U.S. patent application number 17/098974 was filed with the patent office on 2021-03-04 for site-specific enzymes and methods of use.
The applicant listed for this patent is Poseida Therapeutics, Inc.. Invention is credited to Eric M. OSTERTAG, Tseten YESHI.
Application Number | 20210062170 17/098974 |
Document ID | / |
Family ID | 1000005222419 |
Filed Date | 2021-03-04 |
![](/patent/app/20210062170/US20210062170A1-20210304-D00001.png)
![](/patent/app/20210062170/US20210062170A1-20210304-D00002.png)
![](/patent/app/20210062170/US20210062170A1-20210304-P00001.png)
![](/patent/app/20210062170/US20210062170A1-20210304-P00002.png)
![](/patent/app/20210062170/US20210062170A1-20210304-P00003.png)
United States Patent
Application |
20210062170 |
Kind Code |
A1 |
OSTERTAG; Eric M. ; et
al. |
March 4, 2021 |
SITE-SPECIFIC ENZYMES AND METHODS OF USE
Abstract
The present invention provides polypeptides related t Ralstonia
proteins, nucleic acids encoding the same, compositions comprising
the same, kits comprising the same, non-human transgenic animals
comprising the same, and methods of using the same.
Inventors: |
OSTERTAG; Eric M.; (San
Diego, CA) ; YESHI; Tseten; (Lexington, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Poseida Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005222419 |
Appl. No.: |
17/098974 |
Filed: |
November 16, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16518126 |
Jul 22, 2019 |
10844361 |
|
|
17098974 |
|
|
|
|
14443361 |
May 15, 2015 |
10415024 |
|
|
PCT/US2013/070636 |
Nov 18, 2013 |
|
|
|
16518126 |
|
|
|
|
61802066 |
Mar 15, 2013 |
|
|
|
61727652 |
Nov 16, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C07K
14/195 20130101 |
International
Class: |
C12N 9/22 20060101
C12N009/22; C07K 14/195 20060101 C07K014/195 |
Claims
1. A fusion protein comprising (a) at least one amino acid sequence
having a sequence at least 95% identical to the sequence of
LSTEQVVAIASX.sub.1X.sub.2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO: 1),
wherein X.sub.1X.sub.2 comprises a repeat variable diresidue (RVD),
and wherein the RVD consists of SI, SN, SH, NP, NH, NT, NK, ND, HN,
HY, HD, HH, RN, RS, NG or GS, and wherein the amino acids recited
at positions 2 and 3 of SEQ ID NO: 1 are unchanged; and (b) an
effector domain, wherein the effector domain comprises a nuclease,
a nickase, a transcriptional activator, a transcriptional
repressor, a methyltransferase, a deacetylase or any functional
fragment thereof and wherein the effector domain is neither
isolated nor derived from a Ralstonia TALEN.
2. The fusion protein of claim 1, wherein the at least one amino
acid sequence of (a) or the second amino acid sequence comprises
the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.
3. The fusion protein of claim 1, wherein the effector domain
comprises at least one nuclease.
4. The fusion protein of claim 1, wherein the nuclease comprises an
endonuclease.
5. The fusion protein of claim 4, wherein the endonuclease
comprises a Fok I endonuclease.
6. The fusion protein of claim 5, wherein the endonuclease
comprises an I-SceI endonuclease.
7. The fusion protein of claim 1, wherein the effector domain
comprises a zinc-finger nuclease.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/518,126, filed Jul. 22, 2019 (now allowed);
which is a continuation of U.S. patent application Ser. No.
14/443,361, filed May 15, 2015 (now U.S. Pat. No. 10,415,024,
issued Sep. 17, 2019), which is a national phase application under
35 U.S.C. .sctn. 371 of International Patent Application No.
PCT/US2013/070636, filed Nov. 18, 2013, which claims the benefit of
priority to U.S. Provisional Patent Application No. 61/802,066,
filed Mar. 15, 2013, and U.S. Provisional Application No.
61/727,652, filed Nov. 16, 2012, the entire contents of each of
which are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
POTH-006_C02US_SeqList_Corr_ST25.txt. The text file is about 162
kb, was filed Nov. 16, 2020, and is being submitted electronically
via EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention is directed, in part, to Ralstonia
proteins, nucleic acids encoding the same, compositions comprising
the same, kits comprising the same, as well as methods of using the
same.
BACKGROUND OF THE INVENTION
[0004] Transcription factors with programmable DNA binding domains
offer one potential approach toward creating an exogenous
biological circuit in an endogenous system and creating designer
proteins that bind to pre-determined DNA sequences or individual
nucleic acids. Transcriptional activator-like (TAL) proteins have
been recently demonstrated to have modular and predictable DNA
binding domains, thereby allowing for the de novo creation of
synthetic transcription factors that bind to DNA sequences of
interest and, if desirable, allow a second domain present on the
protein or polypeptide to perform an activity related to DNA. TAL
proteins have been derived from the organism Xanthomonas. Provided
herein, however, are Ralstonia proteins or polypeptides that
function in a fashion similar to TAL proteins derived from
Xanthomonas. The invention relates to polypeptides derived from
Ralstonia amino acid sequences or amino acid sequences related
thereto, nucleic acids encoding the same, compositions comprising
the same, kits comprising the same, non-human transgenic animals
comprising the same, and methods of using the same.
SUMMARY OF THE INVENTION
[0005] The present invention is based in part on the fact that the
repeat variable diresidues (RVDs) of Ralstonia effectors correspond
to the nucleotides in their target sites in a direct, linear
fashion, one RVD to one nucleotide, with some degeneracy and no
apparent context dependence. This finding represents a mechanism
for protein-DNA recognition that enables target site prediction for
new target-specific Ralstonia effectors. As described herein, these
proteins may be useful in research and biotechnology as part of a
larger targeted chimeric protein with accessory activities related
to nucleic acids such as nuclease activity. For instance, in some
embodiments, the polypeptide or pronucleases that can facilitate
homologous recombination in genome engineering (e.g., to add or
enhance traits useful for bio fuels or biorenewables in plants or
bacteria, or animal genomes). These proteins also may be useful as,
for example, transcription factors, and especially for therapeutic
applications requiring a very high level of specificity such as
therapeutics against pathogens (e.g., viruses) as non limiting
examples. In some embodiments, the polypeptide or proteins of the
invention comprise at least a first domain and a second domain,
wherein the first domain comprises at least one coding sequence for
a nucleic acid recognition element and the second domain comprises
at least one coding sequence for a nucleic acid effector
element.
[0006] The present invention provides proteins comprising at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ
ID NO:1:
[0007] LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO:1);
[0008] wherein X.sub.1=naturally occurring or non naturally amino
acid.
[0009] X.sub.2=naturally occurring or non naturally amino acid.
[0010] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to SEQ ID NO:1:
[0011] LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE;
[0012] wherein, in any combination, X.sub.1 and X.sub.2 are
independently variable, and X.sub.1=A, N, H, R or G; and X.sub.2=I,
N, H, K, Y, T, D, S, or P.
[0013] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to SEQ ID NO:1:
[0014] LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO:1);
[0015] wherein X.sub.1=S and X.sub.2=I.
[0016] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to SEQ ID NO:1:
[0017] LSTEQVVAIASX1X2GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO:1);
[0018] wherein X.sub.1=S and X.sub.2=N.
[0019] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASSIGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:2).
[0020] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASSNGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:3).
[0021] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASSHGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:4).
[0022] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNPGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:5).
[0023] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNHGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:6).
[0024] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNTGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:7).
[0025] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNKGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:8).
[0026] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNPGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:9).
[0027] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNNGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:10).
[0028] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASNDGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:11).
[0029] In some embodiments, the polypeptide of the present
invention comprises at least
[0030] 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity
to LSTEQVVAIASNGGGKQALEAVKAQLLVLRAAPYE (SEQ ID NO:12).
[0031] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASHNGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:13).
[0032] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASHYGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:14).
[0033] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASHDGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:15).
[0034] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASHHGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:16).
[0035] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASRNGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:17).
[0036] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASRSGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:18).
[0037] In some embodiments, the polypeptide of the present
invention comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% sequence identity to LSTEQVVAIASGSGGKQALEAVKAQLLVLRAAPYE (SEQ
ID NO:19).
[0038] In some embodiments, the polypeptide or proteins of the
invention comprise at least a first domain and a second domain,
wherein the first domain comprises at least one a nucleic acid
recognition element and wherein the second domain comprises at
least one nucleic acid effector element.
[0039] The present invention relates to nucleic acid sequences that
encode any protein or polypeptide described herein.
[0040] The present invention relates to compositions that comprise
any one or a plurality of nucleic acid sequences that encode any
protein or polypeptide described herein. The present invention
relates to compositions that comprises any one or a plurality of
amino acid sequences described herein.
[0041] In some embodiments, the polypeptide of the present
invention comprises SEQ ID NO:1. In some embodiments, the
polypeptide of the present invention consists essentially of SEQ ID
NO:1. In some embodiments, the polypeptide of the present invention
consists of SEQ ID NO:1. In some embodiments, the polypeptide of
the present invention comprises SEQ ID NO:1, wherein X1X2 bind to a
single nucleic acid. In some embodiments, the polypeptide of the
present invention comprises SEQ ID NO:1, wherein X.sub.1X.sub.2
bind to at least one nucleic acid. In some embodiments, the
polypeptide of the present invention consists essentially of SEQ ID
NO:1, wherein X.sub.1X.sub.2 bind to a nucleic acid. In some
embodiments, the polypeptide of the present invention consists of
SEQ ID NO: 1, wherein X.sub.1X.sub.2 bind to a nucleic acid.
[0042] In some embodiments, the polypeptide of the present
invention comprises one or more of any combination of a polypeptide
sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the polypeptides chosen
from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO:20.
[0043] In some embodiments, the polypeptide of the present
invention comprises one or more of any combination of a polypeptide
sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the polypeptides chosen
from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO 14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
SEQ ID NO:20, wherein the 12th and 13th amino acid of at least one
of the polypeptide sequences binds at least one nucleic acid.
[0044] In some embodiments, the polypeptide of the present
invention comprises one or a plurality of any combination of a
polypeptide sequences with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the polypeptides chosen
from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO 17, SEQ ID NO:18, and SEQ ID
NO:19.
[0045] In some embodiments, the polypeptide of the present
invention comprises a first domain and a second domain, wherein the
first domain is a nucleic acid recognition domain that comprises
one or a plurality of any combination of a polypeptide sequences
with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the polypeptides chosen from: SEQ ID NO: 1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID N: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.
[0046] In some embodiments, the polypeptide of the present
invention comprises a first domain and a second domain, wherein the
first domain is a nucleic acid recognition domain that comprises
one or a plurality of any combination of a polypeptide sequences
with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the polypeptides chosen from: SEQ ID NO: 1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; wherein the
12th and 13th amino acid of at least one polypeptide sequence bind
a nucleic acid.
[0047] In some embodiments, the polypeptide comprises at least 80%
sequence identity to SEQ ID NO:1. In some embodiments, the
polypeptide comprises at least 90% sequence identity to SEQ ID
NO:1. In some embodiments, the polypeptide comprises at least 91%
sequence identity to SEQ ID NO 1. In some embodiments, the
polypeptide comprises at least 92% sequence identity to SEQ ID
NO:1. In some embodiments, the polypeptide comprises at least 93%
sequence identity to SEQ ID NO:. In some embodiments, the
polypeptide comprises at least 94% sequence identity to SEQ ID
NO:1. In some embodiments, the polypeptide comprises at least 95%
sequence identity to SEQ ID NO:1. In some embodiments, the
polypeptide comprises at least 96% sequence identity to SEQ ID NO:
1. In some embodiments, the polypeptide comprises at least 97%
sequence identity to SEQ ID NO:1. In some embodiments, the
polypeptide comprises at least 98% sequence identity to SEQ ID
NO:1. In some embodiments, the polypeptide comprises at least 99%
sequence identity to SEQ ID NO:1.
[0048] In some embodiments, the protein comprises at least 80%
sequence identity to SEQ ID NO:1, and comprises more than one of
the amino acid substitutions in any of the polypeptides chosen from
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some
embodiments, the protein comprises at least 90% sequence identity
to SEQ ID NO: 1, and comprises more than one of the amino acid
substitutions in any of the polypeptides chosen from SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19.
[0049] In some embodiments, the protein comprises at least 95%
sequence identity to SEQ ID NO:1, and comprises more than one of
the amino acid substitutions in any of the polypeptides chosen from
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO: 19. In some
embodiments, the protein comprises at least 99% sequence identity
to SEQ ID NO:1, and comprises more than one of the amino acid
substitutions in any of the polypeptides chosen from SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19.
[0050] In some embodiments, the protein or polypeptide comprises at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
or SEQ ID NO:19. In some embodiments, the protein or polypeptide
comprises at least one, two, three, or four polypeptide sequences
selected from polypeptides comprising at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ
ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ
ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, or SEQ ID NO:19.
[0051] The present invention also provides nucleic acids encoding
any of the proteins described above. In some embodiments, the
nucleic acid comprises nucleic acid sequences that encode at least
2, 3, 4, 5 or more polypeptides chosen from polypeptides comprising
at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
or SEQ ID NO:19.
[0052] The present invention also provides vectors comprising any
of the nucleic acid sequences described above encoding any of the
proteins described above. In some embodiments, the vector is a
plasmid. In some embodiments, the vector is a retrovirus. In some
embodiments, the retrovirus comprises long terminal repeats, a psi
packaging signal, a cloning site, and a sequence encoding a
selectable marker.
[0053] The present invention also provides cells comprising any of
the nucleic acids or vectors described herein. In some embodiments,
the cell is a sperm or an egg.
[0054] The present invention also provides kits comprising: a
vector comprising a nucleic acid encoding any of the proteins
described herein.
[0055] The present invention also provides non-human, transgenic
animals comprising a nucleic acid molecule encoding any of the
proteins described herein.
[0056] The present invention also provides methods of modifying
genetic material of a cell or at least one cell of a multicellular
or unicellular organism, the method comprising administering
directly to the cell or at least one cell of a multicellular or
unicellular organism any one or more of nucleic acids described
herein or any polypeptide described herein. In some embodiments,
the protein is administered as a nucleic acid encoding the protein.
In some embodiments, nucleic acid encoding the protein is
administered with a second nucleic acid sequence that encodes an
effector. In some embodiments, the multicellular or unicellular
organism is a vertebrate. In some embodiments, the vertebrate
animal is a mammal. In some embodiments, the vertebrate animal is a
non-human mammal. In some embodiments, the administering is
administering systemically.
[0057] The present invention also provides methods of generating a
non-human, transgenic animal comprising a germline mutation
comprising: introducing a vector comprising a nucleotide sequence
encoding any of the proteins described herein into a cell of the
non-human, transgenic animal.
[0058] The present invention also provides methods of mutagenizing
the germ line of a non-human, transgenic animal comprising:
introducing a nucleic acid molecule encoding any of the proteins
described herein into a cell under conditions sufficient to
generate a transgenic animal.
BRIEF DESCRIPTION OF DRAWINGS
[0059] FIG. 1 depicts a consensus sequence of a DNA-binding protein
from Xanthamonas aligned via BLAST to methyltransferase sequences
from bacterial strains. Based upon sequence alignment, DNA binding
function of the sequences is predicted. The sequences in the
alignment have the following SEQ ID NOs, in order from top to
bottom: SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 33, SEQ ID NO: 32,
SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ
ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID
NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO:
136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO:
136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO:
136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO: 136, SEQ ID NO:
137, SEQ ID NO: 138, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 56,
SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58. The consensus sequence
at the bottom reads LTTERIVAIGTSTGGTQALEAVLTALPRVCQXHG (SEQ ID NO:
209), wherein X is either an aspartic acid (D) or an alanine
(A).
[0060] FIG. 2 depicts a gel demonstrating RTN functionality.
DETAILED DESCRIPTION OF EMBODIMENTS
[0061] In some embodiments, the polypeptide or proteins of the
invention comprise at least a first domain and a second domain,
wherein the first domain comprises at least one coding sequence for
a nucleic acid recognition element and wherein the second domain
comprises at least one coding sequence for a nucleic acid effector
element. In some embodiments, the polypeptide or proteins of the
invention comprise at least a first domain wherein the first domain
comprises at least one coding sequence for a nucleic acid
recognition element derived from a amino acid sequence derived from
Ralstonia or a variant thereof. In some embodiments, the
polypeptide or proteins of the invention comprise at least a first
domain wherein the first domain comprises at least one coding
sequence for a nucleic acid recognition element derived from an
amino acid sequence derived from Ralstonia.
[0062] The term "RTN" refers to a polypeptide or proteins of the
invention that comprise at least a first domain wherein the first
domain comprises at least one coding sequence for a nucleic acid
recognition element derived from an amino acid sequence derived
from Ralstonia. In some embodiments, the term "RTN" refers to a
polypeptide or proteins of the invention that comprise at least a
first domain and a second domain, wherein the first domain
comprises at least one coding sequence for a nucleic acid
recognition element derived from an amino acid sequence derived
from Ralstonia and the second domain comprises a amino acid that is
an effector protein. In some embodiments, the term "RTN" refers to
a polypeptide or proteins of the invention that comprise at least a
first domain and a second domain, wherein the first domain
comprises at least one coding sequence for a nucleic acid
recognition element derived from an amino acid sequence derived
from Ralstonia and the second domain comprises a amino acid that is
a nuclease. RTN DNA binding specificity depends on the number and
order of repeat domains in the DNA binding domain. Repeats are
generally composed of from about 30 to about 40 amino acids. In
some embodiments, the repeat domains comprise from about 32 to
about 38 amino acids. In some embodiments, nucleotide binding
specificity is determined by the 12 and 13 amino acids of each
repeat domain. In some embodiments, the repeat domains comprise
from about 33 to about 37 amino acids. In some embodiments, the
repeat domains comprise from about 34 to about 35 amino acids. In
some embodiments, the repeat domains comprise from about 33 to
about 36 amino acids. In some embodiments, the repeat domains
comprise from about 33 to about 35 amino acids. In some
embodiments, the repeat domains consist of 34 to 35 amino acids. In
some embodiments, the repeat domains consist of 33 to 35 amino
acids. In some embodiments, the repeat domains consist of 34 to 36
amino acids.
[0063] "Nucleic acid" or "oligonucleotide" or "polynucleotide" as
used herein means at least two nucleotides covalently linked
together. The depiction of a single strand also defines the
sequence of the complementary strand. Thus, a nucleic acid also
encompasses the complementary strand of a depicted single strand.
Many variants of a nucleic acid can be used for the same purpose as
a given nucleic acid. Thus, a nucleic acid also encompasses
substantially identical nucleic acids and complements thereof. A
single strand provides a probe that can hybridize to a target
sequence under stringent hybridization conditions. Thus, a nucleic
acid also encompasses a probe that hybridizes under stringent
hybridization conditions. Nucleic acids can be single stranded or
double stranded, or can contain portions of both double stranded
and single stranded sequence. The nucleic acid can be DNA, both
genomic and cDNA, RNA, or a hybrid, where the nucleic acid can
contain combinations of deoxyribo- and ribo-nucleotides, and
combinations of bases including uracil, adenine, thymine, cytosine,
guanine, inosine, xanthine hypoxanthine, isocytosine and
isoguanine. In some embodiments, is synthesized, comprises
non-natural amino acid modifications. Nucleic acids can be obtained
by chemical synthesis methods or by recombinant methods.
[0064] "Operably linked" as used herein means that expression of a
gene is under the control of a promoter with which it is spatially
connected. A promoter can be positioned 5' (upstream) or 3'
(downstream) of a gene under its control. The distance between the
promoter and a gene can be approximately the same as the distance
between that promoter and the gene it controls in the gene from
which the promoter is derived. As is known in the art, variation in
this distance can be accommodated without loss of promoter
function.
[0065] "Promoter" as used herein means a synthetic or
naturally-derived molecule which is capable of conferring,
activating or enhancing expression of a nucleic acid in a cell. A
promoter can comprise one or more specific transcriptional
regulatory sequences to further enhance expression and/or to alter
the spatial expression and/or temporal expression of same. A
promoter can also comprise distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A promoter can be derived from
sources including viral, bacterial, fungal, plants, insects, and
animals. A promoter can regulate the expression of a gene component
constitutively, or differentially with respect to cell, the tissue
or organ in which expression occurs or, with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, pathogens, metal
ions, or inducing agents. Representative examples of promoters
include the bacteriophage T7 promoter, bacteriophage T3 promoter,
SP6 promoter, lac operator-promoter, tac promoter, SV40 late
promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter,
SV40 early promoter or SV40 late promoter and the CMV IE
promoter,
[0066] "Substantially complementary" as used herein means that a
first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98% or 99% identical to the complement of a second sequence
over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino
acids, or that the two sequences hybridize under stringent
hybridization conditions.
[0067] "Substantially identical" as used herein means that a first
and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450,
540 or more nucleotides or amino acids, or with respect to nucleic
acids, if the first sequence is substantially complementary to the
complement of the second sequence.
[0068] "Variant" used herein with respect to a nucleic acid means
(i) a portion or fragment of a referenced nucleotide sequence; (ii)
the complement of a referenced nucleotide sequence or portion
thereof; (iii) a nucleic acid that is substantially identical to a
referenced nucleic acid or the complement thereof; or (iv) a
nucleic acid that hybridizes under stringent conditions to the
referenced nucleic acid, complement thereof, or a sequences
substantially identical thereto.
[0069] As used herein "variant" with respect to a peptide or
polypeptide that differs in amino acid sequence by the insertion,
deletion, or conservative substitution of amino acids, but retain
at least one biological activity. Variant can also mean a protein
with an amino acid sequence that is substantially identical to a
referenced protein with an amino acid sequence that retains at
least one biological activity. A conservative substitution of an
amino acid, i.e., replacing an amino acid with a different amino
acid of similar properties (e.g., hydrophilicity, degree and
distribution of charged regions) is recognized in the art as
typically involving a minor change. These minor changes can be
identified, in part, by considering the hydropathic index of amino
acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:
105-132 (1982). The hydropathic index of an amino acid is based on
a consideration of its hydrophobicity and charge. It is known in
the art that amino acids of similar hydropathic indexes can be
substituted and still retain protein function. In one aspect, amino
acids having hydropathic indexes of .+-.2 are substituted. The
hydrophilicity of amino acids can also be used to reveal
substitutions that would result in proteins retaining biological
function. A consideration of the hydrophilicity of amino acids in
the context of a peptide permits calculation of the greatest local
average hydrophilicity of that peptide, a useful measure that has
been reported to correlate well with antigenicity and
immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein
by reference.
[0070] Substitution of amino acids having similar hydrophilicity
values can result in peptides retaining biological activity, for
example immunogenicity, as is understood in the art. Substitutions
can be performed with amino acids having hydrophilicity values
within .+-.2 of each other. Both the hyrophobicity index and the
hydrophilicity value of amino acids are influenced by the
particular side chain of that amino acid. Consistent with that
observation, amino acid substitutions that are compatible with
biological function are understood to depend on the relative
similarity of the amino acids, and particularly the side chains of
those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties.
Vector
[0071] "Vector" as used herein means a nucleic acid sequence
containing an origin of replication. A vector can be a viral
vector, bacteriophage, bacterial artificial chromosome or yeast
artificial chromosome. A vector can be a DNA or RNA vector. A
vector can be a self-replicating extrachromosomal vector, and
preferably, is a DNA plasmid.
[0072] The present invention provides polypeptide, proteins and
nucleic acid sequences that encode any of the polypeptides or
proteins of the claimed invention. In some embodiments, the protein
comprises at least 75% sequence identity to SEQ ID NO:1. In some
embodiments, the protein comprises at least 75% sequence identity
to SEQ ID NO: 1 and comprises a nucleic acid binding domain at the
12th and 13th amino acids of SEQ ID NO:1. In some embodiments, the
proteins or polypeptides of the present invention comprise at least
one RVD sequence selected from the following: SI, SN, SH, NP, NH,
NT, NK, NN, ND, HN, HY, HD, HH, RN, RS, and GS. In some
embodiments, the proteins or polypeptides of the present invention
comprise at least one or a plurality of RVD sequences in any
combination selected from the following: SI, SN, SH, NP, NH, NT,
NK, NN, ND, HN, HY, HD, HH, RN, RS, NG and GS; wherein SI, SN, SH,
NP, and NH bind any nucleic acid base;
[0073] wherein NT, NK, and NN bind adenine; wherein ND, HN, HY, HD,
and HH bind adenine and/or guanine; wherein NG binds thymine;
wherein RN, RS, and GS bind guanine. In some embodiments, the
proteins or polypeptides of the present invention comprise at least
one or a plurality of RVD sequences in any combination selected
from the following: SI, SN, SH, NP, NH, NT, NK, NN, ND, HN, HY, HD,
HH, RN, RS, NG and GS; wherein SI, SN, SH, NP, and NH bind any
nucleic acid base; wherein NK binds guanine, and NN binds adenine
or guanine; wherein ND, HN, HY, HD, and HH bind cytosine; wherein
NG binds thymine; wherein RN, RS, and GS bind guanine. In some
embodiments, the proteins or polypeptides of the present invention
comprise at least one or a plurality of RVD sequences in any
combination selected from the following: SI, SN, SH, NP, NH, NT,
NK, NN, ND, HN, HY, HD, HH, RN, RS, NG and GS; wherein SI binds
adenine; SN binds guanine and/or adenine, SH, NP, and NH bind any
nucleic acid base; wherein NK binds guanine; and NN binds adenine
and/or guanine; wherein ND binds cytosine, HN binds guanine, HY,
HD, and HH bind cytosine; wherein NG binds thymine; wherein RN
binds guanine and/or adenine;
[0074] wherein RS and GS binds guanine. In some embodiments, the
the proteins or polypeptides of the present invention comprise at
least one or a plurality of RVD sequences in any combination
wherein at least one of the RVD sequences is NP, ND, or HN; and
wherein NP binds cytosine, adenine, and guanine; wherein ND binds
cytosine; and wherein HN binds adenine and/or guanine.
[0075] In some embodiments, the protein comprises at least 75%
sequence identity to SEQ ID NO:1, and comprises a conservative
amino acid substitution in at least one of the following amino acid
positions in SEQ ID NO:1: position 12 and position 13. In some
embodiments, the protein comprises at least 80% sequence identity
to SEQ ID NO:1, and comprises a conservative amino acid
substitution in at least one of the following amino acid positions
in SEQ ID NO:1: position 12 and position 13. In some embodiments,
the protein comprises at least 85% sequence identity to SEQ ID
NO:1, and comprises a conservative amino acid substitution in at
least one of the following amino acid positions in SEQ ID NO:1:
position 12 and position 13. In some embodiments, the protein
comprises at least 90% sequence identity to SEQ ID NO:1, and
comprises a conservative amino acid substitution in at least one of
the following amino acid positions in SEQ ID NO:1: position 12 and
position 13. In some embodiments, the protein comprises at least
91% sequence identity to SEQ ID NO:1, and comprises a conservative
amino acid substitution in at least one of the following amino acid
positions in SEQ ID NO:1: position 12 and position 13. In some
embodiments, the protein comprises at least 92% sequence identity
to SEQ ID NO:1, and comprises a conservative amino acid
substitution in at least one of the following amino acid positions
in SEQ ID NO:1: position 12 and position 13. In some embodiments,
the protein comprises at least 93% sequence identity to SEQ ID
NO:1, and comprises a conservative amino acid substitution in at
least one of the following amino acid positions in SEQ ID NO:1:
position 12 and position 13. In some embodiments, the protein
comprises at least 94% sequence identity to SEQ ID NO:1, and
comprises a conservative amino acid substitution in at least one of
the following amino acid positions in SEQ ID NO:1: position 12 and
position 13. In some embodiments, the protein comprises at least
95% sequence identity to SEQ ID NO:1, and comprises a conservative
amino acid substitution in at least one of the following amino acid
positions in SEQ ID NO:1: position 12 and position 13. In some
embodiments, the protein comprises at least 96% sequence identity
to SEQ ID NO:1, and comprises a conservative amino acid
substitution in at least one of the following amino acid positions
in SEQ ID NO:1: position 12 and position 13. In some embodiments,
the protein comprises at least 97% sequence identity to SEQ ID
NO:1, and comprises a conservative amino acid substitution in at
least one of the following amino acid positions in SEQ ID NO:1:
position 12 and position 13. In some embodiments, the protein
comprises at least 98% sequence identity to SEQ ID NO:1, and
comprises a conservative amino acid substitution in at least one of
the following amino acid positions in SEQ ID NO:1: position 12 and
position 13. In some embodiments, the protein comprises at least
99% sequence identity to SEQ ID NO:1, and comprises a conservative
amino acid substitution in at least one of the following amino acid
positions in SEQ ID NO:1: position 12 and position 13.
[0076] In some embodiments, the protein comprises at least 75%
sequence identity to SEQ ID NO:1.
[0077] In some embodiments, the protein (as nucleic acid, as
nucleic acid in a vector, or as purified recombinant protein)
comprises at least 80% sequence identity to SEQ ID NO:2, and
comprises at least one of the aforementioned amino acid
substitutions in SEQ ID NO:2. In some embodiments, the protein (as
nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 85% sequence identity to
SEQ ID NO:2, and comprises at least one of the aforementioned amino
acid substitutions in SEQ ID NO:2. In some embodiments, the protein
(as nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 90% sequence identity to
SEQ ID NO:2, and comprises at least one of the aforementioned amino
acid substitutions in SEQ ID NO:2. In some embodiments, the protein
(as nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 95% sequence identity to
SEQ ID NO:2, and comprises at least one of the aforementioned amino
acid substitutions in SEQ ID NO:2. In some embodiments, the protein
(as nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 99% sequence identity to
SEQ ID NO:2, and comprises at least one of the aforementioned amino
acid substitutions in SEQ ID NO:2.
[0078] In some embodiments, the protein (encoded by a nucleic acid
or as nucleic acid in a vector, or as purified recombinant protein)
comprises at least 80% sequence identity to SEQ ID NO:3, and
comprises at least one of the aforementioned amino acid
substitutions in SEQ ID NO:3. In some embodiments, the protein
(encoded by a nucleic acid or as nucleic acid in a vector, or as
purified recombinant protein) comprises at least 85% sequence
identity to SEQ ID NO:3, and comprises at least one of the
aforementioned amino acid substitutions in SEQ ID NO:3. In some
embodiments, the protein (encoded by a nucleic acid or as nucleic
acid in a vector, or as purified recombinant protein) comprises at
least 90% sequence identity to SEQ ID NO:3, and comprises at least
one of the aforementioned amino acid substitutions in SEQ ID NO:3.
In some embodiments, the protein (encoded by a nucleic acid or as
nucleic acid in a vector, or as purified recombinant protein)
comprises at least 95% sequence identity to SEQ ID NO:3, and
comprises at least one of the aforementioned amino acid
substitutions in SEQ ID NO:3. In some embodiments, the protein
(encoded by a nucleic acid or as nucleic acid in a vector, or as
purified recombinant protein) comprises at least 99% sequence
identity to SEQ ID NO:3, and comprises at least one of the
aforementioned amino acid substitutions in SEQ ID NO:3.
[0079] In some embodiments, the protein (encoded by a nucleic acid
or as nucleic acid in a vector, or as purified recombinant protein)
comprises at least 75% sequence identity to any of SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO 14, SEQ
ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, and comprises more than one of the aforementioned amino acid
substitutions in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19. In some embodiments, the
protein (encoded by a nucleic acid or as nucleic acid in a vector,
or as purified recombinant protein) comprises at least 80% sequence
identity to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, and comprises more than one of the
aforementioned amino acid substitutions in SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19.
In some embodiments, the protein (as nucleic acid, as nucleic acid
in a vector, or as purified recombinant protein) comprises at least
85% sequence identity to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ
ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ
ID NO:12, SEQ ID NO 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and comprises more than
one of the aforementioned amino acid substitutions in any one or
more of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19. In some embodiments, the protein (as
nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 90% sequence identity to
any one or more of, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and comprises more than one
of the aforementioned amino acid substitutions in any one or more
of, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19. In some embodiments, the protein (as
nucleic acid, as nucleic acid in a vector, or as purified
recombinant protein) comprises at least 95% sequence identity to
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, and comprises more than one of the
aforementioned amino acid substitutions in any one or more of SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19. In some embodiments, the protein (as nucleic acid, as
nucleic acid in a vector, or as purified recombinant protein)
comprises at least 99% sequence identity to any one or more of SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, and comprises more than one of the aforementioned
amino acid substitutions in any one or more of SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19.
[0080] As used herein, "sequence identity" is determined by using
the stand-alone executable BLAST engine program for blasting two
sequences (bl2seq), which can be retrieved from the National Center
for Biotechnology Information (NCBI) ftp site, using the default
parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174,
247-250; which is incorporated herein by reference in its
entirety). "Identical" or "identity" as used herein in the context
of two or more nucleic acids or polypeptide sequences, means that
the sequences have a specified percentage of residues that are the
same over a specified region. The percentage can be calculated by
optimally aligning the two sequences, comparing the two sequences
over the specified region, determining the number of positions at
which the identical residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the specified region,
and multiplying the result by 100 to yield the percentage of
sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered
ends and the specified region of comparison includes only a single
sequence, the residues of single sequence are included in the
denominator but not the numerator of the calculation. When
comparing DNA and RNA, thymine (T) and uracil (U) can be considered
equivalent. Identity can be performed manually or by using a
computer sequence algorithm such as BLAST or BLAST 2.0.
[0081] As used herein, "conservative" amino acid substitutions may
be defined as set out in Tables A, B, or C below. In some
embodiments, fusion polypeptides and/or nucleic acids encoding such
fusion polypeptides include conservative substitutions have been
introduced by modification of polynucleotides encoding polypeptides
of the invention. Amino acids can be classified according to
physical properties and contribution to secondary and tertiary
protein structure. A conservative substitution is recognized in the
art as a substitution of one amino acid for another amino acid that
has similar properties. Exemplary conservative substitutions are
set out in Table A.
TABLE-US-00001 TABLE A Conservative Substitutions I Side Chain
Characteristics Amino Acid Aliphatic Non - polar G A P I L V F
Polar - uncharged C S T M N Q Polar - charged D E K R Aromatic H F
W Y Other N Q D E
[0082] Alternately, conservative amino acids can be grouped as
described in Lehninger, (Biochemistry, Second Edition; Worth
Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table
B.
TABLE-US-00002 TABLE B Conservative Substitutions II Side Chain
Characteristic Amino Acid Non - polar (hydrophobic) Aliphatic: A L
I V P. Aromatic: F W Y Sulfur-containing: M Borderline: G Y
Uncharged-polar Hydroxyl: S T Y Amides: N Q Sulfhydryl: C
Borderline: G Y Positively Charged (Basic): K R H Negatively
Charged (Acidic): D E
[0083] Alternately, exemplary conservative substitutions are set
out in Table C.
TABLE-US-00003 TABLE C Conservative Substitutions III Original
Residue Exemplary Substitution Ala (A) Val Leu Ile Met Arg (R) Lys
His Asn (N) Gln Asp (D) Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp
Gly (G) Ala Val Leu Pro His (H) Lys Arg Ile (I) Leu Val Met Ala Phe
Leu (L) Ile Val Met Ala Phe Lys (K) Arg His Met (M) Leu Ile Val Ala
Phe (F) Trp Tyr Ile Pro (P) Gly Ala Val Leu Ile Ser (S) Thr Thr (T)
Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp Phe Thr Ser Val (V) Ile Leu Met
Ala
[0084] It should be understood that the polypeptides described
herein are intended to include polypeptides bearing one or more
insertions, deletions, or substitutions, or any combination
thereof, of amino acid residues as well as modifications other than
insertions, deletions, or substitutions of amino acid residues. In
some embodiments, the polypeptides or nucleic acids disclosed
herein contain one or more conservative substitution. In some
embodiments, the polypeptides or nucleic acids disclosed herein
contain more than one conservative substitution.
[0085] As used herein, "more than one" of the aforementioned amino
acid substitutions means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid
substitutions. In some embodiments, "more than one" means 2, 3, 4,
or 5 of the recited amino acid substitutions. In some embodiments,
"more than one" means 2, 3, or 4 of the recited amino acid
substitutions. In some embodiments, "more than one" means 2 or 3 of
the recited amino acid substitutions. In some embodiments, "more
than one" means 2 of the recited amino acid substitutions.
[0086] As used herein, "endogenous" refers to nucleic acid or
protein sequence naturally associated with a target gene or a host
cell into which it is introduced.
[0087] As used herein, "exogenous" refers to nucleic acid or
protein sequence not naturally associated with a target gene or a
host cell into which it is introduced, including non-naturally
occurring multiple copies of a naturally occurring nucleic acid,
e.g., DNA sequence, or naturally occurring nucleic acid sequence
located in a non-naturally occurring genome location.
[0088] As used herein, "genetically modified plant (or transgenic
plant)" refers to a plant which comprises within its genome an
exogenous polynucleotide. Generally, and preferably, the exogenous
polynucleotide is stably integrated within the genome such that the
polynucleotide is passed on to successive generations. The
exogenous polynucleotide may be integrated into the genome alone or
as part of a recombinant expression cassette. "Transgenic" is used
herein to include any cell, cell line, callus, tissue, plant part
or plant, the genotype of which has been altered by the presence of
exogenous nucleic acid including those trans genies initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0089] The term "modifying" as used herein is intended to mean that
the sequence is considered modified simply by the binding of the
polypeptide. It is not intended to suggest that the sequence of
nucleotides is changed, although such changes (and others) could
ensue following binding of the polypeptide to the nucleic acid of
interest. In some embodiments, the nucleic acid sequence is DNA.
Modification of the nucleic acid of interest (in the sense of
binding thereto by a polypeptide modified to contain modular repeat
units) could be detected in any of a number of methods (e.g. gel
mobility shift assays, use of labelled polypeptides--labels could
include radioactive, fluorescent, enzyme or biotin/streptavidin
labels). Modification of the nucleic acid sequence of interest (and
detection thereof) may be all that is required (e.g. in diagnosis
of disease). Desirably, however, further processing of the sample
is performed. Conveniently the polypeptide (and nucleic acid
sequences specifically bound thereto) is separated from the rest of
the sample. Advantageously the polypeptide-DNA complex is bound to
a solid phase support, to facilitate such separation. For example,
the polypeptide may be present in an acrylamide or agarose gel
matrix or, more preferably, is immobilised on the surface of a
membrane or in the wells of a microtitre plate.
[0090] In some embodiments, the fusion proteins of the invention
comprise at least two domains, wherein the first domain is a
Ralstonia DNA binding element and the second domain is a
methylase.
[0091] The DNA sequences of the invention can be provided in
expression cassettes for expression in any prokaryotic or
eukaryotic cell and/or organism of interest including, but not
limited to, bacteria, fungi, algae, plants, and animals. The
cassette will include 5' and 3' regulatory sequences operably
linked to a DNA sequence of the invention. "Operably linked" is
intended to mean a functional linkage between two or more elements.
For example, an operable linkage between a polynucleotide or gene
of interest and a regulatory sequence (i.e., a promoter) is
functional link that allows for expression of the polynucleotide of
interest. Operably linked elements may be contiguous or
non-contiguous. When used to refer to the joining of two protein
coding regions, by operably linked is intended that the coding
regions are in the same reading frame. The cassette may
additionally contain at least one additional gene to be
cotransformed into the organism. Alternatively, the additional
gene(s) can be provided on multiple expression cassettes. Such an
expression cassette is provided with a plurality of restriction
sites and/or recombination sites for insertion of the DNA sequence
to be under the transcriptional regulation of the regulatory
regions. The expression cassette may additionally contain
selectable marker genes.
[0092] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region (i.e., a promoter), a DNA sequence of the invention, and a
transcriptional and translational termination region (i.e.,
termination region) functional in plants or other organism or
non-human host cell. The regulatory regions (i.e., promoters,
transcriptional regulatory regions, and translational termination
regions) and/or the DNA sequence of the invention may be
native/analogous to the host cell or to each other. Alternatively,
the regulatory regions and/or DNA sequence of the invention may be
heterologous to the host cell or to each other. As used herein,
"heterologous" in reference to a sequence is a sequence that
originates from a foreign species, or, if from the same species, is
substantially modified from its native form in composition and/or
genomic locus by deliberate human intervention. For example, a
promoter operably linked to a heterologous polynucleotide is from a
species different from the species from which the polynucleotide
was derived, or, if from the same/analogous species, one or both
are substantially modified from their original form and/or genomic
locus, or the promoter is not the native promoter for the operably
linked polynucleotide. As used herein, a chimeric gene comprises a
coding sequence operably linked to a transcription initiation
region that is heterologous to the coding sequence.
[0093] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, may be native with the host, or
may be derived from another source (i.e., foreign or heterologous)
to the promoter, the DNA sequence of interest, the plant host, or
any combination thereof. Convenient termination regions for use in
plants are available from the Ti-plasmid of A. tumefaciens, such as
the octopine synthase and nopaline synthase termination regions.
See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et
al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids
Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.
15:9627-9639.
[0094] Where appropriate, the polynucleotides may be optimized for
increased expression in a transformed organism. That is, the
polynucleotides can be synthesized using codons preferred by the
host for improved expression. See, for example, Campbell and Gown
(1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred
codon usage. Methods are available in the art for synthesizing
host-preferred gene, particularly plant-preferred genes. See, for
example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.
(1989) Nucleic Acids Res. 17:477-498, herein incorporated by
reference.
[0095] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0096] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238),
MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and
human immunoglobulin heavy-chain binding protein (BiP) (Macejak et
al. (1991) Nature 353:90-94); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Tabling et al.
(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)
(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,
New York), pp. 237-256); and maize chlorotic mottle virus leader
(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also,
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.
[0097] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0098] The invention encompasses isolated or substantially purified
polynucleotide or protein compositions. An "isolated" or "purified"
polynucleotide or protein, or biologically active portion thereof,
is substantially or essentially free from components that normally
accompany or interact with the polynucleotide or protein as found
in its naturally occurring environment. Thus, an isolated or
purified polynucleotide or protein is substantially free of other
cellular material or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Optimally, an "isolated"
polynucleotide is free of sequences (optimally protein encoding
sequences) that naturally flank the polynucleotide (i.e., sequences
located at the 5' and 3' ends of the polynucleotide) in the genomic
DNA of the organism from which the polynucleotide is derived. For
example, in various embodiments, the isolated polynucleotide can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or
0.1 kb of nucleotide sequence that naturally flank the
polynucleotide in genomic DNA of the cell from which the
polynucleotide is derived. A protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating
protein. When the protein of the invention or biologically active
portion thereof is recombinantly produced, optimally culture medium
represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight)
of chemical precursors or non-protein-of-interest chemicals.
[0099] Fragments and variants of the disclosed DNA sequences and
proteins encoded thereby are also encompassed by the present
invention. By "fragment" is intended a portion of the DNA sequence
or a portion of the amino acid sequence and hence protein encoded
thereby. Fragments of a DNA sequence comprising coding sequences
may encode protein fragments that retain biological activity of the
native protein and hence DNA recognition or binding activity to a
target DNA sequence as herein described. Alternatively, fragments
of a DNA sequence that are useful as hybridization probes generally
do not encode proteins that retain biological activity or do not
retain promoter activity. Thus, fragments of a DNA sequence may
range from at least about 20 nucleotides, about 50 nucleotides,
about 100 nucleotides, and up to the full-length polynucleotide of
the invention.
[0100] In some embodiments, the protein comprises SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ
ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:18, and/or SEQ ID NO:19. In some
embodiments any one of the polypeptide sequences SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, and/or SEQ ID NO:19 is repeated at least
once. In some embodiments, the polypeptide does not comprise any of
the sequences in Table 1. In some embodiments, the polypeptide
comprises a single sequence in Table 1 but does not comprise at
least one or more of the alternative sequences in Table 1. In some
embodiments, the alternative sequence comprises SEQ ID NO: 2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, or SEQ ID NO:19.
[0101] The present invention also provides nucleic acids encoding
any one of the polypeptide proteins described herein. Thus, the
present invention provides nucleic acids encoding a protein that
comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence
identity to SEQ ID NO:1.
[0102] In some embodiments, the nucleic acid encodes a protein that
comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence
identity to SEQ ID NO:1, where X1X2 are any combination of
naturally occurring to non-naturally occurring amino acids.
[0103] Given the redundancy in the genetic code, one skilled in the
art could generate numerous nucleotide sequences that encode any
particular protein. All such nucleotides sequences are contemplated
herein.
[0104] The present invention also provides vectors comprising any
of the aforementioned nucleic acids. Thus, the present invention
provides vectors comprising a nucleic acid that encodes a protein
that comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%)
sequence identity to SEQ ID NO:1. The present invention provides
vectors comprising a nucleic acid that encodes at least one protein
that comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%)
sequence identity to SEQ ID NO:1, and comprises at least one RVD
sequence at its 12th and 13th amino acids of SEQ ID NO: 1 selected
from the following: SI, SN, SH, NP, NH, NT, NK, NN, ND, HN, HY, HD,
HH, RN, RS, and GS. In some embodiments, the proteins or
polypeptides of the present invention comprise at least one or a
plurality of RVD sequences in any combination selected from the
following: SI, SN, SH, NP, NH, NT, NK, NN, ND, HN, HY, HD, HH, RN,
RS, NG and GS; wherein SI, SN, SH, NP, and NH bind any nucleic acid
base; wherein NT, NK, and NN bind adenine; wherein ND, HN, HY, HD,
and HH bind adenine and/or guanine; wherein NG binds thymine;
wherein RN, RS, and GS bind guanine. In some embodiments, NK binds
to guanine, NG binds to thymine, NN binds to guanine or adenine,
and or/HD binds cytosine. In some embodiments SI binds adenine, SN
binds guanine or adenine, ND binds cytosine, HN binds guanine,
and/or RN binds guanine or adenine.
[0105] In some embodiments, the polypeptide comprises at least a
first and a second domain, wherein the first domain comprises at
least one polypeptide monomer that comprises a single RVD sequence
described above. In some embodiments, the first domain comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, or
20 or more monomers, wherein each monomer comprises a single
nucleic acid binding domain consisting of two amino acids, or RVD.
In some embodiments of the invention, the first domain comprises at
least two monomers wherein each monomer is separated by a spacer of
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 or more amino acids.
[0106] In some embodiments, the second domain comprises another
function complementary to the nucleic acid binding conferred by the
presence first domain. In some embodiments, the second domain is a
nuclease or functional fragment of a nuclease. In some embodiments,
the second domain is an endonuclease or functional fragment of an
endonuclease. In some embodiments, the second domain is a nickase
or functional fragment of a nickase. In some embodiments, the
second domain is a repressor or functional fragment of a repressor.
In some embodiments, the second domain is a transcriptional
activator or functional fragment of a transcriptional
activator.
[0107] In some embodiments, the vector comprises a nucleic acid
that comprises a sequence that encodes one or more of each
polypeptide described herein. In some embodiments, the vector
comprises a nucleic acid that comprises a sequence that encodes one
or more of each polypeptides chosen from: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19.
[0108] In some embodiments, the vector is a plasmid. In other
embodiments, the vector is a retrovirus. In some embodiments, the
vector is a linear DNA molecule. In some embodiments, the
retrovirus comprises long terminal repeats, a psi packaging signal,
a cloning site, and a sequence encoding a selectable marker. In
some embodiments, the vector is a viral vector, such as pLXIN
(Clontech).
[0109] The present invention also provides cells or organisms
comprising any of the aforementioned nucleic acids. Thus, the
present invention provides cells or organisms comprising a nucleic
acid that encodes a protein that comprises at least 75% (or 80%,
85%, 90%, 95%, or 99%) sequence identity to SEQ ID NO:1. The
present invention provides cells or organisms comprising a nucleic
acid that encodes a protein that comprises at least 75% (or 80%,
85%, 90%, 95%, or 99%) sequence identity to any one or more of the
following polypeptides in any combination: SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO
17, SEQ ID NO:18, or SEQ ID NO:19. The present invention provides
cells or organisms comprising a nucleic acid that encodes a protein
that comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%)
sequence identity to any one or more of the following polypeptides
in any combination: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
or SEQ ID NO:19; and comprises at least one mutation at at least
one of the RVD domains at position 12 and 13 of any of SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO 10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. The present
invention provides cells or organisms comprising a nucleic acid
mutated by contact with a protein that comprises at least 75% (or
80%, 85%, 90%, 95%, or 99%) sequence identity to any one or more of
the following polypeptides in any combination: SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, or SEQ ID NO:19; and comprises at least one
mutation at at least one of the RVD domains at position 12 and 13
of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID
NO:19.
[0110] In some embodiments, the cells or organisms comprise a
nucleic acid that encodes a protein that comprises a nucleic acid
sequence with at least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence
identity to SEQ ID NO:1. In some embodiments, the polypeptide or
protein of the claimed invention comprises multiple repeat domains,
wherein at least one repeat domain comprises a nucleic acid
sequence with at least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence
identity to SEQ ID NO:1-19 or a variant thereof that has 75% (or
80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:1-19.
[0111] In some embodiments, the cell comprises any of the
aforementioned vectors or nucleic acid sequences.
[0112] In one aspect of the invention, the polypeptides of the
invention comprise monomer subunits, wherein at least one monomer
subunit comprises at least one amino acid sequence derived from
Ralstonia that comprises a nucleotide recognition element. In some
embodiments, the polypeptides of the invention comprise monomer
subunits, wherein at least one monomer subunit comprises at least
one amino acid sequence derived from Ralstonia that comprises a
nucleotide recognition element that comprises at least 75% (or 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one
or more of the following polypeptides: SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the
polypeptides of the invention comprise monomer subunits, wherein at
least one monomer subunit comprises at least one amino acid
sequence derived from Ralstonia that comprises a nucleotide
recognition element that consist of at least 75% (or 80%, 85%, 90%,
95%, 96%, 97%, 98% or 99%) sequence identity to any one or more of
the following polypeptides: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, or SEQ ID NO:19. In some embodiments, nucleic acid
molecules of the present invention comprise a nucleic acid sequence
that encodes one or more monomer subunits, wherein the one or more
monomer subunits, when encoded, comprise two or more, three of
more, four or more, five or more, six or more, seven or more, eight
or more, nine or more, ten or more, eleven or more, or twelve or
more sequential monomers, each monomer comprising at least one
amino acid sequence derived from Ralstonia that comprises a
nucleotide recognition element. In some embodiments, nucleic acid
molecules of the present invention comprise a nucleic acid sequence
that encodes one or more monomer subunits, wherein the one or more
monomer subunits, when encoded, comprise two or more, three of
more, four or more, five or more, six or more, seven or more, eight
or more, nine or more, ten or more, eleven or more, or twelve or
more sequential monomers, each monomer comprising at least one
amino acid sequence derived from Ralstonia that comprises a
nucleotide recognition element consisting of at least 75% (or 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one
or more of the following polypeptides: SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19 or any variant or analog
thereof. In some embodiments, the protein comprises one or more
nucleic acid sequences that encode one or more polypeptide
sequences comprising a nucleic acid recognition element derived
from Ralstonia and one or more nucleic acid sequences that encode
one or more polypeptide sequences comprising a nucleic acid
recognition element derived from Xanthamonus. In some embodiments,
the fusion protein comprises successive, independently variable
monomer subunits that are DNA recognition elements, wherein at
least one or more of the monomer subunits are derived from a
Ralstonia sequence disclosed herein. In some embodiments, the
invention relates to a fusion protein comprising successive,
independently variable monomer subunits that are DNA recognition
elements, wherein at least one or more of the monomer subunits are
derived from a Ralstonia sequence disclosed in Table 1. In some
embodiments, the invention relates to a fusion protein comprising
successive, monomer subunits that are DNA recognition elements,
wherein the fusion protein comprises a combination of successive
polypeptides chosen from at least one of the following polypeptide
sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or
SEQ ID NO:19 or any variant or analog thereof. In some embodiments,
the invention relates to a fusion protein comprising successive,
monomer subunits that are DNA recognition elements, wherein the
fusion protein comprises a combination of successive polypeptides
chosen from at least one of the following polypeptide sequences:
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19 or
any variant or analog thereof, wherein each monomer binds one
nucleotide of a DNA target sequence. In some embodiments, the
invention relates to a fusion protein comprising successive,
monomer subunits that are DNA recognition elements, wherein the
fusion protein comprises a combination of successive polypeptides
chosen from at least one of the following polypeptide sequences:
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19 or
any variant or analog thereof that is at least 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% percent homologous thereof, wherein
each monomer binds one nucleotide of a DNA target sequence in the
presence of a nucleic acid sequence. In some embodiments, the
invention relates to a fusion protein comprising successive,
monomer subunits that are DNA recognition elements, wherein the
fusion protein comprises a combination of successive polypeptides
chosen from at least one of the following polypeptide sequences:
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19 or
any variant or analog thereof that is at least 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% percent homologous thereof, wherein
each monomer binds one nucleotide of a DNA target sequence in the
presence of a nucleic acid sequence, wherein the fusion protein
further comprises at least one polypeptide sequence that is an
effector protein/polypeptide. In some embodiments, the fusion
protein comprises a first domain that binds a DNA target sequence
and a second domain that has an effector function. In some
embodiments, the invention relates to a fusion protein comprising
successive, monomer subunits that are DNA recognition elements,
wherein the fusion protein comprises a combination of successive
polypeptides chosen from at least one of the following polypeptide
sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or
SEQ ID NO:19 or any variant or analog thereof that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent homologous
thereof, wherein each monomer binds one nucleotide of a DNA target
sequence in the presence of a nucleic acid sequence, wherein the
fusion protein further comprises at least one polypeptide sequence
that is an effector protein/polypeptide. In some embodiments, the
fusion protein comprises a first domain that binds a DNA target
sequence and a second domain that has a nuclease function. In some
embodiments, the invention relates to a fusion protein comprising
successive, monomer subunits that are DNA recognition elements,
wherein the fusion protein comprises a combination of successive
polypeptides chosen from at least one of the following polypeptide
sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or
SEQ ID NO:19 or any variant or analog thereof that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent homologous
thereof, wherein each monomer binds one nucleotide of a DNA target
sequence in the presence of a nucleic acid sequence, wherein the
fusion protein further comprises at least one polypeptide sequence
that is an effector protein/polypeptide. In some embodiments, the
fusion protein comprises a first domain that binds a DNA target
sequence and a second domain that has a nickase or ligase function.
In some embodiments, the fusion protein comprises a first domain
that binds a DNA target sequence and a second domain that has a
nuclease function. In some embodiments, the invention relates to a
fusion protein comprising successive, monomer subunits that are DNA
recognition elements, wherein the fusion protein comprises any
combination of successive polypeptides chosen from at least one of
the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19 or any variant or analog
thereof that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% percent homologous thereof, wherein each monomer binds
one nucleotide of a DNA target sequence in the presence of a
nucleic acid sequence, and wherein the fusion protein further
comprises at least one polypeptide sequence that is an effector
protein/polypeptide. In some embodiments, the invention relates to
a fusion protein comprising successive, monomer subunits that are
DNA recognition elements, wherein the fusion protein comprises any
combination of successive polypeptides chosen from at least one of
the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, or SEQ ID NO:19 or any variant or analog
thereof that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% percent homologous thereof, wherein each monomer binds
one nucleotide of a DNA target sequence in the presence of a
nucleic acid sequence, and wherein the fusion protein further
comprises at least one polypeptide sequence that is an effector
protein/polypeptide and further comprises at least one polypeptide
sequence that has any disclosed effector protein function. In some
embodiments, the invention relates to a fusion protein comprising
successive, monomer subunits that are DNA recognition elements,
wherein the fusion protein comprises any combination of successive
polypeptides chosen from at least one of the following polypeptide
sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or
SEQ ID NO:19 or any variant or analog thereof that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent homologous
thereof, wherein each monomer binds one nucleotide of a DNA target
sequence in the presence of a nucleic acid sequence, and wherein
the fusion protein further comprises at least two polypeptide
sequences that comprise an effector protein/polypeptide function or
that are effector proteins or variants thereof. In some
embodiments, the invention relates to a fusion protein comprising
successive, monomer subunits that are DNA recognition elements,
wherein the fusion protein comprises any combination of successive
polypeptides chosen from at least one of the following polypeptide
sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or
SEQ ID NO:19 or any variant or analog thereof that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent homologous
thereof, wherein each monomer binds one nucleotide of a DNA target
sequence in the presence of a nucleic acid sequence, and wherein
the fusion protein further comprises at least three or more
polypeptide sequences that comprise an effector/protein function.
In some embodiments, the invention relates to a fusion protein
comprising successive, monomer subunits that are DNA recognition
elements, wherein the fusion protein comprises any combination of
successive polypeptides chosen from at least one of the following
polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
or SEQ ID NO:19 or any variant or analog thereof that is at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% percent
homologous thereof, wherein each monomer binds one nucleotide of a
DNA target sequence in the presence of a nucleic acid sequence, and
wherein the fusion protein further comprises at least four or more
polypeptide sequences that comprise effector
protein/polypeptide.
[0113] Nucleic acids or proteins of the present invention can be
constructed by a modular approach by preassembling monomer units
and/or repeat units in target vectors that can subsequently be
assembled into a final destination vector. In one aspect of the
invention, the polypeptides of the invention comprise repeat
monomers of the present invention and can be constructed by a
modular approach by preassembling repeat units in target vectors
that can subsequently be assembled into a final destination vector.
The invention provides the polypeptide produced this method as well
as nucleic acid sequences encoding the polypeptides and host
organisms and cells comprising such DNA sequences.
[0114] Techniques to specifically modify DNA sequences in order to
obtain a specified codon for a specific amino acid are known in the
art. Methods for mutagenesis and polynucleotide alterations have
been widely described. See, for example, Kunkel (1985) Proc. Nat!.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.
154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.
(1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. All these
publications are herein incorporated by reference.
[0115] The following examples provide methods for constructing new
repeat units and testing the specific binding activities of
artificially constructed repeat units specifically recognizing base
pairs in a target DNA sequence. The number of repeat units to be
used in a repeat domain can be ascertained by one skilled in the
art by routine experimentation.
[0116] Generally, at least 1.5 repeat units are considered as a
minimum, although typically at least about 8 repeat units will be
used. The repeat units do not have to be complete repeat units, as
repeat units of half the size can be used. Moreover, the methods
and polypeptides disclosed herein do depend on repeat domains with
a particular number of repeat units. Thus, a polypeptide of the
invention can comprise, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,
5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,
12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,
19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,
25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5,
32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38,
38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5,
46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5 or more repeat
units.
[0117] In the present invention, polypeptides can be designed which
comprise a repeat domain with repeat units wherein in the repeat
units hypervariable regions are included which determine
recognition of a base pair in a target DNA sequence. In one
embodiment of the invention, each repeat unit includes a
hypervariable region which determine recognition of one base pair
in a target DNA sequence. In a further embodiment, 1 or 2 repeat
units in a repeat domain are included which do not specifically
recognize a base pair in a target DNA sequence. Considering the
recognition code found by the inventors, a modular arrangement of
repeat units is feasible wherein each repeat unit is responsible
for the specific recognition of one base pair in a target DNA
sequence. Consequently, a sequence of repeat units corresponds to a
sequence of base pairs in a target DNA sequence so that 1 repeat
unit matches to one base pair.
[0118] The present invention provides a method for selectively
recognizing a base pair in a target DNA sequence by a polypeptide
wherein said polypeptide comprises at least one repeat domain
comprising repeat units wherein in said repeat units each comprise
at least one RVD region which determines recognition of a base pair
or nucleotide in said target DNA sequence. More specifically, the
inventors have determined those amino acids in a DNA-binding
polypeptide responsible for selective recognition of base pairs in
a target DNA sequence. With elucidation of the recognition code, a
general principle for recognizing specific base pairs in a target
DNA sequence by selected amino acids in a polypeptide has been
determined. The inventors have found that distinct types of
monomers that are part of a repeat unit array (or polymer) of
varying amino acid length have the capacity to recognize one
defined/specific base pair. Within each repeat unit forming a
repeat domain, a RVD region is responsible for the specific
recognition of a base pair in a target DNA sequence.
[0119] Thus, the present invention provides not only a method for
selectively recognizing a base pair in a target DNA sequence by a
polypeptide comprising at least one repeat domain comprising repeat
units but also methods wherein target DNA sequences can be
generated which are selectively recognized by repeat domains in a
polypeptide. These polypeptides are useful for molecular biology
tools in order to clone, mutagenize or otherwise alter an isolated
nucleic acid sequence or other in vivo sequence in a laboratory.
This provides an efficient means of selective mutagenesis.
[0120] The invention also provides for a method for constructing
and/or making polypeptides that recognize specific DNA sequences.
These polypeptides of the invention comprise repeat monomers of the
present invention and can be constructed by a modular approach by
preassembling repeat units in target vectors that can subsequently
be assembled into a final destination vector. In some embodiments,
the DNA constructs are codon optimized to recombinantly produce
and/or secrete the polypeptides disclosed herein. Any recombinant
system in the art can be used to produce the recombinant protein.
Examples include baculovirus cells, other eukaryotic cells such as
mammalian cells, or bacterial cells.
[0121] Provided that a target DNA sequence is known and to which
recognition by a protein is desired, the person skilled in the art
is able to specifically construct a modular series of repeat units,
including specific recognition amino acid sequences, and assemble
these repeat units into a polypeptide in the appropriate order to
enable recognition of and binding to the desired target DNA
sequence. Any polypeptide can be modified by being combined with a
modular repeat unit DNA-binding domain of the present invention.
Such examples include polypeptides that are transcription activator
and repressor proteins, resistance-mediating proteins, nucleases,
topoisomerases, ligases, integrases, recombinases, resolvases,
methylases, acetylases, demethylases, deacetylases, and any other
polypeptide capable of modifying DNA, RNA, or proteins.
[0122] The modular repeat unit DNA-binding domain of the present
invention can be combined with cell compartment localisation
signals such as nuclear localisation signals, to function at any
other regulatory regions, including but not limited to,
transcriptional regulatory regions and translational termination
regions.
[0123] In a further embodiment of the invention, these modularly
designed repeat units are combined with an endonuclease domain
capable of cleaving DNA when brought into proximity with DNA as a
result of binding by the repeat domain. Such endonucleolytic breaks
are known to stimulate the rate of homologous recombination in
eukaryotes, including fungi, plants, and animals. The ability to
simulate homologous recombination at a specific site as a result of
a site-specific endonucleolytic break allows the recovery of
transformed cells that have integrated a DNA sequence of interest
at the specific site, at a much higher frequency than is possible
without having made the site-specific break. In addition,
endonucleolytic breaks such as those caused by polypeptides formed
from a repeat domain and an endonuclease domain are sometimes
repaired by the cellular DNA metabolic machinery in a way that
alters the sequence at the site of the break, for instance by
causing a short insertion or deletion at the site of the break
compared to the unaltered sequence. These sequence alterations can
cause inactivation of the function of a gene or protein, for
instance by altering a protein-coding sequence to make a
non-functional protein, modifying a splice site so that a gene
transcript is not properly cleaved, making a non-functional
transcript, changing the promoter sequence of a gene so that it can
no longer by appropriately transcribed, etc.
[0124] Breaking DNA using site specific endonucleases can increase
the rate of homologous recombination in the region of the breakage.
In some embodiments, the Fok I (Flavobacterium okeanokoites)
endonuclease may be utilized in an effector to induce DNA breaks.
The Fok I endonuclease domain functions independently of the DNA
binding domain and cuts a double stranded DNA typically as a dimer
(Li et al. (1992) Proc. Natl. Acad. Sci. U.S.A 89 (10):4275-4279,
and Kim et al. (1996) Proc. Natl. Acad. Sci. U.S.A 93 (3):
1156-1160; the disclosures of which are incorporated herein by
reference in their entireties). A single-chain FokI dimer has also
been developed and could also be utilized (Mino et al. (2009) J.
Biotechnol. 140: 156-161). An effector could be constructed that
contains a repeat domain for recognition of a desired target DNA
sequence as well as a FokI endonuclease domain to induce DNA
breakage at or near the target DNA sequence similar to previous
work done employing zinc finger nucleases (Townsend et al. (2009)
Nature 459:442-445; Shukla et al. (2009) Nature 459, 437-441, all
of which are herein incorporated by reference in their entireties).
Utilization of such effectors could enable the generation of
targeted changes in genomes which include additions, deletions and
other modifications, analogous to those uses reported for zinc
finger nucleases as per Bibikova et al. (2003) Science 300, 764;
Urnov et al. (2005) Nature 435, 646; Wright et al. (2005) The Plant
Journal 44:693-705; and U.S. Pat. Nos. 7,163,824 and 7,001,768, all
of which are herein incorporated by reference in their
entireties.
[0125] The FokI endonuclease domain can be cloned by PCR from the
genomic DNA of the marine bacteria Flavobacterium okeanokoites
(ATCC) prepared by standard methods.
[0126] The sequence of the FokI endonuclease is available on Pubmed
(Ace. No. M28828 and Acc. No J04623, the disclosures of which are
incorporated herein by reference in their entireties). The I-Sce I
endonuclease from the yeast Saccharomyces cerevisiae has been used
to produce DNA breaks that increase the rate of homologous
recombination. I-Sce I is an endonuclease encoded by a
mitochondrial intron which has an 18 bp recognition sequence, and
therefore a very low frequency of recognition sites within a given
DNA, even within large genomes (Thierry et al. (1991) Nucleic Acids
Res. 19 (1): 189-190; the disclosure of which is incorporated
herein by reference in its entirety). The infrequency of cleavage
sites recognized by I-SceI makes it suitable to use for enhancing
homologous recombination. Additional description regarding the use
of I-Sce I to induce said DNA breaks can be found in U.S. Pat.
Appl. 20090305402, which is incorporated herein by reference in its
entirety.
[0127] The recognition site for I-Sce I has been introduced into a
range of different systems. Subsequent cutting of this site with
I-Sce I increases homologous recombination at the position where
the site has been introduced. Enhanced frequencies of homologous
recombination have been obtained with I-Sce I sites introduced into
the extra-chromosomal DNA in Xenopus oocytes, the mouse genome, and
the genomic DNA of the tobacco plant Nicotiana plumbaginifolia.
See, for example, Segal et al. (1995) Proc. Natl. Acad. Sci. U.S.A.
92 (3):806-810; Choulika et al. (1995) Mol. Cell Biol. 15 (4):
1968-1973; and Puchta et al. (1993) Nucleic Acids Res. 21
(22):5034-5040; the disclosures of which are incorporated herein by
reference in their entireties. It will be appreciated that any
other endonuclease domain that works with heterologous DNA binding
domains can be utilized in an effector and that the I-Sce I
endonuclease is one such non-limiting example. The limitation of
the use of endonucleases that have a DNA recognition and binding
domain such as I-Sce I is that the recognition site has to be
introduced by standard methods of homologous recombination at the
desired location prior to the use of said endonuclease to enhance
homologous recombination at that site, if such site is not already
present in the desired location. Methods have been reported that
enable the design and synthesis of novel endonucleases, such as by
modifying known endonucleases or making chimeric versions of one or
more such endonucleases, that recognize novel target DNA sequences,
thus paving the way for generation of such engineered endonuclease
domains to cleave endogenous target DNA sequences of interest
(Chevalier et al. (2002) Molecular Cell 10:895-905; WO2007/060495;
WO2009/095793; Fajardo-Sanchez et al. (2008) Nucleic Acids Res.
36:2163-2173, both of which are incorporated by reference in their
entireties). As such, it could be envisioned that such endonuclease
domains could be similarly engineered so as to render the
DNA-binding activity non-functional but leaving the DNA cleaving
function active and to utilize said similarly engineered
endonuclease cleavage domain in an effector to induce DNA breaks
similar to the use of FokI above. In such applications, target DNA
sequence recognition would preferably be provided by the repeat
domain of the effector but DNA cleavage would be accomplished by
the engineered endonuclease domain.
[0128] As mentioned above, an effector includes a repeat domain
with specific recognition for a desired specific target sequence.
In preferred embodiments, the effector specifically binds to an
endogenous chromosomal DNA sequence. The specific nucleic acid
sequence or more preferably specific endogenous chromosomal
sequence can be any sequence in a nucleic acid region where it is
desired to enhance homologous recombination. For example, the
nucleic acid region may be a region which contains a gene in which
it is desired to introduce a mutation, such as a point mutation or
deletion, or a region into which it is desired to introduce a gene
conferring a desired phenotype.
[0129] Further embodiments relate to methods of generating a
modified plant in which a desired addition has been introduced. The
methods can include obtaining a plant cell that includes an
endogenous target DNA sequence into which it is desired to
introduce a modification; generating a double-stranded cut within
the endogenous target DNA sequence with an effector that includes a
repeat domain that binds to an endogenous target DNA sequence and
an endonuclease domain; introducing an exogenous nucleic acid that
includes a sequence homologous to at least a portion of the
endogenous target DNA into the plant cell under conditions which
permit homologous recombination to occur between the exogenous
nucleic acid and the endogenous target DNA sequence; and generating
a plant from the plant cell in which homologous recombination has
occurred. Other embodiments relate to genetically modified cells
and plants made according to the method described above and herein.
It should be noted that the target DNA sequence could be artificial
or naturally occurring. It will be appreciated that such methods
could be used in any organism (such non-limiting organisms to
include animals, humans, fungi, oomycetes bacteria and viruses)
using techniques and methods known in the art and utilized for such
purposes in such organisms.
[0130] In a further embodiment of the invention, these modularly
designed repeat domains are combined with one or more domains
responsible for the modulation or control of the expression of a
gene, for instance of plant genes, animal genes, fungal genes,
oomycete genes, viral genes, or human genes. Methods for modulating
gene expression by generating DNA-binding polypeptides containing
zinc finger domains is known in the art (U.S. Pat. Nos. 7,285,416,
7,521,241, 7,361,635, 7,273,923, 7,262,054, 7,220,719, 7,070,934,
7,013,219, 6,979,539, 6,933, 113, 6,824,978, each of which is
hereby herein incorporated by reference in its entirety). For
instance, these effectors of the Ralstonia-like family are modified
in order to bind to specific target DNA sequences. Such
polypeptides might for instance be transcription activators or
repressor proteins of transcription which are modified by the
method of the present invention to specifically bind to genetic
control regions in a promoter of or other regulatory region for a
gene of interest in order to activate, repress or otherwise
modulate transcription of said gene.
[0131] In a still further embodiment of the invention, the target
DNA sequences are modified in order to be specifically recognized
by a naturally occurring repeat domain or by a modified repeat
domain. As one example, the target DNA sequences for members of the
Ralstonia-like family can be inserted into promoters to generate
novel controllable promoters that can be induced by the
corresponding effector. Secondary inducible systems can be
constructed using a trans-activator and a target gene, wherein the
trans-activator is a polypeptide wherein said polypeptide comprises
at least a repeat domain comprising repeat units of the present
invention that bind to said target gene and induce expression. The
trans-activator and the target gene can be introduced into one cell
line but may also be present in different cell lines and later be
introgressed. In a further embodiment, disease-resistant plants can
be constructed by inserting the target DNA sequence of a repeat
domain containing polypeptide of the present invention in front of
a gene which after expression leads to a defense reaction of the
plant by activating a resistance-mediating gene.
[0132] In a further embodiment, custom DNA-binding polypeptides can
be constructed by rearranging repeat unit types thus allowing the
generation of repeat domains with novel target DNA binding
specificity. Individual repeat units are nearly identical at the
DNA level which precludes classical cloning strategies. The present
invention provides a quick and inexpensive strategy to assemble
custom polypeptides with repeat domains of the present invention.
To improve cloning versatility such polypeptides, a two-step
assembly method was designed. This method was used to assemble
polypeptides with novel repeat types to study their target DNA
recognition and binding specificity.
[0133] Summarily, any DNA sequence can be modified to enable
binding by a repeat domain containing polypeptide of the present
invention by introducing base pairs into any DNA region or specific
regions of a gene or a genetic control element to specifically
target a polypeptide having a repeat domain comprised of repeat
units that will bind said modified DNA sequence in order to
facilitate specific recognition and binding to each other.
[0134] In some embodiments, polypeptides can be synthetically
manufactured using known amino acid chemistries familiar to one of
ordinary skill in organic chemistry synthesis. Such procedures
include both solution and solid phase procedures, e.g., using
either Boc and Fmoc methodologies. The compounds of the invention
may be synthesized using solid phase synthesis techniques.
Fmoc-N-Protected .beta.-amino acids can be used to synthesize
poly-.alpha./.beta.-peptides by conventional manual solid-phase
synthesis procedures under standard conditions on any number of
solid supports, including ortho-chloro-trityl chloride resin.
Esterification of Fmoc-.beta.-amino acids with the
ortho-chloro-trityl resin can be performed according to the method
of Barlos et. al., Tetrahedron Lett, 1989, 30, 3943. The resin (150
mg, 1.05 mmol Cl) is swelled in 2 ml CH.sub.2Cl.sub.2 for 10 min. A
solution of the Fmoc-protected .beta.-amino acid in
CH.sub.2Cl.sub.2 and iPr.sub.2EtN are then added successively and
the suspension is mixed under argon for 4 h. Subsequently, the
resin is filtered and washed with
CH.sub.2Cl.sub.2/MeOH/iPr.sub.2EtN (17:2:1, 3.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), DMF (2.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), and MeOH (2.times.3 min). The
substitution of the resin is determined on a 3 mg sample by
measuring the absorbance of the dibenzofulvene adduct at 300 nm.
The Fmoc group is removed using 20% piperidine in DMF (4 ml,
2.times.20 min) under Ar bubbling. The resin is then filtered and
washed with DMF (6.times.3 min). For each coupling step, a solution
of the .beta.-amino acid (3 equiv.), BOP (3 equiv.) and HOBT (3
equiv.) in DMF (2 ml) and iPr.sub.2EtN (9 eq) are added
successively to the resin and the suspension is mixed for 1 h under
Ar. Monitoring of the coupling reaction is performed with
2,4,6-trinitrobenzene-sulfonic acid (TNBS) (W. S. Hancock and J. E.
Battersby, Anal. Biochem. (1976), 71, 260). In the case of a
positive TNBS test (indicating incomplete coupling), the suspension
is allowed to react for a further 1 h. The resin is then filtered
and washed with DMF (3.times.3 min) prior to the following Fmoc
deprotection step. After the removal of the last Fmoc protecting
group, the resin is washed with DMF (6.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), Et.sub.2O (3.times.3 min) and
dried under vacuum for 3 h. Finally the peptides are cleaved from
the resin using 2% TFA in CH.sub.2Cl.sub.2 (2 ml, 5.times.15 min)
under Ar. The solvent is removed and the oily residues are
triturated in ether to give the crude polypeptides. The compounds
are further purified by HPLC.
[0135] The invention also provides a method for targeted modulation
of gene expression by constructing modular repeat units specific
for a target DNA sequence of interest, modifying a polypeptide by
the addition of said repeat monomers so as to enable said
polypeptide to now recognize the target DNA, introducing or
expressing said modified polypeptide in a prokaryotic or eukaryotic
cell so as to enable said modified polypeptide to recognize the
target DNA sequence, and modulation of the expression of said
target gene in said cell as a result of such recognition.
[0136] The invention also provides a method for directed
modification of a target DNA sequence by the construction of a
polypeptide including at least a repeat domain of the present
invention that recognizes said target DNA sequence and that said
polypeptide also contains a functional domain capable of modifying
the target DNA (such as via site specific recombination,
restriction or integration of donor target sequences) thereby
enabling targeted DNA modifications in complex genomes.
[0137] The invention further provides for the production of
modified polypeptides including at least a repeat domain comprising
repeat units wherein a hypervariable region within each of the
repeat units determines selective recognition of a base pair in a
target DNA sequence. In a further embodiment of the invention, DNA
is provided which encodes for a polypeptide containing a repeat
domain as described above.
[0138] In another embodiment of the invention, DNA is provided
which is modified to include one or more base pairs located in a
target DNA sequence so that each of the said base pairs can be
specifically recognized by a polypeptide including a repeat domain
having corresponding repeat units, each repeat unit comprising a
hypervariable region which determines recognition of the
corresponding base pair in said DNA.
[0139] In a still another embodiment of the invention, uses of
those polypeptides and DNAs are provided. Additionally provided are
plants, plant parts, seeds, plant cells and other non-human host
cells transformed with the isolated nucleic acid molecules of the
present invention and the proteins or polypeptides encoded by the
coding sequences of the present invention. Still further, the
polypeptides and DNA described herein can be introduced into animal
and human cells as well as cells of other organisms like fungi or
plants.
[0140] In summary, the invention focuses on a method for
selectively recognizing base pairs in a target DNA sequence by a
polypeptide wherein said polypeptide comprises at least a repeat
domain comprising repeat units wherein each repeat unit contains a
hypervariable region which determines recognition of a base pair in
said target DNA sequence wherein consecutive repeat units
correspond to consecutive base pairs in said target DNA
sequence.
[0141] In some embodiments, the invention relates to a human cell
comprising any one or combination of proteins or nucleic acid
sequences disclosed herein. In some embodiments, the invention
relates to cells such as a human cell comprising a mutation,
heterologous gene, variant or other genetic modification caused by
introduction of one or more nucleic acids or polypeptides disclosed
herein. In some embodiments, the invention relates to cells such as
a non-human animal cell comprising a mutation, heterologous gene,
variant or other genetic modification caused by introduction of one
or more nucleic acids or polypeptides disclosed herein. In some
embodiments, the invention relates to cells such as an insect cell
comprising a mutation, heterologous gene, variant or other genetic
modification caused by introduction of one or more nucleic acids or
polypeptides disclosed herein. In some embodiments, the invention
relates to cells such as a plant cell comprising a mutation,
heterologous gene, variant or other genetic modification caused by
introduction of one or more nucleic acids or polypeptides disclosed
herein. In some embodiments, the invention relates to cells such as
a fish cell comprising a mutation, heterologous gene, variant or
other genetic modification caused by introduction of one or more
nucleic acids or polypeptides disclosed herein. In some
embodiments, the invention relates to cells such as a mammalian
cell comprising a mutation, heterologous gene, variant or other
genetic modification caused by introduction of one or more nucleic
acids or polypeptides disclosed herein. In some embodiments, the
invention relates to cells such as a eukaryotic cell comprising a
mutation, heterologous gene, variant or other genetic modification
caused by introduction of one or more nucleic acids or polypeptides
disclosed herein.
[0142] In another aspect, a method of modulating expression of a
target gene in a cell is provided. The cell may be preferably a
plant cell, a human cell, animal cell, fungal cell or any other
living cell. The cells contain a polypeptide wherein said
polypeptide comprises at least a repeat domain comprising repeat
units, and these repeat units contain a hypervariable region and
each repeat unit is responsible for the recognition of 1 base pair
in said target DNA sequence. Said polypeptide is introduced either
as DNA encoding for the polypeptide or the polypeptide is
introduced per se into the cell by methods known in the art.
Regardless of how introduced, the polypeptide should include at
least one repeat domain that specifically recognizes and preferably
binds to a target DNA sequence of base pairs and modulates the
expression of a target gene. In a preferred embodiment, all repeat
units contain a hypervariable region which determines recognition
of base pairs in a target DNA sequence.
[0143] Examples of peptide sequences which can be linked to an
polypeptide or RTN of the present invention, for facilitating
uptake of effectors into cells, include, but are not limited to: an
11 amino acid peptide of the tat protein of HIV; a 20 residue
peptide sequence which corresponds to amino acids 84 103 of the p
16 protein (see Fahraeus et al. (1996) Current Biology 6:84); the
third helix of the 60-amino acid long homeodomain of Antennapedia
(Derossi et al. (1994) J. Biol. Chem. 269: 10444); the h region of
a signal peptide such as the Kaposi fibroblast growth factor
(K-FGF) h region; or the VP22 translocation domain from HSV (Elliot
& O'Hare (1997) Cell 88:223 233). Other suitable chemical
moieties that provide enhanced cellular uptake may also be
chemically linked to effectors. As described herein, effectors can
be designed to recognize any suitable target site, for regulation
of expression of any endogenous gene of choice. Examples of
endogenous genes suitable for regulation include VEGF, CCR5,
ER.alpha., Her2/Neu, Tat, Rev, HBV C, S, X, and P, LDL-R, PEPCK,
CYP7, Fibrinogen, ApoB, Apo E, Apo(a), renin, NF-.kappa.B,
I-.kappa.B, TNF-.alpha., FAS ligand, amyloid precursor protein,
atrial naturetic factor, ob-leptin, ucp-1, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-12, G-CSF, GM-CSF, Epo, PDGF, PAF, p53, Rb, fetal
hemoglobin, dystrophin, eutrophin, GDNF, NGF, IGF-1, VEGF receptors
fit and flk, topoisomerase, telomerase, bcl-2, cyclins,
angiostatin, IGF, ICAM-1, STATS, c-myc, c-myb, TH, PTI-1,
polygalacturonase, EPSP synthase, FAD2-1, delta-12 desaturase,
delta-9 desaturase, delta-15 desaturase, acetyl-CoA carboxylase,
acyl-ACP-thioesterase, ADP-glucose pyrophosphorylase, starch
synthase, cellulose synthase, sucrose synthase,
senescence-associated genes, heavy metal chelators, fatty acid
hydroperoxide lyase, viral genes, protozoal genes, fungal genes,
and bacterial genes. In general, suitable genes to be regulated
include cytokines, lymphokines, growth factors, mitogenic factors,
chemotactic factors, onto-active factors, receptors, potassium
channels, G-proteins, signal transduction molecules, disease
resistance genes, and other disease-related genes.
[0144] Toxin molecules also have the ability to transport
polypeptides across cell membranes. Often, such molecules are
composed of at least two parts (called "binary toxins"): a
translocation or binding domain or polypeptide and a separate toxin
domain or polypeptide. Typically, the translocation domain or
polypeptide binds to a cellular receptor, and then the toxin is
transported into the cell. Several bacterial toxins, including
Clostridium perfringens iota toxin, diphtheria toxin (DT),
Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus
anthracis toxin, and pertussis adenylate cyclase (CYA), have been
used in attempts to deliver peptides to the cell cytosol as
internal or amino-terminal fusions (Arora et al. (1993) J. Biol.
Chem. 268:3334 3341; Perelle et al. (1993) Infect. Immun. 61:5147
5156 (1993); Stenmark et al. (1991) J. Cell Biol. 113:1025 1032
(1991); Donnelly et al. (1993) Proc. Natl. Acad. Sci. USA 90:3530
3534; Carbonetti et al. (1995) Abstr. Annu. Meet. Am. Soc.
Microbiol. 95:295; Sebo et al. (1995) Infect. Immun. 63:3851 3857;
Klimpel et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10277 10281;
and Novak et al. (1992) J. Biol. Chem. 267: 17186 17193).
[0145] Effectors can also be introduced into an animal cell,
preferably a mammalian cell, via liposomes and liposome derivatives
such as immunoliposomes. The term "liposome" refers to vesicles
comprised of one or more concentrically ordered lipid bilayers,
which encapsulate an aqueous phase. The aqueous phase typically
contains the compound to be delivered to the cell, in this case an
effector. The liposome fuses with the plasma membrane, thereby
releasing the effector into the cytosol. Alternatively, the
liposome is phagocytosed or taken up by the cell in a transport
vesicle. Once in the endosome or phagosome, the liposome either
degrades or fuses with the membrane of the transport vesicle and
releases its contents.
[0146] The invention particularly relates to the field of plant and
agricultural technology. In one aspect, the present invention is
directed to a method to modulate the expression of a target gene in
plant cells, which method comprises providing plant cells with a
polypeptide modified according to the invention, said polypeptide
being capable of specifically recognizing a target nucleotide
sequence, or a complementary strand thereof, within a target gene,
and allowing said polypeptide to recognize and particularly bind to
said target nucleotide sequence, whereby the expression of said
target gene in said plant cells is modulated.
[0147] The polypeptide can be provided to the plant cells via any
suitable methods known in the art. For example, the protein can be
exogenously added to the plant cells and the plant cells are
maintained under conditions such that the polypeptide is introduced
into the plant cell, binds to the target nucleotide sequence and
regulates the expression of the target gene in the plant cells.
Alternatively, a nucleotide sequence, e.g., DNA or RNA, encoding
the polypeptide can be expressed in the plant cells and the plant
cells are maintained under conditions such that the expressed
polypeptide binds to the target nucleotide sequence and regulates
the expression of the target gene in the plant cells.
[0148] A preferred method to modulate the expression of a target
gene in plant cells comprises the following steps: a) providing
plant cells with an expression system for a polypeptide modified
according to the invention, said polypeptide being capable of
specifically recognizing, and preferably binding, to a target
nucleotide sequence, or a complementary strand thereof, within an
expression control element of a target gene, preferably a promoter;
and b) culturing said plant cells under conditions wherein said
polypeptide is produced and binds to said target nucleotide
sequence, whereby expression of said target gene in said plant
cells is modulated.
[0149] Any target nucleotide sequence can be modulated by the
present method. For example, the target nucleotide sequence can be
endogenous or exogenous to the target gene. In an embodiment of the
invention the target nucleotide sequence can be present in a living
cell or present in vitro. In a specific embodiment, the target
nucleotide sequence is endogenous to the plant. The target
nucleotide sequence can be located in any suitable place in
relation to the target gene. For example, the target nucleotide
sequence can be upstream or downstream of the coding region of the
target gene. Alternatively, the target nucleotide sequence is
within the coding region of the target gene. Preferably, the target
nucleotide sequence is a promoter of a gene.
[0150] Any target gene can be modulated by the present method. For
example, the target gene can encode a product that affects
biosynthesis, modification, cellular trafficking, metabolism and
degradation of a peptide, a protein, an oligonucleotide, a nucleic
acid, a vitamin, an oligosaccharide, a carbohydrate, a lipid, or a
small molecule. Furthermore, effectors can be used to engineer
plants for traits such as increased disease resistance,
modification of structural and storage polysaccharides, flavors,
proteins, and fatty acids, fruit ripening, yield, color,
nutritional characteristics, improved storage capability, and the
like.
[0151] Therefore, the invention provides a method of altering the
expression of a gene of interest in a target cell, comprising:
determining (if necessary) at least part of the DNA sequence of the
structural region and/or a regulatory region of the gene of
interest; designing a polypeptide including the repeat units
modified in accordance with the invention to recognize specific
base pairs on the DNA of known sequence, and causing said modified
polypeptide to be present in the target cell, (preferably in the
nucleus thereof). (It will be apparent that the DNA sequence need
not be determined if it is already known.)
[0152] The present invention also provides kits comprising: (1) any
of the aforementioned vectors or (2) any of the aforementioned
proteins or polypeptides. The present invention also provides kits
comprising: (1) any of the aforementioned vectors or (2) any of the
aforementioned proteins or polypeptides; and (3) any of the
aforementioned cells (either modified or not modified) disclosed
herein.
[0153] In another embodiments, the invention relates to kits that
are used to produce site specific-mutations in stem cells, which
can be used to generate genetically modified organisms. The kits
typically include one or more site-specific genetic engineering
technology, such as RTNs. The kit may also contain one or more sets
of stem cells or embryonic cells for site-specific modification.
The stem cells may include, but is not limited to spermatogonial
stem cells (SSCs), as well as media and conditions necessary for
growing SSCs. The kits may include exogenous sequences for
site-specific genomic introduction, such as but not limited to
reporter genes or selectable markers. The kits may include
instructions for (i) introducing the RTNs into the stem cells (ii)
identifying stem cells which have been site specifically modified
(iii) growing site-specifically modified stem cells in media or
conditions necessary and to numbers required for stem cells to
produce genetically modified organisms (iv) using the grown stem
cells to produce a genetically modified organism (v) identifying
which organisms or progeny harbor the site-specific mutation of
interest.
[0154] In some embodiment, the invention provides a kit which
includes a mixed population of different or distinct genetically
modified SSCs which may be custom made. The mixed population of
genetically modified SSCs may be provided in suitable quantities
for direct injection into a sterile male recipient for the
production of multiple genetically modified organisms in a single
step. The mixed population of separate or distinct genetically
modified SSCs may consist of at least two genetically modified
SSCs, at least two genetically modified SSCs, at least three
genetically modified SSCs, at least four genetically modified SSCs,
at least five genetically modified SSCs, at least six genetically
modified SSCs, at least seven genetically modified SSCs, at least
eight genetically modified SSCs, at least nine genetically modified
SSCs, at least ten genetically modified SSCs, at least twenty
genetically modified SSCs, at least thirty genetically modified
SSCs, at least forty genetically modified SSCs, at least fifty
genetically modified SSCs, at least one hundred genetically
modified SSCs, at least one thousand genetically modified SSCs, at
least ten thousand genetically modified SSCs, at least thirty
thousand genetically modified SSCs or with genetically modified
SSCs which harbor genetic modification within every gene in the
organisms genome.
[0155] In some embodiment, the invention provides a kit which
includes a mixed population of different or distinct genetically
modified stem cells or embryonic cells which may be custom made by
any of the methods disclosed herein. The mixed population of
genetically modified stem cells or embryonic cells may be provided
in suitable quantities for direct injection into a sterile male
recipient for the production of multiple genetically modified
organisms in a single step. The mixed population of separate or
distinct genetically modified stem cells or embryonic cells may
consist of at least two genetically modified stem cells or
embryonic cells, at least two genetically modified stem cells or
embryonic cells, at least three genetically modified stem cells or
embryonic cells, at least four genetically modified stem cells or
embryonic cells, at least five genetically modified stem cells or
embryonic cells, at least six genetically modified stem cells or
embryonic cells, at least seven genetically modified stem cells or
embryonic cells, at least eight genetically modified stem cells or
embryonic cells, at least nine genetically modified stem cells or
embryonic cells, at least ten genetically modified stem cells or
embryonic cells, at least twenty genetically modified stem cells or
embryonic cells, at least thirty genetically modified stem cells or
embryonic cells, at least forty genetically modified stem cells or
embryonic cells, at least fifty genetically modified stem cells or
embryonic cells, at least one hundred genetically modified stem
cells or embryonic cells, at least one thousand genetically
modified stem cells or embryonic cells, at least ten thousand
genetically modified stem cells or embryonic cells, at least thirty
thousand genetically modified stem cells or embryonic cells or with
genetically modified stem cells or embryonic cells which harbor
genetic modification within every gene in the organisms genome.
[0156] In some embodiment, the invention provides a kit which
includes one or more sets of stem cells or embryonic cells or SSCs
for site-specific modification. The sets of SSCs may be derived
from well-characterized organisms having different disease states.
The SSCs may contain multiple mutations, which may be derived from
genetic modification or naturally or by any method. The kit may
include the media and conditions to grow disease state SSCs, as
well as the sterile recipient male for the production of
genetically modified organisms.
[0157] In some embodiment, the invention provides a kit which
includes the necessary tools for the derivation of SSC lines from
an organism or tissue sample, as well as the necessary tools to
genetically modify the derived SSC and produce a genetically
modified organism from the derived SSCs. The kit may include cell
collection tools such as spermatocytes for harvest, and SSC
selection tools such as laminin selection, and SSC propagation and
cryopreservation tools as well as SSC validation tools which may
include cell surface marker staining. The kit may also include
media and conditions for growing the SSCs, tools for genetic
modification of the SSCs as well as sterile recipient males for
production of genetically modified organisms from the SSCs.
[0158] In some embodiment, the invention provides a kit, which
includes SSCs which have been generated from induced pluripotent
stem (iPS) cells. The iPS cells may be derived from well
characterized different genetic backgrounds including disease
states as well as regional, strain, ethnic genetic backgrounds. The
kit may also include media and conditions for growing the iPS s,
tools for genetic modification of the iPSs as well as sterile
recipient males for production of genetically modified organisms
from the iPSs.
[0159] Further methods for transformation and generation of
transgenic animals or modified cells appear in PCT Application
Serial No. PCT/US2012/038465, the contents of which are
incorporated by reference in its entirety.
[0160] One aspect of the present invention relates to a method for
delivering fusion proteins into a target cell wherein the fusion
protein comprises an effector protein. Creating a fusion protein of
the present invention involves isolating one or more polypeptide
components from media and subsequently ligating the free amino
terminus of one polypeptide component to the carboxy terminus of a
second polypeptide component, in other embodiments, fusion proteins
may be made by simple polypeptide synthesis and or expressed
through cloning a nucleic acid sequence into an expression vector.
In the case of protein purification from cell-based recombinant
systems that express expression constructs of the present
invention, one of ordinary skill in the art can identify compatible
secretion signals can readily be determined for any particular type
III secretion system that is to be employed if such expression
constructs are transformed into bacterial host cells for protein
production. By identifying proteins that are normally secreted by
the type III secretion system, it is possible to prepare deletion
mutants missing various fragments of the full length protein that
is normally secreted by the secretion system. Using labeled
antibodies raised against epitopes of the various deletion
fragments that are expressed (i.e., N-terminal epitopes, C-terminal
epitopes, etc.), it is possible to identify deletion mutants that
are secreted and those that are not secreted. Thus, protein domains
necessary for secretion of the full length protein can be readily
identified. Once the protein domains have been identified and
sequenced, they can be utilized as secretion signals in fusion
proteins of the present invention.
[0161] Typically, the secretion signal is an N-terminal domain of a
protein that is normally secreted by the particular type III
secretion system, for example, a 201 amino acid sequence from the
N-terminal domain of the DspE protein of Erwinia amylovora (see,
e.g., U.S. patent application Ser. No. 09/350,852, filed Jul. 9,
1999, which is hereby incorporated by reference). The 201 amino
acid secretion signal of Erwinia amylovora DspE is compatible with
the hacpin secretion system of Erwinia amylovora. Other secretion
signals that are compatible with various type III secretion systems
have been described in the art and others are continually being
identified.
[0162] Purified effector protein may be obtained by several
methods. The protein or polypeptide is preferably produced in
purified form (at least about 80%, 90%, pure) by conventional
techniques. Because recombinant host cells express a type III
secretion system, the protein or polypeptide is secreted into the
growth medium of recombinant host cells. In such cases, to isolate
the protein, the recombinant host cells are propagated, the growth
medium is centrifuged to separate cellular components from
supernatant containing the secreted protein or polypeptide, and the
supernatant is removed. The supernatant is then subjected to
sequential ammonium sulfate precipitation. The fraction containing
the polypeptide or protein is subjected to gel filtration in an
appropriately sized dextran or polyacrylamide column to separate
the proteins. If necessary, the protein fraction may be further
purified by HPLC.
[0163] Effector proteins carrying protein transduction domains may
also be prepared independently of the type III secretion system by
using current state-of-the-art techniques for preparing large
amounts of purified proteins from recombinant E. coli cells. Such
techniques employ strong, inducible promoters and peptide tags,
such as His6, for one-step affinity purification of the recombinant
protein from E. coli cell lysates.
[0164] In one embodiment, the target cell is a eucaryote cell. The
eucaryote cells include those in tissue culture, such as HeLa
cells, or in whole animals, such as those delivered to mouse via
intraperitoneal injection (Schwarze et al., "Protein Transduction:
Unrestricted Delivery into all Cells?" Trends Cell Biol. 10:290-295
(2000), which is hereby incorporated by reference).
[0165] In one aspect of the invention the DNA binding or
recognition elements of the present invention may be fused together
in a modular fashion to create a string of amino acids that bind to
a DNA target sequence of choice. In another aspect of the invention
the one or more DNA binding recognition elements may be bound to
one or more effector proteins.
[0166] The effector protein may be produced by a bacterial plant
pathogen, animal pathogen, or a rhizosphere bacteria, including,
but not limited to enteropathogenic Escherichia coli, Salmonella
typhimurium, Shigella spp., Yersinia spp., Pseudomonas syringae,
Xanthomonas campestris, Ralstonia solanacearum, Envinia amylovora,
Pseudomonas fluorescens, and Pseudomonas aeruginosa. Suitable
effector proteins include a hypersensitive response elicitor, an
avirulence protein, a hypersensitive response and
pathogenicity-dependent outer protein, a virulence protein, and a
pathogenicity protein. Examples of effector proteins include
HopPsyA AAF71481 (P. syringae), HopPtoA AF232006 (P. syringae), Tir
BAA96815 (E. coli), ExoS AAG07228 (P. aeruginosa), ExoT AAG03434
(P. aeruginosa), ExoY AAG05579 (P. aeruginosa), SopE AAC02071 (S.
typhimurium), SopB AAF21057 (SigA) (S. typhimurium), SipA CAA63302
(S. typhimurium), SptP AAC44349 (S. typhimurium), IpaB A34965
(Shigella spp.), IpaA AAA26525 (Shigella spp.), IpaD SI 5579
(Shigella spp.), YopE SI 4242 (Yersinia spp.), YopH AAC69768
(Yersinia spp.), YpkA AAC69765 (Yersinia spp.), YopJ AAC69766
(YopP) (Yersinia spp.), AvrPto AAA25728 (P. syringae), AvrBs2 AAD 1
1434 (X. campestris), and AvrBs3 CAA34257 (X. campestris) (see,
e.g., Galan et al., "Type III Secretion Machines: Bacterial Devices
for Protein Delivery into Host Cells," Science 284: 1322-1328
(1999), which is hereby incorporated by reference). In one
embodiment, the effector protein is heterologous (i.e., not
normally present) to the target cell.
[0167] In accordance with the purposes of this invention, as
embodied and broadly described herein, this invention relates to
methods for site-specific genetic engineering using RTNs of stem
cells and gametes, including but not limited to pluripotent cells,
totipotent cells, somatic stem cells, spermatogonial stem cells
(SSCs), embryonic stem (ES) cells, induced pluripotent stem (iPS)
cells, embryos, germ cells, primordial germ cells (PGCs), plant
tube cells, pollen cells, and spores. Methods for site-specific
engineering of stem cells include, but are not limited to using
site specific DNA binding and cleaving proteins such as RTNs.
[0168] Site-specific engineering of stem cells results in altered
function of gene(s) or gene product(s) and genetically modified
organisms, and cell or tissue culture models are produced from
these engineered stem cells. Modified stem cells and organisms
include knockout and knockin cells and organisms.
[0169] In another aspect, the invention relates to genetically
modified organisms created by site-specific engineering using RTNs
including but not limited to mammals, including rats, mice, pigs,
rabbits, guinea pigs, dogs, non-human primates, mini-pigs, as well
as plants, including but not limited to maize, soybean, rice,
potato, wheat, tobacco, tomato, and Arabidopsis, as well as the
descendants and ancestors of such organisms.
[0170] In another embodiment, the invention provides kits that are
used to produce site specific-mutations in stem cells, which can be
used to generate genetically modified organisms. The kits typically
include one or more site-specific genetic engineering technology,
such as RTNs. The kit may also comprise one or more sets of stem
cells for site-specific modification. In some embodiments of the
invention, the stem cells may include, but are not limited to,
spermatogonial stem cells (SSCs), as well as media and conditions
necessary for growing SSCs. In some embodiments, the kit comprise
exogenous sequences for site-specific genomic introduction, such as
but not limited to reporter genes or selectable markers. In some
embodiments, the kit comprises instructions for (i) introducing the
RTNs (or nucleic acid sequence encoding the RTN) into the stem
cells (ii) identifying stem cells which have been site specifically
modified by the XTN (iii) growing site-specifically modified stem
cells in media or conditions necessary and to numbers required for
stem cells to produce genetically modified organisms or effect
germline transmission in an animal; (iv) using or transplanting the
grown stem cells to produce a genetically modified organism; and/or
(v) identifying which organisms or progeny comprise the
site-specific mutation of interest. In some embodiments of the
invention, a composition comprises one or more stem cells or one or
more embryos, the one or more stem cells or one or more embryos
comprise one or more of the following mutations: (i) a deletion
mutation; (ii) a knockout mutation; and/or (iii) an addition of a
heterologous nucleic acid sequence; the one or more mutations of
(i), (ii), and/or (iii) are site-specific mutations caused by a
RTN.
[0171] In some embodiments of the invention, the heterologous
nucleic acid sequence is chosen from a selectable marker or an
orthologous gene. In some embodiments of the invention, the one or
more stem cells is chosen from a spermatogonial stem cell (SSC), an
embryonic stem cell, or an induced pluripotent stem cell.
[0172] In some embodiments of the invention, the one or more stem
cells is derived from the germline lineage of an animal or plant.
In some embodiments of the invention, the one or more stem cells or
the one or more embryos further comprise at least one inverted
tandem repeat of a transposon or a variant thereof.
[0173] In some embodiments of the invention, the one or more stem
cells is a somatic stem cell. In some embodiments of the invention,
an organism comprising one or more stem cells, the one or more stem
cells comprise one or more of the following mutations: (i) a
deletion mutation; (ii) a knockout mutation; and/or (iii) an
addition of a heterologous nucleic acid sequence; the one or more
mutations of (i), (ii), and/or (iii) are site-specific mutations
caused by a RTN. In some embodiments of the invention, the one or
more stem cells comprises an SSC.
[0174] In the present invention, the effector protein is fused to
at least one DNA recognition elements disclosed herein or
derivatives or functional analogs thereof to produce a fusion
protein.
[0175] One aspect of the present invention relates to a method for
delivering effector proteins into a target cell. This method
involves introducing into the target cell an effector protein fused
to a polypeptide including at least one repeat domain or DNA
recognition element of the present invention that recognizes said
target DNA or derivatives or functional analogs thereof. Another
aspect of the present invention relates to a DNA construct
including a first DNA molecule encoding an effector protein and a
second DNA molecule operatively associated with the first DNA
molecule and encoding a polypeptide including at least one repeat
domain of the present invention that recognizes said target DNA or
derivatives or functional analogs thereof.
[0176] The method of the present invention allows efficient
delivery of effector proteins into cells, in particular, mammalian
cells. This method also allows for delivery of effector proteins
for use in pharmaceutical, insecticide, fungicide, herbicide, and
other applications. In particular, the present invention will allow
the delivery of effector proteins into patients in the form of
protein therapy. Therapy with biologically active full-length
proteins will allow access to the built-in evolutionary specificity
of these proteins for their targets, thereby potentially avoiding
the nonspecific effects sometimes seen with small-molecule
therapies. Moreover, when used in conjunction with tissue-specific
viral vectors, use of the present invention allows the targeted
delivery of effector proteins to particular cells with the added
benefit of secondary redistribution of the effector protein
subsequent to the initial targeting.
[0177] A precedent for this approach can be found in an experiment
wherein the VP22 protein transduction domain was fused to the p53
tumor suppressor protein (Phelan et al, "Intercellular Delivery of
Functional p53 by the Herpesvirus Protein VP22," Nat. Biotechnol.
16:440-443 (1998), which is hereby incorporated by reference).
[0178] In some embodiments, the invention relates to a composition
comprising one or more nucleic acid sequences with an inserted
nucleic acid sequence. In some embodiments, the inserted nucleic
acid comprises at least one transcriptionally active gene, which is
a coding sequence that is capable of being expressed under
intracellular conditions, e.g. a coding sequence in combination
with any requisite expression regulatory elements that are required
for expression in the intracellular environment of the target cell
whose genome is modified by binding and subsequent action by any of
the polypeptides described herein. The transcriptionally active
genes of the nucleic acids can comprise a domain of nucleotides,
i.e., an expression module that includes a coding sequence of
nucleotides operably linked with requisite transcriptional
mediation or regulatory element(s). Requisite transcriptional
mediation elements that may be present in the expression module
include, but are not limited to, promoters, enhancers, termination
and polyadenylation signal elements, splicing signal elements, and
the like. In some embodiments of the invention, the one or more
stem cells further comprise at least one inverted terminal repeat
of a transposon or variant thereof.
[0179] In some embodiments, the expression module includes
transcription regulatory elements that provide for expression of
the gene in a broad host range. A variety of such combinations are
known, where specific transcription regulatory elements include,
but are not limited to: SV40 elements, transcription regulatory
elements derived from the LTR of the Rous sarcoma virus,
transcription regulatory elements derived from the LTR of human
cytomegalovirus (CMV), hsp70 promoters, and the like.
[0180] In some embodiments, at least one transcriptionally active
gene or expression module present in the inserted nucleic acid acts
as a selectable marker. A variety of different genes have been
employed as selectable markers, and the particular gene employed in
the vectors described herein as a selectable marker is chosen
primarily as a matter of convenience. Known selectable marker genes
include, but are not limited to: thymidine kinase gene,
dihydrofolate reductase gene, xanthine-guanine phosphoribosyl
transferase gene, CAD, adenosine deaminase gene, asparagine
synthetase gene, numerous antibiotic resistance genes
(tetracycline, ampicillin, kanamycin, neomycin, and the like),
aminoglycoside phosphotransferase genes, hygromycin B
phosphotransferase gene, and genes whose expression provides for
the presence of a detectable product, either directly or
indirectly, such as, for example, beta-galactosidase, GFP, and the
like.
[0181] In some embodiments, the nucleic acids of the present
invention comprise least one transcriptionally active gene, the
portion of the nucleic acid also comprises at least one restriction
endonuclease recognized site, e.g. restriction site which serves as
a site for insertion of an exogenous nucleic acid. A variety of
restriction sites are known in the art and include, but are not
limited to: HindIII, PstI, SalI, AccI, HincII, XbaI, BamHI, SmaI,
XmaI, KpnI, SacI, EcoRI, and the like. In some embodiments, the
vector includes a polylinker, i.e. a closely arranged series or
array of sites recognized by a plurality of different restriction
enzymes, such as those disclosed herein. In other embodiments, the
inserted exogenous nucleic acid could comprise recombinase
recognition sites, such as LoxP, FRT, or AttB/AttP sites, which are
recognized by the Cre, Flp, and PhiC31 recombinases,
respectively.
[0182] In another aspect, the present invention relates to a method
for generating a nucleic acid encoding a polypeptide specific for
binding a selected nucleotide sequence, comprising: (1) linearizing
a starter plasmid with PspX1 or nuclease, the starter plasmid
comprising a nucleotide sequence that encodes a first monomer
comprising a RVD specific for the first nucleotide of the selected
nucleotide sequence, wherein the first monomer has a unique PspXl
or nuclease site at its 3' end; (2) ligating into the starter
plasmid PspX1 site a DNA module encoding one or more monomers that
comprise RVDs specific for the next nucleotide(s) of the selected
nucleotide sequence, wherein the DNA module has Xho1 sticky ends;
and (3) repeating steps (1) and (2) until the nucleic acid encodes
a polypeptide capable of binding to the selected nucleotide
sequence. The method can further comprise, after the ligating,
determining the orientation of the DNA module in the PspX1 site or
nuclease site. The method can comprise repeating steps (1) and (2)
from one to 30 times.
[0183] Where the source of DNA-binding domain is a nucleic acid
that encodes the polypeptides of the present invention, the nucleic
acid encoding the polypeptide or protein is generally part of an
expression module, as described above, where the additional
elements provide for expression of the transposase as required.
[0184] In some embodiments, multicellular organisms can be made
using cells mytogenized by the compositions disclosed herein. In
some embodiments, the multicellular or unicellular organism is a
plant or animal. In some embodiments, the multicellular or
unicellular organism is a vertebrate. In some embodiments, the
vertebrate animal is a mammal, such as for example, a rodent (mouse
or rat), livestock (pig, horse, cow, etc.), pets (dog or cat), and
primates, such as, for example, a human.
[0185] The methods described herein can be used in a variety of
applications in which it is desired to introduce and stably
integrate an exogenous nucleic acid into the genome of a target
cell. In vivo methods of integrating exogenous nucleic acid into a
target cell are known. The route of administration of the nucleic
acid-binding system to the multicellular or unicellular organism
depends on several parameters, including: the nature of the vectors
that carry the system components, the nature of the delivery
vehicle, the nature of the multicellular or unicellular organism,
and the like, where a common feature of the mode of administration
is that it provides for in vivo delivery of the nucleic
acid-binding system components to the target cell(s). In certain
embodiments, linear or circularized DNA, such as a plasmid, is
employed as the vector for delivery of the nucleic acid-binding
system to the target cell. In such embodiments, the plasmid may be
administered in an aqueous delivery vehicle, such as a saline
solution. Alternatively, an agent that modulates the distribution
of the vector in the multicellular or unicellular organism can be
employed. For example, where the vectors comprising the subject
system components are plasmid vectors, lipid-based such as a
liposome, vehicles can be employed, where the lipid-based vehicle
may be targeted to a specific cell type for cell or tissue specific
delivery of the vector. Alternately, polylysine-based peptides can
be employed as carriers, which may or may not be modified with
targeting moieties, and the like (Brooks et al., J. Neurosci.
Methods, 1998, 80, 137-47; and Muramatsu et al., Int. J. Mol. Med.,
1998, 1, 55-62). The system components can also be incorporated
onto viral vectors, such as adenovirus-derived vectors,
sindbis-virus derived vectors, retrovirus-derived vectors, hybrid
vectors, and the like. The above vectors and delivery vehicles are
merely representative. Any vector/delivery vehicle combination can
be employed, so long as it provides for in vivo administration of
the nucleic acid-binding system to the multicellular or unicellular
organism and target cell.
[0186] The amount of vector nucleic acid comprising the nucleic
acid-binding element, and in many embodiments the amount of vector
nucleic acid encoding the polypeptide, which is introduced into the
cell is sufficient to provide for the desired excision and
insertion of the nucleic acid-binding nucleic acid into the target
cell genome. As such, the amount of vector nucleic acid introduced
should provide for a sufficient amount of DNA-binding activity and
a sufficient copy number of the nucleic acid that is desired to be
inserted into the target cell. The amount of vector nucleic acid
that is introduced into the target cell varies depending on the
efficiency of the particular introduction protocol that is
employed, such as the particular in vivo administration protocol
that is employed.
[0187] The particular dosage of each component of the system that
is administered to the multicellular or unicellular organism varies
depending on the nature of the nucleic acid-binding nucleic acid,
e.g. the nature of the expression module and gene, the nature of
the vector on which the component elements are present, the nature
of the delivery vehicle and the like. Dosages can readily be
determined empirically by those of skill in the art. For example,
in mice where the nucleic acid-binding system components are
present on separate plasmids which are intravenously administered
to a mammal in a saline solution vehicle, the amount of nucleic
acid-binding plasmid that is administered in many embodiments
typically ranges from about 0.5 to 40 .mu.g and is typically about
25 while the amount of nucleic acid-binding system encoding plasmid
that is administered typically ranges from about 0.5 to 25 .mu.g
and is usually about 1 .mu.g.
[0188] The subject methods may be used to bind and effect nucleic
acids of various sizes. Generally, the size of DNA that is inserted
into a target cell genome using the subject methods ranges from
about 0.5 kb to 100.0 kb, usually from about 1.0 kb to about 60.0
kb, or from about 1.0 kb to about 10.0 kb.
[0189] The present invention can be used in, for example, germline
mutagenesis in a rat, mouse, or other vertebrate; somatic
mutagenesis in a rat, mouse, or other vertebrate; transgenesis in a
rat, mouse, or other vertebrate; and use in human gene therapy. In
each of these, the composition can be delivered as DNA, RNA, or
protein.
[0190] Transformed cells and/or transgenic organisms, such as those
containing the DNA inserted into the host cell's DNA, can be
selected from untransformed cells and/or transformed organisms if a
selectable marker is included as part of the introduced DNA
sequences. Selectable markers include, for example, genes that
provide antibiotic resistance; genes that modify the physiology of
the host, such as for example green fluorescent protein, to produce
an altered visible phenotype. Cells and/or organisms containing
these genes are capable of surviving in the presence of antibiotic,
insecticides or herbicide concentrations that kill untransformed
cells/organisms or producing an altered visible phenotype. Using
standard techniques known to those familiar with the field,
techniques such as, for example, Southern blotting and polymerase
chain reaction, DNA can be isolated from transgenic cells and/or
organisms to confirm that the introduced DNA has been inserted.
[0191] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner.
[0192] Throughout these examples, molecular cloning reactions, and
other standard recombinant DNA techniques, were carried out
according to methods described in Maniatis et al., Molecular
Cloning--A Laboratory Manual, 2nd ed., Cold Spring Harbor Press
(1989), using commercially available reagents, except where
otherwise noted.
[0193] In other aspects of the invention, the invention relates to
viral vectors comprising any one or more than one nucleic acid
sequence disclosed herein. The viral vector is optionally selected
from the group comprising a retroviral vector, an adenoviral
vector, an adeno-associated viral vector, spumaviral, a lentiviral
vector and a plasmid or other vector, such as transposons,
described in the application. The retroviral vector optionally
comprises an oncoretroviral vector. The retroviral vector
optionally comprises a lentiviral vector.
[0194] The application includes compositions and methods for
providing a RTN coding nucleic acid molecule to a subject such that
expression of the molecule in the cells provides the biological
activity of the polypeptide encoded by the coding nucleic acid
molecule to those cells. A coding nucleic acid as used herein means
a nucleic acid that comprises nucleotides which specify an RTN
amino acid sequence, or a portion thereof, of the corresponding
Ralstonia amino acid sequence. A coding sequence may comprise a
start codon and/or a termination sequence.
[0195] In some embodiments, the compositions of the present
invention are pharmaceutical compositions. The pharmaceutical
compositions of this application used to treat patients having
diseases, disorders or abnormal physical states could include an
acceptable carrier, auxiliary or excipient.
[0196] The pharmaceutical compositions are optionally administered
by ex vivo and in vivo methods such as electroporation, DNA
microinjection, liposome DNA delivery, and virus vectors that have
RNA or DNA genomes including retrovirus vectors, lentivirus
vectors, Adenovirus vectors and Adeno-associated virus (AAV)
vectors, Semliki Forest Virus. Derivatives or hybrids of these
vectors are also useful.
[0197] Dosages to be administered depend on patient needs, on the
desired effect and on the chosen route of administration. The
expression cassettes are optionally introduced into the cells or
their precursors using ex vivo or in vivo delivery vehicles such as
liposomes or DNA or RNA virus vectors. They are also optionally
introduced into these cells using physical techniques such as
microinjection or chemical methods such as coprecipitation. The
pharmaceutical compositions are typically prepared by known methods
for the preparation of pharmaceutically acceptable compositions
which are administered to patients, and such that an effective
quantity of the nucleic acid molecule is combined in a mixture with
a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA). Any selectable marker gene can be used in the
present invention.
[0198] On this basis, the pharmaceutical compositions could include
an active compound or substance, such as a nucleic acid molecule,
in association with one or more pharmaceutically acceptable
vehicles or diluents, and contained in buffered solutions with a
suitable pH and isoosmotic with the physiological fluids. The
methods of combining the expression cassettes with the vehicles or
combining them with diluents is well known to those skilled in the
art. The composition could include a targeting agent for the
transport of the active compound to specified sites within cells.
The expression cassette can also comprise a selectable marker gene
for the selection of transformed cells. Selectable marker genes are
utilized for the selection of transformed cells or tissues. Marker
genes include genes encoding antibiotic resistance, such as those
encoding neomycin phosphotransferase II (NEO) and hygromycin
phosphotransferase (HPT), as well as genes conferring resistance to
herbicidal compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional
selectable markers include phenotypic markers such as
.beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng
85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan
florescent protein (CYP) (Bolte et al. (2004) J. Cell Science
117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and
yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte et
al. (2004) J. Cell Science 117:943-54). For additional selectable
markers, see generally, Yarranton (1992) Curr. Opin. Biotech.
3:506-511; Christophers on et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:
1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl Acad. Sci. USA 89:3952-3956;
Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:
1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104;
Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)
Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985)
Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag,
Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures
are herein incorporated by reference.
[0199] Numerous plant transformation vectors and methods for
transforming plants are available. See, for example, An, G. et al.
(1986) Plant Pysiol, 81:301-305; Fry, J., et al. (1987) Plant Cell
Rep. 6:321-325; Block, M. (1988) Theor. Appl Genet. 76:767-774;
Hinchee, et al. (1990) Stadler. Genet. Symp. 203212.203-212;
Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P.
P. and Slightom, J. L. (1992) Gene. 118:255-260; Christou, et al.
(1992) Trends. Biotechnol. 10:239-246; D'Halluin, et al. (1992)
Bio/Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol.
99:81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:
11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant;
29P: 119-124; Davies, et al. (1993) Plant Cell Rep. 12: 180-183;
Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91: 139-148;
Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102: 167;
Golovkin, et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull.
38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M.
and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo,
et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J.
5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230;
Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et
al. (1994) Plant Cell Rep. 13:582-586; Hartman, et al. (1994)
Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol.
24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol.
104:3748.
[0200] The methods of the invention involve introducing a
polynucleotide construct comprising a DNA sequence into a host
cell. By "introducing" is intended presenting to the plant the
polynucleotide construct in such a manner that the construct gains
access to the interior of the host cell. The methods of the
invention do not depend on a particular method for introducing a
polynucleotide construct into a host cell, only that the
polynucleotide construct gains access to the interior of one cell
of the host. Methods for introducing polynucleotide constructs into
bacteria, plants, fungi and animals are known in the art including,
but not limited to, stable transformation methods, transient
transformation methods, and virus-mediated methods.
[0201] By "stable transformation" is intended that the
polynucleotide construct introduced into a plant integrates into
the genome of the host and is capable of being inherited by progeny
thereof. By "transient transformation" is intended that a
polynucleotide construct introduced into the host does not
integrate into the genome of the host.
[0202] The application includes methods and compositions for
providing a coding nucleic acid molecule to the cells of an
individual such that expression of the coding nucleic acid molecule
in the cells provides the biological activity or phenotype of the
polypeptide encoded by the coding nucleic acid molecule. The method
also relates to a method for providing an individual having a
disease, disorder or abnormal physical state with a biologically
active polypeptide by administering a nucleic acid molecule of the
present application. The method may be performed ex vivo or in
vivo. Gene therapy methods and compositions are demonstrated, for
example, in U.S. Pat. Nos. 5,869,040, 5,639,642, 5,928,214,
5,911,983, 5,830,880, 5,910,488, 5,854,019, 5,672,344, 5,645,829,
5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346
and 5,670,488, 5,240,846. The amount of polypeptide will vary with
the subject's needs. The optimal dosage of vector may be readily
determined using empirical techniques, for example by escalating
doses (see U.S. Pat. No. 5,910,488 for an example of escalating
doses). Vectors containing the nucleic acid molecules of the
application are typically administered to mammals, preferably
humans, in gene therapy using techniques described below. The
polypeptides produced from the nucleic acid molecules are also
optionally administered to mammals, preferably humans. The
application relates to a method of medical treatment of a mammal in
need thereof, preferably a human, by administering to the mammal a
vector of the application or a cell containing a vector of the
application. A recipient, preferably human, who develops an adverse
event, such as graft versus host disease, is typically administered
a drug, such as AZT, that is a substrate for the modified tmpk
molecules of the application. Diseases, such as blood diseases or
neural diseases (neurodegenerative), that are readily treated are
described in this application and known in the art (i.e., eg.
diseases, such as thalassemia or sickle cell anemia that are
treated by administering a globin gene as described in Canadian
patent application no. 2,246,005).
[0203] Blood diseases treatable by stem cell transplant include
leukemias, myelodysplastic syndromes, stem cell disorders,
myeloproliferative disorders, lymphoproliferative disorders
phagocyte disorders, inherited metabolic disorders, histiocytic
disorders, inherited erythrocyte abnormalities, inherited immune
system disorders, inherited platelet abnormalities, plasma cell
disorders, malignancies (See also, Medical Professional's Guide to
Unrelated Donor Stem Cell Transplants, 4th Edition). Stem cell
nerve diseases to be treated by neural stem cell transplantation
include diseases resulting in neural cell damage or loss, eg.
paralysis, Parkinson's disease, Alzheimer's disease, ALS, multiple
sclerosis). The vector of the application is useful as a stem cell
marker and to express genes that cause stem cells to differentiate
(e.g. growth factor).
[0204] Various approaches to gene therapy may be used. The
application includes a process for providing a human with a
therapeutic polypeptide including: introducing human cells into a
human, said human cells having been treated in vitro or ex vivo to
insert therein a vector of the application, the human cells
expressing in vivo in said human a therapeutically effective amount
of said therapeutic polypeptide.
[0205] The method also relates to a method for producing a stock of
recombinant virus by producing virus suitable for gene therapy
comprising modified DNA encoding globin. This method preferably
involves transfecting cells permissive for virus replication (the
virus containing modified globin) and collecting the virus
produced.
[0206] Cotransfection (DNA and marker on separate molecules) may be
employed (see U.S. Pat. Nos. 5,928,914 and 5,817,492). As well, a
detection cassette or marker (such as Green Fluorescent Protein
marker or a derivative, CD 19 or CD25) may be used within the
vector itself (preferably a viral vector).
[0207] The methods of the present invention can be used to mutate
any eukaryotic stem cell, including, but not limited to, haploid,
diploid, triploid, tetraploid, or aneuploid. In one embodiment, the
cell is diploid. Stem cells in which the methods of the present
invention can be advantageously used include, but are not limited
to stem cells such as somatic stem cells, SSCs, ES cells, iPS
cells, embryos, or any cell capable of developing into one or more
organisms.
[0208] In one embodiment, the invention relates to a method to
produce a site-specific knockout, knock-in or otherwise genetically
modified stem cell. The site-specific mutation is generated using a
RTN which cleaves the desired site, followed by NHEJ, resulting in
deletion mutations. The site-specific mutation can be produced in
spermatogonial stem cells (SSCs) which are used to generate
heterozygous or homozygous genetically modified organisms.
[0209] In another embodiment, the invention relates to a method to
produce a site-specific knockout, knock-in or otherwise genetically
modified stem cell. The site-specific mutation is generated using a
RTN which cleaves the desired site resulting in deletion mutations.
The site specific mutation is produced in embryonic stem (ES)
cells, which are used to generate heterozygous or homozygous
genetically modified organisms.
[0210] In another embodiment, the invention comprises of methods to
produce a site-specific knockout, knock-in or otherwise genetically
modified stem cell. The site specific mutation is generated using a
RTN which cleaves the desired site resulting in deletion mutations.
The site-specific mutation is produced in induced pluripotent stem
(iPS) cells, which are used to generate heterozygous or homozygous
genetically modified organisms.
[0211] In another embodiment, the invention comprises of methods to
produce a site-specific knockout, knockin or otherwise genetically
modified stem cell. The site specific mutation is generated using a
RTN which cleaves the desired site resulting in deletion mutations.
The site-specific mutation is produced in embryos which are used to
generate heterozygous or homozygous genetically modified
organisms.
[0212] In certain embodiments of the invention, cells can be
mutated within the organism or within the native environment as in
tissue explants (e.g., in vivo or in situ). Alternatively, tissues
or stem cells isolated from the organism using art-known methods
and genes can be mutated according to the present methods. The
tissues or stem cells are either maintained in culture (e.g., in
vitro), or re-implanted into a tissue or organism (e.g., ex
vivo).
[0213] RTNs comprising effector protein function, such as a
nuclease.
[0214] In some embodiments, the invention relates to compositions
comprising any one of the nucleic acids or polypeptides or
fragments thereof described in Example 2.
[0215] In some embodiments, the invention relates to compositions
comprising any one of the nucleic acids or polypeptides described
herein.
[0216] In some embodiment of the invention, genetic modification of
SSCs using RTNs relates to generating multiple mutations in
separate SSCs or SSC lines followed by pooling or combining
separate SSCs or SSC lines and injecting into a single recipient
male, which relates to generating multiple genetically modified
organisms containing one or more mutations is fewer experimental
steps and in a shorter timeframe than is possible with other
systems. The separate stem cells or stem cell lines may be fifteen
or more. In some embodiment of the invention, genetic modification
of stem cells using RTNs relates to generating multiple mutations
in separate stem cells or stem cell lines followed by pooling or
combining separate stem cells or stem cell lines and injecting into
a single recipient male, which relates to generating multiple
genetically modified organisms containing one or more mutations is
fewer experimental steps and in a shorter timeframe than is
possible with other systems. The separate stem cells or stem cell
lines may be fifteen or more.
[0217] In some embodiment of the invention, increasing the number
of distinct or separate pools or lines of genetically modified SSCs
or modified stem cells or embryonic cells, which may be used to
generate a genetically modified organism, does not increase the
amount of effort, time, and resources used, as well as does not
decrease the efficiency of genetically modified organism
production. Multiple separate and distinct genetically modified
SSCs may be transplanted into a single sterile recipient. The mixed
population of distinct genetically modified cells (SSCs or stem
cells), which are derived from separate cell pools from two or more
pools to fifteen or more pools mature within the sterile recipient.
The sterile recipient is then bred with multiple wild type females
which may be two or more, three or more, four or more, five or
more, six or more, seven or more, eight or more, nine or more, ten
or more, eleven or more, twelve or more, thirteen or more, fourteen
or more, fifteen or more, sixteen or more, seventeen or more,
eighteen or more, nineteen or more, twenty or more. These multiple
females produce offspring which have incorporated the desired
mutation into their germline.
[0218] In some embodiment of the invention, increasing the number
distinct or separate pools or lines of genetically modified cells,
which may be used to generate a genetically modified organism, does
not increase the amount of effort, time, and resources used, as
well as does not decrease the efficiency of genetically modified
organism production. The sterile recipient rat may be a recipient
for multiple rounds of separate or distinct genetically modified
cells. The sterile rat may be a recipient of fifteen or more
different genetically modified cells and breed with twenty or more
wild type females to produce fifteen or more separately genetically
modified organisms. Following the first round of breeding, the
sterile male may be treated to eliminate the first round of
genetically modified cells and become a recipient of another round
of fifteen or more separately or distinct genetically modified
cells, breed with twenty or more wild type females to produce
fifteen or more separate genetically modified organisms. The
sterile male may be a recipient of mixed populations of fifteen or
more genetically modified cells and breed twenty or more wild type
females two times or more, three times or more, four times or more,
or five times or more.
[0219] In some embodiment of the invention, increasing the number
distinct or separate pools or lines of genetically modified cells,
which may be used to generate a genetically modified organism, does
not increase the amount of effort, time, and resources used, as
well as does not decrease the efficiency of genetically modified
organism production. Increasing the number of genetically modified
cells does not require the effort and resources of other cell
systems such as embryonic stem (ES) cells or embryos. Increasing
the amount of genetically modified ES cells for genetically
modified organism production requires an increase in the number of
technical steps such as blastocyst injections, as well as the
number of oviduct transfer surgeries. In some embodiments of the
invention, the method does not comprise blastocyst injection,
oviduct transfer, DNA microinjection reimplantation of injected
zygotes, or breeding of chimeric progeny. The cell system may
produce fifteen or more separate genetically modified stem cell
populations for genetically modified organism production in a
single step, while in order to produce fifteen or more separately
genetically modified ES cells, fifteen or more separate steps must
be performed on all levels of the procedure, which include but are
not limited to blastocyst injection, oviduct transfer, zygote
production, preparation of DNA, DNA microinjection, reimplantation
of injected zygotes or breeding chimeric progeny.
[0220] In some embodiment of the invention, genetic modification of
SSCs using RTNs relates to generating genetically modified
organisms without requiring the steps required in producing
genetically modified organisms from alternative stem cells
including but not limited to embryonic stem cells, embryo's,
induced pluripotent stem (iPS) cells, somatic stem cells. Genetic
modification in alternative stem cells includes but is not limited
to zygote production, preparation of DNA, DNA microinjection,
reimplantation of injected zygotes or breeding chimeric
progeny.
[0221] In some embodiments, the stem cells of the present invention
comprise one or more transposons, one or more inverted terminal
repeats (ITRs) of a transposon of variants thereof.
[0222] In some embodiments, the stem cells of the present invention
comprise one or more transposons, one or more inverted terminal
repeats (ITRs) of a transposon of variants derived from the
sequences of Table 2.
[0223] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 70% homologous to known ITRs and known transposon elements
(shown in table 2).
[0224] In some embodiments, the present invention comprise one or
more transposons, one or more inverted tandem repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 75% homologous to known ITRs and known transposon elements
(shown in table 2).
[0225] In some embodiments, the present invention comprise one or
more transposons, one or more inverted tandem repeats (ITRs) of a
transposon wherein the variant sequence inverted terminal repeats
are at least 80% homologous to known ITRs and known transposon
elements (shown in table 2).
[0226] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 85% homologous to known ITRs and known transposon elements
(shown in table 2).
[0227] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 90% homologous to known ITRs and known transposon elements
(shown in table 2).
[0228] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 95% homologous to known ITRs and known transposon elements
(shown in table 2).
[0229] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 96% homologous to known ITRs and known transposon elements
(shown in table 2).
[0230] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 97% homologous to known ITRs and known transposon elements
(shown in table 2).
[0231] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 98% homologous to known ITRs and known transposon elements
(shown in table 2).
[0232] In some embodiments, the present invention comprise one or
more transposons, one or more inverted terminal repeats (ITRs) of a
transposon wherein the variant sequence inverted tandem repeats are
at least 99% homologous to known ITRs and known transposon elements
(shown in table 2).
TABLE-US-00004 TABLE 2 Transposon ITRs Sleeping Beauty 5' Inverted
Tandem Repeat: CAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCATTAAAACTC
GTTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGGCA
AGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACA
ATTGTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCC
AGTGGGTCAGAAGTTTACATACACTAAGT (SEQ ID NO: 68) 3' Inverted Tandem
Repeat: ATTGAGTGTATGTAAACTTCTGACCCACTGGGAATGTGATGAAAGAAATA
AAAGCTGAAATGAATCATTCTCTCTACTATTATTCTGATATTTCACATTC
TTAAAATAAAGTGGTGATCCTAACTGACCTAAGACAGGGAATTTTTACTA
GGATTAAATGTCAGGAATTGTGAAAAAGTGAGTTTAAATGTATTTGGCTA
AGGTGTATGTAAACTTCCGACTTCAACTG (SEQ ID NO: 69) piggyBac 5' Inverted
Tandem Repeat: CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATT
GCTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATC
TCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGT
GTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACGCATGAT
TATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTT
ATTTCATGTTCTACTTACGTGATAACTTATTATATATATATTTTCTTGTT ATAGATATC (SEQ
ID NO: 70, minimal sequence is underlined and bold, i.e., first 35
bp) 3' Inverted Tandem Repeat:
TAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTT
AATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTAT
GTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAA
CCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTA
TCTTTAACGTACGTCACAATATGATTATCTTTCTAGGG (SEQ ID NO: 71, minimal
sequence is underlined and bold, i.e., first 35 bp)
[0233] The invention also comprises DNA which encodes for any one
of the polypeptides described herein with any number of repeat
units disclosed herein.
[0234] Any polypeptides of the invention or nucleic acids that
encode the polypeptides of the present invention can be used in the
methods described in PCT Application No. PCT/IB2010/000154, which
is incorporated by reference in its entirety.
[0235] In an aspect of the invention, the invention relates to a
composition comprising one or more cells modified by one or more
polypeptides disclosed herein. In some embodiments of the
invention, the composition of the present invention comprise one or
more stem cells. In some embodiments of the invention, the
composition of the present invention comprises one or more
mammalian stem cells modified by one or more polypeptides disclosed
herein. In some embodiments of the invention, the composition of
the present invention comprises one or more iPSC cells modified by
one or more polypeptides disclosed herein. In some embodiments of
the invention, the composition of the present invention comprise
one or more human stem cells modified by one or more polypeptides
disclosed herein. In some embodiments of the invention, the
composition of the present invention comprises one or more
spermatogonial stem cells modified by one or more polypeptides
disclosed herein. In some embodiments of the invention, the cell is
derived from a mammal, in some embodiments, the cell is from a rat
or mini pig. In some embodiments of the invention, the mammal is a
sterile male rat or sterile male mini pig. In some embodiments of
the invention, the rat or mini pig is DAZL deficient or DAZL-/-. In
some embodiments of the invention, the invention relates to a
colony of genetically modified organisms comprising:
[0236] at least one organism comprising one or more stem cells, the
one or more stem cells comprise one or more of the following
mutations: (i) a deletion mutation; (ii) a knockout mutation;
and/or (iii) an addition of a heterologous nucleic acid sequence;
the one or more mutations of (i), (ii), and/or (iii) are
site-specific mutations caused by by one or more polypeptides
disclosed herein (one or more RTNs); and (b) progeny of the
organism of subpart (a).
[0237] In some embodiments of the invention, the cell or transgenic
animal, colony or progeny thereof comprises a heterologous nucleic
acid sequence that comprises a selectable marker or an orthologous
gene. In some embodiments of the invention, the at least one
organism and the progeny further comprise at least one inverted
terminal repeat of a transposon or variant thereof.
[0238] In some embodiments of the invention, the at least one
organism and the progeny further comprise a nucleic acid that
comprises a a nucleic acid sequence that is at least 70% homologous
to any or or combination of: SEQ ID NO: 1 through 19, or any
sequence of Table 1, or any variants or functional fragments
thereof. In some embodiments of the invention, the invention
relates to a method of generating one or more genetically modified
organisms comprising: (a) contacting at least one stem cell derived
from the germline lineage of an animal or plant by the stem cell
with: (i) at least one RTN that mutates a gene of interest; or (ii)
at least one expression vector that encodes a RTN that mutates a
gene of interest, thereby creating at least one stem cell
comprising at least one mutation at a gene of interest; (b)
expanding an in vitro culture of the at least one stem cell
comprising at least one mutation at a gene of interest; (c)
implanting one or more stem cells from the culture of step (b) into
an organism.
[0239] In some embodiments of the invention, the invention relates
to a method of generating one or more genetically modified
organisms comprising: (a) contacting at least a first and second
set of stem cell derived from the germline lineage of an animal or
plant with: (i) at least one RTN that mutates a gene of interest;
or (ii) at least one expression vector that encodes a RTN that
mutates a gene of interest, thereby creating at least a first and
second set of stem cells comprising at least one mutation at a gene
of interest; (b) expanding an in vitro culture of the at least one
stem cell comprising at least one mutation at a gene of interest;
(c) implanting one or more sets of stem cells from the culture of
step (b) into an organism. In some embodiments, the method further
comprises a third, fourth, fifth, sixth, seventh, eighth, ninth, or
ten or more sets of stem cells which have been mutated in a
site-specific fashion by a RTN, and, in which case, after expanding
each of the third, fourth, fifth, sixth, seventh, eighth, ninth, or
ten or more sets of mutated stem cells, each set of transplanted
into a single organism. In some embodiments, the single organism
that comprises a set of mutated stem cells is a sterile male.
[0240] In some embodiments of the invention, the organism is
capable of passing at least one mutation at a gene of interest to
progeny by germline transmission. In some embodiments of the
invention, the genetically modified organism is a mammal. In some
embodiments of the invention, the genetically modified organism is
a rat or mini pig. In some embodiments of the invention, the
genetically modified organism is a sterile male rat or sterile male
mini pig.
[0241] In some embodiments of the invention, the method further
comprises: breeding the organism implanted with the one or more
stem cells with another animal to generate one or more progeny that
comprise the mutated gene of interest. In some embodiments of the
invention, the method further comprises: breeding the organism
implanted with the one or more set of stem cells with another
animal to generate one or more progeny that comprise the one or
more mutated genes of interest that correspond to each of the
mutated stem cell lines.
[0242] In some embodiments of the invention, the progeny are
mammals.
[0243] In some embodiments of the invention, a method of breeding a
colony of genetically modified organisms comprising:
[0244] (a) contacting at least one stem cell derived from the
germline lineage of an animal or plant by the stem cell with: (i)
at least one RTN that mutates a gene of interest; or (ii) at least
one expression vector that encodes a RTN that mutates a gene of
interest, thereby creating a stem cell comprising at least one
mutation at a gene of interest;
[0245] (b) expanding an in vitro culture of the stem cell
comprising at least one mutation at a gene of interest;
[0246] (c) implanting the at least one stem cell comprising at
least one mutation at a gene of interest from the culture of step
(b) into a first organism.
[0247] (d) breeding the first organism with a second organism of
the same species;
[0248] (e) selecting progeny of the first and second organism that
comprise the at least one mutation at a gene of interest; and
[0249] (f) breeding the progeny to create a colony of organisms
that comprise the at least one mutation at a gene of interest.
[0250] In some embodiments of the invention, the first and second
organisms are mammals.
[0251] In some embodiments of the invention, the first and second
organisms are rats or mini pigs.
[0252] In some embodiments of the invention, the invention relates
to a method of manufacturing a first filial generation of
genetically modified organisms comprising two or more distinct
subsets of organisms, the method comprising:
[0253] (a) contacting a first stem cell with: (i) a RTN that
mutates a first gene of interest; or (ii) an expression vector that
encodes a RTN that mutates a first gene of interest; thereby
creating a first stem cell comprising a first mutation;
[0254] (b) contacting a second stem cell with a modifying agent,
thereby creating a second stem cell comprising a second
mutation;
[0255] (c) expanding an in vitro culture of each of the first and
the second stem cells;
[0256] (d) implanting a mixed population of stem cells comprising
the first and the second stem cells into an organism;
[0257] (e) breeding the organism with another organism of the same
species.
[0258] In some embodiments of the invention, the first filial
generation of genetically modified organisms comprises two or more
sets of organisms, each set comprising a distinct mutation of
interest derived from a haplotype of distinct stem cells
transplanted into a parent of the organism.
[0259] In some embodiments of the invention, at least one stem cell
of the mixed population is a spermatogonial stem cell of a
mammal.
[0260] In some embodiments of the invention, the organism is a
mammal.
[0261] In some embodiments of the invention, a kit comprising:
[0262] (a) a RTN or a nucleic acid sequence that encodes a RTN that
cleaves a nucleic acid sequence at a gene of interest; and
[0263] (b) an instruction manual comprising directions; and,
optionally
[0264] In some embodiments of the invention, a kit comprising:
[0265] (a) In some embodiments of the invention; and,
optionally
[0266] (b) culture media for the one or more stem cells or one or
more embryos.
[0267] In some embodiments of the invention, the kit comprises:
[0268] (a) an RTN or a nucleic acid sequence that encodes an RTN
that cleaves a nucleic acid sequence at a gene of interest; and
optionally
[0269] (b) culture media for the one or more stem cells or one or
more embryos.
[0270] In some embodiments of the invention, the kit comprises:
[0271] (a) an RTN or a nucleic acid sequence that encodes an RTN
that cleaves a nucleic acid sequence at a gene of interest; and
[0272] (b) one or more stem cell lines derived from a germline
lineage of animal or plant; and, optionally
[0273] (c) culture media for the one or more stem cells or one or
more embryos; and, optionally
[0274] (d) an instruction manual that comprises instructions on how
to mutate the one or more stem cells with the RTN or a nucleic acid
sequence that encodes the RTN that cleaves a nucleic acid sequence
at a gene of interest.
TABLE-US-00005 TABLE 1 lsteqvvaia snkggkqale avkahlldll gapyv (SEQ
ID NO: 72) lsteqvvava snkggkqala aveaqllrlr aapye (SEQ ID NO: 73)
lnteqvvava snkggkqale avgaqllalr avpya (SEQ ID NO: 74) lsteqvvava
snkggkqvle avgaqllalr avpye (SEQ ID NO: 75) lsteqvvaia snkggkqale
avkahlldll gapyv (SEQ ID NO: 76) lsteqvvaia snkggkqale avkahlldll
gapyv (SEQ ID NO: 77) lsteqvvaia snkggkqale avkahlldll gapyv (SEQ
ID NO: 78) lsteqvvaia snkggkqale avkaqllelr gapya (SEQ ID NO: 79)
lsteqvvava snkggkqala aveaqllrlr aapye (SEQ ID NO: 80) lsteqvvaia
snkggkqale avkahlldll gapyv (SEQ ID NO: 81) lsteqvvaia snkggkqale
avkahlldll gapyv (SEQ ID NO: 82) lsteqvvaia snnggkqale avkaqlldlr
gapya (SEQ ID NO: 83) lsteqvvaia snnggkqale avkaqlpvlr rapyg (SEQ
ID NO: 84) lspeqvvaia snnggkpale avkaqllelr aapye (SEQ ID NO: 85)
lspeqvvaia snnggkpale avkalllalr aapye (SEQ ID NO: 86) lsteqvvaia
snnggkpale avkalllelr aapye (SEQ ID NO: 87) lsteqvvaia snnggkqale
avktqllalr tapye (SEQ ID NO: 88) lsteqvvaia snnggkqale avkaqlpalr
aapye (SEQ ID NO: 89) lsteqvvaia snnggkqale avkaqllvlr aapyg (SEQ
ID NO: 90) lstaqvvaia annggkqale avrallpvlr vapye (SEQ ID NO: 91)
lspeqvvaia snnggkqale avkaqllelr aapye (SEQ ID NO: 92) lsteqvvaia
snnggkqale avkaqllelr aapye (SEQ ID NO: 93) lspeqvvaia snnggkqale
avkaqllelr aapye (SEQ ID NO: 94) lsteqvvaia snnggkqale avkaqllelr
aapye (SEQ ID NO: 95) lspeqvvaia snnggkqale avkaqllelr aapye (SEQ
ID NO: 96) lsteqvvvia snnggkqale avkaqllalr aapye (SEQ ID NO: 97)
lsteqvvaia snnggkqale avkalllelr aapye (SEQ ID NO: 98) lsteqvvaia
snnggkqale avktqllalr tapye (SEQ ID NO: 99) lsteqvvaia snnggkqale
avkaqlpalr aapye (SEQ ID NO: 100) lspeqvvaia snnggkqale avrallpvlr
vapye (SEQ ID NO: 101) lstaqvvaia sngggkqale gigeqllklr tapyg (SEQ
ID NO: 102) lsteqvvaia sngggkqale gigkqlqelr aaphg (SEQ ID NO: 103)
lstgqvvaia sngggrqale avreqllalr avpye (SEQ ID NO: 104) lstgqvvaia
sngggrqale avreqllalr avpye (SEQ ID NO: 105) lstaqvvaia sngggkqale
gigeqllklr tapyg (SEQ ID NO: 106) lstaqvvaia sngggkqale gigeqllklr
tapyg (SEQ ID NO: 107) lstaqvvaia sngggkqale gigeqlrklr tapyg (SEQ
ID NO: 108) lstaqvvaia sngggkqale gigeqllklr tapyg (SEQ ID NO: 109)
lsteqvvaia sngggkqale gigkqlqelr aaphg (SEQ ID NO: 110) lntaqvvaia
shdggkpale avwaklpvlr gvpye (SEQ ID NO: 111) lntaqvvaia shdggkpale
avwaklpvlr gvpye (SEQ ID NO: 112) lntaqvvaia shdggkpale avwaklpvlr
gvpya (SEQ ID NO: 113) lsteqvvaia shdggkqale avgaqlvalr aapya (SEQ
ID NO: 114) lstaqvvaia shdggnqale avgtqlvalr aapya (SEQ ID NO: 115)
lsteqvvaia shdggkqale avgaqlvalr aapya (SEQ ID NO: 116) lntaqivaia
shdggkpale avwaklpvlr gapya (SEQ ID NO: 117) lstaqvvava shdggkpale
avrkqlpvlr gvphq (SEQ ID NO: 118) lstaqvvaia shdggkpale avwaklpvlr
gapya (SEQ ID NO: 119) lntaqvvaia shdggkpale avwaklpvlr gvpye (SEQ
ID NO: 120) lntaqvvaia shdggkpale avwaklpvlr gvpya (SEQ ID NO: 121)
lstaqvvaia shdggkqale avgaqlvelr aapya (SEQ ID NO: 122) lsteqvvaia
shdggkqale avgaqlvalr aapya (SEQ ID NO: 123) lntaqvvaia shdggkpale
avraklpvlr gvpya (SEQ ID NO: 124) ltpqqvvaia shdggkpale avwaklpvlr
gvpya (SEQ ID NO: 125) ltpqqvvaia shdggkpale avwaklpvlr gvpya (SEQ
ID NO: 126) lstaqvatia ssiggrqale avkvqlpvlr aapyg (SEQ ID NO: 127)
lsteqvvvia nsiggkqale avkvqlpvlr aapye (SEQ ID NO: 128) lsteqvvvia
nsiggkqale avkvqlpvlr aapye (SEQ ID NO: 129) lstaqvatia ssiggrqale
avkvqlpvlr aapyg (SEQ ID NO: 130)
[0275] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, and the
like) cited in the present application is incorporated herein by
reference in its entirety.
EXAMPLES
Example 1: Generating Nucleic Acid Vectors with Ralstonia TALs
(RTALs) with Functional Analysis
[0276] Cluster analysis and review of sequence homologies of
Ralstonia genome revealed the sequence of SEQ ID NO: 1 which is
homolgous to known TAL sequences.
[0277] Nucleic acid sequences that encode the polypeptides of the
claimed invention will be made through molecular biology techniques
known to those with ordinary skill in the art. DNA sequences, for
instance, will be synthesized with the XbaI and/or Sail restriction
sites flanking each nucleic acid sequence that encodes the
polyproteins of the present invention. Polymerase chain reaction
will be performed to amplify the DNA with certain restriction
endonuclease sites. Sequences will be gel-purified, isolated, and
reconstituted in water or suitable buffer for ligation reactions. A
plasmid that encodes a protein with effector function (such as
nuclease function) that comprises requisite regulatory elements
will be ligated to one or more of the nucleic acid sequences that
encode the following sequences at the plasmid multiple cloning
sites:
TABLE-US-00006 a. (SEQ ID NO: 72) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV b. (SEQ ID NO: 93) LSTEQVVAIAS NN
GGKQALEAVKAQLLELRAAPYE c. (SEQ ID NO: 102) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG d. (SEQ ID NO: 134) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA e. (SEQ ID NO: 135) LSTEQVVTIAS SI
GGKQALEAVKVQLPVLRAAPYE
[0278] Plasmid sequences will be transformed in suitable bacteria
for production of high copy numbers of plasmid. Plasmids containing
at least one polypeptide above can be selected using antibiotic
selection, isolated and purified from bacterial cells using
techniques known to those skilled in the art.
[0279] Plasmids will also be built and in-vitro testing of
expressed DNA-binding polypeptides will be validated using the
methods described in Nature Biotechnology 2012 May; 30(5):460-5.
"FLASH assembly of TALENs for high-throughput genome editing."
Reyon D, Tsai S Q, Khayter C, Foden J A, Sander J D, Joung J K,
which is incorporated by reference in its entirety.
Construction of a Plasmid Archive Encoding Pre-Assembled TALE
Repeats
[0280] We sought to construct TALE repeat arrays using the same
architecture first described by Miller, J. C. et al. "A TALE
nuclease architecture for efficient genome editing.", Nat
Biotechnol. 2011; 29: 143-148 in which distinct TALE repeat
backbones that differ slightly in their amino acid and DNA
sequences occur in a repeated pattern. In some embodiments, we
designated the first, amino-terminal TALE repeat in an array as the
.alpha. unit. This is followed by .beta., , and .delta. units and
then an .epsilon. unit that is essentially identical to the .alpha.
unit except for the different positioning of a Type IIS restriction
site on the 5' end (required to enable creation of a unique
overhang on the .alpha. unit needed for cloning). The c unit is
then followed again by repeats of .beta., , .delta. and .epsilon.
units. Due to constraints related to creation of a 3'end required
for cloning, slightly modified DNA sequences were required for TALE
repeat arrays that end with a carboxyterminal or .epsilon.
unit.
Preparation of TALE Repeat-Encoding DNA Fragments for FLASH
Assembly
[0281] To prepare DNA fragments encoding .alpha. units for use in
FLASH assembly, we will perform 20 rounds of PCR with each .alpha.
unit plasmid as a template using primers
oJS2581(5'-Biotin-TCTAGAGAAGACAAGAACCTGACC-3', SEQ ID NO: 20) and
oJS2582(5'-GGATCCGGTCTCTTAAGGCCGTGG-3', SEQ ID NO: 21). The
resulting PCR products will be biotinylated on the 5' end. Each
.alpha. PCR product will then be digested with 40 units of BsaI-HF
restriction enzyme to generate 4 bp overhangs, purified using the
QIAquick PCR purification kit (QIAGEN) according to manufacturer's
instructions except that the final product will be eluted in 50
.mu.l of 0.1.times.EB.
[0282] To prepare DNA fragments encoding polypeptide repeats, we
will digest 10 .mu.g of each of these plasmids with 50 units of
BsaI restriction enzyme in NEBuffer 2 for 2 hours at 37.degree. C.
followed by serial restriction digests performed in NEBuffer 4 at
37.degree. C. using 100 units each of XbaI, BamHI-HF, and SalI-HF
enzymes that will be added at 5 minute intervals. The latter set of
restriction digestions will be designed to cleave the plasmid
backbone to ensure that this larger DNA fragment will not interfere
with subsequent ligations performed during the FLASH assembly
process. These restriction digest reactions will then be purified
using the QIAquick PCR purification kit (QIAGEN) according to
manufacturer's instructions except that the final product will be
eluted in 180 .mu.l of 0.1.times.EB.
Automated FLASH Assembly
[0283] All steps of FLASH assembly will be performed using a
Sciclone G3 liquid-handling workstation (Caliper) or similar device
sold by another company in 96-well plates and using a SPRIplate
96-ring magnet (Beckman Coulter Genomics) and a DynaMag-96 Side
magnet (Life Technologies). In the first step of FLASH, a
biotinylated a unit fragment will be ligated to the first
.beta..delta..epsilon. fragment and then the resulting
.alpha..beta..delta..epsilon. fragments will be bound to Dynabeads
MyOne C1 streptavidin-coated magnetic beads (Life Technologies) in
2.times.B&W Buffer. Beads will then be drawn to the side of the
well by placing the plate on the magnet and then will be washed
with 100 .mu.l B&W buffer with 0.005% Tween 20 (Sigma) and
again with 100 .mu.l 0.1 mg/ml bovine serum albumin (BSA) (New
England Biolabs). Additional .beta..delta..epsilon. fragments will
be ligated by removing the plate from the magnet, resuspending the
beads in solution in each well, digesting the bead-bound fragment
with Bsal-HF restriction enzyme, placing the plate on the magnet,
washing with 100 .mu.l B&W/Tween20 followed by 100 .mu.I of 0.1
mg/ml BSA, and then ligating the next fragment. This process will
be repeated multiple times with additional .beta..delta..epsilon.
units to extend the bead-bound fragment. The last fragment to be
ligated is always a .beta., .beta.*, .beta..delta., or
.delta..epsilon.* unit to enable cloning of the full-length
fragment into expression vectors (note that fragments that end with
a .delta..epsilon.* unit will always be preceded by ligation of a
.beta. unit).
[0284] The final full-length bead-bound fragment will be digested
with 40 units of Bsal-HF restriction enzyme followed by 25 units of
BsaI restriction enzyme (New England Biolabs). Digestion with BsaI
will release the fragment from the beads and generates a unique 5'
overhang for cloning of the fragment. Digestion with Bsal-HF
results in creation of a unique 3' overhang for cloning.
Subcloning of TALE Repeat Array-Encoding DNA Fragments into TALEN
Expression Vectors
[0285] We will subclone DNA fragments encoding our FLASH assembled
TALE repeat arrays into TALE expression vectors. In some
embodiments, there will be 4 or more separate plasmids. In some
embodiments, vectors will include a CMV promoter, a translational
start codon optimized for mammalian cell expression, a triple FLAG
epitope tag, a nuclear localization signal, amino acids 153 to 288
from the TALE 13 protein (as numbered by Miller et al. 6), two
unique and closely positioned Type IIS BsmBI restriction sites, a
0.5
[0286] TALE repeat domain encoding RVDs, amino acids 715 to 777
from the TALE 13 protein, and the wild-type FokI cleavage
domain.
[0287] All DNA fragments assembled by FLASH will possess overhangs
that enable directional cloning into any of the expression vectors
that will be digested with BsmBI. Standard TALEN expression vectors
(each possessing a different 0.5 TALE repeat) are available from
suppliers such as Addgene and full sequences of these plasmids are
freely available on a web page dedicated to these constructs:
[[http://www.]]addgene.org/talengineering/expressionvectors/for
synthetic construction.
[0288] To prepare a TALEN expression vector for subcloning, we will
digest 5 .mu.g of plasmid DNA with 50 units of BsmBI restriction
enzyme (New England Biolabs) in NEBuffer 3 for 8 hours at 55
degrees C. Digested DNA will be purified using 90 .mu.l of Ampure
XP beads (Agencourt) according to manufacturer's instructions and
will be diluted to a final concentration of 5 ng/.mu.l in 1 mM
TrisHCl. FLASH-assembled TALE repeat arrays will be ligated into
TALEN expression vectors using 400 U of T4 DNA Ligase (New England
Biolabs). Ligation products will be transformed into chemically
competent XL-1 Blue cells. Typically, six colonies will be picked
for each ligation and plasmid DNA will be isolated by an alkaline
lysis miniprep procedure. Simultaneously, the same colonies will be
screened by PCR using primers oSQT34
(5'-GACGGTGGCTGTCAAATACCAAGATATG-3', SEQ ID NO: 22) and oSQT35
(5'-TCTCCTCCAGTTCACTTTTGACTAGTTGGG-3', SEQ ID NO: 23). PCR products
will be analyzed on a QIAxcel capillary electrophoresis system
(Qiagen). Miniprep DNA from clones that contaid correctly sized PCR
products will be sent for DNA sequence confirmation with primers
oSQT1 (5'-AGTAACAGCGGTAGAGGCAG-3', SEQ ID NO: 24), oSQT3
(5'-ATTGGGCTACGATGGACTCC-3', SEQ ID NO: 210), and oJS2980
(5'-TTAATTCAATATATTCATGAGGCAC-3', SEQ ID NO: 25); oSQT1 anneals at
the 5' end of the TALE repeat array coding sequence and will enable
sequencing of the amino-terminal half of the assembled array, oSQT3
anneals at the 3' end of the TALE repeat array coding sequence and
enables sequencing of the carboxy-terminal half of the assembled
array, and oJS2980 primes within the coding sequence of the FokI
domain (downstream of oSQT3) and will enable sequencing and
verification of the carboxy-terminal 0.5 TALE repeat domain.
[0289] We will screen six colonies for each assembly as described
above, followed by six additional colonies if necessary. With this
approach, one or more sequence-verified clones for >90% of
assembly reactions. These percentages will be derived primarily
from experiments designed to construct DNA fragments encoding 16.5
TALE repeats.
EGFP TALEN Activity and Toxicity Assays
[0290] EGFP reporter assays will be performed in a clonal U2OS
human cell line bearing an integrated construct that constitutively
expresses an EGFP-PEST fusion protein. This clonal line will be
derived from a polyclonal U2OS EGFP-PEST reporter line. Clonal U2OS
EGFP-PEST cells will be cultured in Advanced DMEM (Life
Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life
Technologies), penicillin/streptomycin, and 400 .mu.g/ml G418.
Cells will be transfected in triplicate with 500 ng of each TALEN
plasmid DNA and 50 ng ptdTomato-N1 plasmid DNA using a Lonza
4D-Nucleofector System, Solution SE, and program DN-100 according
to manufacturer's instructions. 1 .mu.g of ptdTomato-N1 plasmid
alone will be transfected in triplicate as a negative control.
Cells will be assayed for EGFP and tdTomato expression at 2 and 5
days post-transfection using a BD FACSAriall flow cytometer.
PCR Amplification and Sequence Verification of Endogenous Human
Genes
[0291] PCR reactions to amplify targeted loci will be performed
using the primers shown in Supplementary Table 5. Standard PCR
conditions with Phusion Hot Start II high-fidelity DNA polymerase
(Thermo-Fisher) will be performed according to manufacturer's
instructions for 35 cycles (98.degree. C., 10 s denaturation;
68.degree. C., 15 s annealing; 72.degree. C., 30 s extension). For
loci that do not amplify under standard conditions we will use one
of the following modifications: 1) the addition of betaine to a
final concentration of 1.8M, 2) touchdown PCR ([98.degree. C., 10
s; 72-62.degree. C., -1.degree. C./cycle, 15 s; 72.degree. C., 30
s]10 cycles, [98.degree. C., 10 s; 62.degree. C., -1.degree.
C./cycle, 15 s; 72.degree. C., 30 s]25 cycles) with 1.8M betaine,
and 3) the addition of 3% or 5% DMSO and an annealing temperature
of 65.degree. C. PCR products will be analyzed for correct size on
a QIAxcel capillary electrophoresis system. Correctly sized
products will be treated with ExoSap-IT (Affymetrix) to remove
unincorporated nucleotides or primers and sent for DNA sequencing
to confirm the endogenous gene sequence.
T7 Endonuclease I Assay for Quantifying NHEJ-Mediated Mutation of
Endogenous Human Genes
[0292] U2OS-EGFP cells will be cultured and transfected in
duplicate as described above. Genomic DNA was isolated from cells
transfected with TALEN-encoding or control plasmids using a
high-throughput magnetic-bead based purification system (Agencourt
DNAdvance) according to the manufacturer's instructions. PCR to
amplify endogenous loci will be performed for 35 cycles as
described above and fragments were purified with Ampure XP
(Agencourt) according to manufacturer's instructions. 200 ng of
purified PCR product will be denatured and reannealed in NEBuffer 2
(New England Biolabs) using a thermocycler with the following
protocol (95.degree. C., 5 min; 95-85.degree. C. at -2.degree.
C./s; 85-25.degree. C. at -0.1.degree. C./s; hold at 4.degree. C.).
33 Hybridized PCR products were treated with 10 U of T7
Endonuclease I at 37.degree. C. for 15 minutes in a reaction volume
of 20 .mu.l. Reactions were stopped by the addition of 2 .mu.l 0.5
M EDTA, purified with Ampure XP, and quantified on a QIAxcel
capillary electrophoresis system using method OM500. The sum of the
area beneath TALEN-specific cleavage peaks (expressed as a
percentage of the parent amplicon peak, denoted fraction cleaved)
is used to estimate gene modification levels using the following
equation as previously described.
(% gene modification=100.times.(1-(1-fraction cleaved)1/2)
Example 2
[0293] Five fragments shown below were synthesized and each cloned
into a modified pUC57: pUC57-.DELTA.Bsal (vectors as disclosed in
Juong et. al. FLASH assembly paper). It contains single basepair
change to disrupt a Bsal site) with Xbal and BamHI.
TABLE-US-00007 RTNI EBEs: NK: XbaI BbsI ATGCA T{circumflex over (
)}CTAGA-GAAGACAA{circumflex over ( )}CTGA-
GCACCGAGCAGGTGGTGGCCATCGCCAGCAACAAGGGCGGCAAGCAGGCCCTG
GAGGCCGTGAAGGCCCACCTGCTGGACCTGCTGGGCGCCCCCTACGAG- CTGA{circumflex
over ( )}AGAGACC-G{circumflex over ( )}GATCC(CGGGC) SEQ ID NO: 26)
BsaI BamHI NN: ATGCA
TCTAGAGAAGACAACTGAGCACCGAGCAGGTGGTGGCCATCGCCAGCAACAACG
GCGGCAAGCAGGCCCTGGAGGCCGTGAAGGCCCAGCTGCTGGAGCTGAGGGCCG
CCCCCTACGAGCTGAAGAGACCGGATCC CGGGC (SEQ ID NO: 27) NG: ATGCA
TCTAGAGAAGACAACTGAGCACCGagCAGGTGGTGGCCATCGCCAGCAACGGCG
GCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACC
GCCCCCTACGAGCTGAAGAGACCGGATCC CGGGC (SEQ ID NO: 28) HD: ATGCA
TCTAGAGAAGACAACTGAGCACCGagCAGGTGGTGGCCATCGCCAGCCACGACG
GCGGCAAGCCCGCCCTGGAGGCCGTGTGGGCCAAGCTGCCCGTGCTGAGGGGCG
TGCCCTACGAGCTGAAGAGACCGGATCC CGGGC (SEQ ID NO: 29) SI: ATGCA
TCTAGAGAAGACAACTGAGCACCGAGCAGGTGGTGACCATCGCCAGCAGCATCG
GCGGCAAGCAGGCCCTGGAGGCCGTGAAGGTGCAGCTGCCCGTGCTGAGGGCCG
CCCCCTACGAGCTGAAGAGACCGGATCC CGGGC (SEQ ID NO: 30)
[0294] For proof of principle, these cloned fragments will be used
to generate chimeric proteins of six repeat units fused to FokI
nuclease, following the exact protocol in Joung's FLASH TALEN
paper, i.e. a chimeric protein that targets a string of A (C, T and
G) nucleotides. These chimeric proteins will then be tested for
binding/targeting efficiency to desired DNA bases using a reporter
construct.
[0295] Once the binding efficiency of these units are confirmed, a
library of Ralstonia EBEs will be generated that will be an exact
copy of FLASH TALEN's Xanthomonas EBE library. This library can
then be used to generate Ralstonia TALENs following the exact
protocol of the FLASH TALEN system.
Example 3. Generating Nucleic Acid Vectors with Methylesterase
[0296] Additional sequences cloned from other species or cloned
from the same species may be used functionally as an enzyme either
by itself or in series as a monomer or polymer (protein fusion) for
performing any of the experiments disclosed herein with DNA
recognition. A RVD identification consensus sequence was created
using sequence optimization techniques known in the art. A BLAST
search was performed across methlyesterase sequences in bacterial
species (see FIG. 1). The following polypeptides were identified as
having DNA base pair recognition capability similar to the nucleic
acid sequences and polypeptides disclosed herein SEQ ID NO:
1-19:
TABLE-US-00008 TAL EBE against methylesterases #1 Xanthomonas
Consensus EBEs LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 31)
LTPEQVVAIANNNGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 32)
LTPDQVVAIASHDGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 33)
LTPAQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 34) EJO92907 166
TTDRVVALGTSTGGTQALEVVLRQLPVDC 194 (SEQ ID NO: 35) YP_001187060 166
TTDRVVALGTSTGGTQALEVVLRQLPVDC 194 (SEQ ID NO: 35) YP_003847734 169
MTSEQIVAIGTSTGGTQALEAVLTALPRVC 198 (SEQ ID NO: 36) ZP_08780698 167
TTDRVVAIGTSTGGTQALEVVLTALPRVC 195 (SEQ ID NO: 37) YP_004846745 185
TTERIVAIGTSTGGTQALETVLHRLPATC 213 (SEQ ID NO: 38) YP_005027668 186
TTDKIIAIGTSTGGTQALEAVLTKLPAVC 214 (SEQ ID NO: 39) ZP_10991552 174
TTERIVAIGTSTGGTQALETVLTALPRVC 202 (SEQ ID NO: 40) YP_001792820 162
TTERVVALGTSTGGTQALEVVLRTLPRVC 190 (SEQ ID NO: 58) EKE17764 172
TTDQLIAIGTSTGGTQALEAILTKLPATC 200 (SEQ ID NO: 62) ZP_03698248 178
TTERIV AIGTSTGGTQALETVLPRLP A TC 206 (SEQ ID NO: 44) EGH48032 11
TTERIVAIGTSTGGTQALEAVLTALPRVC 39 (SEQ ID NO: 136) ZP_06495900 1
TTERIV AIGTSTGGTQALEAVLTALPRVC 29 (SEQ ID NO: 136) ZP_10381001 76
TTERIVAIGTSTGGTQALEAVLTALPRVC 104 (SEQ ID NO: 136) ZP_10442431 158
TSDKVVAIGASTGGTQALELLLTGLPAVC 186 (SEQ ID NO: 45) #2 ZP_10991552
174 TTERIVAIGTSTGGTQALETVLTALPRVC 202 (SEQ ID NO: 138) EGH48032 11
TTERIVAIGTSTGGTQALEAVLTALPRVC 39 (SEQ ID NO: 136) ZP_06495900 1
TTERIVAIGTSTGGTQALEAVLTALPRVC 29 (SEQ ID NO: 136) EGH61007 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH06695 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH31878 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH66597 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_07003572 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_06457223 172
TTERIV AIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_04590480 172
TTERIV AIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_07251539 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) NP_790747 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH77388 172
TTERIV AIGTSTGGTQALEA VLTALPRVC 200 (SEQ ID NO: 136) EFW86187 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH54563 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) YP_233877 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH23390 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_05638023 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) EGH71924 16
TTERIVAIGTSTGGTQALEAVLTALPRVC 134 (SEQ ID NO: 136) EFW82095 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) ZP_07265841 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) YP_273082 172
TTERIVAIGTSTGGTQALEAVLTALPRVC 200 (SEQ ID NO: 136) YP-004030667 117
FSQADIVRIADNIGGAQALKAVLEHGPTL 145 (SEQ ID NO: 46) YP_004030667 186
ADIVKIASNGGGAQALEAVAMEIGSTLCE 213 (SEQ ID NO: 46) YP_004030667 153
ADIVKIAGNGGGARALKAVVMHGPTLCE 180 (SEQ ID NO: 48) ZP_10995147 155
TTDRVVALGCSTGGTQALEFILRQLPRDC 183 (SEQ ID NO: 49) EGH56182 30
ALAAAVGGKGALEVPANLIPANCE 53 (SEQ ID NO: 50) YP_003907367 173
RIVAIGTSTGGTQALEVVLTALP 195 (SEQ ID NO: 51) EBE1
LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG 34 (SEQ ID NO: 52) EBE4 LTPAQVV
AIASNIGGKQALETVQRLLPVLCQDHG 34 (SEQ ID NO: 53) EBE3
LTPDQVVAIASHDGGKQALETVQRLLPVLCQAHG 34 (SEQ ID NO: 54) EBE2 LTPEQVV
AIANNNGGKQALETVQRLLPVLCQAHG 34 (SEQ ID NO: 55) ZP_07265841_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) YP_273082_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EFW82095_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EGH71924_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136)
ZP_05638023_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) EGH23390_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) YP_233877_2 -TTERIVAIGTSTGGTQALEA VL TALPRVC---- 29 (SEQ ID
NO: 136) EGH54563_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID
NO: 136) EFW86187_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID
NO: 136) EGH77388_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID
NO: 136) NP_790747_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID
NO: 136) ZP_07251539_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ
ID NO: 136) ZP_04590480_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29
(SEQ ID NO: 136) ZP_06457223_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC----
29 (SEQ ID NO: 136) ZP_07003572_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EGH66597_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EGH31878_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EGH06695_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136) EGH61007_2
-TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO: 136)
ZP_06495900_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) EGH48032_2 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) ZP_10381001 -TTERIV AIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) ZP_06495900 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) EGH48032 -TTERIVAIGTSTGGTQALEAVLTALPRVC---- 29 (SEQ ID NO:
136) YP_003847734 MTSEQIVAIGTSTGGTQALEAVLTALPRVC---- 30 (SEQ ID NO:
137) ZP_10991552 -TTERIV AIGTSTGGTQALETVLTALPRVC---- 29 (SEQ ID NO:
138) ZP_10991552_2 -TTERIVAIGTSTGGTQALETVLTALPRVC---- 29 (SEQ ID
NO: 40) YP_003907367_2 ----RIVAIGTSTGGTQALEVVLTALP------- 23 (SEQ
ID NO: 51) EJO92907 -TTDRVVALGTSTGGTQALEVVLRQLPVDC---- 29 (SEQ ID
NO: 56) YP_001187060 -TTDRVVALGTSTGGTQALEVVLRQLPVDC---- 29 (SEQ ID
NO: 56) ZP_10995147_2 -TTDRVVALGCSTGGTQALEFILRQLPRDC---- 29 (SEQ ID
NO: 57) YP_001792820 -TTERVVALGTSTGGTQALEVVLRTLPRVC---- 29 (SEQ ID
NO: 58) ZP_08780698 -TTDRVVAIGTSTGGTQALEVVLTALPRVC---- 29 (SEQ ID
NO: 59) YP_004846745 -TTERIVAIGTSTGGTQALETVLHRLPATC---- 29 (SEQ ID
NO: 38) ZP_03698248 -TTERIVAIGTSTGGTQALETVLPRLPATC---- 29 (SEQ ID
NO: 44) YP_00527668 -TTDKIIAIGTSTGGTQALEAVLTKLPAVC---- 29 (SEQ ID
NO: 61) EKE17764 -TTDQLIAIGTSTGGTQALEAILTKLPATC---- 29 (SEQ ID NO:
62) ZP_10442431 -TSDKVVAIGASTGGTQALELLLTGLPAVC---- 29 (SEQ ID NO:
63) YP_004030667_2b ---ADIVKIASNGGGAQALEAVAMHGSTLCE--- 28 (SEQ ID
NO: 64) YP_004030667_2c ---ADIVKIAGNGGGARALKAVVMHGPTLCE--- 28 (SEQ
ID NO: 65) YP_004030667_2a FSQADIVRIADNIGGAQALKAVLEHGPTL----- 29
(SEQ ID NO: 66) EGH56182_2 -------ALAAAVGGKGALEVPANLIPANCE--- 24
(SEQ ID NO: 67)
Example 4
[0297] A pair of Bmpr2 specific EBEs (Ralstonia DNA binding domain,
16EBEs each) were gene synthesized and cloned into XTN-BB
(Xanthomonas TAL backbone fused to FokI). These constructs were
co-transfected into Rat C6 cells and gDNA extracted after 48 hrs
for Cell surveyor nuclease assay. If successful, the assay should
produce 240 bp and 150 bp subpopulations from the original 400 bp
amplicon of the locus. The results are shown in the FIG. 2.
[0298] The assay reveals the expected 250 bp and 150 bp bands in
the Ralstonia and Xanthomonas TALEN transfected cells, which are
absent in the WT negative control. This indicates that the
Ralstonia EBEs target this locus and the fusion of FokI nuclease to
Ralstonia EBEs lead to targeted digestion of genomic DNA. Using the
250 bp band, 5.75% for XTN, 1.82% for RTN. Using the 150 bp band,
3.66% for XTN, 5.43% for RTN.
TABLE-US-00009 BmpR2 Target
T-T-GATA-GTCG-CCTT-ATG-TtttggatacagaatgtT-GAC-AGGT- site
AAAC-GAAA-T-A (SEQ ID NO: 141) Fwd RTN TGATAGTCGCCTTATG (SEQ ID NO:
142) Rev RTN ATTTGGTTTACCTGTC (SEQ ID NO: 143) Note: the first and
last nucleotide of the targeted site (underlined) are not specified
by ht eRTNs. These are specified by the Xanthomonas TALEN
backbone.
TABLE-US-00010 Bmpr2 FWD RTN EBEs' amino acid sequence: LSTAQVVAIAS
NG GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) LSTEQVVAIAS NN
GGKQALEAVKAQLLELRAAPYE (SEQ ID NO: 93) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NN
GGKQALEAVKAQLLELRAAPYE (SEQ ID NO: 93) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NN
GGKQALEAVKAQLLELRAAPYE (SEQ ID NO: 93) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) Bmpr2 FWD RTN DNA sequence:
(Bolded font: synthesized Ralstonia EBEs) This sequence is
Contiguous (SEQ ID NO: 145):
GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTG
ATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGT
AGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATG
AAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATAT
ACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA
GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT
GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA
GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT
GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC
AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT
CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT
GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC
ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA
AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT
ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCACCATGGACT
ACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGAT
GACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTAT
GGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTA
AGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACT
CATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTC
AAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGG
GGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGG
GTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCG
AAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCA
CCGGGGCCCCCTTGAAC CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG GGC GGCAAGC AGGC C C T
GGAGGC C GTGAAGGC C CAC C TGC TGGAC C TGC TGGGC GC CCCCTACGTG
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAC GGC GGCAAGC AGGC C C T
GGAGGC C GTGAAGGC C CAGC TGC TGGAGC T GAGGGC C GC CCCCTACGAG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAC GGC GGCAAGC AGGC C C T
GGAGGC C GTGAAGGC C CAGC TGC TGGAGC T GAGGGC C GC CCCCTACGAG
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG GGC GGCAAGC AGGC C C T
GGAGGC C GTGAAGGC C CAC C TGC TGGAC C TGC TGGGC GC CCCCTACGTG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC GGC GGCAAGC C C GC C C
TGGAGGC C GT GT GGGC CAAGC TGC CC GTGC TGAGGGGC GT GCCCTACGCC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG GGC GGCAAGC AGGC C C T
GGAGGC C GTGAAGGC C CAC C TGC TGGAC C TGC TGGGC GC CCCCTACGTG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC GGC GGCAAGC C C GC C C
TGGAGGC C GT GT GGGC CAAGC TGC CC GTGC TGAGGGGC GT GCCCTACGCC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC GGC GGCAAGC C C GC C C
TGGAGGC C GT GT GGGC CAAGC TGC CC GTGC TGAGGGGC GT GCCCTACGCC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAC GGC GGCAAGC AGGC CC T
GGAGGCC GTGAAGGC CCAGC TGC TGGAGC T GAGGGCC GC CCCCTACGAG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG GGC GGCAAGC AGGC CC T
GGAGGCC GTGAAGGC CCAC C TGC TGGACC TGC TGGGC GC CCCCTACGTG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGCAACGGCGGAGGACGGCCAGCCTT
GGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAA
TGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAA
AAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCG AGAGAAC TT
CC CAT C GAGT C GC GGGATC C CAAC TAGT CAAAAGTGAAC TGGAGGAG
AAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTA AT TGAAAT
TGCCAGAAATT CC AC T CAGGATAGAAT TC TT GAAATGAAGGTAAT GGA
ATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACC GGAC
GGAGCAAT TTATAC TGT C GGATC T CC TATT GATTAC GGTGT GATC GT GGATAC
TAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAAC
GATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGG
AAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTA AAGGAAAC
TAC AAAGC TC AGC T TAC AC GATTAAAT CATATC AC TAAT TGTAAT GGA
GCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACA
TTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTTAAGG
GCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACC
GGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCT
TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG
GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA
GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT
TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGC
GGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCAT
TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC
CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC
CCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA
CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTG
ATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTG
TTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGA
TTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG
CGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCA
GGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAA
AGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCA
GCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCC
GCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCG
CCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCT
TTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGT
TGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGA
ACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCA
ACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGC
TCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGA
CCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTG
ACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACG
GTGTCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCGATAGTGAAGGACA
GTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGT
GGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTT
CGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGC
CGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAA
CTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC
AAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTA
TCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCAT
AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCG
GAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATT
GCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAA
TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC
TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC
TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT
GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG
TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG
AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC
CCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC
CCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTG
TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC
TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG
CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGC
TACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGG
TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC
CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC
GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGA
TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATA
TGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC
GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG
ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACG
CTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA
GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG
CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG
GCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG
ATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG
TCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGC
AGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGT
GAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC
CCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT
CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATC
CAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC
AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA
GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCA
TTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA
AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC Bmpr2 REV RTN
EBEs' amino acid sequence: LSTEQVVAIAS NN GGKQALEAVKAQLLELRAAPYE
(SEQ ID NO: 93) LSTAQVVAIAS NG GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO:
102) LSTAQVVAIAS NG GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102)
LSTAQVVAIAS NG GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS
HD GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NN
GGKQALEAVKAQLLELRAAPYE (SEQ ID NO: 93) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTEQVVAIAS NK
GGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 72) LSTAQVVAIAS NG
GGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 102) LSTAQVVAIAS HD
GGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 134) Bmpr2 REV RTN DNA Sequence:
(Bolded Font: synthesized Ralstonia EBEs)this sequence is
Contiguous (SEQ ID NO: 147):
GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTG
ATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGT
AGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATG
AAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATAT
ACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA
GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT
GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA
GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT
GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC
AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT
CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT
GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC
ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA
AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT
ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCACCATGGACT
ACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGAT
GACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTAT
GGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTA
AGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACT
CATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTC
AAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGG
GGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGG
GTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCG
AAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCA
CCGGGGCCCCCTT CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAC
GGCGGCAAGCAGGCCCTGGAGGCCGTGAAGGCCCAGCTGCTGGAGCTGAGGGCCGC CCCCTACGAG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC
GGCGGCAAGCCCGCCCTGGAGGCCGTGTGGGCCAAGCTGCCCGTGCTGAGGGGCGT GCCCTACGCC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG
GGCGGCAAGCAGGCCCTGGAGGCCGTGAAGGCCCACCTGCTGGACCTGCTGGGCGC CCCCTACGTG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAC
GGCGGCAAGCAGGCCCTGGAGGCCGTGAAGGCCCAGCTGCTGGAGCTGAGGGCCGC CCCCTACGAG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC
GGCGGCAAGCCCGCCCTGGAGGCCGTGTGGGCCAAGCTGCCCGTGCTGAGGGGCGT GCCCTACGCC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC
GGCGGCAAGCCCGCCCTGGAGGCCGTGTGGGCCAAGCTGCCCGTGCTGAGGGGCGT GCCCTACGCC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGAGCAGGTGGTGGCCATCGCCAGC AACAAG
GGCGGCAAGCAGGCCCTGGAGGCCGTGAAGGCCCACCTGCTGGACCTGCTGGGCGC CCCCTACGTG
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC AACGGC
GGCGGCAAGCAGGCCCTGGAGGGCATCGGCGAGCAGCTGCTGAAGCTGAGGACCG CCCCCTACGGC
CTGAGCACCGCCCAGGTGGTGGCCATCGCCAGC CACGAC
GGCGGCAAGCCCGCCCTGGAGGCCGTGTGGGCCAAGCTGCCCGTGCTGAGGGGCGT GCCCTACGCC
CTGAGCACCGAGCAGGTGGTGACCATCGCCAGC
AGCATCGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGA
TCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGG
ACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAA
AAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAAC
TAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATAT
GTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGA
ATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAA
CATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATT
GATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATT
GGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAAC
ATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGT
TTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAA
ATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTG
GAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAAT
AACGGCGAGATAAACTTTTAAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTC
CTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACC
CGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC
CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG
AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGC
GGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATC
CCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC
GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT
TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGG
GTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG
TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCC
ACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG
GTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATG
AGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGG
GTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTC
AATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT
GCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCAT
CCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTT
TTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGA
GGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCC
ATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCA
TAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAA
GAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGA
AGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGT
CAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGC
TGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACA
GGGGCATCTTGAGCCCCTGCGGACGGTGTCGACAGGTGCTTCTCGATCTGCATCCTG
GGATCAAAGCGATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGT
GAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCA
GGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGG
CTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT
GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA
AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT
GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCT
AGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC
AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT
GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA
ACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTG
CGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC
TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA
GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC
GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATC
ACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTA
CCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACG
CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGA
ACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAA
CCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA
GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC
TACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA
AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT
TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT
GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT
GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAG
TTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTT
AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGA
CTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT
GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCC
AGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC
GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG
CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT
GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG
CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC
CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG
TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACA
TAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC
AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG
ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA
AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCT
TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT
ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA
AGTGCCACCTGACGTC
Example 5
[0299] A library of Ralstonia EBEs and backbone vectors were made
which could be used to assemble full length Ralstonia DNA binding
domains into Ralstonia or Xanthomonas TALEN backbones, utilizing
the golden gate assembly method. The RTNs were co-transfected into
the Rat C6 cell line and gDNA extracted for analysis 48 hrs post
transfection. A 420 bp gDNA fragment containing the RTN binding
site was amplified by PCR. This amplicon was then subjected to the
Cell assay using Surveyor Mutation Detection Kit (Transgenomic) as
per manufacturer protocol. In brief, the amplicon is denatured into
single stranded DNA and slowly re-annealed back to double stranded
DNA. During this process, considering the original pool was a
mixture of WT and mutated sequences, there will be cross
hybridization between WT and mutant strands leading to formation of
heteroduplexes. Upon treatment of this re-annealed pool with the
Surveyor Nuclease, it recognizes the heteroduplexes and cleaves
them, which generated two shorter fragments from (255 bp and 165
bp) the original amplicon (420 bp).
Terminology
[0300] pRVD: plasmid containing a single Ralstonia EBE. Individual
EBEs were gene synthesized and cloned in FLASH-XTN sub-array
backbone (XbaI, BamHI). pFus X: a sub-array plasmid that holds the
first 10 EBEs of any given RTN. The required piece was gene
synthesized and cloned into pHSG-298 (Sacl, Sbfl). pFUS Z: a
sub-array plasmid that holds EBE 11 upto the second-last EBE of any
given RTN. Eg: Z4 holds EBEs 11-14, Z5 holds EBEs 11-15 and Z6
holds EBEs 11-16. Gene synthesized and cloned into pHSG-298 (Sacl,
Sbfl). XTN-bb: Xanthomonas TAL backbone that contains the
N-terminal and C-terminal Xanthomonas TAL domains fused to FokI
nuclease. This backbone specifies a T nucleotide 5' of the target
sequence specified by the EBEs. It also contains the last half EBE
that specifies the last nucleotide of the targeted sequence.
Therefore there are four XTN-bb plasmids, each specifying a
different final nucleotide of the targeted sequence (same as FLASH
XTN backbones).
[0301] All plasmids are stored at 150 ng/ul in 0.1.times.TE
buffer.
[0302] Methods: (building a 16EBE DNA binding domain and cloning it
into a Xanthomonas TALEN backbone).
[0303] Assembly of a custom TALEN or TAL effector construct
involves two steps: (i) assembly of repeat modules (pRVDs) into
sub-arrays of 1-10 repeats and (ii) joining of the sub-arrays into
a backbone to make the final construct.
[0304] We constructed of a TALEN monomer with a 17 RVD array
(5'-TGATAGTCGC-CTTATG-T-3', SEQ ID NO: 208). Select from the pRVD
plasmids those that encode RVDs 1-10 in the array using plasmids
numbered in that order. For example, the plasmid for the first RVD
would be gRTN-1T, the second gRTN-2G, the third gRTN-3A etc.
Modules from these plasmids will be cloned into sub-array plasmid
pFUS-X. Next, select modules for RVDs 11-16 in the 16 RVD array
again starting with plasmids numbered from 1. Thus for RVD 11
gRTN-1C would be used, for RVD 12 gRTN-2T, etc. The pFUS-Z plasmids
are numbered 1-10 and should be selected according to the number of
EBEs going in. Thus, in our example, pFUS-Z6 should be used.
[0305] The pRVDs and sub-array plasmids (150 ng each) are subjected
to digestion and ligation in a single 20 ul reaction containing 1
ul BsaI (10 U, New England BioLabs) and 1 ul T4 DNA Ligase (2000 U,
New England BioLabs) in T4 DNA ligase buffer (New England BioLabs).
The reaction is incubated in a thermocycler for 10 cycles of 5 min
at 37 C and 10 min at 16 C, then heated to 50 C for 5 min and then
80 C for 5 min. Then, 1 ul 25 mM ATP and 1 ul Plasmid Safe DNase
(10 U, Epicentre) are added. The mixture is incubated at 37 C for 1
h, then used to transform Escherichia coli cells. Cells are plated
on LB agar containing 50 mg/ml Kanamycin, overnight at 37.degree.
C.
[0306] Up to six colonies from each transformation were screened
with M13 fwd and rev primers, via colony PCR, to identify clones
that contain a full-length sub-array. Full length pFUS-X sub-array
clones should produce a 1.1 kb band and full-length pFUS-Z6 clones
should produce a 700 bp band (add or subtract 105 bp for each EBE
more or less). Cultures were started overnight cultures of a
full-length pFUS-X and a full-length pFUS-Z6 clone.
[0307] We isolated plasmid DNA from your pFUS-X and pFUS-Z
cultures. Sub-arrays were joined into one of the four backbone
plasmids. A 20 ul digestion and ligation reaction mixture is
prepared with 150 ng each of the pFUS-X and pFUS-Z plasmids, 150 ng
of the backbone plasmid, in this case XTN-bbT, 1 ul Esp3I (10 U,
Thermo Scientific) and 1 ul T4 DNA Ligase (2000 U, New England
Biolabs) in T4 DNA ligase buffer. The reaction is then incubated in
a thermocycler for 3 cycles of 10 min at 37 C and 15 min at 16 C.
The reaction is then incubated at 37 C for an additional 30 min and
heated to 50 C for 5 min, then 80 C for 5 min. After cooling to
room temperature, 1 ul 25 mM ATP and 1 ul Plasmid Safe DNase (10 U,
Epicenter) were added and incubated at 37 C for 1 hr. The reaction
is then used to transform E. coli as above, except that Plasmid
Safe. Also, in this step, ampicillin (100 mg/ml) is used in place
of Kanamycin for selection of transformants.
[0308] We screened up to three colonies from each transformation
via colony PCR with XTN-VF and XTN-VR2 primers and started
overnight cultures of 1 full length clone for each RTN (2.1 kb band
indicates 17EBE array). We then isolated plasmid DNA and identify
clones containing the final, full-length repeat array by DNA
sequencing with XTN-VF, XTN-VR1 and XTN-VR2.
TABLE-US-00011 XbaI and BamHI digested XTN sub-array backbone
(sites underlined): SEQ ID NO: 148:
(BamHI)GGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATC
ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA
CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCAC
ATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT
GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC
CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATC
AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA
AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT
GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC
AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG
GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC
CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT
TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC
GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC
TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAG
TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT
CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC
AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT
CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA
GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTA
TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT
AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG
AGCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC
GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC
CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT
GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTT
CCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCT
CCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG
TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGT
GACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTG
CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT
GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT
GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT
TTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGG
AATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG
AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAA
AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT
AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT
TTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGG
AGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGC
GCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA
GATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGA
GAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG
CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCA
AGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAC
GGCCAGTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGA (XbaI) XTN-bb (BsmBI
digested, sites are self excised from the backbone during
digestion): Undelined sequences overlap with sub-arrays pFUS-X and
pFUS-Z. XTN-bbA: NNNNNNNNN is replaced with TCTAACATC XTN-bbC:
NNNNNNNNN is replaced with TCCCACGAC XTN-bbG: NNNNNNNNN is replaced
with AATAATAAC XTN-bbT: NNNNNNNNN is replaced with TCTAATGGG SEQ ID
NO 149: pFUS-Z overlap CTGACACCCGAACAGGTGGTCGCCATTGCTNNNNNNNNN
GGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGC
GTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACC
CGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAA
CCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAACTAGTC
AAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCC
TCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCT
TGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTT
GGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTA
CGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCA
AGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCA
ACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATT
TGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATAT
CACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAAT
GATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCG
AGATAAACTTTTAAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTC
TCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGAT
CAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC
TTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT
TGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGA
CAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC
TCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGC
GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG
CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCC
ACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGA
TTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCT
TTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC
TTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATT
TAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAA
AGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTC
AGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCA
TGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC
TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTT
TTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGG
ATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAA
TACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCAC
CCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACA
GCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTAT
ATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTG
CGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATC
TTGAGCCCCTGCGGACGGTGTCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAA
GCGATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCT
GCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGA
CACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGA
ATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAG
TTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA
GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC
CAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTG
GCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC
ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGC
TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT
GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGG
CGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG
CGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC
GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG
CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC
GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT
CCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC
TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTA
TCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT
TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG
ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT
ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG
GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCA
AGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA
CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA
TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA
ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG
GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCG
TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC
CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA
AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT
TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT
GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT
CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG
GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA
CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT
TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC
CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT
TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC
CGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCAT
CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA
AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT
ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT
TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG ACGTC
GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTG
ATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGT
AGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATG
AAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATAT
ACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA
GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT
GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA
GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT
GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC
AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT
CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT
GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC
ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA
AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTT
ATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCACCATGGACT
ACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGAT
GACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTAT
GGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTA
AGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACT
CATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTC
AAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGG
GGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGG
GTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCG
AAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCA
CCGGGGCCCCCTTGAAC pFUS-X overlap BamHI and XbaI flanked pRVD
fragments (gene synthesized, BamHI-EBE-XbaI)): gXTN-1C:
TCTAGAGGTCTCATTGACCCCAGACCAGGTAGTCGCAATCGCGTCAcatgacGGGGG
AAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACG
GCAGAGACCGGATCC (SEQ ID NO: 154) gXTN-2C: TCTAGAGGTC
TCACGGCctgactcccgatcaagttgtagcgattgcgtcgCATGACggagggaaacaagcatt-
ggag actgtccaacggctccttcccgtgttgtgtcaagcccacggAGAGACCGGATCC (SEQ ID
NO: 155) gXTN-3C:
TCTAGAGGTCTCAacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAGCcatgatGGCG
GTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCAT
GAGAGACCGGATCC (SEQ ID NO: 156) gXTN-4C:
TCTAGAGGTCTCACATGGActgaccccagaccaggtagtcgcaatcgcgtcaCATGACgggggaaagcaagccc
tggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacAGAGACCGGATCC (SEQ ID
NO: 157) gXTN-5C:
TCTAGAGGTCTCAccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAcatgacGG
GGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACC
AAGAGACCGGATCC (SEQ ID NO: 158) gXTN-6C:
TCTAGAGGTCTCAACCACGGCctgactcccgatcaagttgtagcgattgcgtcgCATGACggagggaaaca
agcattggagactgtccaacggctccttcccgtgttgtgtcaagcccAGAGACCGGATCC (SEQ
ID NO: 159) gXTN-7C:
TCTAGAGGTCTCAgcccacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAGCCAT
GATGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGC
CAGGATAGAGACCGGATCC (SEQ ID NO: 160) gXTN-8C:
TCTAGAGGTCTCAGGATCATGGActgaccccagaccaggtagtcgcaatcgcgtcacatgacgggggaaagc
aagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaAGAGACCGGATCC (SEQ ID
NO: 161) gXTN-9C:
TCTAGAGGTCTCAaagaccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAca
tgacGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTC
AAGAGAGACCGGATCC (SEQ ID NO: 162) gXTN-10C:
TCTAGAGGTCTCACAAGACCACGGCctgactcccgatcaagttgtagcgattgcgtcgcatgacggagggaa
acaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccaTggAAGAGACCGGATCC
(SEQ ID NO: 163) gXTN-1T :
TCTAGAGGTCTCATTGACCCCAGACCAGGTAGTCGCAATCGCGTCAAACGGAGG
GGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGA
CCACGGCAGAGACCGGATCC (SEQ ID NO: 164) gXTN-2T:
TCTAGAGGTCTCACGGCctgactcccgatcaagttgtagcgattgcgtcgAACGGTggagggaaacaagcattg
gagactgtccaacggctccttcccgtgttgtgtcaagcccacggAGAGACCGGATCC (SEQ ID
NO: 165) gXTN-3T:
TCTAGAGGTCTCAacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCTCGAATGGC
GGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAG
GATCATGAGAGACCGGATCC (SEQ ID NO: 166)
gXTN-4T:
TCTAGAGGTCTCACATGGActgaccccagaccaggtagtcgcaatcgcgtcaaacggagggggaaagcaagccc
tggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacAGAGACCGGATCC (SEQ ID
NO: 167) gXTN-5T:
TCTAGAGGTCTCAccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAaacgga
GGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAA
GACCAAGAGACCGGATCC (SEQ ID NO: 168) gXTN-6T:
TCTAGAGGTCTCAACCACGGCctgactcccgatcaagttgtagcgattgcgtcgAACGGTggagggaaaca
agcattggagactgtccaacggctccttcccgtgttgtgtcaagcccAGAGACCGGATCC (SEQ
ID NO: 169) gXTN-7T:
TCTAGAGGTCTCAgcccacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAGCaatgg
cGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCA
GGATAGAGACCGGATCC (SEQ ID NO: 170) gXTN-8T:
TCTAGAGGTCTCAGGATCATGGActgaccccagaccaggtagtcgcaatcgcgtcaAACGGAggggga
aagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaAGAGACCGGATCC
(SEQ ID NO: 371) gXTN-9T:
TCTAGAGGTCTCAaagaccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAA
ACGGAGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTT
GTCAAGAGAGACCGGATCC (SEQ ID NO: 172) gXTN-10T:
TCTAGAGGTCTCACAAGACCACGGCctgactcccgatcaagttgtagcgattgcgtccaacggtggagggaa
acaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccaTggAAGAGACCGGATCC
(SEQ ID NO: 173) gXTN-1A:
TCTAGAGGTCTCATTGACCCCAGACCAGGTAGTCGCAATCGCGTCAaacattGGGGG
AAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCA
CGGCAGAGACCGGATCC (SEQ ID NO: 174) gXTN-2A:
TCTAGAGGTCTCACGGCctgactcccgatcaagttgtagcgattgcgtcgaacattggagggaaacaagcattg-
gaga ctgtccaacggctccttcccgtgttgtgtcaagcccacggAGAGACCGGATCC (SEQ ID
NO: 175) gXTN-3A:
TCTAGAGGTCTCAacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAGCaatattGG
CGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGA
TCATGAGAGACCGGATCC (SEQ ID NO: 176) gXTN-4A:
TCTAGAGGTCTCACATGGActgaccccagaccaggtagtcgcaatcgcgtcaAACATTgggggaaagcaag
ccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacAGAGACCGGATCC (SEQ
ID NO: 177) gXTN-5A:
TCTAGAGGTCTCAccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACA
TTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTC
AAGACCAAGAGACCGGATCC (SEQ ID NO: 178) gXTN-6A:
TCTAGAGGTCTCAACCAtGGCctgactcccgatcaagttgtagcgattgcgtcgaacattggagggaaacaagc-
att ggagactgtccaacggctccttcccgtgttgtgtcaagcccAGAGACCGGATCC (SEQ ID
NO: 179) gXTN-7A:
TCTAGAGGTCTCAgcccacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAAT
ATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGC
CAGGATAGAGACCGGATCC (SEQ ID NO: 180) gXTN-8A:
TCTAGAGGTCTCAGGATCATGGActgaccccagaccaggtagtcgcaatcgcgtcgaacattgggggaaagc
aagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaAGAGACCGGATCC (SEQ ID
NO: 181) gXTN-9A:
TCTAGAGGTCTCAaagaccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGA
ACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTT
GTCAAGAGAGACCGGATCC (SEQ ID NO: 182) gXTN-10A:
TCTAGAGGTCTCACAAGACCACGGCctgactcccgatcaagttgtagcgattgcgtcgAACATTggagg
gaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccaTggAAGAGACCGGATCC
(SEQ ID NO: 183) gXTN-1G:
TCTAGAGGTCTCATTGACCCCAGACCAGGTAGTCGCAATCGCGaacaataatGGGGGA
AAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCAC
GGCAGAGACCGGATCC (SEQ ID NO: 184) gXTN-2G:
TCTAGAGGTCTCACGGCctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaacaagcattg-
gag actgtccaacggctccttcccgtgttgtgtcaagcccacggAGAGACCGGATCC (SEQ ID
NO: 185) gXTN-3G:
TCTAGAGGTCTCAacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAA
CGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCA
GGATCATGAGAGACCGGATCC (SEQ ID NO: 186) gXTN-4G:
TCTAGAGGTCTCACATGGActgaccccagaccaggtagtcgcaatcgcgaacaataatgggggaaagcaagccc-
t ggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacAGAGACCGGATCC (SEQ ID
NO: 187) gXTN-5G:
TCTAGAGGTCTCAccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGAACAATA
ATGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTC
AAGACCAAGAGACCGGATCC (SEQ ID NO: 188) gXTN-6G:
TCTAGAGGTCTCAACCAtGGCctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaacaagc-
at tggagactgtccaacggctccttcccgtgttgtgtcaagcccAGAGACCGGATCC (SEQ ID
NO: 189) gXTN-7G:
TCTAGAGGTCTCAgcccacggtTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAAC
AACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGC
CAGGATAGAGACCGGATCC (SEQ ID NO: 190) gXTN-8G:
TCTAGAGGTCTCAGGATCATGGActgaccccagaccaggtagtcgcaatcgcgaacaataatgggggaaagc
aagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaAGAGACCGGATCC (SEQ ID
NO: 191) gXTN-9G:
TCTAGAGGTCTCAaagaccacggcCTGACCCCAGACCAGGTAGTCGCAATCGCGAACA
ATAATGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTT
GTCAAGAGAGACCGGATCC (SEQ ID NO: 192) gXTN-10G:
TCTAGAGGTCTCACAAGACCACGGCctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaa
caagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccaTggAAGAGACCGGATCC
(SEQ ID NO: 193) Sbjl and Sacl flanked pFUS fragments (gene
synthesized, Sbfl-pFUS-SacI) pFUS-X:
(Sbfl)CCTGCAGGTCGACCGTCTCAGAACTTGAAGAGACCGTACGTGATCGTGGTC
TCATggaTTGAAGAGACGGGTACCGAGCTC(Sacl) (SEQ ID NO: 194) pFUS-Z1:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCACGGCctgaA
GAGACGGGTACCGAGCTC (SEQ ID NO: 195) pFUS-Z2:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAacggtctgaA
GAGACGGGTACCGAGCTC (SEQ ID NO: 196) pFUS-Z3:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCACATGGAct
gaAGAGACGGGTACCGAGCTC (SEQ ID NO: 197) pFUS-Z4:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAccacggcctga
AGAGACGGGTACCGAGCTC (SEQ ID NO: 198) pFUS-Z5:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAACCACGG
CctgaAGAGACGGGTACCGAGCTC (SEQ ID NO: 199) pFUS-Z6:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAgcccacggtct
gaAGAGACGGGTACCGAGCTC (SEQ ID NO: 200) pFUS-Z7:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAGGATCAT
GGActgaAGAGACGGGTACCGAGCTC (SEQ ID NO: 201) pFUS-Z8:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCAaagaccacggc
ctgaAGAGACGGGTACCGAGCTC (SEQ ID NO: 202) pFUS-Z9:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCACAAGACC
ACGGCctgaAGAGACGGGTACCGAGCTC (SEQ ID NO: 203) pFUS-Z10:
CCTGCAGGTCGACCGTCTCATTGAAGAGACCGTACTGgatcgtGGTCTCATggActgaA
GAGACGGGTACCGAGCTC (SEQ ID NO: 204)
Example 7
TABLE-US-00012 [0309] Methylesterases and Methyltransferases 34 aa
Consensus EBE (nn is replaced with relevant RVD): QTTERIVAIGL (SEQ
ID NO: 211) nn GGTQALEAVLTALPRVCPGMV (SEQ ID NO: 212) Backtranseq
of 34aa QTTERIVAIGT (SEQ ID NO: 211) SH GGTQALEAVLTALPRVCPGMV (SEQ
ID NO: 212) (SH is a non-specific RVD)
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCT
GGAGGCCGTGCTGACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG (SEQ ID NO: 102)
Methylesterase EB (14EBEs in XTN backbone): Bold Font:
Methylesterase EBEs. All with non-specific RVD SH in this example.
Black Font: FLASH XTN Backbone. The sequence is contiguous (SEQ ID
NO: 207):
GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC-
CA
GTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG-
GC
TTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATA-
TA
CGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG-
GAG
TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAT-
AA
TGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAgTATTTACGGTAAACT-
GC
CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG-
CC
TGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT-
TAC
CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC-
AC
CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGC-
CC
CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAA-
C
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCACCATGGACTA-
CA
AAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAG-
A
AGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAG-
G
AGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCAT-
G
CGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCG-
GC
CCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGC-
T
GCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGA-
G
AGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAAC
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CAGACCACCGAGAGGATCGTGGCCATCGGCACCAGCCACGGCGGCACCCAGGCCCTGGAGGCCGTGCT
GACCGCCCTGCCCAGGGTGTGCCCCGGCATGGTG
CTGACACCCGAACAGGTGGTCGCCATTGCTAATAATAACGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCA-
A
TTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACG-
AC
CCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCC-
G AGAGAACTTCCCATCGAGTCGCGGGATCCCAACTAGTC
Sequence CWU 1
1
237135PRTArtificial SequenceRalstonia DNA-binding peptide
1MISC_FEATURE(12)..(12)Xaa = A, N, H, R or
GMISC_FEATURE(13)..(13)Xaa = I, N, H, K, Y, T, D, S, or P 1Leu Ser
Thr Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys1 5 10 15Gln
Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25
30Pro Tyr Glu 35235PRTArtificial SequenceRalstonia DNA-binding
peptide 2 2Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Ser Ile Gly
Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu
Arg Ala Ala 20 25 30Pro Tyr Glu 35335PRTArtificial
SequenceRalstonia DNA-binding peptide 3 3Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Ser Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
35435PRTArtificial SequenceRalstonia DNA-binding peptide 4 4Leu Ser
Thr Glu Gln Val Val Ala Ile Ala Ser Ser His Gly Gly Lys1 5 10 15Gln
Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25
30Pro Tyr Glu 35535PRTArtificial SequenceRalstonia DNA-binding
peptide 5 5Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Pro Gly
Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu
Arg Ala Ala 20 25 30Pro Tyr Glu 35635PRTArtificial
SequenceRalstonia DNA-binding peptide 6 6Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn His Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
35735PRTArtificial SequenceRalstonia DNA-binding peptide 7 7Leu Ser
Thr Glu Gln Val Val Ala Ile Ala Ser Asn Thr Gly Gly Lys1 5 10 15Gln
Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25
30Pro Tyr Glu 35835PRTArtificial SequenceRalstonia DNA-binding
peptide 8 8Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly
Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu
Arg Ala Ala 20 25 30Pro Tyr Glu 35935PRTArtificial
SequenceRalstonia DNA-binding peptide 9 9Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Pro Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
351035PRTArtificial SequenceRalstonia DNA-binding peptide 10 10Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala
20 25 30Pro Tyr Glu 351135PRTArtificial SequenceRalstonia
DNA-binding peptide 11 11Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu 351235PRTArtificial
SequenceRalstonia DNA-binding peptide 12 12Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
351335PRTArtificial SequenceRalstonia DNA-binding peptide 13 13Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser His Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala
20 25 30Pro Tyr Glu 351435PRTArtificial SequenceRalstonia
DNA-binding peptide 14 14Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser His Tyr Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu 351535PRTArtificial
SequenceRalstonia DNA-binding peptide 15 15Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
351635PRTArtificial SequenceRalstonia DNA-binding peptide 16 16Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser His His Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala
20 25 30Pro Tyr Glu 351735PRTArtificial SequenceRalstonia
DNA-binding peptide 17 17Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Arg Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu 351835PRTArtificial
SequenceRalstonia DNA-binding peptide 18 18Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Arg Ser Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
351935PRTArtificial SequenceRalstonia DNA-binding peptide 19 19Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Gly Ser Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala
20 25 30Pro Tyr Glu 352024DNAArtificial SequenceoJS2581 primer
20tctagagaag acaagaacct gacc 242124DNAArtificial SequenceoJS2582
primer 21ggatccggtc tcttaaggcc gtgg 242228DNAArtificial
SequenceoSQT34 primer 22gacggtggct gtcaaatacc aagatatg
282330DNAArtificial SequenceoSQT35 primer 23tctcctccag ttcacttttg
actagttggg 302420DNAArtificial SequenceoSQT1 primer 24agtaacagcg
gtagaggcag 202525DNAArtificial SequenceoJS2980 primer 25ttaattcaat
atattcatga ggcac 2526146DNAArtificial SequenceDNA-binding peptide
insert 1 26atgcatctag agaagacaac tgagcaccga gcaggtggtg gccatcgcca
gcaacaaggg 60cggcaagcag gccctggagg ccgtgaaggc ccacctgctg gacctgctgg
gcgcccccta 120cgagctgaag agaccggatc ccgggc 14627146DNAArtificial
SequenceDNA-binding peptide insert 2 27atgcatctag agaagacaac
tgagcaccga gcaggtggtg gccatcgcca gcaacaacgg 60cggcaagcag gccctggagg
ccgtgaaggc ccagctgctg gagctgaggg ccgcccccta 120cgagctgaag
agaccggatc ccgggc 14628146DNAArtificial SequenceDNA-binding peptide
insert 3 28atgcatctag agaagacaac tgagcaccga gcaggtggtg gccatcgcca
gcaacggcgg 60cggcaagcag gccctggagg gcatcggcga gcagctgctg aagctgagga
ccgcccccta 120cgagctgaag agaccggatc ccgggc 14629146DNAArtificial
SequenceDNA-binding peptide insert 4 29atgcatctag agaagacaac
tgagcaccga gcaggtggtg gccatcgcca gccacgacgg 60cggcaagccc gccctggagg
ccgtgtgggc caagctgccc gtgctgaggg gcgtgcccta 120cgagctgaag
agaccggatc ccgggc 14630146DNAArtificial SequenceDNA-binding peptide
insert 5 30atgcatctag agaagacaac tgagcaccga gcaggtggtg accatcgcca
gcagcatcgg 60cggcaagcag gccctggagg ccgtgaaggt gcagctgccc gtgctgaggg
ccgcccccta 120cgagctgaag agaccggatc ccgggc 1463134PRTArtificial
SequenceDNA-binding fragment 1 31Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25 30His
Gly3234PRTArtificial SequenceDNA-binding fragment 2 32Leu Thr Pro
Glu Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly Lys1 5 10 15Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30His
Gly3334PRTArtificial SequenceDNA-binding fragment 3 33Leu Thr Pro
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30His
Gly3434PRTArtificial SequenceDNA-binding fragment 4 34Leu Thr Pro
Ala Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys1 5 10 15Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25 30His
Gly3529PRTPseudomonas mendocina DLHK 35Thr Thr Asp Arg Val Val Ala
Leu Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Val Val Leu
Arg Gln Leu Pro Val Asp Cys 20 253630PRTGallionella
capsiferriformans ES-2 36Met Thr Ser Glu Gln Ile Val Ala Ile Gly
Thr Ser Thr Gly Gly Thr1 5 10 15Gln Ala Leu Glu Ala Val Leu Thr Ala
Leu Pro Arg Val Cys 20 25 303729PRTMethylobacter tundripaludum SV96
37Thr Thr Asp Arg Val Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1
5 10 15Ala Leu Glu Val Val Leu Thr Ala Leu Pro Arg Val Cys 20
253829PRTPseudogulbenkiania sp. NH8B 38Thr Thr Glu Arg Ile Val Ala
Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Thr Val Leu
His Arg Leu Pro Ala Thr Cys 20 253929PRTDechlorosoma suillum PS
39Thr Thr Asp Lys Ile Ile Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1
5 10 15Ala Leu Glu Ala Val Leu Thr Lys Leu Pro Ala Val Cys 20
254029PRTPseudomonas fuscovaginae UPB0736 40Thr Thr Glu Arg Ile Val
Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Thr Val
Leu Thr Ala Leu Pro Arg Val Cys 20 254129PRTArtificial
SequenceDNA-binding fragment 11 41Thr Thr Glu Arg Val Val Ala Leu
Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Val Val Leu Arg
Thr Leu Pro Arg Val Cys 20 254229PRTArtificial SequenceDNA-binding
fragment 12 42Thr Thr Asp Gln Leu Ile Ala Ile Gly Thr Ser Thr Gly
Gly Thr Gln1 5 10 15Ala Leu Glu Ala Ile Leu Thr Lys Leu Pro Ala Thr
Cys 20 254329PRTArtificial SequenceDNA-binding fragment 13 43Thr
Thr Asp Gln Leu Ile Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10
15Ala Leu Glu Ala Ile Leu Thr Lys Leu Pro Ala Thr Cys 20
254429PRTPseudogulbenkiania ferrooxidans 2002 44Thr Thr Glu Arg Ile
Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Thr
Val Leu Pro Arg Leu Pro Ala Thr Cys 20 254529PRTJanthinobacterium
lividum PAMC 25724 45Thr Ser Asp Lys Val Val Ala Ile Gly Ala Ser
Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Leu Leu Leu Thr Gly Leu Pro
Ala Val Cys 20 254629PRTBurkholderia rhizoxinica HKI 454 46Phe Ser
Gln Ala Asp Ile Val Arg Ile Ala Asp Asn Ile Gly Gly Ala1 5 10 15Gln
Ala Leu Lys Ala Val Leu Glu His Gly Pro Thr Leu 20
254728PRTArtificial SequenceDNA-binding fragment 17 47Ala Asp Ile
Val Lys Ile Ala Ser Asn Gly Gly Gly Ala Gln Ala Leu1 5 10 15Glu Ala
Val Ala Met His Gly Ser Thr Leu Cys Glu 20 254828PRTBurkholderia
rhizoxinica HKI 454 48Ala Asp Ile Val Lys Ile Ala Gly Asn Gly Gly
Gly Ala Arg Ala Leu1 5 10 15Lys Ala Val Val Met His Gly Pro Thr Leu
Cys Glu 20 254929PRTPseudomonas fuscovaginae UPB0736 49Thr Thr Asp
Arg Val Val Ala Leu Gly Cys Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu
Glu Phe Ile Leu Arg Gln Leu Pro Arg Asp Cys 20 255024PRTPseudomonas
syringae Cit 7 50Ala Leu Ala Ala Ala Val Gly Gly Lys Gly Ala Leu
Glu Val Pro Ala1 5 10 15Asn Leu Ile Pro Ala Asn Cys Glu
205123PRTBurkholderia sp. CCGE1003 51Arg Ile Val Ala Ile Gly Thr
Ser Thr Gly Gly Thr Gln Ala Leu Glu1 5 10 15Val Val Leu Thr Ala Leu
Pro 205234PRTArtificial SequenceDNA-binding fragment 22 52Leu Thr
Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1 5 10 15Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25
30His Gly5334PRTArtificial SequenceDNA-binding fragment 23 53Leu
Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys1 5 10
15Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
20 25 30His Gly5434PRTArtificial SequenceDNA-binding fragment 24
54Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala 20 25 30His Gly5534PRTArtificial SequenceDNA-binding fragment
25 55Leu Thr Pro Glu Gln Val Val Ala Ile Ala Asn Asn Asn Gly Gly
Lys1 5 10 15Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala 20 25 30His Gly5629PRTPseudomonas mendocina DLHK 56Thr Thr
Asp Arg Val Val Ala Leu Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala
Leu Glu Val Val Leu Arg Gln Leu Pro Val Asp Cys 20
255729PRTPseudomonas fuscovaginae UPB0736 57Thr Thr Asp Arg Val Val
Ala Leu Gly Cys Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Phe Ile
Leu Arg Gln Leu Pro Arg Asp Cys 20 255829PRTLeptothrix cholodnii
SP-6 58Thr Thr Glu Arg Val Val Ala Leu Gly Thr Ser Thr Gly Gly Thr
Gln1 5 10 15Ala Leu Glu Val Val Leu Arg Thr Leu Pro Arg Val Cys 20
255929PRTArtificial SequenceDNA-binding fragment 29 59Thr Thr Asp
Arg Val Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu
Glu Val Val Leu Thr Ala Leu Pro Arg Val Cys 20 256029PRTArtificial
SequenceDNA-binding fragment 30 60Thr Thr Glu Arg Ile Val Ala Ile
Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Thr Val Leu Pro
Arg Leu Pro Ala Thr Cys 20 256129PRTDechlorosoma suillum PS 61Thr
Thr Asp Lys Ile Ile Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10
15Ala Leu Glu Ala Val Leu Thr Lys Leu Pro Ala Val Cys 20
256229PRTUnknownuncultured bacterium 62Thr Thr Asp Gln Leu Ile Ala
Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Ile Leu
Thr Lys Leu Pro Ala Thr Cys 20 256329PRTJanthinobacterium lividum
PAMC 25724 63Thr Ser Asp Lys Val Val Ala Ile Gly Ala Ser Thr Gly
Gly Thr Gln1 5 10 15Ala Leu Glu Leu Leu Leu Thr Gly Leu Pro Ala Val
Cys 20 256428PRTBurkholderia rhizoxinica HKI 454 64Ala Asp Ile Val
Lys Ile Ala Ser Asn Gly Gly Gly Ala Gln Ala Leu1 5 10 15Glu Ala Val
Ala Met His Gly Ser Thr Leu Cys Glu 20 256528PRTBurkholderia
rhizoxinica HKI 454 65Ala Asp Ile Val Lys Ile Ala Gly Asn Gly Gly
Gly Ala Arg Ala Leu1 5 10 15Lys Ala Val Val Met His Gly Pro Thr Leu
Cys Glu 20 256629PRTBurkholderia rhizoxinica HKI 454 66Phe Ser Gln
Ala Asp Ile Val Arg Ile Ala Asp Asn Ile Gly Gly Ala1 5 10 15Gln Ala
Leu Lys Ala Val Leu Glu His Gly Pro Thr Leu 20 256724PRTPseudomonas
syringae Cit 7 67Ala Leu Ala Ala Ala Val Gly Gly Lys Gly Ala Leu
Glu Val Pro Ala1 5 10 15Asn Leu Ile Pro Ala Asn Cys Glu
2068229DNAArtificial SequenceSleeping Beauty 5' Inverted Tandem
Repeat 68cagttgaagt cggaagttta catacactta agttggagtc attaaaactc
gtttttcaac
60tactccacaa atttcttgtt aacaaacaat agttttggca agtcagttag gacatctact
120ttgtgcatga cacaagtcat ttttccaaca attgtttaca gacagattat
ttcacttata 180attcactgta tcacaattcc agtgggtcag aagtttacat acactaagt
22969229DNAArtificial SequenceSleeping Beauty 3' Inverted Tandem
Repeat 69attgagtgta tgtaaacttc tgacccactg ggaatgtgat gaaagaaata
aaagctgaaa 60tgaatcattc tctctactat tattctgata tttcacattc ttaaaataaa
gtggtgatcc 120taactgacct aagacaggga atttttacta ggattaaatg
tcaggaattg tgaaaaagtg 180agtttaaatg tatttggcta aggtgtatgt
aaacttccga cttcaactg 22970309DNAArtificial SequencepiggyBac 5'
Inverted Tandem Repeat 70ccctagaaag atagtctgcg taaaattgac
gcatgcattc ttgaaatatt gctctctctt 60tctaaatagc gcgaatccgt cgctgtgcat
ttaggacatc tcagtcgccg cttggagctc 120ccgtgaggcg tgcttgtcaa
tgcggtaagt gtcactgatt ttgaactata acgaccgcgt 180gagtcaaaat
gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
240ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata
ttttcttgtt 300atagatatc 30971238DNAArtificial SequencepiggyBac 3'
Inverted Tandem Repeat 71taaaagtttt gttactttat agaagaaatt
ttgagttttt gttttttttt aataaataaa 60taaacataaa taaattgttt gttgaattta
ttattagtat gtaagtgtaa atataataaa 120acttaatatc tattcaaatt
aataaataaa cctcgatata cagaccgata aaacacatgc 180gtcaatttta
cgcatgatta tctttaacgt acgtcacaat atgattatct ttctaggg
2387235PRTArtificial SequenceRalstonia DNA-binding peptide 20 72Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu Leu Gly Ala
20 25 30Pro Tyr Val 357335PRTArtificial SequenceRalstonia
DNA-binding peptide 21 73Leu Ser Thr Glu Gln Val Val Ala Val Ala
Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Ala Ala Val Glu Ala Gln
Leu Leu Arg Leu Arg Ala Ala 20 25 30Pro Tyr Glu 357435PRTArtificial
SequenceRalstonia DNA-binding peptide 22 74Leu Asn Thr Glu Gln Val
Val Ala Val Ala Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Gly Ala Gln Leu Leu Ala Leu Arg Ala Val 20 25 30Pro Tyr Ala
357535PRTArtificial SequenceRalstonia DNA-binding peptide 24 75Leu
Ser Thr Glu Gln Val Val Ala Val Ala Ser Asn Lys Gly Gly Lys1 5 10
15Gln Val Leu Glu Ala Val Gly Ala Gln Leu Leu Ala Leu Arg Ala Val
20 25 30Pro Tyr Glu 357635PRTArtificial SequenceRalstonia
DNA-binding peptide 24 76Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala His
Leu Leu Asp Leu Leu Gly Ala 20 25 30Pro Tyr Val 357735PRTArtificial
SequenceRalstonia DNA-binding peptide 25 77Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala His Leu Leu Asp Leu Leu Gly Ala 20 25 30Pro Tyr Val
357835PRTArtificial SequenceRalstonia DNA-binding peptide 26 78Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu Leu Gly Ala
20 25 30Pro Tyr Val 357935PRTArtificial SequenceRalstonia
DNA-binding peptide 27 79Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Glu Leu Arg Gly Ala 20 25 30Pro Tyr Ala 358035PRTArtificial
SequenceRalstonia DNA-binding peptide 28 80Leu Ser Thr Glu Gln Val
Val Ala Val Ala Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Ala Ala
Val Glu Ala Gln Leu Leu Arg Leu Arg Ala Ala 20 25 30Pro Tyr Glu
358135PRTArtificial SequenceRalstonia DNA-binding peptide 29 81Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu Leu Gly Ala
20 25 30Pro Tyr Val 358235PRTArtificial SequenceRalstonia
DNA-binding peptide 30 82Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Lys Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala His
Leu Leu Asp Leu Leu Gly Ala 20 25 30Pro Tyr Val 358335PRTArtificial
SequenceRalstonia DNA-binding peptide 31 83Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Asp Leu Arg Gly Ala 20 25 30Pro Tyr Ala
358435PRTArtificial SequenceRalstonia DNA-binding peptide 32 84Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Pro Val Leu Arg Arg Ala
20 25 30Pro Tyr Gly 358535PRTArtificial SequenceRalstonia
DNA-binding peptide 33 85Leu Ser Pro Glu Gln Val Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu 358635PRTArtificial
SequenceRalstonia DNA-binding peptide 34 86Leu Ser Pro Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala
Val Lys Ala Leu Leu Leu Ala Leu Arg Ala Ala 20 25 30Pro Tyr Glu
358735PRTArtificial SequenceRalstonia DNA-binding peptide 35 87Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Pro Ala Leu Glu Ala Val Lys Ala Leu Leu Leu Glu Leu Arg Ala Ala
20 25 30Pro Tyr Glu 358835PRTArtificial SequenceRalstonia
DNA-binding peptide 36 88Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Thr Gln
Leu Leu Ala Leu Arg Thr Ala 20 25 30Pro Tyr Glu 358935PRTArtificial
SequenceRalstonia DNA-binding peptide 37 89Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Pro Ala Leu Arg Ala Ala 20 25 30Pro Tyr Glu
359035PRTArtificial SequenceRalstonia DNA-binding peptide 38 90Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Val Leu Arg Ala Ala
20 25 30Pro Tyr Gly 359135PRTArtificial SequenceRalstonia
DNA-binding peptide 39 91Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ala Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Arg Ala Leu
Leu Pro Val Leu Arg Val Ala 20 25 30Pro Tyr Glu 359235PRTArtificial
SequenceRalstonia DNA-binding peptide 40 92Leu Ser Pro Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu
359335PRTArtificial SequenceRalstonia DNA-binding peptide 41 93Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Glu Leu Arg Ala Ala
20 25 30Pro Tyr Glu 359435PRTArtificial SequenceRalstonia
DNA-binding peptide 42 94Leu Ser Pro Glu Gln Val Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu 359535PRTArtificial
SequenceRalstonia DNA-binding peptide 43 95Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Gln Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu
359635PRTArtificial SequenceRalstonia DNA-binding peptide 44 96Leu
Ser Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Glu Leu Arg Ala Ala
20 25 30Pro Tyr Glu 359735PRTArtificial SequenceRalstonia
DNA-binding peptide 45 97Leu Ser Thr Glu Gln Val Val Val Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Ala Leu Arg Ala Ala 20 25 30Pro Tyr Glu 359835PRTArtificial
SequenceRalstonia DNA-binding peptide 46 98Leu Ser Thr Glu Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala
Val Lys Ala Leu Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu
359935PRTArtificial SequenceRalstonia DNA-binding peptide 47 99Leu
Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1 5 10
15Gln Ala Leu Glu Ala Val Lys Thr Gln Leu Leu Ala Leu Arg Thr Ala
20 25 30Pro Tyr Glu 3510035PRTArtificial SequenceRalstonia
DNA-binding peptide 48 100Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Pro Ala Leu Arg Ala Ala 20 25 30Pro Tyr Glu
3510135PRTArtificial SequenceRalstonia DNA-binding peptide 49
101Leu Ser Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys1
5 10 15Gln Ala Leu Glu Ala Val Arg Ala Leu Leu Pro Val Leu Arg Val
Ala 20 25 30Pro Tyr Glu 3510235PRTArtificial SequenceRalstonia
DNA-binding peptide 50 102Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln
Leu Leu Lys Leu Arg Thr Ala 20 25 30Pro Tyr Gly
3510335PRTArtificial SequenceRalstonia DNA-binding peptide 51
103Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1
5 10 15Gln Ala Leu Glu Gly Ile Gly Lys Gln Leu Gln Glu Leu Arg Ala
Ala 20 25 30Pro His Gly 3510435PRTArtificial SequenceRalstonia
DNA-binding peptide 52 104Leu Ser Thr Gly Gln Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Arg1 5 10 15Gln Ala Leu Glu Ala Val Arg Glu Gln
Leu Leu Ala Leu Arg Ala Val 20 25 30Pro Tyr Glu
3510535PRTArtificial SequenceRalstonia DNA-binding peptide 53
105Leu Ser Thr Gly Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg1
5 10 15Gln Ala Leu Glu Ala Val Arg Glu Gln Leu Leu Ala Leu Arg Ala
Val 20 25 30Pro Tyr Glu 3510635PRTArtificial SequenceRalstonia
DNA-binding peptide 54 106Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln
Leu Leu Lys Leu Arg Thr Ala 20 25 30Pro Tyr Gly
3510735PRTArtificial SequenceRalstonia DNA-binding peptide 55
107Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1
5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu Lys Leu Arg Thr
Ala 20 25 30Pro Tyr Gly 3510835PRTArtificial SequenceRalstonia
DNA-binding peptide 56 108Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln
Leu Arg Lys Leu Arg Thr Ala 20 25 30Pro Tyr Gly
3510935PRTArtificial SequenceRalstonia DNA-binding peptide 57
109Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1
5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu Lys Leu Arg Thr
Ala 20 25 30Pro Tyr Gly 3511035PRTArtificial SequenceRalstonia
DNA-binding peptide 58 110Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Gly Gly Gly Lys1 5 10 15Gln Ala Leu Glu Gly Ile Gly Lys Gln
Leu Gln Glu Leu Arg Ala Ala 20 25 30Pro His Gly
3511135PRTArtificial SequenceRalstonia DNA-binding peptide 59
111Leu Asn Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Val 20 25 30Pro Tyr Glu 3511235PRTArtificial SequenceRalstonia
DNA-binding peptide 60 112Leu Asn Thr Ala Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys
Leu Pro Val Leu Arg Gly Val 20 25 30Pro Tyr Glu
3511335PRTArtificial SequenceRalstonia DNA-binding peptide 61
113Leu Asn Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Val 20 25 30Pro Tyr Ala 3511435PRTArtificial SequenceRalstonia
DNA-binding peptide 62 114Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Gly Ala Gln
Leu Val Ala Leu Arg Ala Ala 20 25 30Pro Tyr Ala
3511535PRTArtificial SequenceRalstonia DNA-binding peptide 63
115Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Asn1
5 10 15Gln Ala Leu Glu Ala Val Gly Thr Gln Leu Val Ala Leu Arg Ala
Ala 20 25 30Pro Tyr Ala 3511635PRTArtificial SequenceRalstonia
DNA-binding peptide 64 116Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Gly Ala Gln
Leu Val Ala Leu Arg Ala Ala 20 25 30Pro Tyr Ala
3511735PRTArtificial SequenceRalstonia DNA-binding peptide 65
117Leu Asn Thr Ala Gln Ile Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Ala 20 25 30Pro Tyr Ala 3511835PRTArtificial SequenceRalstonia
DNA-binding peptide 66 118Leu Ser Thr Ala Gln Val Val Ala Val Ala
Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Arg Lys Gln
Leu Pro Val Leu Arg Gly Val 20 25 30Pro His Gln
3511935PRTArtificial SequenceRalstonia DNA-binding peptide 67
119Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Ala 20 25 30Pro Tyr Ala 3512035PRTArtificial SequenceRalstonia
DNA-binding peptide 68 120Leu Asn Thr Ala Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys
Leu Pro Val Leu Arg Gly Val 20 25 30Pro Tyr Glu
3512135PRTArtificial SequenceRalstonia DNA-binding peptide 69
121Leu Asn Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Val 20 25 30Pro Tyr Ala 3512235PRTArtificial SequenceRalstonia
DNA-binding peptide 70 122Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Gly Ala Gln
Leu Val Glu Leu Arg Ala Ala 20 25 30Pro Tyr Ala
3512335PRTArtificial SequenceRalstonia DNA-binding peptide 71
123Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Gly Ala Gln Leu Val
Ala Leu Arg Ala Ala 20 25 30Pro Tyr Ala 3512435PRTArtificial
SequenceRalstonia DNA-binding peptide 72 124Leu Asn Thr Ala Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala
Val Arg Ala Lys Leu Pro Val Leu Arg Gly Val 20 25 30Pro Tyr Ala
3512535PRTArtificial SequenceRalstonia DNA-binding peptide 73
125Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys1
5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
Val 20 25 30Pro Tyr Ala 3512635PRTArtificial SequenceRalstonia
DNA-binding peptide 74 126Leu Thr Pro Gln Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys
Leu Pro Val Leu Arg Gly Val 20 25 30Pro Tyr Ala
3512735PRTArtificial SequenceRalstonia DNA-binding peptide 75
127Leu Ser Thr Ala Gln Val Ala Thr Ile Ala Ser Ser Ile Gly Gly Arg1
5 10 15Gln Ala Leu Glu Ala Val Lys Val Gln Leu Pro Val Leu Arg Ala
Ala 20 25 30Pro Tyr Gly 3512835PRTArtificial SequenceRalstonia
DNA-binding peptide 76 128Leu Ser Thr Glu Gln Val Val Val Ile Ala
Asn Ser Ile Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Val Gln
Leu Pro Val Leu Arg Ala Ala 20 25 30Pro Tyr Glu
3512935PRTArtificial SequenceRalstonia DNA-binding peptide 77
129Leu Ser Thr Glu Gln Val Val Val Ile Ala Asn Ser Ile Gly Gly Lys1
5 10 15Gln Ala Leu Glu Ala Val Lys Val Gln Leu Pro Val Leu Arg Ala
Ala 20 25 30Pro Tyr Glu 3513035PRTArtificial SequenceRalstonia
DNA-binding peptide 78 130Leu Ser Thr Ala Gln Val Ala Thr Ile Ala
Ser Ser Ile Gly Gly Arg1 5 10 15Gln Ala Leu Glu Ala Val Lys Val Gln
Leu Pro Val Leu Arg Ala Ala 20 25 30Pro Tyr Gly
3513135PRTArtificial SequenceRalstonia DNA-binding peptide 79
131Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly Lys1
5 10 15Gln Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu Leu Gly
Ala 20 25 30Pro Tyr Val 3513235PRTArtificial SequenceRalstonia
DNA-binding peptide 80 132Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Asn Gly Gly Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln
Leu Leu Glu Leu Arg Ala Ala 20 25 30Pro Tyr Glu
3513335PRTArtificial SequenceRalstonia DNA-binding peptide 81
133Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys1
5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu Lys Leu Arg Thr
Ala 20 25 30Pro Tyr Gly 3513435PRTArtificial SequenceRalstonia
DNA-binding peptide 82 134Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly Lys1 5 10 15Pro Ala Leu Glu Ala Val Trp Ala Lys
Leu Pro Val Leu Arg Gly Val 20 25 30Pro Tyr Ala
3513535PRTArtificial SequenceRalstonia DNA-binding peptide 83
135Leu Ser Thr Glu Gln Val Val Thr Ile Ala Ser Ser Ile Gly Gly Lys1
5 10 15Gln Ala Leu Glu Ala Val Lys Val Gln Leu Pro Val Leu Arg Ala
Ala 20 25 30Pro Tyr Glu 3513629PRTPseudomonas syringae pv. pisi
str. 1704B 136Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly
Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val
Cys 20 2513730PRTGallionella capsiferriformans ES-2 137Met Thr Ser
Glu Gln Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr1 5 10 15Gln Ala
Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20 25
3013829PRTPseudomonas fuscovaginae UPB0736 138Thr Thr Glu Arg Ile
Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Thr
Val Leu Thr Ala Leu Pro Arg Val Cys 20 2513923PRTArtificial
SequenceDNA-binding fragment 41 139Arg Ile Val Ala Ile Gly Thr Ser
Thr Gly Gly Thr Gln Ala Leu Glu1 5 10 15Val Val Leu Thr Ala Leu Pro
2014029PRTArtificial SequenceDNA-binding fragment 42 140Thr Thr Glu
Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu
Glu Thr Val Leu His Arg Leu Pro Ala Thr Cys 20 2514152DNAArtificial
SequenceBmpr2 Target site 141ttgatagtcg ccttatgttt tggatacaga
atgttgacag gtaaacgaaa ta 5214216DNAArtificial SequenceForward RTN
142tgatagtcgc cttatg 1614316DNAArtificial SequenceReverse RTN
143atttggttta cctgtc 16144560PRTArtificial SequenceBmpr2 FWD RTN
EBE 144Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
Lys1 5 10 15Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu Lys Leu Arg
Thr Ala 20 25 30Pro Tyr Gly Leu Ser Thr Glu Gln Val Val Ala Ile Ala
Ser Asn Lys 35 40 45Gly Gly Lys Gln Ala Leu Glu Ala Val Lys Ala His
Leu Leu Asp Leu 50 55 60Leu Gly Ala Pro Tyr Val Leu Ser Thr Glu Gln
Val Val Ala Ile Ala65 70 75 80Ser Asn Asn Gly Gly Lys Gln Ala Leu
Glu Ala Val Lys Ala Gln Leu 85 90 95Leu Glu Leu Arg Ala Ala Pro Tyr
Glu Leu Ser Thr Ala Gln Val Val 100 105 110Ala Ile Ala Ser Asn Gly
Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly 115 120 125Glu Gln Leu Leu
Lys Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr Glu 130 135 140Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu145 150 155
160Ala Val Lys Ala Gln Leu Leu Glu Leu Arg Ala Ala Pro Tyr Glu Leu
165 170 175Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly
Lys Gln 180 185 190Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu
Leu Gly Ala Pro 195 200 205Tyr Val Leu Ser Thr Ala Gln Val Val Ala
Ile Ala Ser Asn Gly Gly 210 215 220Gly Lys Gln Ala Leu Glu Gly Ile
Gly Glu Gln Leu Leu Lys Leu Arg225 230 235 240Thr Ala Pro Tyr Gly
Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser 245 250 255His Asp Gly
Gly Lys Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro 260 265 270Val
Leu Arg Gly Val Pro Tyr Ala Leu Ser Thr Glu Gln Val Val Ala 275 280
285Ile Ala Ser Asn Lys Gly Gly Lys Gln Ala Leu Glu Ala Val Lys Ala
290 295 300His Leu Leu Asp Leu Leu Gly Ala Pro Tyr Val Leu Ser Thr
Ala Gln305 310 315 320Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
Pro Ala Leu Glu Ala 325 330 335Val Trp Ala Lys Leu Pro Val Leu Arg
Gly Val Pro Tyr Ala Leu Ser 340 345 350Thr Ala Gln Val Val Ala Ile
Ala Ser His Asp Gly Gly Lys Pro Ala 355 360 365Leu Glu Ala Val Trp
Ala Lys Leu Pro Val Leu Arg Gly Val Pro Tyr 370 375 380Ala Leu Ser
Thr Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly385 390 395
400Lys Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu Lys Leu Arg Thr
405 410 415Ala Pro Tyr Gly Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn 420 425 430Gly Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly Glu
Gln Leu Leu Lys 435 440 445Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr
Glu Gln Val Val Ala Ile 450 455 460Ala Ser Asn Asn Gly Gly Lys Gln
Ala Leu Glu Ala Val Lys Ala Gln465 470 475 480Leu Leu Glu Leu Arg
Ala Ala Pro Tyr Glu Leu Ser Thr Ala Gln Val 485 490 495Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Gly Ile 500 505 510Gly
Glu Gln Leu Leu Lys Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr 515 520
525Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly Lys Gln Ala Leu
530 535 540Glu Ala Val Lys Ala His Leu Leu Asp Leu Leu Gly Ala Pro
Tyr Val545 550 555 5601458106DNAArtificial SequenceBmpr2 FWD RTN
145gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc
tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg
atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
gggactttcc 420attgacgtca atgggtggag tatttacggt aaactgccca
cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg
tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta
tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc
cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa
gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa
attaatacga ctcactatag ggagacccaa gctggctagc 900accatggact
acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
960gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg
cggggtacct 1020atggtggact tgaggacact cggttattcg caacagcaac
aggagaaaat caagcctaag 1080gtcaggagca ccgtcgcgca acaccacgag
gcgcttgtgg ggcatggctt cactcatgcg 1140catattgtcg cgctttcaca
gcaccctgcg gcgcttggga cggtggctgt caaataccaa 1200gatatgattg
cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
1260tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct
tagggggcct 1320ccgctccagc tcgacaccgg gcagctgctg aagatcgcga
agagaggggg agtaacagcg 1380gtagaggcag tgcacgcctg gcgcaatgcg
ctcaccgggg cccccttgaa cctgagcacc 1440gcccaggtgg tggccatcgc
cagcaacggc ggcggcaagc aggccctgga gggcatcggc 1500gagcagctgc
tgaagctgag gaccgccccc tacggcctga gcaccgagca ggtggtggcc
1560atcgccagca acaagggcgg caagcaggcc ctggaggccg tgaaggccca
cctgctggac 1620ctgctgggcg ccccctacgt gctgagcacc gagcaggtgg
tggccatcgc cagcaacaac 1680ggcggcaagc aggccctgga ggccgtgaag
gcccagctgc tggagctgag ggccgccccc 1740tacgagctga gcaccgccca
ggtggtggcc atcgccagca acggcggcgg caagcaggcc 1800ctggagggca
tcggcgagca gctgctgaag ctgaggaccg ccccctacgg cctgagcacc
1860gagcaggtgg tggccatcgc cagcaacaac ggcggcaagc aggccctgga
ggccgtgaag 1920gcccagctgc tggagctgag ggccgccccc tacgagctga
gcaccgagca ggtggtggcc 1980atcgccagca acaagggcgg caagcaggcc
ctggaggccg tgaaggccca cctgctggac 2040ctgctgggcg ccccctacgt
gctgagcacc gcccaggtgg tggccatcgc cagcaacggc 2100ggcggcaagc
aggccctgga gggcatcggc gagcagctgc tgaagctgag gaccgccccc
2160tacggcctga gcaccgccca ggtggtggcc atcgccagcc acgacggcgg
caagcccgcc 2220ctggaggccg tgtgggccaa gctgcccgtg ctgaggggcg
tgccctacgc cctgagcacc 2280gagcaggtgg tggccatcgc cagcaacaag
ggcggcaagc aggccctgga ggccgtgaag 2340gcccacctgc tggacctgct
gggcgccccc tacgtgctga gcaccgccca ggtggtggcc 2400atcgccagcc
acgacggcgg caagcccgcc ctggaggccg tgtgggccaa gctgcccgtg
2460ctgaggggcg tgccctacgc cctgagcacc gcccaggtgg tggccatcgc
cagccacgac 2520ggcggcaagc ccgccctgga ggccgtgtgg gccaagctgc
ccgtgctgag gggcgtgccc 2580tacgccctga gcaccgccca ggtggtggcc
atcgccagca acggcggcgg caagcaggcc 2640ctggagggca tcggcgagca
gctgctgaag ctgaggaccg ccccctacgg cctgagcacc 2700gcccaggtgg
tggccatcgc cagcaacggc ggcggcaagc aggccctgga gggcatcggc
2760gagcagctgc tgaagctgag gaccgccccc tacggcctga gcaccgagca
ggtggtggcc 2820atcgccagca acaacggcgg caagcaggcc ctggaggccg
tgaaggccca gctgctggag 2880ctgagggccg ccccctacga gctgagcacc
gcccaggtgg tggccatcgc cagcaacggc 2940ggcggcaagc aggccctgga
gggcatcggc gagcagctgc tgaagctgag gaccgccccc 3000tacggcctga
gcaccgagca ggtggtggcc atcgccagca acaagggcgg caagcaggcc
3060ctggaggccg tgaaggccca cctgctggac ctgctgggcg ccccctacgt
gctgagcacc 3120gcccaggtgg tggccatcgc cagcaacggc ggaggacggc
cagccttgga gtccatcgta 3180gcccaattgt ccaggcccga tcccgcgttg
gctgcgttaa cgaatgacca tctggtggcg 3240ttggcatgtc ttggtggacg
acccgcgctc gatgcagtca aaaagggtct gcctcatgct 3300cccgcattga
tcaaaagaac caaccggcgg attcccgaga gaacttccca tcgagtcgcg
3360ggatcccaac tagtcaaaag tgaactggag gagaagaaat ctgaacttcg
tcataaattg 3420aaatatgtgc ctcatgaata tattgaatta attgaaattg
ccagaaattc cactcaggat 3480agaattcttg aaatgaaggt aatggaattt
tttatgaaag tttatggata tagaggtaaa 3540catttgggtg gatcaaggaa
accggacgga gcaatttata ctgtcggatc tcctattgat 3600tacggtgtga
tcgtggatac taaagcttat agcggaggtt ataatctgcc aattggccaa
3660gcagatgaaa tgcaacgata tgtcgaagaa aatcaaacac gaaacaaaca
tatcaaccct 3720aatgaatggt ggaaagtcta tccatcttct gtaacggaat
ttaagttttt atttgtgagt 3780ggtcacttta aaggaaacta caaagctcag
cttacacgat taaatcatat cactaattgt 3840aatggagctg ttcttagtgt
agaagagctt ttaattggtg gagaaatgat taaagccggc 3900acattaacct
tagaggaagt cagacggaaa tttaataacg gcgagataaa cttttaaggg
3960cccttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac
gcgtaccggt 4020catcatcacc atcaccattg agtttaaacc cgctgatcag
cctcgactgt gccttctagt 4080tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga aggtgccact 4140cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag taggtgtcat 4200tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
4260aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
cagctggggc 4320tctagggggt atccccacgc gccctgtagc ggcgcattaa
gcgcggcggg tgtggtggtt 4380acgcgcagcg tgaccgctac acttgccagc
gccctagcgc ccgctccttt cgctttcttc 4440ccttcctttc tcgccacgtt
cgccggcttt ccccgtcaag ctctaaatcg gggcatccct 4500ttagggttcc
gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
4560ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
gttggagtcc 4620acgttcttta atagtggact cttgttccaa actggaacaa
cactcaaccc tatctcggtc 4680tattcttttg atttataagg gattttgggg
atttcggcct attggttaaa aaatgagctg 4740atttaacaaa aatttaacgc
gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa 4800agtccccagg
ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
4860accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
catgcatctc 4920aattagtcag caaccatagt cccgccccta actccgccca
tcccgcccct aactccgccc 4980agttccgccc attctccgcc ccatggctga
ctaatttttt ttatttatgc agaggccgag 5040gccgcctctg cctctgagct
attccagaag tagtgaggag gcttttttgg aggcctaggc 5100ttttgcaaaa
agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
5160acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
aggaactaaa 5220ccatggccaa gcctttgtct caagaagaat ccaccctcat
tgaaagagca acggctacaa 5280tcaacagcat ccccatctct gaagactaca
gcgtcgccag cgcagctctc tctagcgacg 5340gccgcatctt cactggtgtc
aatgtatatc attttactgg gggaccttgt gcagaactcg 5400tggtgctggg
cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
5460gaaatgagaa caggggcatc ttgagcccct gcggacggtg tcgacaggtg
cttctcgatc 5520tgcatcctgg gatcaaagcg atagtgaagg acagtgatgg
acagccgacg gcagttggga 5580ttcgtgaatt gctgccctct ggttatgtgt
gggagggcta agcacttcgt ggccgaggag 5640caggactgac acgtgctacg
agatttcgat tccaccgccg ccttctatga aaggttgggc 5700ttcggaatcg
ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
5760gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
ataaagcaat 5820agcatcacaa atttcacaaa taaagcattt ttttcactgc
attctagttg tggtttgtcc 5880aaactcatca atgtatctta tcatgtctgt
ataccgtcga cctctagcta gagcttggcg 5940taatcatggt catagctgtt
tcctgtgtga aattgttatc cgctcacaat tccacacaac 6000atacgagccg
gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
6060ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
ccagctgcat 6120taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
ttgggcgctc ttccgcttcc 6180tcgctcactg actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc agctcactca 6240aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa catgtgagca 6300aaaggccagc
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
6360ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg 6420acaggactat aaagatacca ggcgtttccc cctggaagct
ccctcgtgcg ctctcctgtt 6480ccgaccctgc cgcttaccgg atacctgtcc
gcctttctcc cttcgggaag cgtggcgctt 6540tctcaatgct cacgctgtag
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 6600tgtgtgcacg
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
6660gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
taacaggatt 6720agcagagcga ggtatgtagg cggtgctaca gagttcttga
agtggtggcc taactacggc 6780tacactagaa
ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
6840agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 6900tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 6960acggggtctg acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgagatta 7020tcaaaaagga tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa 7080agtatatatg
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
7140tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact 7200acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg agacccacgc 7260tcaccggctc cagatttatc agcaataaac
cagccagccg gaagggccga gcgcagaagt 7320ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 7380agtagttcgc
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
7440tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
aaggcgagtt 7500acatgatccc ccatgttgtg caaaaaagcg gttagctcct
tcggtcctcc gatcgttgtc 7560agaagtaagt tggccgcagt gttatcactc
atggttatgg cagcactgca taattctctt 7620actgtcatgc catccgtaag
atgcttttct gtgactggtg agtactcaac caagtcattc 7680tgagaatagt
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
7740gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
ggggcgaaaa 7800ctctcaagga tcttaccgct gttgagatcc agttcgatgt
aacccactcg tgcacccaac 7860tgatcttcag catcttttac tttcaccagc
gtttctgggt gagcaaaaac aggaaggcaa 7920aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat actcttcctt 7980tttcaatatt
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
8040tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
agtgccacct 8100gacgtc 8106146560PRTArtificial SequenceBmpr2 REV RTN
EBE 146Leu Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
Lys1 5 10 15Gln Ala Leu Glu Ala Val Lys Ala Gln Leu Leu Glu Leu Arg
Ala Ala 20 25 30Pro Tyr Glu Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn Gly 35 40 45Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly Glu Gln
Leu Leu Lys Leu 50 55 60Arg Thr Ala Pro Tyr Gly Leu Ser Thr Ala Gln
Val Val Ala Ile Ala65 70 75 80Ser Asn Gly Gly Gly Lys Gln Ala Leu
Glu Gly Ile Gly Glu Gln Leu 85 90 95Leu Lys Leu Arg Thr Ala Pro Tyr
Gly Leu Ser Thr Ala Gln Val Val 100 105 110Ala Ile Ala Ser Asn Gly
Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly 115 120 125Glu Gln Leu Leu
Lys Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr Ala 130 135 140Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys Pro Ala Leu Glu145 150 155
160Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly Val Pro Tyr Ala Leu
165 170 175Ser Thr Glu Gln Val Val Ala Ile Ala Ser Asn Lys Gly Gly
Lys Gln 180 185 190Ala Leu Glu Ala Val Lys Ala His Leu Leu Asp Leu
Leu Gly Ala Pro 195 200 205Tyr Val Leu Ser Thr Ala Gln Val Val Ala
Ile Ala Ser Asn Gly Gly 210 215 220Gly Lys Gln Ala Leu Glu Gly Ile
Gly Glu Gln Leu Leu Lys Leu Arg225 230 235 240Thr Ala Pro Tyr Gly
Leu Ser Thr Ala Gln Val Val Ala Ile Ala Ser 245 250 255Asn Gly Gly
Gly Lys Gln Ala Leu Glu Gly Ile Gly Glu Gln Leu Leu 260 265 270Lys
Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr Ala Gln Val Val Ala 275 280
285Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly Glu
290 295 300Gln Leu Leu Lys Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr
Glu Gln305 310 315 320Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
Gln Ala Leu Glu Ala 325 330 335Val Lys Ala Gln Leu Leu Glu Leu Arg
Ala Ala Pro Tyr Glu Leu Ser 340 345 350Thr Ala Gln Val Val Ala Ile
Ala Ser His Asp Gly Gly Lys Pro Ala 355 360 365Leu Glu Ala Val Trp
Ala Lys Leu Pro Val Leu Arg Gly Val Pro Tyr 370 375 380Ala Leu Ser
Thr Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly385 390 395
400Lys Pro Ala Leu Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly
405 410 415Val Pro Tyr Ala Leu Ser Thr Ala Gln Val Val Ala Ile Ala
Ser Asn 420 425 430Gly Gly Gly Lys Gln Ala Leu Glu Gly Ile Gly Glu
Gln Leu Leu Lys 435 440 445Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr
Glu Gln Val Val Ala Ile 450 455 460Ala Ser Asn Lys Gly Gly Lys Gln
Ala Leu Glu Ala Val Lys Ala His465 470 475 480Leu Leu Asp Leu Leu
Gly Ala Pro Tyr Val Leu Ser Thr Ala Gln Val 485 490 495Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Gly Ile 500 505 510Gly
Glu Gln Leu Leu Lys Leu Arg Thr Ala Pro Tyr Gly Leu Ser Thr 515 520
525Ala Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Pro Ala Leu
530 535 540Glu Ala Val Trp Ala Lys Leu Pro Val Leu Arg Gly Val Pro
Tyr Ala545 550 555 5601478102DNAArtificial SequenceBmpr2 REV RTN
147gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc
tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg
atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
gggactttcc 420attgacgtca atgggtggag tatttacggt aaactgccca
cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg
tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta
tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc
cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa
gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa
attaatacga ctcactatag ggagacccaa gctggctagc 900accatggact
acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
960gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg
cggggtacct 1020atggtggact tgaggacact cggttattcg caacagcaac
aggagaaaat caagcctaag 1080gtcaggagca ccgtcgcgca acaccacgag
gcgcttgtgg ggcatggctt cactcatgcg 1140catattgtcg cgctttcaca
gcaccctgcg gcgcttggga cggtggctgt caaataccaa 1200gatatgattg
cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
1260tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct
tagggggcct 1320ccgctccagc tcgacaccgg gcagctgctg aagatcgcga
agagaggggg agtaacagcg 1380gtagaggcag tgcacgcctg gcgcaatgcg
ctcaccgggg cccccttctg agcaccgagc 1440aggtggtggc catcgccagc
aacaacggcg gcaagcaggc cctggaggcc gtgaaggccc 1500agctgctgga
gctgagggcc gccccctacg agctgagcac cgcccaggtg gtggccatcg
1560ccagcaacgg cggcggcaag caggccctgg agggcatcgg cgagcagctg
ctgaagctga 1620ggaccgcccc ctacggcctg agcaccgccc aggtggtggc
catcgccagc aacggcggcg 1680gcaagcaggc cctggagggc atcggcgagc
agctgctgaa gctgaggacc gccccctacg 1740gcctgagcac cgcccaggtg
gtggccatcg ccagcaacgg cggcggcaag caggccctgg 1800agggcatcgg
cgagcagctg ctgaagctga ggaccgcccc ctacggcctg agcaccgccc
1860aggtggtggc catcgccagc cacgacggcg gcaagcccgc cctggaggcc
gtgtgggcca 1920agctgcccgt gctgaggggc gtgccctacg ccctgagcac
cgagcaggtg gtggccatcg 1980ccagcaacaa gggcggcaag caggccctgg
aggccgtgaa ggcccacctg ctggacctgc 2040tgggcgcccc ctacgtgctg
agcaccgccc aggtggtggc catcgccagc aacggcggcg 2100gcaagcaggc
cctggagggc atcggcgagc agctgctgaa gctgaggacc gccccctacg
2160gcctgagcac cgcccaggtg gtggccatcg ccagcaacgg cggcggcaag
caggccctgg 2220agggcatcgg cgagcagctg ctgaagctga ggaccgcccc
ctacggcctg agcaccgccc 2280aggtggtggc catcgccagc aacggcggcg
gcaagcaggc cctggagggc atcggcgagc 2340agctgctgaa gctgaggacc
gccccctacg gcctgagcac cgagcaggtg gtggccatcg 2400ccagcaacaa
cggcggcaag caggccctgg aggccgtgaa ggcccagctg ctggagctga
2460gggccgcccc ctacgagctg agcaccgccc aggtggtggc catcgccagc
cacgacggcg 2520gcaagcccgc cctggaggcc gtgtgggcca agctgcccgt
gctgaggggc gtgccctacg 2580ccctgagcac cgcccaggtg gtggccatcg
ccagccacga cggcggcaag cccgccctgg 2640aggccgtgtg ggccaagctg
cccgtgctga ggggcgtgcc ctacgccctg agcaccgccc 2700aggtggtggc
catcgccagc aacggcggcg gcaagcaggc cctggagggc atcggcgagc
2760agctgctgaa gctgaggacc gccccctacg gcctgagcac cgagcaggtg
gtggccatcg 2820ccagcaacaa gggcggcaag caggccctgg aggccgtgaa
ggcccacctg ctggacctgc 2880tgggcgcccc ctacgtgctg agcaccgccc
aggtggtggc catcgccagc aacggcggcg 2940gcaagcaggc cctggagggc
atcggcgagc agctgctgaa gctgaggacc gccccctacg 3000gcctgagcac
cgcccaggtg gtggccatcg ccagccacga cggcggcaag cccgccctgg
3060aggccgtgtg ggccaagctg cccgtgctga ggggcgtgcc ctacgccctg
agcaccgagc 3120aggtggtgac catcgccagc agcatcggag gacggccagc
cttggagtcc atcgtagccc 3180aattgtccag gcccgatccc gcgttggctg
cgttaacgaa tgaccatctg gtggcgttgg 3240catgtcttgg tggacgaccc
gcgctcgatg cagtcaaaaa gggtctgcct catgctcccg 3300cattgatcaa
aagaaccaac cggcggattc ccgagagaac ttcccatcga gtcgcgggat
3360cccaactagt caaaagtgaa ctggaggaga agaaatctga acttcgtcat
aaattgaaat 3420atgtgcctca tgaatatatt gaattaattg aaattgccag
aaattccact caggatagaa 3480ttcttgaaat gaaggtaatg gaatttttta
tgaaagttta tggatataga ggtaaacatt 3540tgggtggatc aaggaaaccg
gacggagcaa tttatactgt cggatctcct attgattacg 3600gtgtgatcgt
ggatactaaa gcttatagcg gaggttataa tctgccaatt ggccaagcag
3660atgaaatgca acgatatgtc gaagaaaatc aaacacgaaa caaacatatc
aaccctaatg 3720aatggtggaa agtctatcca tcttctgtaa cggaatttaa
gtttttattt gtgagtggtc 3780actttaaagg aaactacaaa gctcagctta
cacgattaaa tcatatcact aattgtaatg 3840gagctgttct tagtgtagaa
gagcttttaa ttggtggaga aatgattaaa gccggcacat 3900taaccttaga
ggaagtcaga cggaaattta ataacggcga gataaacttt taagggccct
3960tcgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt
accggtcatc 4020atcaccatca ccattgagtt taaacccgct gatcagcctc
gactgtgcct tctagttgcc 4080agccatctgt tgtttgcccc tcccccgtgc
cttccttgac cctggaaggt gccactccca 4140ctgtcctttc ctaataaaat
gaggaaattg catcgcattg tctgagtagg tgtcattcta 4200ttctgggggg
tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
4260atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
tggggctcta 4320gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
ggcgggtgtg gtggttacgc 4380gcagcgtgac cgctacactt gccagcgccc
tagcgcccgc tcctttcgct ttcttccctt 4440cctttctcgc cacgttcgcc
ggctttcccc gtcaagctct aaatcggggc atccctttag 4500ggttccgatt
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
4560cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
gagtccacgt 4620tctttaatag tggactcttg ttccaaactg gaacaacact
caaccctatc tcggtctatt 4680cttttgattt ataagggatt ttggggattt
cggcctattg gttaaaaaat gagctgattt 4740aacaaaaatt taacgcgaat
taattctgtg gaatgtgtgt cagttagggt gtggaaagtc 4800cccaggctcc
ccaggcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
4860ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
catctcaatt 4920agtcagcaac catagtcccg cccctaactc cgcccatccc
gcccctaact ccgcccagtt 4980ccgcccattc tccgccccat ggctgactaa
ttttttttat ttatgcagag gccgaggccg 5040cctctgcctc tgagctattc
cagaagtagt gaggaggctt ttttggaggc ctaggctttt 5100gcaaaaagct
cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa
5160ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
actaaaccat 5220ggccaagcct ttgtctcaag aagaatccac cctcattgaa
agagcaacgg ctacaatcaa 5280cagcatcccc atctctgaag actacagcgt
cgccagcgca gctctctcta gcgacggccg 5340catcttcact ggtgtcaatg
tatatcattt tactggggga ccttgtgcag aactcgtggt 5400gctgggcact
gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
5460tgagaacagg ggcatcttga gcccctgcgg acggtgtcga caggtgcttc
tcgatctgca 5520tcctgggatc aaagcgatag tgaaggacag tgatggacag
ccgacggcag ttgggattcg 5580tgaattgctg ccctctggtt atgtgtggga
gggctaagca cttcgtggcc gaggagcagg 5640actgacacgt gctacgagat
ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 5700gaatcgtttt
ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
5760tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
agcaatagca 5820tcacaaattt cacaaataaa gcattttttt cactgcattc
tagttgtggt ttgtccaaac 5880tcatcaatgt atcttatcat gtctgtatac
cgtcgacctc tagctagagc ttggcgtaat 5940catggtcata gctgtttcct
gtgtgaaatt gttatccgct cacaattcca cacaacatac 6000gagccggaag
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
6060ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
ctgcattaat 6120gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
gcgctcttcc gcttcctcgc 6180tcactgactc gctgcgctcg gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg 6240cggtaatacg gttatccaca
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6300gccagcaaaa
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
6360gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
aacccgacag 6420gactataaag ataccaggcg tttccccctg gaagctccct
cgtgcgctct cctgttccga 6480ccctgccgct taccggatac ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc 6540aatgctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6600tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
6660ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
aggattagca 6720gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac tacggctaca 6780ctagaaggac agtatttggt atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag 6840ttggtagctc ttgatccggc
aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6900agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
6960ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
agattatcaa 7020aaaggatctt cacctagatc cttttaaatt aaaaatgaag
ttttaaatca atctaaagta 7080tatatgagta aacttggtct gacagttacc
aatgcttaat cagtgaggca cctatctcag 7140cgatctgtct atttcgttca
tccatagttg cctgactccc cgtcgtgtag ataactacga 7200tacgggaggg
cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
7260cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
agaagtggtc 7320ctgcaacttt atccgcctcc atccagtcta ttaattgttg
ccgggaagct agagtaagta 7380gttcgccagt taatagtttg cgcaacgttg
ttgccattgc tacaggcatc gtggtgtcac 7440gctcgtcgtt tggtatggct
tcattcagct ccggttccca acgatcaagg cgagttacat 7500gatcccccat
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
7560gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
tctcttactg 7620tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
ctcaaccaag tcattctgag 7680aatagtgtat gcggcgaccg agttgctctt
gcccggcgtc aatacgggat aataccgcgc 7740cacatagcag aactttaaaa
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 7800caaggatctt
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
7860cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
aggcaaaatg 7920ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
actcatactc ttcctttttc 7980aatattattg aagcatttat cagggttatt
gtctcatgag cggatacata tttgaatgta 8040tttagaaaaa taaacaaata
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 8100tc
81021482706DNAArtificial SequenceXTN sub-array backbone
148ggatcccggg cccgtcgact gcagaggcct gcatgcaagc ttggcgtaat
catggtcata 60gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
gagccggaag 120cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
ctcacattaa ttgcgttgcg 180ctcactgccc gctttccagt cgggaaacct
gtcgtgccag ctgcattaat gaatcggcca 240acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 300gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
360gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
gccagcaaaa 420ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
cataggctcc gcccccctga 480cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga aacccgacag gactataaag 540ataccaggcg tttccccctg
gaagctccct cgtgcgctct cctgttccga ccctgccgct 600taccggatac
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
660ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
tgcacgaacc 720ccccgttcag cccgaccgct gcgccttatc cggtaactat
cgtcttgagt ccaacccggt 780aagacacgac ttatcgccac tggcagcagc
cactggtaac aggattagca gagcgaggta 840tgtaggcggt gctacagagt
tcttgaagtg gtggcctaac tacggctaca ctagaagaac 900agtatttggt
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
960ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
agcagcagat 1020tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc 1080tcagtggaac gaaaactcac gttaagggat
tttggtcatg agattatcaa aaaggatctt 1140cacctagatc cttttaaatt
aaaaatgaag ttttaaatca atctaaagta tatatgagta 1200aacttggtct
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
1260atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
tacgggaggg 1320cttaccatct ggccccagtg ctgcaatgat accgcgagag
ccacgctcac cggctccaga 1380tttatcagca ataaaccagc cagccggaag
ggccgagcgc agaagtggtc ctgcaacttt 1440atccgcctcc atccagtcta
ttaattgttg ccgggaagct agagtaagta gttcgccagt 1500taatagtttg
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
1560tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
gatcccccat 1620gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
gttgtcagaa gtaagttggc 1680cgcagtgtta tcactcatgg ttatggcagc
actgcataat tctcttactg tcatgccatc 1740cgtaagatgc ttttctgtga
ctggtgagta ctcaaccaag tcattctgag aatagtgtat 1800gcggcgaccg
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
1860aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
caaggatctt 1920accgctgttg agatccagtt cgatgtaacc cactcgtgca
cccaactgat cttcagcatc 1980ttttactttc
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
2040gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
aatattattg 2100aagcatttat cagggttatt gtctcatgag cggatacata
tttgaatgta tttagaaaaa 2160taaacaaata ggggttccgc gcacatttcc
ccgaaaagtg ccacctgacg tctaagaaac 2220cattattatc atgacattaa
cctataaaaa taggcgtatc acgaggccct ttcgtctcgc 2280gcgtttcggt
gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
2340ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
cgggtgttgg 2400cgggtgtcgg ggctggctta actatgcggc atcagagcag
attgtactga gagtgcacca 2460tatgcggtgt gaaataccgc acagatgcgt
aaggagaaaa taccgcatca ggcgccattc 2520gccattcagg ctgcgcaact
gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2580ccagctggcg
aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
2640ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtacct
cgcgaatgca 2700tctaga 27061496426DNAArtificial
SequenceXTN-bbmisc_feature(31)..(39)n is a, c, g, or t
149ctgacacccg aacaggtggt cgccattgct nnnnnnnnng gaggacggcc
agccttggag 60tccatcgtag cccaattgtc caggcccgat cccgcgttgg ctgcgttaac
gaatgaccat 120ctggtggcgt tggcatgtct tggtggacga cccgcgctcg
atgcagtcaa aaagggtctg 180cctcatgctc ccgcattgat caaaagaacc
aaccggcgga ttcccgagag aacttcccat 240cgagtcgcgg gatcccaact
agtcaaaagt gaactggagg agaagaaatc tgaacttcgt 300cataaattga
aatatgtgcc tcatgaatat attgaattaa ttgaaattgc cagaaattcc
360actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
ttatggatat 420agaggtaaac atttgggtgg atcaaggaaa ccggacggag
caatttatac tgtcggatct 480cctattgatt acggtgtgat cgtggatact
aaagcttata gcggaggtta taatctgcca 540attggccaag cagatgaaat
gcaacgatat gtcgaagaaa atcaaacacg aaacaaacat 600atcaacccta
atgaatggtg gaaagtctat ccatcttctg taacggaatt taagttttta
660tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
aaatcatatc 720actaattgta atggagctgt tcttagtgta gaagagcttt
taattggtgg agaaatgatt 780aaagccggca cattaacctt agaggaagtc
agacggaaat ttaataacgg cgagataaac 840ttttaagggc ccttcgaagg
taagcctatc cctaaccctc tcctcggtct cgattctacg 900cgtaccggtc
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
960ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa 1020ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt 1080aggtgtcatt ctattctggg gggtggggtg
gggcaggaca gcaaggggga ggattgggaa 1140gacaatagca ggcatgctgg
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1200agctggggct
ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
1260gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc 1320gctttcttcc cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg 1380ggcatccctt tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat 1440tagggtgatg gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg 1500ttggagtcca
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
1560atctcggtct attcttttga tttataaggg attttgggga tttcggccta
ttggttaaaa 1620aatgagctga tttaacaaaa atttaacgcg aattaattct
gtggaatgtg tgtcagttag 1680ggtgtggaaa gtccccaggc tccccaggca
ggcagaagta tgcaaagcat gcatctcaat 1740tagtcagcaa ccaggtgtgg
aaagtcccca ggctccccag caggcagaag tatgcaaagc 1800atgcatctca
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1860actccgccca gttccgccca ttctccgccc catggctgac taattttttt
tatttatgca 1920gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
agtgaggagg cttttttgga 1980ggcctaggct tttgcaaaaa gctcccggga
gcttgtatat ccattttcgg atctgatcag 2040cacgtgttga caattaatca
tcggcatagt atatcggcat agtataatac gacaaggtga 2100ggaactaaac
catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
2160cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
gcagctctct 2220ctagcgacgg ccgcatcttc actggtgtca atgtatatca
ttttactggg ggaccttgtg 2280cagaactcgt ggtgctgggc actgctgctg
ctgcggcagc tggcaacctg acttgtatcg 2340tcgcgatcgg aaatgagaac
aggggcatct tgagcccctg cggacggtgt cgacaggtgc 2400ttctcgatct
gcatcctggg atcaaagcga tagtgaagga cagtgatgga cagccgacgg
2460cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
gcacttcgtg 2520gccgaggagc aggactgaca cgtgctacga gatttcgatt
ccaccgccgc cttctatgaa 2580aggttgggct tcggaatcgt tttccgggac
gccggctgga tgatcctcca gcgcggggat 2640ctcatgctgg agttcttcgc
ccaccccaac ttgtttattg cagcttataa tggttacaaa 2700taaagcaata
gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
2760ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
ctctagctag 2820agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 2880ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta atgagtgagc 2940taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3000cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
3060tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca 3120gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 3180atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt gctggcgttt 3240ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3300cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
3360tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
ttcgggaagc 3420gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 3480aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt atccggtaac 3540tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc agccactggt 3600aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
3660aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
gccagttacc 3720ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 3780ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga agatcctttg 3840atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3900atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
3960tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
aatcagtgag 4020gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact ccccgtcgtg 4080tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat gataccgcga 4140gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg aagggccgag 4200cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
4260gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
tgctacaggc 4320atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
gctccggttc ccaacgatca 4380aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt cggtcctccg 4440atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca tggttatggc agcactgcat 4500aattctctta
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
4560aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
gtcaatacgg 4620gataataccg cgccacatag cagaacttta aaagtgctca
tcattggaaa acgttcttcg 4680gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta acccactcgt 4740gcacccaact gatcttcagc
atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4800ggaaggcaaa
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
4860ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
gagcggatac 4920atatttgaat gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa 4980gtgccacctg acgtcgacgg atcgggagat
ctcccgatcc cctatggtcg actctcagta 5040caatctgctc tgatgccgca
tagttaagcc agtatctgct ccctgcttgt gtgttggagg 5100tcgctgagta
gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
5160gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
cgggccagat 5220atacgcgttg acattgatta ttgactagtt attaatagta
atcaattacg gggtcattag 5280ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 5340gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 5400caatagggac
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
5460cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 5520ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 5580tctacgtatt agtcatcgct attaccatgg
tgatgcggtt ttggcagtac atcaatgggc 5640gtggatagcg gtttgactca
cggggatttc caagtctcca ccccattgac gtcaatggga 5700gtttgttttg
gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
5760tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
gctctctggc 5820taactagaga acccactgct tactggctta tcgaaattaa
tacgactcac tatagggaga 5880cccaagctgg ctagcaccat ggactacaaa
gaccatgacg gtgattataa agatcatgac 5940atcgattaca aggatgacga
tgacaagatg gcccccaaga agaagaggaa ggtgggcatt 6000caccgcgggg
tacctatggt ggacttgagg acactcggtt attcgcaaca gcaacaggag
6060aaaatcaagc ctaaggtcag gagcaccgtc gcgcaacacc acgaggcgct
tgtggggcat 6120ggcttcactc atgcgcatat tgtcgcgctt tcacagcacc
ctgcggcgct tgggacggtg 6180gctgtcaaat accaagatat gattgcggcc
ctgcccgaag ccacgcacga ggcaattgta 6240ggggtcggta aacagtggtc
gggagcgcga gcacttgagg cgctgctgac tgtggcgggt 6300gagcttaggg
ggcctccgct ccagctcgac accgggcagc tgctgaagat cgcgaagaga
6360gggggagtaa cagcggtaga ggcagtgcac gcctggcgca atgcgctcac
cggggccccc 6420ttgaac 64261506426DNAArtificial SequenceXTN-bbA
150ctgacacccg aacaggtggt cgccattgct tctaacatcg gaggacggcc
agccttggag 60tccatcgtag cccaattgtc caggcccgat cccgcgttgg ctgcgttaac
gaatgaccat 120ctggtggcgt tggcatgtct tggtggacga cccgcgctcg
atgcagtcaa aaagggtctg 180cctcatgctc ccgcattgat caaaagaacc
aaccggcgga ttcccgagag aacttcccat 240cgagtcgcgg gatcccaact
agtcaaaagt gaactggagg agaagaaatc tgaacttcgt 300cataaattga
aatatgtgcc tcatgaatat attgaattaa ttgaaattgc cagaaattcc
360actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
ttatggatat 420agaggtaaac atttgggtgg atcaaggaaa ccggacggag
caatttatac tgtcggatct 480cctattgatt acggtgtgat cgtggatact
aaagcttata gcggaggtta taatctgcca 540attggccaag cagatgaaat
gcaacgatat gtcgaagaaa atcaaacacg aaacaaacat 600atcaacccta
atgaatggtg gaaagtctat ccatcttctg taacggaatt taagttttta
660tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
aaatcatatc 720actaattgta atggagctgt tcttagtgta gaagagcttt
taattggtgg agaaatgatt 780aaagccggca cattaacctt agaggaagtc
agacggaaat ttaataacgg cgagataaac 840ttttaagggc ccttcgaagg
taagcctatc cctaaccctc tcctcggtct cgattctacg 900cgtaccggtc
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
960ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa 1020ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt 1080aggtgtcatt ctattctggg gggtggggtg
gggcaggaca gcaaggggga ggattgggaa 1140gacaatagca ggcatgctgg
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1200agctggggct
ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
1260gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc 1320gctttcttcc cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg 1380ggcatccctt tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat 1440tagggtgatg gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg 1500ttggagtcca
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
1560atctcggtct attcttttga tttataaggg attttgggga tttcggccta
ttggttaaaa 1620aatgagctga tttaacaaaa atttaacgcg aattaattct
gtggaatgtg tgtcagttag 1680ggtgtggaaa gtccccaggc tccccaggca
ggcagaagta tgcaaagcat gcatctcaat 1740tagtcagcaa ccaggtgtgg
aaagtcccca ggctccccag caggcagaag tatgcaaagc 1800atgcatctca
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1860actccgccca gttccgccca ttctccgccc catggctgac taattttttt
tatttatgca 1920gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
agtgaggagg cttttttgga 1980ggcctaggct tttgcaaaaa gctcccggga
gcttgtatat ccattttcgg atctgatcag 2040cacgtgttga caattaatca
tcggcatagt atatcggcat agtataatac gacaaggtga 2100ggaactaaac
catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
2160cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
gcagctctct 2220ctagcgacgg ccgcatcttc actggtgtca atgtatatca
ttttactggg ggaccttgtg 2280cagaactcgt ggtgctgggc actgctgctg
ctgcggcagc tggcaacctg acttgtatcg 2340tcgcgatcgg aaatgagaac
aggggcatct tgagcccctg cggacggtgt cgacaggtgc 2400ttctcgatct
gcatcctggg atcaaagcga tagtgaagga cagtgatgga cagccgacgg
2460cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
gcacttcgtg 2520gccgaggagc aggactgaca cgtgctacga gatttcgatt
ccaccgccgc cttctatgaa 2580aggttgggct tcggaatcgt tttccgggac
gccggctgga tgatcctcca gcgcggggat 2640ctcatgctgg agttcttcgc
ccaccccaac ttgtttattg cagcttataa tggttacaaa 2700taaagcaata
gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
2760ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
ctctagctag 2820agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 2880ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta atgagtgagc 2940taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3000cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
3060tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca 3120gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 3180atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt gctggcgttt 3240ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3300cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
3360tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
ttcgggaagc 3420gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 3480aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt atccggtaac 3540tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc agccactggt 3600aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
3660aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
gccagttacc 3720ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 3780ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga agatcctttg 3840atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3900atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
3960tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
aatcagtgag 4020gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact ccccgtcgtg 4080tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat gataccgcga 4140gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg aagggccgag 4200cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
4260gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
tgctacaggc 4320atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
gctccggttc ccaacgatca 4380aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt cggtcctccg 4440atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca tggttatggc agcactgcat 4500aattctctta
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
4560aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
gtcaatacgg 4620gataataccg cgccacatag cagaacttta aaagtgctca
tcattggaaa acgttcttcg 4680gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta acccactcgt 4740gcacccaact gatcttcagc
atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4800ggaaggcaaa
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
4860ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
gagcggatac 4920atatttgaat gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa 4980gtgccacctg acgtcgacgg atcgggagat
ctcccgatcc cctatggtcg actctcagta 5040caatctgctc tgatgccgca
tagttaagcc agtatctgct ccctgcttgt gtgttggagg 5100tcgctgagta
gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
5160gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
cgggccagat 5220atacgcgttg acattgatta ttgactagtt attaatagta
atcaattacg gggtcattag 5280ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 5340gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 5400caatagggac
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
5460cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 5520ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 5580tctacgtatt agtcatcgct attaccatgg
tgatgcggtt ttggcagtac atcaatgggc 5640gtggatagcg gtttgactca
cggggatttc caagtctcca ccccattgac gtcaatggga 5700gtttgttttg
gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
5760tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
gctctctggc 5820taactagaga acccactgct tactggctta tcgaaattaa
tacgactcac tatagggaga 5880cccaagctgg ctagcaccat ggactacaaa
gaccatgacg gtgattataa agatcatgac 5940atcgattaca aggatgacga
tgacaagatg gcccccaaga agaagaggaa ggtgggcatt 6000caccgcgggg
tacctatggt ggacttgagg acactcggtt attcgcaaca gcaacaggag
6060aaaatcaagc ctaaggtcag gagcaccgtc gcgcaacacc acgaggcgct
tgtggggcat 6120ggcttcactc atgcgcatat tgtcgcgctt tcacagcacc
ctgcggcgct tgggacggtg 6180gctgtcaaat accaagatat gattgcggcc
ctgcccgaag ccacgcacga ggcaattgta 6240ggggtcggta aacagtggtc
gggagcgcga gcacttgagg cgctgctgac tgtggcgggt 6300gagcttaggg
ggcctccgct ccagctcgac accgggcagc tgctgaagat cgcgaagaga
6360gggggagtaa cagcggtaga ggcagtgcac gcctggcgca atgcgctcac
cggggccccc 6420ttgaac 64261516426DNAArtificial SequenceXTN-bbC
151ctgacacccg aacaggtggt cgccattgct tcccacgacg gaggacggcc
agccttggag 60tccatcgtag cccaattgtc caggcccgat cccgcgttgg ctgcgttaac
gaatgaccat 120ctggtggcgt tggcatgtct tggtggacga cccgcgctcg
atgcagtcaa aaagggtctg 180cctcatgctc ccgcattgat caaaagaacc
aaccggcgga ttcccgagag aacttcccat 240cgagtcgcgg gatcccaact
agtcaaaagt gaactggagg agaagaaatc tgaacttcgt 300cataaattga
aatatgtgcc tcatgaatat attgaattaa ttgaaattgc cagaaattcc
360actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
ttatggatat 420agaggtaaac atttgggtgg atcaaggaaa ccggacggag
caatttatac tgtcggatct 480cctattgatt acggtgtgat cgtggatact
aaagcttata gcggaggtta taatctgcca 540attggccaag cagatgaaat
gcaacgatat gtcgaagaaa atcaaacacg aaacaaacat 600atcaacccta
atgaatggtg gaaagtctat ccatcttctg taacggaatt taagttttta
660tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
aaatcatatc 720actaattgta atggagctgt tcttagtgta gaagagcttt
taattggtgg agaaatgatt 780aaagccggca cattaacctt agaggaagtc
agacggaaat ttaataacgg cgagataaac 840ttttaagggc ccttcgaagg
taagcctatc cctaaccctc tcctcggtct cgattctacg 900cgtaccggtc
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
960ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa 1020ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt 1080aggtgtcatt ctattctggg gggtggggtg
gggcaggaca gcaaggggga ggattgggaa 1140gacaatagca ggcatgctgg
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1200agctggggct
ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
1260gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc 1320gctttcttcc cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg 1380ggcatccctt tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat 1440tagggtgatg gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg 1500ttggagtcca
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
1560atctcggtct attcttttga tttataaggg attttgggga tttcggccta
ttggttaaaa 1620aatgagctga tttaacaaaa atttaacgcg aattaattct
gtggaatgtg tgtcagttag 1680ggtgtggaaa gtccccaggc tccccaggca
ggcagaagta tgcaaagcat gcatctcaat 1740tagtcagcaa ccaggtgtgg
aaagtcccca ggctccccag caggcagaag tatgcaaagc 1800atgcatctca
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1860actccgccca gttccgccca ttctccgccc catggctgac taattttttt
tatttatgca 1920gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
agtgaggagg cttttttgga 1980ggcctaggct tttgcaaaaa gctcccggga
gcttgtatat ccattttcgg atctgatcag 2040cacgtgttga caattaatca
tcggcatagt atatcggcat agtataatac gacaaggtga 2100ggaactaaac
catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
2160cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
gcagctctct 2220ctagcgacgg ccgcatcttc actggtgtca atgtatatca
ttttactggg ggaccttgtg 2280cagaactcgt ggtgctgggc actgctgctg
ctgcggcagc tggcaacctg acttgtatcg 2340tcgcgatcgg aaatgagaac
aggggcatct tgagcccctg cggacggtgt cgacaggtgc 2400ttctcgatct
gcatcctggg atcaaagcga tagtgaagga cagtgatgga cagccgacgg
2460cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
gcacttcgtg 2520gccgaggagc aggactgaca cgtgctacga gatttcgatt
ccaccgccgc cttctatgaa 2580aggttgggct tcggaatcgt tttccgggac
gccggctgga tgatcctcca gcgcggggat 2640ctcatgctgg agttcttcgc
ccaccccaac ttgtttattg cagcttataa tggttacaaa 2700taaagcaata
gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
2760ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
ctctagctag 2820agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 2880ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta atgagtgagc 2940taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3000cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
3060tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca 3120gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 3180atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt gctggcgttt 3240ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3300cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
3360tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
ttcgggaagc 3420gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 3480aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt atccggtaac 3540tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc agccactggt 3600aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
3660aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
gccagttacc 3720ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 3780ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga agatcctttg 3840atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3900atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
3960tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
aatcagtgag 4020gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact ccccgtcgtg 4080tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat gataccgcga 4140gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg aagggccgag 4200cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
4260gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
tgctacaggc 4320atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
gctccggttc ccaacgatca 4380aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt cggtcctccg 4440atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca tggttatggc agcactgcat 4500aattctctta
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
4560aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
gtcaatacgg 4620gataataccg cgccacatag cagaacttta aaagtgctca
tcattggaaa acgttcttcg 4680gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta acccactcgt 4740gcacccaact gatcttcagc
atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4800ggaaggcaaa
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
4860ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
gagcggatac 4920atatttgaat gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa 4980gtgccacctg acgtcgacgg atcgggagat
ctcccgatcc cctatggtcg actctcagta 5040caatctgctc tgatgccgca
tagttaagcc agtatctgct ccctgcttgt gtgttggagg 5100tcgctgagta
gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
5160gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
cgggccagat 5220atacgcgttg acattgatta ttgactagtt attaatagta
atcaattacg gggtcattag 5280ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 5340gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 5400caatagggac
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
5460cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 5520ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 5580tctacgtatt agtcatcgct attaccatgg
tgatgcggtt ttggcagtac atcaatgggc 5640gtggatagcg gtttgactca
cggggatttc caagtctcca ccccattgac gtcaatggga 5700gtttgttttg
gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
5760tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
gctctctggc 5820taactagaga acccactgct tactggctta tcgaaattaa
tacgactcac tatagggaga 5880cccaagctgg ctagcaccat ggactacaaa
gaccatgacg gtgattataa agatcatgac 5940atcgattaca aggatgacga
tgacaagatg gcccccaaga agaagaggaa ggtgggcatt 6000caccgcgggg
tacctatggt ggacttgagg acactcggtt attcgcaaca gcaacaggag
6060aaaatcaagc ctaaggtcag gagcaccgtc gcgcaacacc acgaggcgct
tgtggggcat 6120ggcttcactc atgcgcatat tgtcgcgctt tcacagcacc
ctgcggcgct tgggacggtg 6180gctgtcaaat accaagatat gattgcggcc
ctgcccgaag ccacgcacga ggcaattgta 6240ggggtcggta aacagtggtc
gggagcgcga gcacttgagg cgctgctgac tgtggcgggt 6300gagcttaggg
ggcctccgct ccagctcgac accgggcagc tgctgaagat cgcgaagaga
6360gggggagtaa cagcggtaga ggcagtgcac gcctggcgca atgcgctcac
cggggccccc 6420ttgaac 64261526426DNAArtificial SequenceXTN-bbG
152ctgacacccg aacaggtggt cgccattgct aataataacg gaggacggcc
agccttggag 60tccatcgtag cccaattgtc caggcccgat cccgcgttgg ctgcgttaac
gaatgaccat 120ctggtggcgt tggcatgtct tggtggacga cccgcgctcg
atgcagtcaa aaagggtctg 180cctcatgctc ccgcattgat caaaagaacc
aaccggcgga ttcccgagag aacttcccat 240cgagtcgcgg gatcccaact
agtcaaaagt gaactggagg agaagaaatc tgaacttcgt 300cataaattga
aatatgtgcc tcatgaatat attgaattaa ttgaaattgc cagaaattcc
360actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
ttatggatat 420agaggtaaac atttgggtgg atcaaggaaa ccggacggag
caatttatac tgtcggatct 480cctattgatt acggtgtgat cgtggatact
aaagcttata gcggaggtta taatctgcca 540attggccaag cagatgaaat
gcaacgatat gtcgaagaaa atcaaacacg aaacaaacat 600atcaacccta
atgaatggtg gaaagtctat ccatcttctg taacggaatt taagttttta
660tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
aaatcatatc 720actaattgta atggagctgt tcttagtgta gaagagcttt
taattggtgg agaaatgatt 780aaagccggca cattaacctt agaggaagtc
agacggaaat ttaataacgg cgagataaac 840ttttaagggc ccttcgaagg
taagcctatc cctaaccctc tcctcggtct cgattctacg 900cgtaccggtc
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
960ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa 1020ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt 1080aggtgtcatt ctattctggg gggtggggtg
gggcaggaca gcaaggggga ggattgggaa 1140gacaatagca ggcatgctgg
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1200agctggggct
ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
1260gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc 1320gctttcttcc cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg 1380ggcatccctt tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat 1440tagggtgatg gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg 1500ttggagtcca
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
1560atctcggtct attcttttga tttataaggg attttgggga tttcggccta
ttggttaaaa 1620aatgagctga tttaacaaaa atttaacgcg aattaattct
gtggaatgtg tgtcagttag 1680ggtgtggaaa gtccccaggc tccccaggca
ggcagaagta tgcaaagcat gcatctcaat 1740tagtcagcaa ccaggtgtgg
aaagtcccca ggctccccag caggcagaag tatgcaaagc 1800atgcatctca
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1860actccgccca gttccgccca ttctccgccc catggctgac taattttttt
tatttatgca 1920gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
agtgaggagg cttttttgga 1980ggcctaggct tttgcaaaaa gctcccggga
gcttgtatat ccattttcgg atctgatcag 2040cacgtgttga caattaatca
tcggcatagt atatcggcat agtataatac gacaaggtga 2100ggaactaaac
catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
2160cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
gcagctctct 2220ctagcgacgg ccgcatcttc actggtgtca atgtatatca
ttttactggg ggaccttgtg 2280cagaactcgt ggtgctgggc actgctgctg
ctgcggcagc tggcaacctg acttgtatcg 2340tcgcgatcgg aaatgagaac
aggggcatct tgagcccctg cggacggtgt cgacaggtgc 2400ttctcgatct
gcatcctggg atcaaagcga tagtgaagga cagtgatgga cagccgacgg
2460cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
gcacttcgtg 2520gccgaggagc aggactgaca cgtgctacga gatttcgatt
ccaccgccgc cttctatgaa 2580aggttgggct tcggaatcgt tttccgggac
gccggctgga tgatcctcca gcgcggggat 2640ctcatgctgg agttcttcgc
ccaccccaac ttgtttattg cagcttataa tggttacaaa 2700taaagcaata
gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
2760ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
ctctagctag 2820agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 2880ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta atgagtgagc 2940taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3000cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
3060tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca 3120gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 3180atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt gctggcgttt 3240ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3300cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
3360tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
ttcgggaagc 3420gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 3480aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt atccggtaac 3540tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc agccactggt 3600aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
3660aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
gccagttacc 3720ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 3780ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga agatcctttg 3840atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3900atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
3960tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
aatcagtgag 4020gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact ccccgtcgtg 4080tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat gataccgcga 4140gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg aagggccgag 4200cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
4260gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
tgctacaggc 4320atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
gctccggttc ccaacgatca 4380aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt cggtcctccg 4440atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca tggttatggc agcactgcat 4500aattctctta
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
4560aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
gtcaatacgg 4620gataataccg cgccacatag cagaacttta aaagtgctca
tcattggaaa acgttcttcg 4680gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta acccactcgt 4740gcacccaact gatcttcagc
atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4800ggaaggcaaa
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
4860ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
gagcggatac 4920atatttgaat gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa 4980gtgccacctg acgtcgacgg atcgggagat
ctcccgatcc cctatggtcg actctcagta 5040caatctgctc tgatgccgca
tagttaagcc agtatctgct ccctgcttgt gtgttggagg 5100tcgctgagta
gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
5160gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
cgggccagat 5220atacgcgttg acattgatta ttgactagtt attaatagta
atcaattacg gggtcattag 5280ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 5340gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 5400caatagggac
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
5460cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 5520ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 5580tctacgtatt agtcatcgct attaccatgg
tgatgcggtt ttggcagtac atcaatgggc 5640gtggatagcg gtttgactca
cggggatttc caagtctcca ccccattgac gtcaatggga 5700gtttgttttg
gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
5760tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
gctctctggc 5820taactagaga acccactgct tactggctta tcgaaattaa
tacgactcac tatagggaga 5880cccaagctgg ctagcaccat ggactacaaa
gaccatgacg gtgattataa agatcatgac 5940atcgattaca aggatgacga
tgacaagatg gcccccaaga agaagaggaa ggtgggcatt 6000caccgcgggg
tacctatggt ggacttgagg acactcggtt attcgcaaca gcaacaggag
6060aaaatcaagc ctaaggtcag gagcaccgtc gcgcaacacc acgaggcgct
tgtggggcat 6120ggcttcactc atgcgcatat tgtcgcgctt tcacagcacc
ctgcggcgct tgggacggtg 6180gctgtcaaat accaagatat gattgcggcc
ctgcccgaag ccacgcacga ggcaattgta 6240ggggtcggta aacagtggtc
gggagcgcga gcacttgagg cgctgctgac tgtggcgggt 6300gagcttaggg
ggcctccgct ccagctcgac accgggcagc tgctgaagat cgcgaagaga
6360gggggagtaa cagcggtaga ggcagtgcac gcctggcgca atgcgctcac
cggggccccc 6420ttgaac 64261536426DNAArtificial SequenceXTN-bbT
153ctgacacccg aacaggtggt cgccattgct tctaatgggg gaggacggcc
agccttggag 60tccatcgtag cccaattgtc caggcccgat cccgcgttgg ctgcgttaac
gaatgaccat 120ctggtggcgt tggcatgtct tggtggacga cccgcgctcg
atgcagtcaa aaagggtctg 180cctcatgctc ccgcattgat caaaagaacc
aaccggcgga ttcccgagag aacttcccat 240cgagtcgcgg gatcccaact
agtcaaaagt gaactggagg agaagaaatc tgaacttcgt 300cataaattga
aatatgtgcc tcatgaatat attgaattaa ttgaaattgc cagaaattcc
360actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
ttatggatat 420agaggtaaac atttgggtgg atcaaggaaa ccggacggag
caatttatac tgtcggatct 480cctattgatt acggtgtgat cgtggatact
aaagcttata gcggaggtta taatctgcca 540attggccaag cagatgaaat
gcaacgatat gtcgaagaaa atcaaacacg aaacaaacat 600atcaacccta
atgaatggtg gaaagtctat ccatcttctg taacggaatt taagttttta
660tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
aaatcatatc 720actaattgta atggagctgt tcttagtgta gaagagcttt
taattggtgg agaaatgatt 780aaagccggca cattaacctt agaggaagtc
agacggaaat ttaataacgg cgagataaac 840ttttaagggc ccttcgaagg
taagcctatc cctaaccctc tcctcggtct cgattctacg 900cgtaccggtc
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
960ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
gaccctggaa 1020ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
ttgcatcgca ttgtctgagt 1080aggtgtcatt ctattctggg gggtggggtg
gggcaggaca gcaaggggga ggattgggaa 1140gacaatagca ggcatgctgg
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1200agctggggct
ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
1260gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc 1320gctttcttcc cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg 1380ggcatccctt tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat 1440tagggtgatg gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg 1500ttggagtcca
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
1560atctcggtct attcttttga tttataaggg attttgggga tttcggccta
ttggttaaaa 1620aatgagctga tttaacaaaa atttaacgcg aattaattct
gtggaatgtg tgtcagttag 1680ggtgtggaaa gtccccaggc tccccaggca
ggcagaagta tgcaaagcat gcatctcaat 1740tagtcagcaa ccaggtgtgg
aaagtcccca ggctccccag caggcagaag tatgcaaagc 1800atgcatctca
attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1860actccgccca gttccgccca ttctccgccc catggctgac taattttttt
tatttatgca 1920gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
agtgaggagg cttttttgga 1980ggcctaggct tttgcaaaaa gctcccggga
gcttgtatat ccattttcgg atctgatcag 2040cacgtgttga caattaatca
tcggcatagt atatcggcat agtataatac gacaaggtga 2100ggaactaaac
catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
2160cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
gcagctctct 2220ctagcgacgg ccgcatcttc actggtgtca atgtatatca
ttttactggg ggaccttgtg 2280cagaactcgt ggtgctgggc actgctgctg
ctgcggcagc tggcaacctg acttgtatcg 2340tcgcgatcgg aaatgagaac
aggggcatct tgagcccctg cggacggtgt cgacaggtgc 2400ttctcgatct
gcatcctggg atcaaagcga tagtgaagga cagtgatgga cagccgacgg
2460cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
gcacttcgtg 2520gccgaggagc aggactgaca cgtgctacga gatttcgatt
ccaccgccgc cttctatgaa 2580aggttgggct tcggaatcgt tttccgggac
gccggctgga tgatcctcca gcgcggggat 2640ctcatgctgg agttcttcgc
ccaccccaac ttgtttattg cagcttataa tggttacaaa 2700taaagcaata
gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
2760ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
ctctagctag 2820agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 2880ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta atgagtgagc 2940taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3000cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
3060tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca 3120gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 3180atgtgagcaa
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
3240ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
tcagaggtgg 3300cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc 3360tctcctgttc cgaccctgcc gcttaccgga
tacctgtccg cctttctccc ttcgggaagc 3420gtggcgcttt ctcaatgctc
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3480aagctgggct
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
3540tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
agccactggt 3600aacaggatta gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct 3660aactacggct acactagaag gacagtattt
ggtatctgcg ctctgctgaa gccagttacc 3720ttcggaaaaa gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3780ttttttgttt
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
3840atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
gattttggtc 3900atgagattat caaaaaggat cttcacctag atccttttaa
attaaaaatg aagttttaaa 3960tcaatctaaa gtatatatga gtaaacttgg
tctgacagtt accaatgctt aatcagtgag 4020gcacctatct cagcgatctg
tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4080tagataacta
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
4140gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
aagggccgag 4200cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
ctattaattg ttgccgggaa 4260gctagagtaa gtagttcgcc agttaatagt
ttgcgcaacg ttgttgccat tgctacaggc 4320atcgtggtgt cacgctcgtc
gtttggtatg gcttcattca gctccggttc ccaacgatca 4380aggcgagtta
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
4440atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
agcactgcat 4500aattctctta ctgtcatgcc atccgtaaga tgcttttctg
tgactggtga gtactcaacc 4560aagtcattct gagaatagtg tatgcggcga
ccgagttgct cttgcccggc gtcaatacgg 4620gataataccg cgccacatag
cagaacttta aaagtgctca tcattggaaa acgttcttcg 4680gggcgaaaac
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
4740gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
agcaaaaaca 4800ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
ggaaatgttg aatactcata 4860ctcttccttt ttcaatatta ttgaagcatt
tatcagggtt attgtctcat gagcggatac 4920atatttgaat gtatttagaa
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 4980gtgccacctg
acgtcgacgg atcgggagat ctcccgatcc cctatggtcg actctcagta
5040caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
gtgttggagg 5100tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
gcaaggcttg accgacaatt 5160gcatgaagaa tctgcttagg gttaggcgtt
ttgcgctgct tcgcgatgta cgggccagat 5220atacgcgttg acattgatta
ttgactagtt attaatagta atcaattacg gggtcattag 5280ttcatagccc
atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
5340gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
atagtaacgc 5400caatagggac tttccattga cgtcaatggg tggagtattt
acggtaaact gcccacttgg 5460cagtacatca agtgtatcat atgccaagta
cgccccctat tgacgtcaat gacggtaaat 5520ggcccgcctg gcattatgcc
cagtacatga ccttatggga ctttcctact tggcagtaca 5580tctacgtatt
agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
5640gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
gtcaatggga 5700gtttgttttg gcaccaaaat caacgggact ttccaaaatg
tcgtaacaac tccgccccat 5760tgacgcaaat gggcggtagg cgtgtacggt
gggaggtcta tataagcaga gctctctggc 5820taactagaga acccactgct
tactggctta tcgaaattaa tacgactcac tatagggaga 5880cccaagctgg
ctagcaccat ggactacaaa gaccatgacg gtgattataa agatcatgac
5940atcgattaca aggatgacga tgacaagatg gcccccaaga agaagaggaa
ggtgggcatt 6000caccgcgggg tacctatggt ggacttgagg acactcggtt
attcgcaaca gcaacaggag 6060aaaatcaagc ctaaggtcag gagcaccgtc
gcgcaacacc acgaggcgct tgtggggcat 6120ggcttcactc atgcgcatat
tgtcgcgctt tcacagcacc ctgcggcgct tgggacggtg 6180gctgtcaaat
accaagatat gattgcggcc ctgcccgaag ccacgcacga ggcaattgta
6240ggggtcggta aacagtggtc gggagcgcga gcacttgagg cgctgctgac
tgtggcgggt 6300gagcttaggg ggcctccgct ccagctcgac accgggcagc
tgctgaagat cgcgaagaga 6360gggggagtaa cagcggtaga ggcagtgcac
gcctggcgca atgcgctcac cggggccccc 6420ttgaac 6426154128DNAArtificial
SequencegXTN-1C 154tctagaggtc tcattgaccc cagaccaggt agtcgcaatc
gcgtcacatg acgggggaaa 60gcaagccctg gaaaccgtgc aaaggttgtt gccggtcctt
tgtcaagacc acggcagaga 120ccggatcc 128155131DNAArtificial
SequencegXTN-2C 155tctagaggtc tcacggcctg actcccgatc aagttgtagc
gattgcgtcg catgacggag 60ggaaacaagc attggagact gtccaacggc tccttcccgt
gttgtgtcaa gcccacggag 120agaccggatc c 131156131DNAArtificial
SequencegXTN-3C 156tctagaggtc tcaacggttt gacgcctgca caagtggtcg
ccatcgccag ccatgatggc 60ggtaagcagg cgctggaaac agtacagcgc ctgctgcctg
tactgtgcca ggatcatgag 120agaccggatc c 131157131DNAArtificial
SequencegXTN-4C 157tctagaggtc tcacatggac tgaccccaga ccaggtagtc
gcaatcgcgt cacatgacgg 60gggaaagcaa gccctggaaa ccgtgcaaag gttgttgccg
gtcctttgtc aagaccacag 120agaccggatc c 131158131DNAArtificial
SequencegXTN-5C 158tctagaggtc tcaccacggc ctgaccccag accaggtagt
cgcaatcgcg tcacatgacg 60ggggaaagca agccctggaa accgtgcaaa ggttgttgcc
ggtcctttgt caagaccaag 120agaccggatc c 131159131DNAArtificial
SequencegXTN-6C 159tctagaggtc tcaaccacgg cctgactccc gatcaagttg
tagcgattgc gtcgcatgac 60ggagggaaac aagcattgga gactgtccaa cggctccttc
ccgtgttgtg tcaagcccag 120agaccggatc c 131160131DNAArtificial
SequencegXTN-7C 160tctagaggtc tcagcccacg gtttgacgcc tgcacaagtg
gtcgccatcg ccagccatga 60tggcggtaag caggcgctgg aaacagtaca gcgcctgctg
cctgtactgt gccaggatag 120agaccggatc c 131161131DNAArtificial
SequencegXTN-8C 161tctagaggtc tcaggatcat ggactgaccc cagaccaggt
agtcgcaatc gcgtcacatg 60acgggggaaa gcaagccctg gaaaccgtgc aaaggttgtt
gccggtcctt tgtcaagaag 120agaccggatc c 131162131DNAArtificial
SequencegXTN-9C 162tctagaggtc tcaaagacca cggcctgacc ccagaccagg
tagtcgcaat cgcgtcacat 60gacgggggaa agcaagccct ggaaaccgtg caaaggttgt
tgccggtcct ttgtcaagag 120agaccggatc c 131163140DNAArtificial
SequencegXTN-10C 163tctagaggtc tcacaagacc acggcctgac tcccgatcaa
gttgtagcga ttgcgtcgca 60tgacggaggg aaacaagcat tggagactgt ccaacggctc
cttcccgtgt tgtgtcaagc 120ccatggaaga gaccggatcc
140164128DNAArtificial SequencegXTN-1T 164tctagaggtc tcattgaccc
cagaccaggt agtcgcaatc gcgtcaaacg gagggggaaa 60gcaagccctg gaaaccgtgc
aaaggttgtt gccggtcctt tgtcaagacc acggcagaga 120ccggatcc
128165131DNAArtificial SequencegXTN-2T 165tctagaggtc tcacggcctg
actcccgatc aagttgtagc gattgcgtcg aacggtggag 60ggaaacaagc attggagact
gtccaacggc tccttcccgt gttgtgtcaa gcccacggag 120agaccggatc c
131166131DNAArtificial SequencegXTN-3T 166tctagaggtc tcaacggttt
gacgcctgca caagtggtcg ccatcgcctc gaatggcggc 60ggtaagcagg cgctggaaac
agtacagcgc ctgctgcctg tactgtgcca ggatcatgag 120agaccggatc c
131167131DNAArtificial SequencegXTN-4T 167tctagaggtc tcacatggac
tgaccccaga ccaggtagtc gcaatcgcgt caaacggagg 60gggaaagcaa gccctggaaa
ccgtgcaaag gttgttgccg gtcctttgtc aagaccacag 120agaccggatc c
131168131DNAArtificial SequencegXTN-5T 168tctagaggtc tcaccacggc
ctgaccccag accaggtagt cgcaatcgcg tcaaacggag 60ggggaaagca agccctggaa
accgtgcaaa ggttgttgcc ggtcctttgt caagaccaag 120agaccggatc c
131169131DNAArtificial SequencegXTN-6T 169tctagaggtc tcaaccacgg
cctgactccc gatcaagttg tagcgattgc gtcgaacggt 60ggagggaaac aagcattgga
gactgtccaa cggctccttc ccgtgttgtg tcaagcccag 120agaccggatc c
131170131DNAArtificial SequencegXTN-7T 170tctagaggtc tcagcccacg
gtttgacgcc tgcacaagtg gtcgccatcg ccagcaatgg 60cggcggtaag caggcgctgg
aaacagtaca gcgcctgctg cctgtactgt gccaggatag 120agaccggatc c
131171131DNAArtificial SequencegXTN-8T 171tctagaggtc tcaggatcat
ggactgaccc cagaccaggt agtcgcaatc gcgtcaaacg 60gagggggaaa gcaagccctg
gaaaccgtgc aaaggttgtt gccggtcctt tgtcaagaag 120agaccggatc c
131172131DNAArtificial SequencegXTN-9T 172tctagaggtc tcaaagacca
cggcctgacc ccagaccagg tagtcgcaat cgcgtcaaac 60ggagggggaa agcaagccct
ggaaaccgtg caaaggttgt tgccggtcct ttgtcaagag 120agaccggatc c
131173140DNAArtificial SequencegXTN-10T 173tctagaggtc tcacaagacc
acggcctgac tcccgatcaa gttgtagcga ttgcgtccaa 60cggtggaggg aaacaagcat
tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc 120ccatggaaga
gaccggatcc 140174128DNAArtificial SequencegXTN-1A 174tctagaggtc
tcattgaccc cagaccaggt agtcgcaatc gcgtcaaaca ttgggggaaa 60gcaagccctg
gaaaccgtgc aaaggttgtt gccggtcctt tgtcaagacc acggcagaga 120ccggatcc
128175131DNAArtificial SequencegXTN-2A 175tctagaggtc tcacggcctg
actcccgatc aagttgtagc gattgcgtcg aacattggag 60ggaaacaagc attggagact
gtccaacggc tccttcccgt gttgtgtcaa gcccacggag 120agaccggatc c
131176131DNAArtificial SequencegXTN-3A 176tctagaggtc tcaacggttt
gacgcctgca caagtggtcg ccatcgccag caatattggc 60ggtaagcagg cgctggaaac
agtacagcgc ctgctgcctg tactgtgcca ggatcatgag 120agaccggatc c
131177131DNAArtificial SequencegXTN-4A 177tctagaggtc tcacatggac
tgaccccaga ccaggtagtc gcaatcgcgt caaacattgg 60gggaaagcaa gccctggaaa
ccgtgcaaag gttgttgccg gtcctttgtc aagaccacag 120agaccggatc c
131178131DNAArtificial SequencegXTN-5A 178tctagaggtc tcaccacggc
ctgaccccag accaggtagt cgcaatcgcg tcgaacattg 60ggggaaagca agccctggaa
accgtgcaaa ggttgttgcc ggtcctttgt caagaccaag 120agaccggatc c
131179131DNAArtificial SequencegXTN-6A 179tctagaggtc tcaaccatgg
cctgactccc gatcaagttg tagcgattgc gtcgaacatt 60ggagggaaac aagcattgga
gactgtccaa cggctccttc ccgtgttgtg tcaagcccag 120agaccggatc c
131180131DNAArtificial SequencegXTN-7A 180tctagaggtc tcagcccacg
gtttgacgcc tgcacaagtg gtcgccatcg cctccaatat 60tggcggtaag caggcgctgg
aaacagtaca gcgcctgctg cctgtactgt gccaggatag 120agaccggatc c
131181131DNAArtificial SequencegXTN-8A 181tctagaggtc tcaggatcat
ggactgaccc cagaccaggt agtcgcaatc gcgtcgaaca 60ttgggggaaa gcaagccctg
gaaaccgtgc aaaggttgtt gccggtcctt tgtcaagaag 120agaccggatc c
131182131DNAArtificial SequencegXTN-9A 182tctagaggtc tcaaagacca
cggcctgacc ccagaccagg tagtcgcaat cgcgtcgaac 60attgggggaa agcaagccct
ggaaaccgtg caaaggttgt tgccggtcct ttgtcaagag 120agaccggatc c
131183140DNAArtificial SequencegXTN-10A 183tctagaggtc tcacaagacc
acggcctgac tcccgatcaa gttgtagcga ttgcgtcgaa 60cattggaggg aaacaagcat
tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc 120ccatggaaga
gaccggatcc 140184128DNAArtificial SequencegXTN-1G 184tctagaggtc
tcattgaccc cagaccaggt agtcgcaatc gcgaacaata atgggggaaa 60gcaagccctg
gaaaccgtgc aaaggttgtt gccggtcctt tgtcaagacc acggcagaga 120ccggatcc
128185131DNAArtificial SequencegXTN-2G 185tctagaggtc tcacggcctg
actcccgatc aagttgtagc gattgcgaat aacaatggag 60ggaaacaagc attggagact
gtccaacggc tccttcccgt gttgtgtcaa gcccacggag 120agaccggatc c
131186131DNAArtificial SequencegXTN-3G 186tctagaggtc tcaacggttt
gacgcctgca caagtggtcg ccatcgccaa caacaacggc 60ggtaagcagg cgctggaaac
agtacagcgc ctgctgcctg tactgtgcca ggatcatgag 120agaccggatc c
131187131DNAArtificial SequencegXTN-4G 187tctagaggtc tcacatggac
tgaccccaga ccaggtagtc gcaatcgcga acaataatgg 60gggaaagcaa gccctggaaa
ccgtgcaaag gttgttgccg gtcctttgtc aagaccacag 120agaccggatc c
131188131DNAArtificial SequencegXTN-5G 188tctagaggtc tcaccacggc
ctgaccccag accaggtagt cgcaatcgcg aacaataatg 60ggggaaagca agccctggaa
accgtgcaaa ggttgttgcc ggtcctttgt caagaccaag 120agaccggatc c
131189131DNAArtificial SequencegXTN-6G 189tctagaggtc tcaaccatgg
cctgactccc gatcaagttg tagcgattgc gaataacaat 60ggagggaaac aagcattgga
gactgtccaa cggctccttc ccgtgttgtg tcaagcccag 120agaccggatc c
131190131DNAArtificial SequencegXTN-7G 190tctagaggtc tcagcccacg
gtttgacgcc tgcacaagtg gtcgccatcg ccaacaacaa 60cggcggtaag caggcgctgg
aaacagtaca gcgcctgctg cctgtactgt gccaggatag 120agaccggatc c
131191131DNAArtificial SequencegXTN-8G 191tctagaggtc tcaggatcat
ggactgaccc cagaccaggt agtcgcaatc gcgaacaata 60atgggggaaa gcaagccctg
gaaaccgtgc aaaggttgtt gccggtcctt tgtcaagaag 120agaccggatc c
131192131DNAArtificial SequencegXTN-9G 192tctagaggtc tcaaagacca
cggcctgacc ccagaccagg tagtcgcaat cgcgaacaat 60aatgggggaa agcaagccct
ggaaaccgtg caaaggttgt tgccggtcct ttgtcaagag 120agaccggatc c
131193140DNAArtificial SequencegXTN-10G 193tctagaggtc tcacaagacc
acggcctgac tcccgatcaa gttgtagcga ttgcgaataa 60caatggaggg aaacaagcat
tggagactgt ccaacggctc cttcccgtgt tgtgtcaagc 120ccatggaaga
gaccggatcc 14019481DNAArtificial SequencepFUS-X 194cctgcaggtc
gaccgtctca gaacttgaag agaccgtacg tgatcgtggt ctcatggatt 60gaagagacgg
gtaccgagct c 8119577DNAArtificial SequencepFUS-Z1 195cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca cggcctgaag 60agacgggtac
cgagctc 7719678DNAArtificial SequencepFUS-Z2 196cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca acggtctgaa 60gagacgggta
ccgagctc 7819779DNAArtificial SequencepFUS-Z3 197cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca catggactga 60agagacgggt
accgagctc 7919880DNAArtificial SequencepFUS-Z4 198cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca ccacggcctg 60aagagacggg
taccgagctc 8019981DNAArtificial SequencepFUS-Z5 199cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca accacggcct 60gaagagacgg
gtaccgagct c 8120082DNAArtificial SequencepFUS-Z6 200cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca gcccacggtc 60tgaagagacg
ggtaccgagc tc 8220183DNAArtificial SequencepFUS-Z7 201cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca ggatcatgga 60ctgaagagac
gggtaccgag ctc 8320284DNAArtificial SequencepFUS-Z8 202cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca aagaccacgg 60cctgaagaga
cgggtaccga gctc 8420385DNAArtificial SequencepFUS-Z9 203cctgcaggtc
gaccgtctca ttgaagagac cgtactggat cgtggtctca caagaccacg 60gcctgaagag
acgggtaccg agctc 8520477DNAArtificial SequencepFUS-Z10
204cctgcaggtc gaccgtctca ttgaagagac cgtactggat cgtggtctca
tggactgaag 60agacgggtac cgagctc 7720534PRTArtificial
SequenceMethylesterases and Methyltransferases 34aa Consensus EBE
205Gln Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Asn Asn Gly Gly Thr1
5 10 15Gln Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys Pro
Gly 20 25 30Met Val206102DNAArtificial SequenceBacktranseq of 34aa
QTTERIVAIGT SH GGTQALEAVLTALPRVCPGMV 206cagaccaccg agaggatcgt
ggccatcggc accagccacg gcggcaccca ggccctggag 60gccgtgctga ccgccctgcc
cagggtgtgc cccggcatgg tg 1022073123DNAArtificial
SequenceMethylesterase EBE 207gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg
cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca
acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag
gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca
aaatgtcgta acaactccgc
cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa
gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa
attaatacga ctcactatag ggagacccaa gctggctagc 900accatggact
acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
960gacgatgaca agatggcccc caagaagaag aggaaggtgg gcattcaccg
cggggtacct 1020atggtggact tgaggacact cggttattcg caacagcaac
aggagaaaat caagcctaag 1080gtcaggagca ccgtcgcgca acaccacgag
gcgcttgtgg ggcatggctt cactcatgcg 1140catattgtcg cgctttcaca
gcaccctgcg gcgcttggga cggtggctgt caaataccaa 1200gatatgattg
cggccctgcc cgaagccacg cacgaggcaa ttgtaggggt cggtaaacag
1260tggtcgggag cgcgagcact tgaggcgctg ctgactgtgg cgggtgagct
tagggggcct 1320ccgctccagc tcgacaccgg gcagctgctg aagatcgcga
agagaggggg agtaacagcg 1380gtagaggcag tgcacgcctg gcgcaatgcg
ctcaccgggg cccccttgaa ccagaccacc 1440gagaggatcg tggccatcgg
caccagccac ggcggcaccc aggccctgga ggccgtgctg 1500accgccctgc
ccagggtgtg ccccggcatg gtgcagacca ccgagaggat cgtggccatc
1560ggcaccagcc acggcggcac ccaggccctg gaggccgtgc tgaccgccct
gcccagggtg 1620tgccccggca tggtgcagac caccgagagg atcgtggcca
tcggcaccag ccacggcggc 1680acccaggccc tggaggccgt gctgaccgcc
ctgcccaggg tgtgccccgg catggtgcag 1740accaccgaga ggatcgtggc
catcggcacc agccacggcg gcacccaggc cctggaggcc 1800gtgctgaccg
ccctgcccag ggtgtgcccc ggcatggtgc agaccaccga gaggatcgtg
1860gccatcggca ccagccacgg cggcacccag gccctggagg ccgtgctgac
cgccctgccc 1920agggtgtgcc ccggcatggt gcagaccacc gagaggatcg
tggccatcgg caccagccac 1980ggcggcaccc aggccctgga ggccgtgctg
accgccctgc ccagggtgtg ccccggcatg 2040gtgcagacca ccgagaggat
cgtggccatc ggcaccagcc acggcggcac ccaggccctg 2100gaggccgtgc
tgaccgccct gcccagggtg tgccccggca tggtgcagac caccgagagg
2160atcgtggcca tcggcaccag ccacggcggc acccaggccc tggaggccgt
gctgaccgcc 2220ctgcccaggg tgtgccccgg catggtgcag accaccgaga
ggatcgtggc catcggcacc 2280agccacggcg gcacccaggc cctggaggcc
gtgctgaccg ccctgcccag ggtgtgcccc 2340ggcatggtgc agaccaccga
gaggatcgtg gccatcggca ccagccacgg cggcacccag 2400gccctggagg
ccgtgctgac cgccctgccc agggtgtgcc ccggcatggt gcagaccacc
2460gagaggatcg tggccatcgg caccagccac ggcggcaccc aggccctgga
ggccgtgctg 2520accgccctgc ccagggtgtg ccccggcatg gtgcagacca
ccgagaggat cgtggccatc 2580ggcaccagcc acggcggcac ccaggccctg
gaggccgtgc tgaccgccct gcccagggtg 2640tgccccggca tggtgcagac
caccgagagg atcgtggcca tcggcaccag ccacggcggc 2700acccaggccc
tggaggccgt gctgaccgcc ctgcccaggg tgtgccccgg catggtgcag
2760accaccgaga ggatcgtggc catcggcacc agccacggcg gcacccaggc
cctggaggcc 2820gtgctgaccg ccctgcccag ggtgtgcccc ggcatggtgc
tgacacccga acaggtggtc 2880gccattgcta ataataacgg aggacggcca
gccttggagt ccatcgtagc ccaattgtcc 2940aggcccgatc ccgcgttggc
tgcgttaacg aatgaccatc tggtggcgtt ggcatgtctt 3000ggtggacgac
ccgcgctcga tgcagtcaaa aagggtctgc ctcatgctcc cgcattgatc
3060aaaagaacca accggcggat tcccgagaga acttcccatc gagtcgcggg
atcccaacta 3120gtc 312320817DNAArtificial SequenceTALEN monomer
with 17 RVD array 208tgatagtcgc cttatgt 1720934PRTArtificial
Sequenceconsensus sequence Xaa is Ala or AspX(32)..(32)Xaa is Ala
or Asp 209Leu Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly
Gly Thr1 5 10 15Gln Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val
Cys Gln Xaa 20 25 30Gly His21020DNAArtificial SequencePCR primer
210attgggctac gatggactcc 2021111PRTArtificial Sequenceconsensus EBE
fragment 211Gln Thr Thr Glu Arg Ile Val Ala Ile Gly Thr1 5
1021221PRTArtificial Sequenceconsensus EBE fragment 212Gly Gly Thr
Gln Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val1 5 10 15Cys Pro
Gly Met Val 2021329PRTPseudomonas mendocina ymp 213Thr Thr Asp Arg
Val Val Ala Leu Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu
Val Val Leu Arg Gln Leu Pro Val Asp Cys 20 2521429PRTPseudomonas
syringae pv. syringae FF5 214Thr Thr Glu Arg Ile Val Ala Ile Gly
Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala
Leu Pro Arg Val Cys 20 2521529PRTSulfuricella denitrificans skB26
215Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1
5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2521629PRTPseudomonas syringae pv. pisi str. 1704B 216Thr Thr Glu
Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu
Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2521729PRTPseudomonas syringae pv. syringae FF5 217Thr Thr Glu Arg
Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu
Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20 2521829PRTPseudomonas
syringae pv. maculicola str. ES4326 218Thr Thr Glu Arg Ile Val Ala
Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu
Thr Ala Leu Pro Arg Val Cys 20 2521929PRTPseudomonas amygdali pv.
morsprunorum str. M302280 219Thr Thr Glu Arg Ile Val Ala Ile Gly
Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala
Leu Pro Arg Val Cys 20 2522029PRTPseudomonas syringae pv. japonica
str. M301072 220Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly
Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val
Cys 20 2522129PRTPseudomonas syringae pv. actinidiae str. M302091
221Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1
5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2522229PRTPseudomonas savastanoi pv. savastanoi NCPPB 3335 222Thr
Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10
15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2522329PRTPseudomonas syringae pv. aesculi str. NCPPB 3681 223Thr
Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10
15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2522429PRTPseudomonas syringae pv. oryzae str. 1_6 224Thr Thr Glu
Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu
Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2522529PRTPseudomonas syringae pv. tomato K40 225Thr Thr Glu Arg
Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu
Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20 2522629PRTPseudomonas
syringae pv. tomato str. DC3000 226Thr Thr Glu Arg Ile Val Ala Ile
Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr
Ala Leu Pro Arg Val Cys 20 2522729PRTPseudomonas syringae pv.
aptata str. DSM 50252 227Thr Thr Glu Arg Ile Val Ala Ile Gly Thr
Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu
Pro Arg Val Cys 20 2522829PRTPseudomonas savastanoi pv. glycinea
str. race 4 228Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly
Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val
Cys 20 2522929PRTPseudomonas syringae Cit 7 229Thr Thr Glu Arg Ile
Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala
Val Leu Thr Ala Leu Pro Arg Val Cys 20 2523029PRTPseudomonas
syringae pv. syringae B728a 230Thr Thr Glu Arg Ile Val Ala Ile Gly
Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala
Leu Pro Arg Val Cys 20 2523129PRTPseudomonas amygdali pv. mori str.
301020 231Thr Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly
Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys
20 2523229PRTPseudomonas syringae pv. tabaci str. ATCC 11528 232Thr
Thr Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10
15Ala Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2523329PRTPseudomonas syringae pv. aceris str. M302273 233Thr Thr
Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala
Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2523429PRTPseudomonas savastanoi pv. glycinea str. B076 234Thr Thr
Glu Arg Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala
Leu Glu Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20
2523529PRTPseudomonas syringae pv. syringae 642 235Thr Thr Glu Arg
Ile Val Ala Ile Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu
Ala Val Leu Thr Ala Leu Pro Arg Val Cys 20 2523629PRTPseudomonas
syringae pv. phaseolicola 1448A 236Thr Thr Glu Arg Ile Val Ala Ile
Gly Thr Ser Thr Gly Gly Thr Gln1 5 10 15Ala Leu Glu Ala Val Leu Thr
Ala Leu Pro Arg Val Cys 20 2523728PRTBurkholderia rhizoxinica HKI
454 237Ala Asp Ile Val Lys Ile Ala Ser Asn Gly Gly Gly Ala Gln Ala
Leu1 5 10 15Glu Ala Val Ala Met His Gly Ser Thr Leu Cys Glu 20
25
* * * * *
References