Gateway Vectors for BiFC
1. The enhanced YFP (EYFP) are used (Split EYFP).
2. The Fusion fusion gene is expressed by CaMV35S promoter.
3. The N- or C-terminal fragments of EYFP are fused subsequent to the LR reaction.
4. Please read the Gateway instruction manual (Invitrogen) for procedures, etc.

Please note that our BiFC vectors are available for basic research only. If you plan touse them for commercial research, please contact Invitrogen about the licencing ofGatewayTM.
Please do not distribute to other researchers without permission.

Tsuyoshi Nakagawa
Department of Molecular and Functional Genomics
Center for Integrated Research in Science
Shimane University
Matsue 690-8504, Japantnakagaw@life.shimane-u.ac.jp

Construction of BiFC vectors
The vector used as the starting plasmid was pUGW2 and pUGW0 (Nakagawa, T. etal., J. Biosci. Bioeng., 104, 34-41. 2007) which were constructed from pUC119. The
EYFP (239 amino acids) were separated into nEYFP (1-174) and cEYFP (175-239),
then introduced into pUGW2 and pUGW0, respectively, to make four types of vectors.
(1) nEYFP/pUGW2 (35S pro, C-nEYFP)(2) cEYFP/pUGW2 (35S pro, C-cEYFP)(3) nEYFP/pUGW0 (35S pro, N-nEYFP)(4) cEYFP/pUGW0 (35S pro, N-cEYFP)
Antibiotics
All BiFC vectors : ampicillin (50 mg/l) and chloramphenicol (30 mg/l)
All recombinant BiFC vectors (after LR reaction) : ampicillin (50 mg/l)
Amplification of BiFC vectors(1) Use competent cell of DB3.1 (Invitrogen 11782-018) or ccdB Survival T1(Invitrogen C7510-03)(2) Spread on a plate containing ampicillin (50 mg/l) and chloramphenicol (30 mg/l).
We made and use 10 mg/ml (methanol) stock solution of chloramphenicol.
(3) Pick up colony and culture in a liquid medium containing ampicillin (50 mg/l) andchloramphenicol (30 mg/l)(4) Prepare plasmids from E. coli with plasmid purification kit. We usually use Mag
Extractor plasmid (Toyobo NPK-301).
LR reaction and transformation(1) Reaction mixture can be scaled down. We usually use the following reaction:(2) Entry clone

0.5 µl(about 150 ng)

BiFC vector

1.0 µl(about 150 ng)

LR clonase II

0.5 µlH2O

2.0 µl------------------------------------------total

4.0 µl(3) Incubate at 25 ºC for more than 3hr.

(4) 75 ºC for 10 min. We do not use proteinase K solution to stop the reaction.
(5) Add 2 µl of reaction mixture to 5 µl of DH5α competent cell. We usually use
Competent High DH5α(Toyobo DNA-903). On ice for 30 min, 42 ºC for 45 sec,

on ice for 2 min, add 100 µl of SOC, shake for 1hr.
(6) Spread on a plate containing ampicillin (50 mg/l).
Note(1) Linearization of BiFC vectors before performing the LR reaction is not necessary.
If you want to linealize BiFC vectors, use XhoI.
(2) Use the Escherichia coli strain DH5α for cloning the product of the LR reactions,since negative selection with ccdB does not work in the E.coli which has F’ episome(see the Invitrogen instruction manual).
(3) The pUGW2 has the structure of HindIII-XbaI-HindIII-CaMV35S pro-XbaI aroundthe CaMV35S promoter. The CaMV35S promoter is removed from pUGW2 bydigestion of XbaI and following self ligation.
(4) BiFC vectors are used for transient expression experiments. See protocol of particle
gun, protoplast transformation with PEG, etc., on the website.

Structure of BiFC vectors

ori

nYFP / pUGW2 (P35S / C-nEYFP)

Ampr

HindIII XbaI HindIII
XhoI
XbaI
P35S

SacI

ccdB

nEYFP

Cmr

attR1

Ampr

HindIII XbaI HindIII
XhoI
XbaI
P35S

SacI

ccdB

cEYFP

Cmr

attR1

Ampr

HindIII
XbaI
P35S

XhoI

nEYFP

ccdB

Cmr

ori

SacI

EcoRI

Tnos

attR1

cYFP / pUGW0 (P35S / N- cYFP)

EcoRI

Tnos

attR2orinYFP / pUGW0 (P35S / N-nEYFP)

Tnos

attR2

ori

cYFP / pUGW2 (P35S / C- cYFP)

EcoRI

attR2

Ampr

HindIII
XhoI

XbaI
P35S

cEYFPCmr

attR1

ccdB SacI

EcoRI

Tnos
attR2

Structure of the BiFC gateway vectors. ori, ColE1 origin; Ampr, ampicillin resistant marker; Cmr, chloramphenicol resistantmarker.

Sequence of BiFC vectors
The HindIII(AAGCTT), XbaI

(TCTAGA)

andSacI(GAGCTC)sitesareshown. The

attR1-CmR-ccdB-attR2 cassette is underlined. The termination codon in the vector are bold-underlined.

n/cEYFP / pUGW2(35S promoter, C-n/cEYFP)(AAGCTT)GGG(TCTAGA)CCC(AAGCTT) -- (35S promoter) --//(TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA --(CmR,ccdB)--TTC TTG TAC AAA GTG
GTT GAT AAC AGC n/cEYFP GCT TA(G AGCTC)nEYFP (M1-D174) + TAG
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAMVSKGEELFTGVVPILVELDGDVAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGN

GHKF

SVSGE

GEGDATYGKL

TLK

TTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGF

IC

T

T

GKL

PVPW

PT

L

V

T

TF

GY

GL

CAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCQ

CF

A

RYP

DH

MKQ

H

DFF

KSA

M

PEGTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCYVQ

ERTI

FF

KDDGNYKT

R

A

E

V

K

F

GAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTG
E

GDTLVNRI

E

LKGI

DFK

E

DGNI

L

GGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCGHKL

EY

NYNS

HNVYI

M

A

DKQKN

G

ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACTAG
I

KV

NF

KIRH

N

I

E

D

*cEYFP ATG + (G175-TAA)
ATGGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG
M

GSVQ

LA

DHYQ

Q

NTP

I

GDGPVLL

CCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGPDNHYLSYQ

S

A

L

SK

DP

N

E

K

R

D

GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAVLL

EFVTA

A

GITL

GM

DELYK*HMn/cEYFP / pUGE0 (35S promoter, N-n/cEYFP)(AAGCTT) -- 35S promoter --//(TCTAGA) ATG AGC n/cEYFP GCT GTT ATC ACA AGT TTG TAC AAA AAA –-(CmR,ccdB)--TTCTTG TAC AAA GTG GTG ATA ACC TA(G AGCTC)nEYFP M1-D174
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAMVSKGEELFT

GVVPILV

E

L

DG

DV

AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGNGHKF

SVSGE

GE

GDA

TYGK

L

TL

K

TTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGF

IC

T

T

GKL

PVPW

PTL

V

TTF

GYGL

CAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCQ

CF

A

RYP

DH

MKQ

H

DF

F

KS

A

MP

E

G

TACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCYVQ

E

RTIF

FKDDGNYKTR

A

EVK

F

GAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTG
EGDTLVNRIELKGIDFK

E

DGNIL

GGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC
GHK

L

E

YNYNS

HN

VYI

M

A

DKQ

K

N

G

ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGAC
IK

V

NFKI

R

H

N

I

E

cEYFP ATG + (G175-K239)

DK239 is last amino acid of EYFPATGGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG
MGSVQLADHYQQ

NTPIGDGPVLL

CCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG
P

DNHYL

SY

Q

SA

L

SK

DP

N

E

K

R

GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGV

L

L

E

F

V

T

A

A

GI

TL

GM

DELYKDHM

Nucleotide and amino acid sequences around attB sites after LR reactionnEYFP / pUGW2 (CaMV35S promoter; C-fusion to nEYFP)!!"#$$"""$#$!"!###!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!"$$!!
$#!!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$$!"!$!!!#!!"#!!$"%&24567-%!"!#!$!"!"#$!$!"!!"#$#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!;!!!.!!!,!!!(!!!!!!!!!!!!;!!!!
!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=?!!!!!

cEYFP / pUGW2 (CaMV35S promoter; C-fusion to cEYFP)!!"#$$"""$#$!"!###!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!"$$!!
$#!!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$$!"!$!!!#!!"#!!$"%""#%&/4567-%!!!"%$!!!"#$!$!"!!"#$#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!;!!!.!!!,!!!(!!!"!!!!!!!!!!!!:!!!!!
!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=?!!!!!

nEFP / pUGW0 (CaMV35S promoter; N-fusion to nEYFP)!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!
!$"!!"#!!$"%&24567-%"!#!"#$!"$$!!$#!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$"!!$!!!##!$!"!!"#$#(!!!,!!!(!!!!!!!!!!!;!!!!!!!)!!!@!!!$!!!,!!!9!!!5!!!:!!!:!!!!!!!"!!!8!!!!!!!!!!!8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!@!!!$!!!A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=?!!!!

nEFP / pUGW0 (CaMV35S promoter; N-fusion to cEYFP)!!"#$$"#!$"##$"#!""$%&#'()*+,-%#"#!!"!###$$##$#$!$!$!!""!!"$$#!$$$#!$$$""!"!"!!#!#"""""!#$#$!"!
!$"!!"#!!$"%""#%&/4567-%!!"!"#$!"$$!!$#!!#!!!"$!$$"!$!#!!!!!!!!!"#!!""#!$..!&/0123-!.!#!##!!"#$!$$#!$$"!$!#!!!!!"$"!"$"!!$!!!##!$!"!!"#$#(!!!,!!!(!!!"!!!!!!!!!!!:!!!!!!!)!!!@!!!$!!!,!!!9!!!5!!!:!!!:!!!!!!!"!!!8!!!!!!!!!!!8!!!7!!!!!!!6!!!9!!!5!!!:!!!)!!!)!!!@!!!$!!!A!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=>!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '<<=?!!!!

The sequences between HindIII and Sac I sites (see “Structure of BiFC vectors”) are
shown for all vectors. To clearly show the relation of functional elements, linefeeds areirregularly inserted in the sequences. The attB1 and attB2 sites are underlined. Nindicates a nucleotide derived from an entry clone (green) and X indicates an aminoacid residue corresponding to the N-containing codon. The CaMV35S promoter isshown in orange, where the region between -46 and +8 is represented by a nucleotide
sequence with the predicted transcription start site (underlined larger letter). The nEYFPis indicated in red and cYFP is indicated in blue. Only partial sequences are shown for
n/cEYFP, whose whole sequences are indicated in the former section. In C-fusion
vectors, the peptide sequence derived from attB2 region becomes the linker between theentry clone and n/cEYFP. In N-fusion vectors, the peptide sequence derived from attB1
region becomes the linker between n/cEYFP and the entry clone, and the peptide
sequence derived from attB2 region (gray italic) is added at the C-terminus of the fusion
protein only if the entry clone lacks the termination codon in it.