag gtg atc ctg ctg acg 816Leu Ser Gln Ala Glu His Gly
Pro Asp Ser Gln Val Ile Leu Leu Thr 260 265 270cct gat gct gac att
gcc cgc aag gtg gcg gag gcg gta gaa cgt caa 864Pro Asp Ala Asp Ile
Ala Arg Lys Val Ala Glu Ala Val Glu Arg Gln 275 280 285ctg gcg gaa
ctg ccg cgc gcg gac acc gcc cgg cag gcc ctg agc gcc 912Leu Ala Glu
Leu Pro Arg Ala Asp Thr Ala Arg Gln Ala Leu Ser Ala 290 295 300agt
cgt ctg att gtg acc aaa gat tta gcg cag tgc gtc gcc atc tct 960Ser
Arg Leu Ile Val Thr Lys Asp Leu Ala Gln Cys Val Ala Ile Ser305 310
315 320aat cag tat ggg ccg gaa cac tta atc atc cag acg cgc aat gcg
cgc 1008Asn Gln Tyr Gly Pro Glu His Leu Ile Ile Gln Thr Arg Asn Ala
Arg 325 330 335gat ttg gtg gat gcg att acc agc gca ggc tcg gta ttt
ctc ggc gac 1056Asp Leu Val Asp Ala Ile Thr Ser Ala Gly Ser Val Phe
Leu Gly Asp 340 345 350tgg tcg ccg gaa tcc gcc ggt gat tac gct tcc
gga acc aac cat gtt 1104Trp Ser Pro Glu Ser Ala Gly Asp Tyr Ala Ser
Gly Thr Asn His Val 355 360 365tta ccg acc tat ggc tat act gct acc
tgt tcc agc ctt ggg tta gcg 1152Leu Pro Thr Tyr Gly Tyr Thr Ala Thr
Cys Ser Ser Leu Gly Leu Ala 370 375 380gat ttc cag aaa cgg atg acc
gtt cag gaa ctg tcg aaa gcg ggc ttt 1200Asp Phe Gln Lys Arg Met Thr
Val Gln Glu Leu Ser Lys Ala Gly Phe385 390 395 400tcc gct ctg gca
tca acc att gaa aca ttg gcg gcg gca gaa cgt ctg 1248Ser Ala Leu Ala
Ser Thr Ile Glu Thr Leu Ala Ala Ala Glu Arg Leu 405 410 415acc gcc
cat aaa aat gcc gtg acc ctg cgc gta aac gcc ctc aag gag 1296Thr Ala
His Lys Asn Ala Val Thr Leu Arg Val Asn Ala Leu Lys Glu 420 425
430caa gca tga 1305Gln Ala139434PRTSalmonella typhimurium 139Met
Ser Phe Asn Thr Leu Ile Asp Trp Asn Ser Cys Ser Pro Glu Gln1 5 10
15Gln Arg Ala Leu Leu Thr Arg Pro Ala Ile Ser Ala Ser Asp Ser Ile
20 25 30Thr Arg Thr Val Ser Asp Ile Leu Asp Asn Val Lys Thr Arg Gly
Asp 35 40 45Asp Ala Leu Arg Glu Tyr Ser Ala Lys Phe Asp Lys Thr Glu
Val Thr 50 55 60Ala Leu Arg Val Thr Pro Glu Glu Ile Ala Ala Ala Gly
Ala Arg Leu65 70 75 80Ser Asp Glu Leu Lys Gln Ala Met Thr Ala Ala
Val Lys Asn Ile Glu 85 90 95Thr Phe His Ser Ala Gln Thr Leu Pro Pro
Val Asp Val Glu Thr Gln 100 105 110Pro Gly Val Arg Cys Gln Gln Val
Thr Arg Pro Val Ser Ser Val Gly 115 120 125Leu Tyr Ile Pro Gly Gly
Ser Ala Pro Leu Phe Ser Thr Val Leu Met 130 135 140Leu Ala Thr Pro
Ala Arg Ile Ala Gly Cys Gln Lys Val Val Leu Cys145 150 155 160Ser
Pro Pro Pro Ile Ala Asp Glu Ile Leu Tyr Ala Ala Gln Leu Cys 165 170
175Gly Val Gln Glu Ile Phe Asn Val Gly Gly Ala Gln Ala Ile Ala Ala
180 185 190Leu Ala Phe Gly Ser Glu Ser Val Pro Lys Val Asp Lys Ile
Phe Gly 195 200 205Pro Gly Asn Ala Phe Val Thr Glu Ala Lys Arg Gln
Val Ser Gln Arg 210 215 220Leu Asp Gly Ala Ala Ile Asp Met Pro Ala
Gly Pro Ser Glu Val Leu225 230 235 240Val Ile Ala Asp Ser Gly Ala
Thr Pro Asp Phe Val Ala Ser Asp Leu 245 250 255Leu Ser Gln Ala Glu
His Gly Pro Asp Ser Gln Val Ile Leu Leu Thr 260 265 270Pro Asp Ala
Asp Ile Ala Arg Lys Val Ala Glu Ala Val Glu Arg Gln 275 280 285Leu
Ala Glu Leu Pro Arg Ala Asp Thr Ala Arg Gln Ala Leu Ser Ala 290 295
300Ser Arg Leu Ile Val Thr Lys Asp Leu Ala Gln Cys Val Ala Ile
Ser305 310 315 320Asn Gln Tyr Gly Pro Glu His Leu Ile Ile Gln Thr
Arg Asn Ala Arg 325 330 335Asp Leu Val Asp Ala Ile Thr Ser Ala Gly
Ser Val Phe Leu Gly Asp 340 345 350Trp Ser Pro Glu Ser Ala Gly Asp
Tyr Ala Ser Gly Thr Asn His Val 355 360 365Leu Pro Thr Tyr Gly Tyr
Thr Ala Thr Cys Ser Ser Leu Gly Leu Ala 370 375 380Asp Phe Gln Lys
Arg Met Thr Val Gln Glu Leu Ser Lys Ala Gly Phe385 390 395 400Ser
Ala Leu Ala Ser Thr Ile Glu Thr Leu Ala Ala Ala Glu Arg Leu 405 410
415Thr Ala His Lys Asn Ala Val Thr Leu Arg Val Asn Ala Leu Lys Glu
420 425 430Gln Ala1401305DNAArtificialATG-less his sequence 140atg
agc ttc aat acc ctg att gac tgg aac agc tgt agc cct gaa cag 48Met
Ser Phe Asn Thr Leu Ile Asp Trp Asn Ser Cys Ser Pro Glu Gln1 5 10
15cag cgt gcg ctg ctg acg cgt ccg gcg att tcc gcc tct gac agt att
96Gln Arg Ala Leu Leu Thr Arg Pro Ala Ile Ser Ala Ser Asp Ser Ile
20 25 30acc cgg acg gtc agc gat att ctg gat aac gta aaa acg cgc ggt
gac 144Thr Arg Thr Val Ser Asp Ile Leu Asp Asn Val Lys Thr Arg Gly
Asp 35 40 45gac gcc ctg cgt gaa tac agc gct aaa ttt gat aaa aca gaa
gtg aca 192Asp Ala Leu Arg Glu Tyr Ser Ala Lys Phe Asp Lys Thr Glu
Val Thr 50 55 60gcg cta cgc gtc acc cct gaa gag atc gcc gcc gcc ggc
gcg cgt ctg 240Ala Leu Arg Val Thr Pro Glu Glu Ile Ala Ala Ala Gly
Ala Arg Leu65 70 75 80agc gac gaa tta aaa cag gcg att acc gct gcc
gtc aaa aat att gaa 288Ser Asp Glu Leu Lys Gln Ala Ile Thr Ala Ala
Val Lys Asn Ile Glu 85 90 95acg ttc cat tcc gcg cag acg cta ccg cct
gta gac gtg gaa acc cag 336Thr Phe
His Ser Ala Gln Thr Leu Pro Pro Val Asp Val Glu Thr Gln 100 105
110cca ggc gtg cgt tgc cag cag gtt acg cgt ccc gtc tcg tct gtc ggt
384Pro Gly Val Arg Cys Gln Gln Val Thr Arg Pro Val Ser Ser Val Gly
115 120 125ctg tat att ccc ggc ggc tcg gct ccg ctc ttc tca acg gtg
ctg ctg 432Leu Tyr Ile Pro Gly Gly Ser Ala Pro Leu Phe Ser Thr Val
Leu Leu 130 135 140ctg gcg acg ccg gcg cgc att gcg ggt tgc cag aag
gtg gtt ctg tgc 480Leu Ala Thr Pro Ala Arg Ile Ala Gly Cys Gln Lys
Val Val Leu Cys145 150 155 160tcg ccg ccg ccc atc gct gac gaa atc
ctc tac gcg gcg caa ctg tgt 528Ser Pro Pro Pro Ile Ala Asp Glu Ile
Leu Tyr Ala Ala Gln Leu Cys 165 170 175ggc gtg cag gaa atc ttt aac
gtc ggc ggc gcg cag gcg att gcc gct 576Gly Val Gln Glu Ile Phe Asn
Val Gly Gly Ala Gln Ala Ile Ala Ala 180 185 190ctg gcc ttc ggc agc
gag tcc gta ccg aaa gtg gat aaa att ttt ggc 624Leu Ala Phe Gly Ser
Glu Ser Val Pro Lys Val Asp Lys Ile Phe Gly 195 200 205ccc ggc aac
gcc ttt gta acc gaa gcc aaa cgt cag gtc agc cag cgt 672Pro Gly Asn
Ala Phe Val Thr Glu Ala Lys Arg Gln Val Ser Gln Arg 210 215 220ctc
gac ggc gcg gct atc gat att cca gcc ggg ccg tct gaa gta ctg 720Leu
Asp Gly Ala Ala Ile Asp Ile Pro Ala Gly Pro Ser Glu Val Leu225 230
235 240gtg atc gca gac agc ggc gca aca ccg gat ttc gtc gct tct gac
ctg 768Val Ile Ala Asp Ser Gly Ala Thr Pro Asp Phe Val Ala Ser Asp
Leu 245 250 255ctc tcc cag gct gag cac ggc ccg gat tcc cag gtg atc
ctg ctg acg 816Leu Ser Gln Ala Glu His Gly Pro Asp Ser Gln Val Ile
Leu Leu Thr 260 265 270cct gac gct gac att gcc cgc aag gtg gcg gag
gcg gta gaa cgt caa 864Pro Asp Ala Asp Ile Ala Arg Lys Val Ala Glu
Ala Val Glu Arg Gln 275 280 285ctg gcg gaa ctg ccg cgc gcg gac acc
gcc cgg cag gcc ctg agc gcc 912Leu Ala Glu Leu Pro Arg Ala Asp Thr
Ala Arg Gln Ala Leu Ser Ala 290 295 300agt cgt ctg att gtg acc aaa
gat tta gcg cag tgc gtc gcc atc tct 960Ser Arg Leu Ile Val Thr Lys
Asp Leu Ala Gln Cys Val Ala Ile Ser305 310 315 320aat cag tac ggg
ccg gaa cac tta atc atc cag acg cgc aac gcg cgc 1008Asn Gln Tyr Gly
Pro Glu His Leu Ile Ile Gln Thr Arg Asn Ala Arg 325 330 335gat ttg
gtg gac gcg att acc agc gca ggc tcg gta ttt ctc ggc gac 1056Asp Leu
Val Asp Ala Ile Thr Ser Ala Gly Ser Val Phe Leu Gly Asp 340 345
350tgg tcg ccg gaa tcc gcc ggt gat tac gct tcc gga acc aac cac gtt
1104Trp Ser Pro Glu Ser Ala Gly Asp Tyr Ala Ser Gly Thr Asn His Val
355 360 365tta ccg acc tac ggc tat act gct acc tgt tcc agc ctt ggg
tta gcg 1152Leu Pro Thr Tyr Gly Tyr Thr Ala Thr Cys Ser Ser Leu Gly
Leu Ala 370 375 380gat ttc cag aaa cgg att acc gtt cag gaa ctg tcg
aaa gcg ggc ttt 1200Asp Phe Gln Lys Arg Ile Thr Val Gln Glu Leu Ser
Lys Ala Gly Phe385 390 395 400tcc gct ctg gca tca acc att gaa aca
ttg gcg gcg gca gaa cgt ctg 1248Ser Ala Leu Ala Ser Thr Ile Glu Thr
Leu Ala Ala Ala Glu Arg Leu 405 410 415acc gcc cat aaa aac gcc gtg
acc ctg cgc gta aac gcc ctc aag gag 1296Thr Ala His Lys Asn Ala Val
Thr Leu Arg Val Asn Ala Leu Lys Glu 420 425 430caa gca taa 1305Gln
Ala141434PRTArtificialSynthetic Construct 141Met Ser Phe Asn Thr
Leu Ile Asp Trp Asn Ser Cys Ser Pro Glu Gln1 5 10 15Gln Arg Ala Leu
Leu Thr Arg Pro Ala Ile Ser Ala Ser Asp Ser Ile 20 25 30Thr Arg Thr
Val Ser Asp Ile Leu Asp Asn Val Lys Thr Arg Gly Asp 35 40 45Asp Ala
Leu Arg Glu Tyr Ser Ala Lys Phe Asp Lys Thr Glu Val Thr 50 55 60Ala
Leu Arg Val Thr Pro Glu Glu Ile Ala Ala Ala Gly Ala Arg Leu65 70 75
80Ser Asp Glu Leu Lys Gln Ala Ile Thr Ala Ala Val Lys Asn Ile Glu
85 90 95Thr Phe His Ser Ala Gln Thr Leu Pro Pro Val Asp Val Glu Thr
Gln 100 105 110Pro Gly Val Arg Cys Gln Gln Val Thr Arg Pro Val Ser
Ser Val Gly 115 120 125Leu Tyr Ile Pro Gly Gly Ser Ala Pro Leu Phe
Ser Thr Val Leu Leu 130 135 140Leu Ala Thr Pro Ala Arg Ile Ala Gly
Cys Gln Lys Val Val Leu Cys145 150 155 160Ser Pro Pro Pro Ile Ala
Asp Glu Ile Leu Tyr Ala Ala Gln Leu Cys 165 170 175Gly Val Gln Glu
Ile Phe Asn Val Gly Gly Ala Gln Ala Ile Ala Ala 180 185 190Leu Ala
Phe Gly Ser Glu Ser Val Pro Lys Val Asp Lys Ile Phe Gly 195 200
205Pro Gly Asn Ala Phe Val Thr Glu Ala Lys Arg Gln Val Ser Gln Arg
210 215 220Leu Asp Gly Ala Ala Ile Asp Ile Pro Ala Gly Pro Ser Glu
Val Leu225 230 235 240Val Ile Ala Asp Ser Gly Ala Thr Pro Asp Phe
Val Ala Ser Asp Leu 245 250 255Leu Ser Gln Ala Glu His Gly Pro Asp
Ser Gln Val Ile Leu Leu Thr 260 265 270Pro Asp Ala Asp Ile Ala Arg
Lys Val Ala Glu Ala Val Glu Arg Gln 275 280 285Leu Ala Glu Leu Pro
Arg Ala Asp Thr Ala Arg Gln Ala Leu Ser Ala 290 295 300Ser Arg Leu
Ile Val Thr Lys Asp Leu Ala Gln Cys Val Ala Ile Ser305 310 315
320Asn Gln Tyr Gly Pro Glu His Leu Ile Ile Gln Thr Arg Asn Ala Arg
325 330 335Asp Leu Val Asp Ala Ile Thr Ser Ala Gly Ser Val Phe Leu
Gly Asp 340 345 350Trp Ser Pro Glu Ser Ala Gly Asp Tyr Ala Ser Gly
Thr Asn His Val 355 360 365Leu Pro Thr Tyr Gly Tyr Thr Ala Thr Cys
Ser Ser Leu Gly Leu Ala 370 375 380Asp Phe Gln Lys Arg Ile Thr Val
Gln Glu Leu Ser Lys Ala Gly Phe385 390 395 400Ser Ala Leu Ala Ser
Thr Ile Glu Thr Leu Ala Ala Ala Glu Arg Leu 405 410 415Thr Ala His
Lys Asn Ala Val Thr Leu Arg Val Asn Ala Leu Lys Glu 420 425 430Gln
Ala
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