17. Available plasmids

 

The plasmids listed here are available.  To obtain these plasmids, please send your requests

to Mitsuyasu Hasebe (mhasebe@nibb.ac.jp)

 

1. Plasmids for gene deletion

No

Name

marker

Resistance in Physco

Resistance in E. coli

comment

1

pTN182

(AB267706)

nptII

G418

Kanamycin and Ampicillin

Floxed modified nptII cassette

2

pTN186

aph4

Hygromycin

Kanamycin and Ampicillin

Floxed modified aphIV cassette

3

p35S-loxP-Zeo

ble

Zeocin

Kanamycin and Ampicillin

Floxed ble cassette

4

p35S-loxP-BSD

(AB537973)

BSD

Blasticidin S

Kanamycin and Ampicillin

Floxed BSD cassette

* Both pTN182 and pTN186 plasmids are available from addgene.

 

2. Plasmids for a reporter gene assay

 

No

Name

reporter

marker

comment

1

pTN83

uidA

nptII

Modified nptII cassette: to fuse GUS to a C terminal

2

pTN84

uidA

nptII

Floxed modified nptII cassette: to fuse GUS to a C terminal

3

pTN85

uidA

nptII

GUS (M1L), Floxed modified nptII cassette: to fuse GUS to a C terminal

4

pGFPmutNPTII

gfp

nptII

GFP(M1L): to fuse GFP to a C terminal

5

pYHG2

gfp

aph4

GFP(M1L): to fuse GFP to a C terminal

6

plinkerYHG2

gfp

aph4

GFP(M1L), linker sequence just before GFP;  to fuse GFP to a C terminal

7

pHIZ2

gfp

Ble

GFP(M1L): to fuse GFP to a C terminal

8

pHIZ3

gfp

ble

to fuse GFP to a N terminal

 

 

3. Plasmids for overexpression analysis

No

Name

Promoter

Marker in Physco

Marker in E. coli

Target site

Comment & references

1

pPpMAD2 7113

E7113

nptII

 

PpMAD2

 

2

pPpMAD2 7113 rev

E7113

nptII

 

PpMAD2

 

3

pPphb7 7113

E7113

nptII

 

Pphb7

 

4

pPphb7 7113 rev

E7113

nptII

 

Pphb7

 

5

pPpMADS2 Actin (pTFH9119)

Actin

nptII

 

PpMAD2

 

6

pPpMAD2 Actin rev

Actin

nptII

 

PpMAD2

 

7

pTN90

Actin

nptII

 

Pphb7

 

8

pTFH15.3

Actin

nptII

 

Pphb7

 

9

pZAG1

Actin

ble

 

213 locus

GFP-fusion

10

pMAK1

7113

ble

 

213 locus

 

11

pCMAK1

7113

ble

 

213 locus

 

12

pTFH38.9N

CaMV35S

ble

 

213 locus

mRFP-fusion

13

pTFH38.9C

CaMV35S

ble

 

213 locus

mRFP-fusion

14

pKS2

Actin

nptII

 

 

GFP-fusion

15

pActN-GFP

Actin

nptII

 

 

GFP-fusion

16

pTFH22.4

Actin

nptII

GFP

 

 

 

17

pTKM1

Actin

None

 

 

Transient expression

18

pT1OGC

Elongation Factor 1 alpha (EF1alpha)

zeocin

Ampicillin

PTA1

Constitutive expression

of Cerulean-fusion protein

 

 

1-1 pTN182

Constructed by Tomoaki Nishiyama

DDBJ/EMBL/ Genbank accession no: AB267706

Primers:

Pcmv-R: 5f- GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3: 5f- TGT AAA ACG ACG GCC AGT-3f

CMVP-1: 5f-taCCCTTTGTTGAAAAGTgTCAA-3f

CMV-PA-SEQ: 5f-tCCTCACCAAAATACGAAAGAGTT-3f

nptII-F2: 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

 

 

pTN81 (loxP is inserted at each PstI and SpeI site for pTN182)

Constructed by Tomoaki Nishiyama

Primers:

Pcmv-R (2307): 5f- GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3 (1152): 5f- TGT AAA ACG ACG GCC AGT-3f

CMVP-1 (380): 5f-taCCCTTTGTTGAAAAGTgTCAA-3f

CMV-PA-SEQ (220): 5f-tCCTCACCAAAATACGAAAGAGTT-3f

nptII-F2 (658): 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

1-2 pTN186

Constructed by Tomoaki Nishiyama

 

Primers:

M13-21 (1152): 5f- TGT AAA ACG ACG GCC AGT-3f

Reverse (658): 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

Pcmv-R (2307): 5f- GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3 (1152): 5f- TGT AAA ACG ACG GCC AGT-3f

CMV-PA-SEQ (220): 5f-tCCTCACCAAAATACGAAAGAGTT-3f

nptII-F2 (658): 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

 

 

 

pTN86 (loxP is inserted at each PstI and SpeI site for pTN186)

Constructed by Tomoaki Nishiyama

Primers:

M13-21 (1152): 5f- TGT AAA ACG ACG GCC AGT-3f

Reverse (658): 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

Pcmv-R (2307): 5f- GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3 (1152): 5f- TGT AAA ACG ACG GCC AGT-3f

CMV-PA-SEQ (220): 5f-tCCTCACCAAAATACGAAAGAGTT-3f

nptII-F2 (658): 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

 

1-3 p35S-loxP-Zeo   Constructed by Tsuyoshi Aoyama

nptII-F3
 

Pcmv-R
 

nptII-F2
 

 

Primers

Pcmv-R: 5f-GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3: 5f-ACGGAAGGAAGGAGGAAGACAAGGAAGGAT-3f

nptII-F2: 5f-AGGAGGAAGACAAGGAAGGATAAGGTTGCA-3f

 

Sequence

>p35S-Zeo

ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccggGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCATAACTTCGTATAGCATACATTATACGAAGTTATGCCCCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGACACTTTTCAACAAAGGGTCTAGAACTAGTGGATCCCCGTCACCGGTGTGAGGGAACTAGTTTTGATCTTGAAAGATCTTTTATCTTTAGAGTTAAGAACTCTTTCGTATTTTGGTGAGGTTTTATCCTCTTGAGTTTTGGTCATAGACCTATTCATGGCTCTGATACCAATTTTTAAGCGGGGGCTTATGCGGATTATTTCTTAAATTGATAAGGGGTTATTAGGGGGTATAGGGTATAAATACAAGCATTCCCTTAGCGTATAGTATAAGTATAGTAGCGTACCTCTATCAAATTTCCATCTTCTTACCTTGCACAGGGCCTGCAACCTTATCCTTCCTTGTCTTCCTCCTTCCTTCCGTCCACTTCATCATATTTAAACCAAACCTACGGGGGAGTCAACGTAACCAACCCTGCCTTAGCATCTTTTCCCTAACGGCCTCCTGCCTAAGCGGTACTTCTAGCTTCGAACGGCGTCTGGGCTCCAGGTTTAGTCGTCTCGTGTCTGGTTTATATTCACGACAAAGATCTATAGGGACTTTAGGAGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAAGAACCCTAATTTCCCTTATCGGGAAACTACTCACACATTATTTATGGAGAAAATAGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTACCGTCCGAACCCCAGAGTCCCGCTCAGTCCTGCTCCTCGGCCACGAAGTGCACGCAGTTGCCGGCCGGGTCGCGCAGGGCGAACTCCCGCCCCCACGGCTGCTCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGACCTCCGACCACTCGGCGTACAGCTCGTCCAGGCCGCGCACCAACACCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCCTGGACCGCGCTGATGAACAGGGTCACGTCGTCCCGGACCACACCGGCGAAGTCGTCCTCCACGAAGTCCCGGGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCCGGCGACGTCGCGCGCGGTGAGCACCGGAACGGCACTGGTCAACTTGGCCATGGATCCCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTGACGAAGATTTTCTTCTTGTCATTGAGTCGTAAGAGACTCTGTATGAACTGTTCGCCAGTCTTTACGGCGAGTTCTGTTAGGTCCTCTATTTGAATCTTTGACTCCATGGCCTTTGATTCAGTGGGAACTACCTTTTTAGAGACTCCAATCTCTATTACTTGCCTTGGTTTGTGAAGCAAGCCTTGAATCGTCCATACTGGAATAGTACTTCTGATCTTGAGAAATATATCTTTCTCTGTGTTCTTGATGCAGTTAGTCCTGAATCTTTTGACTGCATCTTTAACCTTCTTGGGAAGGTATTTGATTTCCTGGAGATTATTGCTCGGGTAGATCGTCTTGATGAGACCTGCTGCGTAAGCCTCTCTAACCATCTGTGGGTTAGCATTCTTTCTGAAAGGGAATTCCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTATAACTTCGTATAGCATACATTATACGAAGTTATCCCTCACACCGGTGACGGGGATCGCATGCCCGGGTCTAGACATATGGATCctagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

 

Lower letters show pBluescriptSKII sequence.

 

1-4 p35S-loxP-BSD

Constructed by Tsuyoshi Aoyama

Pcmv-R
 

nptII-F3
 

nptII-F2
 

 

 

Primers

Pcmv-R: 5f-GAGGAAGGGTCTTGCGAAGGATAGTG-3f

nptII-F3: 5f-ACGGAAGGAAGGAGGAAGACAAGGAAGGAT-3f

nptII-F2: 5f-AGGAGGAAGACAAGGAAGGATAAGGTTGCA-3f

 

Sequence

>p35S-loxP-BSD

ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgatatCGAATTCCATAACTTCGTATAGCATACATTATACGAAGTTATGCCCCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGACACTTTTCAACAAAGGGATCGAATTCCTGCAGCCCCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGCAAGGAGGTGAGCTTTCTCATTGTCAATATGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACCAGCCATTGATCTTGTACATTCAAGCTACTTTCTGTTACTAGATGAACCGAGCATGAGTTGATGGTGACGCGGGACTCTGGGGTTCGGACGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTGACTCCCCCGTAGGTTTGGTTTAAATATGATGAAGTGGACGGAAGGAAGGAGGAAGACAAGGAAGGATAAGGTTGCAGGCCCTGTGCAAGGTAAGAAGATGGAAATTTGATAGAGGTACGCTACTATACTTATACTATACGCTAAGGGAATGCTTGTATTTATACCCTATACCCCCTAATAACCCCTTATCAATTTAAGAAATAATCCGCATAAGCCCCCGCTTAAAAATTGGTATCAGAGCCATGAATAGGTCTATGACCAAAACTCAAGAGGATAAAACCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTAGTTCCCTCACACCGGTGACGGGGATCGCATGCGATCCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTATAACTTCGTATAGCATACATTATACGAAGTTATCCCTCACACCGGTGACGGGGATCGCATGCCCGGGTCTAGACATATGGATCctagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

 

Lower letters show pBluescriptSKII sequence.

 2. Plasmids for a reporter gene assay

2)-1 pTN83

Constructed by Tomoaki Nishiyama

Primers

GUS-R2 (308): 5f- ATTTCACGGGTTGGGGTTTCTACAGGACGT-3f

GUS-R3 (308): 5f- TCTTGTAACGCGCTTTCCCACCAACGCTGA-3f

Sequence:

>5f MCS

GAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGTGGTCAGTCCCTTATGTTACGTCCTGTAGAAACCCCAACCCGTGAAATCAAAAAACTCGACGGCCTGT

>3f MCS

TCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCG

2)-2 pTN84

Constructed by Tomoaki Nishiyama

Primers

GUS-R2 (308): 5f- ATTTCACGGGTTGGGGTTTCTACAGGACGT-3f

GUS-R3 (308): 5f- TCTTGTAACGCGCTTTCCCACCAACGCTGA-3f

Sequence:

>5f MCS

AGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCTCAGTCCCTTATGTTACGT

 

>3f MCS

ATAGCATACATTATACGAAGTTATCCCTCACACCGGTGACGGGGATCGGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCC

2)-3 pTN85

Constructed by Tomoaki Nishiyama

Primers

GUS-R2 (308): 5f- ATTTCACGGGTTGGGGTTTCTACAGGACGT-3f

GUS-R3 (308): 5f- TCTTGTAACGCGCTTTCCCACCAACGCTGA-3f

M13-21: 5f-TGT AAA ACG ACG GCC AGT-3f

Reverse: 5f-AGC GGA TAA CAA TTT CAC ACA GG-3f

Sequence:

>5f MCS

CGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTTTGTTACGTCCTGTAGAAACCCCAACCCGTGAAATCAAAAAA

>3f MCS

CCGGTGACGGGGATCGGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAAT

2)-4 pGFPmutNPT

Constructed by Rumiko Kofuji

Primers

sGFP- (308): 5f- TCGCCGGACACGCTGAACTTGTG-3f

 

 

Sequence

>5fMCS:

TTG GGT ACC GGG CCC CCC CTC GAG GTC GAC GGT ATC GAT AAG CTT TTG GTG AG

>3fMCS:

AAAGATAAAAGATCTTTCAAGATCAAAACTAGTTCCCTCACACCGGTGACGGGGATCGCATGCGATATCTCGAGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAG

2)-5 pYHG2

Constructed by Yuji Hiwatashi

Primers

sGFP- (308): 5f- TCGCCGGACACGCTGAACTTGTG-3f

 

 

Sequence

>5fMCS:

TTG GGT ACC GGG CCC CCC CTC GAG GTC GAC GGT ATC GAT AAG CTT TTG GTG AG

2)-6 plinkerYHG2

Constructed by Junko Kawai

Primers

sGFP- (308): 5f- TCGCCGGACACGCTGAACTTGTG-3f

 

Sequence

>5fMCS:

TTG GGT ACC GGG CCC CCC CTC GAG GTC GAC GGT ATC GAT TCC CTG CAG GCA GGA GCA GGA GCA GGA GCA GGA GCA GGA GCA GGT CAG CTT TTG GTG AG

 

2)-7 pHIZ2

Constructed by Yuji Hiwatashi

Primers

sGFP- (308): 5f- TCGCCGGACACGCTGAACTTGTG-3f

 

Sequence

>5fMCS:

TTG GGT ACC GGG CCC CCC CTC GAG GTC GAC GGT ATC GAT AAG CTT TTG GTG AG

2)-8 pHIZ3

Constructed by Yuji Hiwatashi

pHIZ3

Primers

sGFP- (308): 5’- TCGCCGGACACGCTGAACTTGTG-3’

 

Sequence

>Cloning site:

 

 

 


 

Comment&Reference

sGFP (stop codon: TAA->TCG in a SalI site) expression vector, zeocin resistang, pBluescriptKSII

 

 

3. Plasmids for overexpression analysis

 

3-1, 2, 3, and 4. pPpMAD2 7113, pPpMAD2 7113 rev, pPphb7 7113, and pPphb7 7113 rev.

Constructed by Rumiko Kofuji

   

Primers:

PMADS2-5: 5f- AGT GGA TTG TAG GGA TGG TCC AGC GAG C-3f

PMADS2-3: 5f- TGA AAA ATC TCC GGC ACT CCA CGC CAA C-3f

PHB9-52: 5f- TAG CGG CCG CGG AAA GGG GAG GGA AGG GTG TAA-3f

PHB9-32: 5f- TAG CGG CCG CTC AGG GAC GCA CAA CAG CGA CAA-3f

The following primers are located outside the genomic region of the PpHB9 platform.  You can check whether this vector was targeted into the PpHB9 locus or not by PCR with these primers

PpHB9-51: 5f- GTG TCG AGC GCG TCG GCA AGA G-3f

PpHB9-31: 5f- AAA GAG GTG CCA CTT TAT TCC AT-3f

3-5 and 6. pTFH9119 and pPpMAD2 Actin rev

Constructed by Tomomichi Fujita

@@

Primers:

PMADS2-5: 5f- AGT GGA TTG TAG GGA TGG TCC AGC GAG C-3f

PMADS2-3: 5f- TGA AAA ATC TCC GGC ACT CCA CGC CAA C-3f

 

3-7. pTN90

Constructed by Tomoaki Nishiyama

Sequence

>pTN90

ggaaaggggagggaagggtgtaaggaggcccacgggcagggtcaaggtgtccaatgcacctgcaagatcaggaagcttgaagtagatcagggaacaaacgatggtagtccctagtttacccgccttcggaggacagaacgccatgctcagacgcaacattgacaacaacaccgacactctgatttctctgcttcaagggtcctgctcccctcgcgtgagcatgcaacaaggtgtgcattatatgctttacactcgatcctacactgggcatacgtgattagatttgcaccatgcgaaggtgcgttggaaatgcgtgcaaggtgcaatgcaggttgcttagccactcttatttttgaagacacttgtttccgagttggtagagcaaagtttctgctaagagagcttgtcgcacgatagccttcagaaaatcggcctagaaatgagcttcgtcaagggaacaactcaatgttgaggacatagccagtccgttggtgatatgctcaactgttgtctgtgccttactcttcttctgcaaaccgctccttgttgaagggcctcgcttgttctacatggtaaaggtacataaatgattccggttgaaaagttcatgtaggtaggtggtgtgaatgtttggctcgctagatatgttgctagttttttcgtcggacgatgttgtcatttttgttctccttcgtctggttacgctgagcatttcgcagtgttgaccggatatgatacacgagcattgcaagaaaagagaattgaagctttttcgatgtcaccttgtgtgaagtgacgtgcggttgattgaacttcgggctgcattttggcccacttgtctctcggtccatctcttcgcataagacttaaagacgcccaacattttaagattaaggattcgtggaaccgtcactatgaatgtgaaaaatgtattcgcatgtgcgacaaatatctttcaccggtattccttttggcactccgctcggcacaacgatttcgcagagcgattctctattagaccttgtattctctctacagaaaacacttcaagcagaatcactaaaaatcccttgatctggtggacaatattatctttgttgtcctcgcctgccatctgaaggagatgctgttcagtcctctcttgttggattttgagttgaacaagtatctaatcgaatctgcaggatatgttgcagtgccgcgttcatcggagagtctcgaaaacatgatgggggcttgtgggcaaaaactgccttacttttcgtcatttgatgggccgagtgtagaagagcaagaggatgtcgacgaaggtatcgacgaattcgcacaccacAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTCAAGCTTCGAGGTCATTCATATGCTTGAGAAGAGAGTCGGGATAGTCCAAAATAAAACAAAGGTAAGATTACCTGGTCAAAAGTGAAAACATCAGTTAAAAGGTGGTATAAAGTAAAATATCGGTAATAAAAGGTGGCCCAAAGTGAAATTTACTCTTTTCTACTATTATAAAAATTGAGGATGTTTTTGTCGGTACTTTGATACGTCATTTTTGTATGAATTGGTTTTTAAGTTTATTCGCTTTTGGAAATGCATATCTGTATTTGAGTCGGGTTTTAAGTTCGTTTGCTTTTGTAAATACAGAGGGATTTGTATAAGAAATATCTTTAAAAAAACCCATATGCTAATTTGACATAATTTTTGAGAAAAATATATATTCAGGCGAATTCTCACAATGAACAATAATAAGATTAAAATAGCTTTCCCCCGTTGCAGCGCATGGGTATTTTTTCTAGTAAAAATAAAAGATAAACTTAGACTCAAAACATTTACAAAAACAACCCCTAAAGTTCCTAAAGCCCAAAGTGCTATCCACGATCCATAGCAAGCCCAGCCCAACCCAACCCAACCCAACCCACCCCAGTCCAGCCAACTGGACAATAGTCTCCACACCCCCCCACTATCACCGTGAGTTGTCCGCACGCACCGCACGTCTCGCAGCCAAAAAAAAAAAAGAAAGAAAAAAAAGAAAAAGAAAAAACAGCAGGTGGGTCCGGGTCGTGGGGGCCGGAAACGCGAGGAGGATCGCGAGCCAGCGACnAGGCCGGCCCTCCCTCCGCTTCCAAAGAAACGCCCCCCATCGCCACTATATACATnCCCCCCCCTCTCCTCCCATCCCCCCAACCCcTACCACCACCACCACCACCACCTCCACCTCCTCCCCCCTCGCTGCCGGACGACGAGCTCCTCCCCCCTCCCCCTCCGCCGCCGCCGCGCCGGTAACCACCCCGCCCCTCTCCTCTTTCTTTCTCCGTTTTTTTTTTCCGTCTCGGTCTCGATCTTTGGCCTTGGTAGTTTGGGTGGGCGAGAGGCGGCTTCGTGCGCGCCCAGATCGGTGCGCGGGAGGGGCGGGATCTCGCGGCTGnGGsTCTCGCCGGCGTGGATCCGGCCCGGATCTCGCGGGGAATGGGGCTCTCGGATGTAGATCTGCGATCCGCCGTTGTTGGGGGAGATGATGGGGGGTTTAAAATTTCCgCCATGcTAAaCAAGATCAGGAAGAGGGGAAAAGGGCACTATGGTTTATATTTTTATATATTTCTGCTGCTTCGTCAGGCTTAGATGTGCTAGATCTTTCTTTCTTCTTTTTGTGGGTAGAATTTGAATCCCTCAGCATTGTTCATCGGTAGTTTTTCTTTTCATGATTTGTGACAAATGCAGCCTCGTGCGGAGCTTTTTTGTAGGTAGAAGATATCAGCTTTCGTCCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCCCACACACCAGAATCCTACTAAGTTTGAGTATTATGGCATTGGAAAAGCTGTTTTCTTCTATCATTTGTTCTGCTTGTAATTTACTGTGTTCTTTCAGTTTTTGTTTTCGGACATCAAAATGCAAATGGATGGATAAGAGTTAATAAATGATATGGTCCTTTTGTTCATTCTCAAATTATTATTATCTGTTGTTTTTACTTTAATGGGTTGAATTTAAGTAAGAAAGGAACTAACAGTGTGATATTAAGGTGCAATGTTAGACATATAAAACAGTCTTTCACCTCTCTTTGGTTATGTCTTGAATTGGTTTGTTTCTTCACTTATCTGTGTAATCAAGTTTACTATGAGTCTATGATCAAGTAATTATGCAATCAAGTTAAGTACAGtATAGGCTTTTTGTGTCGACATTTAAATATCGAATTCCTGCAGCCCCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGACACTTTTCAACAAAGGGTATATAATCGGGAAGACTCCTCGGATTCCATTGCCCAGCTATCTGTCaCTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGCAAGGAGGTGAGCATGATTGAACAAGACGGATTGCACGCAgGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAgCTGTGCTCGACGTTGTCACTGAAgCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAtCTCCTGTCATCTCACCTTGCtCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGACGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCnATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTATCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAgtggagaaaaagaggagattgtcattagaacaagtgcgatcattagaacggaattttgaagtggaaaacaagcttgagcccgagaggaaaatgcaactagctaaggagcttggactgcaacctcgtcaagtggcggtgtggttccagaatagacgggcaaggtggaaaaccaaacagctcgagcgcgactacgagaccctgaagaaagcctacgacaggcttaaagcagacttcgaagccgttactctagacacaagtgctcttaaagctgaggtacttagcatcttaatcctttccgcttgccttttcaaatttgagctcaaggagtgccaagtctcttgtttttgctgtgttcaccatacattaccaaaacctaacctcccatttcgctctgttttgcaggtgagtcgcctcaagggaatctctaatgacgacgtcaagcccgccgaattcgttcagggcaagtgtgacacaacgagtcaccctgcctcccctgcgcaatcggagaggtccgacattgtgtcatcgaggaatcgcacaactcctaccatacatgtggatcccgtggcacccgaggaagccggcgctcacttaaccatgagctcggatagcaattccagcgaggtcatggacgctgatagccctcgcacgagccacaccagcgctagtaggagcactttgtccacaagtgtggtgcagcctgacgagggcctgggagtggcccagtacccccacttttctcccgaaaacttcgtgggtcccaatatgccagagatttgcgctgatcagtcacttgcatctcaagtgaagctggaagagatccacagcttcaatcccgaccaaaccttcctgctcttgcccaactggtgggattgggcttgattcgtttcttcatctgtacccatacactttttccttgaatccaagttgaattcactttaggcagtgttttttcacgatgtaccacttgttattcttccaccatgtgcaatccaacgtcaaccaaagttgcaccatcggcgatcattggtgacgatgtcgagcatcgatcgtcaccatgccagtcggcgcttgtgcctttttgaacagcagcaggaaggcaacagaaaactctgggaaaagcaaccaggagcttcgaagttaggtgctgcaaccttgctagatatatgatcgaatcttgaggaattatgtagttaagtagttggacttatgtaaaaagtcagagtagggacacatagcccctttggaccagagctagaaccgaaacacactaatcggattactacaacttcaattcacttgccttggtgacacagtgatggcgatggtccgtgcatctgcagtattgttgtcggccgtgtaattgtacatatgcgaataagcattggttctcaaggtaggaaaggaggaaaaaatctttcgttgtcgctgttgtgcgtccctgagcggccgc

 

 

Lowercase letters: 5for 3f PpHB9 region

 

    : Sse9793I

    : NotI

    : EcoRV

    : KpnI

 

You can excise a region from Actin promoter (Pact) to the modified nptII cassette from this plasmid with KpnI and Sse9793I.

3-8. pTFH15.3

Constructed by Tomomichi Fujita

Sequence:

>cloning site

aaGATATCGGCCATTCAGGCCGGCGCGCCACCCGGGAGGGCCCGGCCGATTTGGCCatca

 

3-9. pZAG1

Constructed by Yuji Hiwatashi

pZAG1

 

 

Plasmid name: pZAG1

Plasmid size: 9.076 kb

Construction by: Yuji Hiwatashi

Construction date: 12 Feb., 2003

Comment & Reference: sGFP (start codon: ATG ->TTG) expression vector

under control of rice actin promoter. This vector contains a bleomycine (zeocin) resistant gene

and the BS213 fragments as a platform for integration. A kinesin gene fragment (KINID1a_cDNA)

is inserted in front of sGFP CDS fragment to express GFP fused with 1st to 30th amino acids of KINID1a.

You have to replace this fragment with your gene to express the GFP fusion protein.

This vector is in pBluescriptKSII.

 

Primers:

Pact-rev1: 5f- GGAGGAGCTCGTCGTCCGGCA-3f

The following primer is located upstream of BS213-5f region.  You can check the targeting of pZAG1 by PCR with 213F2 and Pact-rev1 primers (amplification of ca. 1.3-kb fragment).

213F2: 5f- AAATCTCATAGGCGTTGCTTG-3f

 

Sequence: The sequence of pZAG1

>pZAG1

TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCATCAGACGTAAATATAAAGTTGTAAGGAAAATTAGTTCTATAAAATGCCTCTATATTAACATTCAATTGTAAAGGTGCTGCGTATTCATGGATTAGTATATAAATTATTATTTTTGTAAAATGTGTGTGTGTGTTGTTAATGCTTTATGTAAAAAGAACTGTGTGCTTCAAAATACTATAGATCCCTTTTAGACAGATTATCTTTATGTAAAAAGAACTGTGTGCTTCAAAATACTATAGATCCCTTTTAGACAGAATATCTATAATATGTAGAAGTTAAATTCGGTTTTAATTCTGTACCATGTATTGTGAACATCTAGATTTTGTGATACAGCTTCATGAAAAACTTCAAAACTTCAAAACATTTTACTTCTATAAAGTTATCAATTTGTTTGACAAACAACCTTATATGGTAATGACAAGAGTCTCACATTAATTCAGCATCTCAAAAATAAATATAAATACTCTTTTATACTCAAAATTGTATTTAATACAAAACATATGCATGTATCAATCCAATGACTTGGGCCAATAATGTAAGTCACTCATTATAGTATTAGTATTTATTATTCCTTTTTGTTTCTCAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTCATCTATGTGATTCGTTCTCAACACTTACACTTCAAAAATATTTATTTACTCATTCATCAATACATCCAAGATAAAATATATATATATAGATATATATATATATATATATATATATTTATCTTTTTCTAACCTCTTCTACAGAGTTTTGTTCAGTGTTGTAAACCCAAACTTGGTGTCGAGGGCCCGAGGTCATTCATATGCTTGAGAAGAGAGTCGGGATAGTCCAAAATAAAACAAAGGTAAGATTACCTGGTCAAAAGTGAAAACATCAGTTAAAAGGTGGTATAAAGTAAAATATCGGTAATAAAAGGTGGCCCAAAGTGAAATTTACTCTTTTCTACTATTATAAAAATTGAGGATGTTTTTGTCGGTACTTTGATACGTCATTTTTGTATGAATTGGTTTTTAAGTTTATTCGCTTTTGGAAATGCATATCTGTATTTGAGTCGGGTTTTAAGTTCGTTTGCTTTTGTAAATACAGAGGGATTTGTATAAGAAATATCTTTAAAAAAACCCATATGCTAATTTGACATAATTTTTGAGAAAAATATATATTCAGGCGAATTCTCACAATGAACAATAATAAGATTAAAATAAGCTTTCCCCCGTTGCAGCGCATGGGTATTTTTTCTAGTAAAAATAAAAGATAAACTTAGACTCAAAACATTTACAAAAACAACCCCTAAAGTTCCTAAAGCCCAAAGTGCTATCCACGATCCATAGCAAGCCCAGCCCAACCCAACCCAACCCAACCCACCCCAGTCCAGCCAACTGGACAATAGTCTCCACACCCCCCCACTATCACCGTGAGTTGTCCGCACGCACCGCACGTCTCGCAGCCAAAAAAAAAAAAGAAAGAAAAAAAAGAAAAAGAAAAAACAGCAGGTGGGTCCGGGTCGTGGGGGCCGGAAACGCGAGGAGGATCGCGAGCCAGCGACGAGGCCGGCCCTCCCTCCGCTTCCAAAGAAACGCCCCCCATCGCCACTATATACATACCCCCCCCTCTCCTCCCATCCCCCCAACCCTACCACCACCACCACCACCACCTCCACCTCCTCCCCCCTCGCTGCCGGACGACGAGCTCCTCCCCCCTCCCCCTCCGCCGCCGCCGCGCCGGTAACCACCCCGCCCCTCTCCTCTTTCTTTCTCCGTTTTTTTTTTCCGTCTCGGTCTCGATCTTTGGCCTTGGTAGTTTGGGTGGGCGAGAGGCGGCTTCGTGCGCGCCCAGATCGGTGCGCGGGAGGGGCGGGATCTCGCGGCTGGGGCTCTCGCCGGCGTGGATCCGGCCCGGATCTCGCGGGGAATGGGGCTCTCGGATGTAGATCTGCGATCCGCCGTTGTTGGGGGAGATGATGGGGGGTTTAAAATTTCCGCCATGCTAAACAAGATCAGGAAGAGGGGAAAAGGGCACTATGGTTTATATTTTTATATATTTCTGCTGCTTCGTCAGGCTTAGATGTGCTAGATCTTTCTTTCTTCTTTTTGTGGGTAGAATTTGAATCCCTCAGCATTGTTCATCGGTAGTTTTTCTTTTCATGATTTGTGACAAATGCAGCCTCGTGCGGAGCTTTTTTGTAGGTAGAAGATATCGGGGATCCTCTAGAGTCGACGAAGCACCTCGCTCAATGTCTCTAAAACGTCAAGCCCCTCAGTCCTGTGGAATGAAACTCTTCTAGCGACCCCAGTGTAGTGTGACAGAGGGCTGGTGAGGACGAGGATTCCAAGAATGTCGTCTTGCTTATGCAGGTAAGGAGCTTATCCTCCAGACATCGACTAGAGGATCCATGCACCTATCATCTGGAATTTGAAATCCACTTTCTTACAGTGTCCTCTACATTGCGGCCTTCACGGTGACCGCTGGAGTGTGCCCACATACTCCCTTAAAACAGAGCCGTCGGGTGCGATGACAACGATGATGTCCCCGAAGCCGAGGGAGAAGAGGCCGTCTGGCAATGTAGGGGGTAATACACCCACGGGTAAGGCCAGGTCTATCGATAAGCTTTTGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGTAAAGCGGCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCCGATGATAAGCTGTCAAACATGAGAATTCCCTTTCAGAAAGAATGCTAACCCACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGGGATCCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTTGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGAGCGGGACTCTGGGGTTCGGACGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTGACTCCCCCGTAGGTTTGGTTTAAATATGATGAAGTGGACGGAAGGAAGGAGGAAGACAAGGAAGGATAAGGTTGCAGGCCCTGTGCAAGGTAAGAAGATGGAAATTTGATAGAGGTACGCTACTATACTTATACTATACGCTAAGGGAATGCTTGTATTTATACCCTATACCCCCTAATAACCCCTTATCAATTTAAGAAATAATCCGCATAAGCCCCCGCTTAAAAATTGGTATCAGAGCCATGAATAGGTCTATGACCAAAACTCAAGAGGATAAAACCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTAGTTCCCTCACACCGGTGACGGGGATCCACTAGTTCTAGAGCGGCCGCAACTTAGAAGTAGCGTGTTTCTCTACATCACGCTTTTATCCCTTTGTAAATTTTAGAGCGTAGGTTGGCATGGGGGGGCTATTGGCTGTGAAAGGGACAGAGATATCTGCTGCACCATGTTCTGTACATAAAGCTGCCGCTCTGTTACGCCGTTTCTTGTGAACCGAGGCAGCTTCTGAGGTTGGCTTTGCTTAAAGTTGCGCTGATGCTGTTTCCCAATGTTTAGAGGAAATCCTGGTGACGAGAACGAAGAACTCGGGAATTACTAGGGAGGATTTGAGGTTTGTATCTGCAGGGCGTGGTTCAAAATTCTTTCATGTCGAACAGGTTGACAAAGTTCCAGAAATTATGAAATCTCAAAGAGGTAAAGGGTCGAAACGTTTGAAGATCTCGAAAGAAAAAGGAGGAGAAAGGTTGAACATCATTTCATTTTAAGGCATTTCGAGGACATAAAATTGGATGGTGCAGAGGGGAGAAAGCAGGCCAATGGGAAGCTAGGTGTCGATGAAAAGCCTGTCGTTACCGAATTGCTTTCGAGTACACCTAGGCAATGAGATCCAGAAGATAATTACAGAGAATTTCAAAAGAATGAAAAGATGAAGAAAAAGAAGAAGAGTTTGAATGGAGAGGCAATGGATGTGGATGTAGGAGGGTTGTACTCGGACATTTTGATCGAGGCCGATGTGGATTGGTACCCTGATATTACGAAAAACATTGTAGAGCATCTGACATAATGTCTTCTAAAGTACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAG

 

 

3-10. pMAK1

Constructed by Masakazu Kitani

@@

 

3-11. pCMAK1

Constructed by Kaoru Hashimoto

3-12 and 13. pTFH38.9N and pTFH38.9C.

Constructed by Tomomichi Fujita)

3-14 and 15. pKS2 and pACtN-GFP.

pKS2 and pActN-GFP was constructed by Keiko Sakakibara and Yuji Hiwatashi, respectively

Sequence

>Cloning site of pKS2

GTC GAC CCT GCA GGG TTT AAA CGG CGC GCC ATC GAT AAG CTT TTG GTG AG

>cloning site of pActN-GFP

TG TAC AAG TCG ACC GCT CCC GCC ATC GAT TAA

 

3-16. pTFH22.4.

Constructed by Tomomichi Fujita

Sequence

>MCS of pTFH22.4

aaGATATCGGCCATTCAGGCCGGCGCGCCACCCGGGAGGGCCCGGCCGATTTGGCCatca

 

3-17. pTKM1

Constructed by Takashi Murata

Sequence

>cloning site of pTKM1

GATATCGGCCATTCAGGCCGGCGCGCCACCCGGGAGGGCCCGGCCGATTTGGCC

 

 

3-18. pT1OGC

Constructed by Minoru Kubo

 

Complimentary DNA fused with Cerulean (modified CFP) gene is targeted to the PTA1 site. The PTA1 is an initiation factor Sui1 locus. Sui1 is redundant to two paralogs and we could not detect any phenotype difference of the deletion mutant to wild type P. patens.

 

This plasmid is unpublished and please acknowledge to Minoru Kubo and Mitsuyasu Hasebe.

 

EST:

estExt_gwp_gw1.C_3100010

 

 

Genomic location:

5' targeting region: scaffold_310: 79837:80871 (1035 bp)

3' targeting region: scaffold_310: 81504:82528 (1025 bp)

 

<General>

>Name: pT1OGC

>Date: 2009”N 12ŒŽ 24“ϊ (–Ψ); 3:34:08 PM

>General Info:

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

<Generations>

>Generation: 0

>Name: 0 (Most Recent Generation) (Oldest Generation)

<Sites>

>Name: KpnI

>Position: 1

>Name: Sse8387I

>Position: 5

>Name: PmeI

>Position: 12

>Name: BstXI

>Position: 3867

>Name: Sse8387I

>Position: 8552

>Name: PmeI

>Position: 8559

>Name: SacI

>Position: 8567

<Segments>

>Name: M13F

>Range: 11371 to 11393

>Name: M13R

>Range: 8615 to 8638

>Name: NONE

>Range: 22 to 1056

>Name: pBSSK+Kokelinker1

>Range: 22 to 1056

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1H

>Range: 1065 to 2436

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1H

>Range: 2437 to 4147

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1O

>Range: 2437 to 4147

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1H

>Range: 7464 to 7507

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1OG

>Range: 7431 to 7520

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

>Name: pT1OG

>Range: 4155 to 4871

pBlueScribe SK(+)
    pBlueScribe  Length: 2964  (today)  Check: 1446  ..

<Regions of Interest>

>Name: AmpR

>Direction:  <--

>Range: 9788 to 10645

>Name:  ColE1 ori

>Direction:  ---

>Range: 9011 to 9625

>Name:  PTA1 5' side

>Direction:  ---

>Range: 22 to 1056

>Name: PTA1 3' side

>Direction:  ---

>Range: 7524 to 8548

>Name: ZeoR

>Direction:  -->

>Range: 6321 to 6692

>Name: loxP

>Direction:  ---

>Range: 5398 to 5431

>Name: P35S

>Direction:  ---

>Range: 5501 to 6239

>Name: TrbcS

>Direction:  ---

>Range: 4890 to 5350

>Name: EF1-a promoter

>Direction:  ---

>Range: 1073 to 2436

>Name: GATEWAY rfcA

>Direction:  -->

>Range: 2437 to 4147

>Name: T35S

>Direction:  ---

>Range: 6693 to 7430

>Name: loxP

>Direction:  ---

>Range: 7430 to 7463

>Name: Cerulean

>Direction:  -->

>Range: 4155 to 4871

 

GGTACCTGCAGGTTTAAACCCACTAGAAAATTTACCCTCTCATTCATAATTAATTATTTAAAATTAAAAATAAACGAACCCACGTGGAAAGAATCCATGATGACGCAACCGTGACGGTGCATGCAACGGCCCGAATGGACCACGACTCGCGGGGGCCACGTCACCAGCTCTTACGCGATCCGCGCCCGGCCCAGCCCCGGCCAAAACCGGCGAACACGAAGAAACTGCGCCGTAAGCTCTTCTCCTTCTCCTTCAAGCGGGGTCCAGAGCATCCAACGTTGCTCATTGTTCCTCGTCCCGCCCGCTTGCTTTCGTCCTCCTCGCGCATCGCTACCGTCTTCTCGTGCACTTCATCTCCCTCGCAGTGCCAGTTCTTTGTTGTTTGCTTGCATTTCTTTTGTGGGTTCGTGAGAAAATCGTGCGTTTCGGAGCCAGGGTTGAGATCAGGTTTCCGCCTTTAGGGTGTTGCTCCGCTCGCCAAAATTACAGATCCGGGGCAGAAGCAGTCCGGCACGTCGGGGTTCATGTTGGAGTTGTCGGACACGGAGATTCCGGCCTCTTTTGGTGCGCTTCTTACTTACCCTGCGCTTTGTGTTCTGGGATGTGGTTGTGACTGCGACGCTATTTTTTTTCTTTGTCTCCTGTGTTTCTTGCCATTTACGGTGTGACCGCGATGTGATTCTCTTCTGTGTTATATCCGGGGCTTGCGTTTCGAGTGTTTGTTCCTTTTGGGGTTTGCCCGTATGGTTTTGCTTGAACAAGTCACATTAGATATTGTGCTTTCATCCGTATGGAAGGACATGACTGGGTTTATGTTCCTTTGTGCATGTTTTTTACACCATGCTTGGTAATGCTTGTCTACTTCGGATATTCGTTACATAGTCAAGTTTTTTATTCCCGGCGACTTGTGCTTACGTTTTTCACCCAATCTTGGACTTGCGTAAACTTCTGTCAGTTTATTGTGTCAATTACCGTCTTTTCCGGTCCTTATGATATTATTTTTTGGGTATGCCGTATGGTCGTTATGACTGAGTCTTGGATTTTGAATTGGTGTTCGGGATgtaGCTCTAGATCACATTTTGATGCACTATGAGCTAATAATTATAAAGTAAATATTCTTATTTTATGTATTGTGCTCATAATTTGATCAAAAATTTGCATTCATTCCTAATTCTATTAAATGATCAAATACTTAATGAATTACTTGATACAAAAATAGTACTAAATATCATGTGAGAAAAAAGAGATTATTGTGTGTTTATCAATACTTCTACACCATGCTAAGTATACAAGTGTTTATCAAAACACATGCAATATCAATTATAAAGAAAGAAAATAATACAAATACACAACTTGGATTTACATATGTGTGTATGTATGTGTGTGTATATATATATATATATATATATATATATAATGGAATGTTAAATAGACCCAAGTTTTTATTAAATCAACCTATCCATGATGAATGTGGTTGGTTTTTATTGGTTTTTTTATCTAGTGGTGAAATTAATCTAGAGGTCATTTGTTTTCATGATAGTTAGGACAGATGGATCATTTTGGTAAAAGCGTGGGTCTATTTAACATTCTCCTATATTTATGCCTACCCAATTTATTCCATAACATTTATGCACAACCTATTTAATCCAATGATAAGAAAATTCAGAATAGAATCGAGAAAGTAAAAGTGACAAAACATTAAGCACGTGGTAAGAACTCAAAAAGTAAAAGTGGACCAAAAACATTAATCGCATATCGAGGGGACATGACAGCTGACACGTGGCGGTGCGGTTTCGCGGGGTGCCTAACTGGGCAGGTGTCGTCCCGACCGCGCGGTTCGCACCGTTTGGATCGCCGATCTTGTGGTAGTTAAGCGCGGTTGGCAATAAAAGGGATGCCCGTGTGGAAGGGCCTCTACAAGAGGAGTGTTTGTGCAGCGAAAGGATCCTCCTCCTCCTTCAGCCTCTCGCAGGTTGGAATTCAGCTGTCCAGGCCCTCGTCCTTCACTTCTCCTCCTCCGCACCCTCTCGCCACCATCACCGGGTATGCATGCTCTTGTTCTACTACTCTTGGGTCTCTTTTCATCCTTCTTTCCAACACACCCCAATTCACAGTGTTCTGTTATTTTGATCTAGCACCCGGTCAACCACTGGTAGAAGGCTTTTTGGTGATTCGTGGTGTGAAGGGGGTGCTTGATGGCTAATTTCCGTTTCGGATCCTTCGTTTAGTTTGCAAGGATTAGCATCAGAAATGTTTTGGTACTGAGTTCTAGGGTTTTGATCTAATTGCTTATTGTGTTTTGACTCGTAGTAAGTGACTTGGTGTTTCGAGCTTTTCCTTAATTTTAAGAATCTGTGTTCTAAGAGTGACAACCGCTACGAGAAGCGTGTGATTTATACCTTAAACTCGAAGAGTTTTTGCAGGTAATCGAAGGTTATCACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTGATAACAGGaGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACGCCATCAGCGACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACCTGTAGATTTGGCCATCagctttcgtccgtatcatcggtttcgacaacgttcgtcaagttcaatgcatcagtttcattgcccacacaccagaatcctactaagtttgagtattatggcattggaaaagctgttttcttctatcatttgttctgcttgtaatttactgtgttctttcagtttttgttttcggacatcaaaatgcaaatggatggataagagttaataaatgatatggtccttttgttcattctcaaattattattatctgttgtttttactttaatgggttgaatttaagtaagaaaggaactaacagtgtgatattaaggtgcaatgttagacatataaaacagtctttcacctctctttggttatgtcttgaattggtttgtttcttcacttatctgtgtaatcaagtttactatgagtctatgatcaagtaattatgcaatcaagttaagtacagtataggcttttgtatctagcTAGACCCGGGCATGCGATCCCCGTCACCGGTGTGAGGGATAACTTCGTATAATGTATGCTATACGAAGTTATAGTTTTGATCTTGAAAGATCTTTTATCTTTAGAGTTAAGAACTCTTTCGTATTTTGGTGAGGAATTCCCTTTCAGAAAGAATGCTAACCCACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGGGATCCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTTGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGAGCGGGACTCTGGGGTTCGGACGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTGACTCCCCCGTAGGTTTGGTTTAAATATGATGAAGTGGACGGAAGGAAGGAGGAAGACAAGGAAGGATAAGGTTGCAGGCCCTGTGCAAGGTAAGAAGATGGAAATTTGATAGAGGTACGCTACTATACTTATACTATACGCTAAGGGAATGCTTGTATTTATACCCTATACCCCCTAATAACCCCTTATCAATTTAAGAAATAATCCGCATAAGCCCCCGCTTAAAAATTGGTATCAGAGCCATGAATAGGTCTATGACCAAAACTCAAGAGGATAAAACCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTATAACTTCGTATAGCATACATTATACGAAGTTATCCCTCACACCGGTGACGGGGATCGCATGCCCGGGTCTAgctagagaaggagATCGATtacAAACCTGTGGACCGCCACGATGCGGTGGCGCTGTCGATTGTATTCATGACATATATGTCTGGAAGGGCACCACCGTAGATATAATGTGGCGTATTATACTTGATGCGGGAAGTAATGGCAGATCTGTGCATGTTGCAGTCAATCGTGGAATAGAACCATTTAGAGCTTATGGTCGCGACTTGTCGTCGTAGGTATCCTTTGGTGTGTATTGCAGCTGGATGGGTAGTAGATGTGGGGATGAATAATATGTACCGCAAACTATGGTGTTCTTTTATTATTGTGAACCTATTTGTTGATTACTACTGAAGTGTTGTCTCTGAGCTTTGGTCTCTCCGCCTATTTTGTTCCTGTTAGATGATAGTGGTTGGCCTTTGGTATCAGTTTCATGGATACTAGCATGTAACTTTTTAAGGTAGAATCTCTCTGCAAAGTTTCTCAGGATCAACGGTCTTGTGTAGCCGTATTATCCATGCTGTGCCTGAATGGATTCGCCTGTTTTTATGCTAGAAATTGTTTATCGGGGCATTGTGGTTACGTTAATTAGAGGAGGTAAATTATACCACAAAATGGTCACAACGTGTTGTATGCTTGCTCACTTCGGATATCCGTACACTTGAAAAGTCGGATAATTATTGTTATAATAAAACTCCTATTTATACAGACATAATGAATCATTCGTGGATTGACATCTCTTTAATGTGAAAGCTCAATTCTAAGTGTCAATCCTAAATACAAAGTTATCTGTTACTTGGGGTGAAGACTTATTTTTCGTTGTTAGAGGTACTGATTTCTGCAATCAGTCTCTAAACTGCATGCACCAATTAAGTTGCACAGCTTCAAAAAAATATGCAGGATATTCCATACTATAAGGTGAAATCTGTAGCTCAATACTTTACATATATATATATATATATTTTTTTTGCTAAGAACGGTGTGTTGCAAAAAAACACAACTCAGTCTGGGTCTCACAGAAGGGTTGGCAGATGAATGATGGTGGGGGCTAAATGCCATCGAAGAGAGAATAACCTCCTGCAGGTTTAAACGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTCCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTG