GATEWAY Binary Vector (pGWB)
1. All pGWB vectors confer resistance to both kanamycin and hygromycin in plant.
2. Some of the vectors contain the 35S promoter upstream of the cloning site whileothers have none.
3. The N- or C-terminal tags are automatically fused subsequent to the LR reaction.
4. Please read the GATEWAY instruction manual (Invitrogen) for procedures,

etc.

Please note that our pGWB seriese are available only for basic research. If you
plan to use these pGWBs for commercial research, please contact Invitrogen about
licencing of GATEWAYTM.
Please do not distribute to other researchers without permission.

Tsuyoshi Nakagawa
Research Institute of Molecular Genetics
Shimane University
Matsue 690, Japantnakagaw@life.shimane-u.ac.jp(July 26, 2002)

The pGWB Series(1) pGWB1 : [ ( no promoter, no tag)

( --R1-CmR-ccdB-R2 -) ](2) pGWB2 : [ ( 35S promoter, no tag)(--35S promoter–R1-CmR-ccdB-R2--)](3) pGWB3 : [ (no promoter, C-GUS)(--R1- CmR-ccdB-R2–GUS--) ](4) pGWB4 : [ (no promoter, C-sGFP)(--R1- CmR-ccdB-R2–sGFP--) ](5) pGWB5: [ (35S promoter, C-sGFP)(--35S promoter–R1- CmR-ccdB-R2–sGFP--) ](6) pGWB6 : [ (35S promoter, N-sGFP)(--35S promoter-sGFP-R1- CmR-ccdB-R2--) ](7) pGWB7 : [ (no promoter, C-6xHis)(--R1- CmR-ccdB-R2-6xHis--) ](8) pGWB8 : [ (35S promoter, C-6xHis)(--35S promoter-R1- CmR-ccdB-R2-6xHis--) ](9) pGWB9 : [ (35S promoter, N-6xHis)(--35S promoter-6xHis-R1- CmR-ccdB-R2--) ](10) pGWB10 : [ (no promoter, C-FLAG)(--R1- CmR-ccdB-R2-FLAG--) ](11) pGBW11 : [ (35S promoter, C-FLAG)(--35S promoter-R1- CmR-ccdB-R2-FLAG--) ](12) pGWB12 : [ (35S promoter, N-FLAG)(--35S promoter-FLAG-R1- CmR-ccdB-R2--) ](13) pGWB13 : [ (no promoter, C-3xHA)(--R1- CmR-ccdB-R2-3xHA--) ](14) pGWB14 : [ (35S promoter, C-3xHA)(--35S promoter-R1- CmR-ccdB-R2-3xHA--) ](15) pGWB15 : [ (35S promoter, N-3xHA)(--35S promoter-3xHA-R1- CmR-ccdB-R2--) ](16) pGWB16 : [ (no promoter, C-4xMyc)(--R1- CmR-ccdB-R2-4xMyc--) ](17) pGWB17 : [ (35S promoter, C-4xMyc)(--35S promoter-R1- CmR-ccdB-R2-4xMyc--) ](18) pGWB18 : [ (35S promoter, N-4xMyc)(--35S promoter-4xMyc-R1- CmR-ccdB-R2--) ](19) pGWB19 : [ (no promoter, C-10xMyc)(- -R1- CmR-ccdB-R2-10xMyc) ](20) pGWB20 : [ (35S promoter, C-10xMyc) (--35S promoter-R1- CmR-ccdB-R2-10xMyc) ](21) pGWB21 : [ (35S promoter, N-10xMyc) (--35S promoter-10xMyc-R1- CmR-ccdB-R2) ](22) pGWB22 : [ (no promoter, C-GST)(--R1- CmR-ccdB-R2-GST--) ](23) pGWB23 : [ (35S promoter, C-GST)(--35S promoter-R1- CmR-ccdB-R2-GST) ](24) pGWB24 : [ (35S promoter, N-GST)(--35S promoter-GST-R1- CmR-ccdB-R2) ](25) pGWB25 : [ (no promoter, C-T7)(--R1- CmR-ccdB-R2-T7--) ](26) pGWB26 : [ (35S promoter, C-T7)(--35S promoter-R1- CmR-ccdB-R2-T7) ](27) pGWB27 : [ (35S promoter, N-T7)(--35S promoter-T7-R1- CmR-ccdB-R2) ](28) pGWB28 : [ (no promoter, C-TAP)(--R1- CmR-ccdB-R2-TAP--) ](29)pGWB29 :[ (35S promoter, C-TAP)
Notes:(--35S promoter-R1- CmR-ccdB-R2-T7) ]1. pGWB1 is used for simple cloning, pGWB2 is for expression driven by the 35S promoter, pGWB3 forpromoter-GUS analysis, pGWB4-6 for fusion of GFP, and pGWB7-24 are for fusion of tags.
2. Tags could be added to either the N- or C- terminus of Your Clone.

For example, in pGWB10 “C-

FLAG” means that after the LR reaction with the vector, FLAG will be fused at the C-terminal of Your
Clone.
3. DNA sequences enclosed in [ ] are introduced into the HindIII-SacI site of the binary vector pABHHm1 [Mita et al. (1995) Plant Physiol. 107: 895-904; see next page].

R = the attR site.

Construction of pGWBs
The binary vector used as the starting plasmid for the construction of pGWBs was
pABH-Hm1 [Mita et al. (1995) Plant Physiol. 107: 895-904].

pABH-Hm1 itself wasmade by introducing the beta amylase promoter and HPT unit into pBI101.

The

HindIII-SacI region in pABH-Hm1 harboring the beta amylase promoter::GUS was
replaced by the GATEWAY cassette and tag in the pGWB as shown below.

Theaverage size of pGWB is 17kb.

Nos-P

NPTII Nos-T-(HindIII)-GATEWAY -(SacI)- Nos-T - Nos-T HPT 35S

The DNA fragment diagrammed above is present between LB and RB of binary
vector.

Note that the HPT unit is in reverse orientation.

All pGWBs contain NPTII gene for kanamycin selection and HPT gene forhygromycin selection in plant.
Antibiotics can be used for selection in bacteria are as below.
pGWBs:

kanamycin, hygromycin, chloramphenicol

recombinant pGWB (after LR reaction) : kanamycin, hygromycin.
The princple behind the GATEWAY technology and the pGWB vector series could be
generalized as follow.
(1) Subsequent to the LR reaction with an Entry Clone and a pGWB vector, the attR1(CmR, ccdB)-attR2 cassette in the pGWB will be replaced by the attB1-(Your
Clone)-attB2.
(2) The tag will be fused automatically in-frame to Your Clone, if the primer was

designed according to the GATEWAY system (see instruction manual by Invitrogen).
Note that the peptide encoded by the attB and linker region will be included in yourconstruct, linking the tag to Your Clone.
(3) N indicates the nucleotide (and X the amino acid) that shold be included in Your

Clone.

Important Note
(1) Linearize pGWBs by digestion with XhoI before performing the LR reaction.

Theefficiency of the LR reaction depends on the topology of the plasmids in thefollowing order (see instruction manual of Invitrogen).
Either or both plasmids linear > both plasmids relaxed >> both plasmids supercoiled

The XhoI linker was introduced into NotI site (of GATEWAY cassette justdownstream of attR1) in all pGWBs. This XhoI site is unique.
P
Nos-P

Xh

BPE

NPTII Nos-T- (HindIII)- GATEWAY -(SacI)- Nos-T - Nos-T HPT 35S

P:PstI, B:BamHI, E:EcoRI, Xh: XhoIThe HindIII、XhoI and SacI sites are unique in pGWBs.

(2)Select with kanamycin (50 ug/ml) AND hygromycin (50 ug/ml) after the LR reaction.
Because the Entry Clone vector pDONR201 is also encode kanamycin resistance,
hygromycin is necessary to screen recombinant pGWBs.

Kanamycin, should be

used in addition as many petite colonies will appear on plate without kanamycin.
(3)Use the Escherichia coli strain DH5alpha for cloning the product of the LR (BP)reactions, since negative selection with ccdB does not work in the E.coli which has F’episome (see instruction manual of Invitrogen).
(4)Sometimes, E.coli with both the Entry Clone and recombinant pGWB are obtained
subsequent to the cloning. I recommend that only the E. coli colonies with only therecombinant pGWB be used.

pGWB1 : [ ( no promoter, no tag) ( --R1-ccdB-R2 --) ](AAGCTT) --//(TCTAGA)TC ACA AGT TTG TAC AAA AAA --(CmR, ccdB) -- TTC TTG TAC AAA GTG
GTG ATC TAG AGG (GAG CTC)

(1) The HindIII (AAGCTT),

XbaI (TCTAGA) and SacI (GAGCTC) sites are shown(2) The attR1-CmR-ccdB-attR2 cassette is underlined.

After the LR reaction, the following sequence will be obtained.

(AAGCTT) --//(TCTAGA)TC ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC CCA GCT
TTC TTG TAC AAA GTG GTG ATC TAG AGG (GAG CTC)

(1) The attB1 and attB2 sequences are underlined.
(2) The in-frame termination codon TAG (double-underlined) is present downstream of
attB2.

pGWB2 : [ ( 35S promoter, no tag) ( --35S promoter-R1-ccdB-R2 --) ](AAGCTT) -- 35S promoter --//(TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA --(CmR,ccdB)--TTC TTG TAC AAA GTG
GTT GAT AAC AGC GCT TA(G AGCTC)(1) The HindIII (AAGCTT),

XbaI (TCTAGA) and SacI (GAGCTC) sites are shown(2) The attR1-CmR-ccdB-attR2 cassette is underlined.
(3) The sequence upstream of the XbaI site is the same as in pBI121

After LR reaction, the following sequence will be obtained(AAGCTT) -- (35S promoter) --//(TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC CCA
M -------(X) (P)GCT TTC TTG TAC AAA GTG GTT GAT AAC AGC GCT TA(G AGCTC)(A) (F) (L) (Y) (K) (V) (V) (D) (N) (S) (A)*(1) Include the initiation codon ATG in Your Clone as pGWB2 does not have one.
(2) The inclusion of a termination codon in Your Clone is not necessary since one ispresent downstream of attB2.

The amino acid residues in ( ) are encoded by the

attB2 and the linker region, and will be included if the termination codon TAG(double-underlined) of the vector is used.
(3) N indicates the nucleotide (and X the amino acid) that should be included in Your
Clone.

pGWB3 : [ (no promoter, C-GUS) (--R1-ccdB-R2–GUS--) ](AAGCTT) --//(TCTAA)T CAA ACA AGT TTG TAC AAA AAA --(CmR, ccdB)-- TTC TTG TAC AAA GTGGTT CGA TCT AGA GGA TCC CCG GGT GGT CAG TCC CTT ATG--(GUS sequence)--(GAGCTC)(1) As a result of a mistake during the fill-in reaction while constructing pGWB3, the
XbaI site TCTAGA was changed to TCTAA.

However, I think it would not affectthe result of your experiments.
(2) The initiation codon ATG (double-underlined) is from the GUS coding sequence.
(3) The SacI site (GAGCTC) is present just upstream of the Nos terminator.

After the LR reaction, the following sequence will be obtained.

(AAGCTT) --//(TCTAA)T CAA ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC CCA GCT(X) (P) (A)TTC TTG TAC AAA GTG GTT CGA TCT AGA GGA TCC CCG GGT GGT CAG TCC CTT ATG(F) (L) (Y) (K) (V) (V) (R) (S) (R) (G) (S) (P) (G) (G) (Q) (S) (V)

M--(GUS sequence) --(GAGCTC)(1) If Your Clone has only a 5’ region/promoter without an initiation codon, translation
will start at the ATG (double-underlined) of GUS.
(2) If Your Clone contains at least a part of an ORF, C-terminal GUS fusion will bemade (if in frame).

In that case, the peptide sequence made up of the amino acids in( ) will link Your Clone and GUS.
(3) N indicates the nucleotide (and X the amiono acid) that should be included in Your
Clone, if C-terminal GUS fusion is to be made.
(4) The DNA sequence from GUS to the SacI site is the same as in pBI121.
.

pGWB4 : [ (no promoter, C-sGFP)(--R1-ccdB-R2–sGFP--) ](AAGCTT) --//(TCTA A)TC ACA AGT TTG TAC AAA AAA --(Cm-R, ccdB)-- TTC TTG TAC AAA GTG
GTG ATC ATG --(GFP sequence) --(GAGCTC)(1) As a result of a mistake during the fill-in reaction while constructing pGWB4, the
XbaI site

TCTAGA was changed to TCTAA.

However, I think it would not affectthe results of your experiments.
(2) The initiation codon ATG (double-underlined) is from the GFP coding sequence.
After the LR reaction, the following sequence will be obtained.

(AAGCTT) --//(TCTA A)TC ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC CCA GCT(X) (P) (A)TTC TTG TAC AAA GTG GTG ATC ATG --(GFP sequence) --(GAGCTC)(F) (L) (Y) (K) (V) (V) (I)M(1) If Your Clone has only a 5’ region/promoter without an initiation codon, tranalation
will start at the ATG (double-underlined) of GFP.
(2) If Your Clone contains at leaset a part of an ORF, C-terminal GFP fusion will bemade.

In that case, the peptide sequence made up of the amino acid in ( ) will linkYour Clone and GFP.
(3) N indicates the nucleotide (and X the amino acid) that should be included in Your
Clone, if C-terminal GUS fusion is to be made.
(4) The sequences from the termination codon of GFP to SacI site (see below) was
generated by the follwing steps: (i) NotI digest, (ii) fill-in, and (iii) SacI linker (8mer) ligation.
TAAAGCGGCCC(GAGCTC)

pGWB5 : [(35S promoter, C-sGFP) (--35Spromoter–R1-ccdB-R2–sGFP--) ](AAGCTT) -- 35S promoter --//
(TCT AA)T CAA ACA AGT TTG TAC AAA AAA --(CmR, ccdB)—TTC TTG TAC AAA GTGGTT CGA TCT AGA GGA TCC ATG --(GFP sequence) --(GAGCTC)(1) As a result of a mistake during the fill-in reaction while constructing pGWB5, the
XbaI site TCTAGA was changed to TCTAA.

However, I think it would not affectthe result of your experiments.
(2) The initiation codon ATG (double-underlined) is from the GFP coding sequence.

After LR reaction, the following sequence will be obtained.

35S promoter –(TCT AA)T CAA ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone)
NAC CCA GCT TTC TTG TAC AAA GTG GTT CGA TCT AGA GGA TCC ATG --X- -P- -A- -F- -L- -Y- -K- -V- -V- -R- -S- -R- -G- -S- -M(GFP sequence) -- (GAGCTC)(1) Include an initiation codon ATG in Your Clone.

Protein fusion with the C-terminal

GFP will be linked by the peptide sequence derived from the attB2 and linker region
as shown in the above sequence.
(2) N indicates the nucleotide (and X the amino acid) that is needed to be included inYour Clone.
(3) The DNA sequence downstream of GFP is the same ase in pGWB4.
(4) Hyphenations flanking the amino acid residues are used for proper spacing.

pGWB6 : [ (35S promoter, N-sGFP) (--35S promoter-sGFP-R1-ccdB-R2--) ](AAGCTT) -- 35S promoter --(GFP sequence)--//--- C(TG TAC A)TC ACA AGT TTG TAC AAA AAA --(CmR, ccdB)—TTC TTG TAC AAA
GTG GTG ATG TAC AAG TAA AGCGGCCC(GAGCTC)(1) The GATEWAY conversion cassette was introduced into the BsrGI (TGTACA) site
(in the 3’ end of GFP).
(2) The termination codon TAA (double-underlined) is derived from GFP.

After LR reaction, the following sequence will be obtained.

(AAGCTT) -- 35S promoter --(GFP sequence)--//--- C(TG TAC A)TC ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC
---

-L-

-Y-

-I- -T- -S- -L- -Y- -K- -K- -A- -G- -X----- -----(X)

CCA GCT TTC TTG TAC AAA GTG GTG ATG TAC AAG TAA AGCGGCCC(GAGCTC)(P) (A) (F) (L) (Y) (K) (V) (V) (M) (Y) (K)*(1) The inclusion of a termination codon in Your Clone is not necessary since one ispresent downstream of attB2.

The amino acid residues in ( ) is encoded by the

attB2 and the linker region, and will be included if the termination codon TAG of thevector is used.
(2) The C-terminus of the original GFP is LYK*.
(3) The 5’ end sequence of GFP is as shown below.
TCTAGAGGATCC ATG --(GFP sequence)-XbaIBamHI M

(4) The sequence upstream of XbaI site is the same as pBI121.

pGWB7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23 , 25, 26, 28, 29(no promoter or 35S promoter, C-tag)
No promoter --- 7, 10, 13, 16, 19, 22, 25, 28
35S promoter --- 8, 11, 14, 17, 20, 23, 26, 29(AAGCTT) -- (no promoter or 35S promoter) --//(TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA --(CmR,ccdB)--TTC TTG TAC AAA GTG
GTT GAT AAC AGC tag GCT TA(G AGCTC)(1) The HindIII (AAGCTT), XbaI (TCTAGA) and SacI (GAGCTC) sites are shown.
(2) The attR1-CmR-ccdB-attR2 cassette is underlined.
(3) The sequence upstream of the XbaI site is the same as in pBI101 or pBI121.
(4) The sequence of each tag is indicated in the “Tags” section.

After LR reaction, the following sequence will be obtained.

(AAGCTT) -- (no promoter or 35S promoter) --//(TCTAGA)GTTA TCA ACA AGT TTG TAC AAA AAA GCA GGC TNN (Your Clone) NAC CCA
M --------X- -P-GCT TTC TTG TAC AAA GTG GTT GAT AAC AGC tag GCT TA(G AGCTC)-A- -F- -L- -Y- -K- -V- -V- -D- -N- -S- tag –A-(1) Include an initiation codon ATG in Your Clone.

*

Protein fusion with the C-terminal

tag will be linked by the peptide sequence derived from the attB2 and linker region
as shown in the above sequence.
(2) Translation will stop at the termination codon TAG (double-underlined) after thetag.

pGWB9, 12, 15, 18,2 1, 24, 27(35S promoter, N-tag)(AAGCTT) -- 35S promoter --//(TCTAGA) ATG AGC tag GCT GTT ATC ACA AGT TTG TAC AAA AAA –-(CmR,ccdB)-TTC TTG TAC AAA GTG GTG ATA ACC TA(G AGCTC)(1) The HindIII (AAGCTT), XbaI (TCTAGA) and SacI (GAGCTC) sites are shown.
(2) The attR1-CmR-ccdB-attR2 cassette is underlined.
(3) Sequence upstream of the XbaI site is the same as in pBI121.
(4) The sequence of each tags is indicated in the “Tags” section.
After the LR reaction, the following sequence will be obtained.

(AAGCTT) -- 35S promoter --//(TCTAGA) ATG AGC tag GCT GTT ATC ACA AGT TTG TAC AAA AAA GCA GGC TNN
M- -S- tag –A- -V- -I- -T- -S- -L- -Y- -K- -K- -A- -G- -X(Your Clone) NAC CCA GCT TTC TTG TAC AAA GTG GTG ATA ACC TA(G AGCTC)--- ---(X) (P) (A) (F) (L) (Y) (K) (V) (V) (I) (T)*(1) The inclusion of a termination codon in Your Clone is not necessary since one ispresent downstream of attB2. The amino acid residues in ( ) is encoded by the attB2
and the linker region, and will be included if the termination codon TAG (doubleunderlined) of the vector is used.

Tags
Hyphenations in the amino acid sequences are used for proper spacing.
pGWB4, 5, 6

(sGFP) 720bp

The sequence of sGFP (ATG to TAA) is as described by Chiu et al., (1996) Current Biol., 6: 325-330 .
Note that the C-terminal K is converted to I in pGWB6 (35S promoter, N-GFP).

(see the section on

pGWB6)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTG
M— V- S— K— G— E— E— L— F— T— G— V— V— P— I— L— V— E— L— D- G- D- VAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAG
N- G- H- K- F- S- V- S- G- E- G- E- G- D- A- T- Y- G- K- L- T- L- KTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTG
F- I- C- T- T- G- K- L- P- V- P- W- P- T- L- V- T- T- F- T- Y- G- VCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGC
Q- C- F- S- R- Y- P- D- H- M- K- Q- H- D- F- F- K- S- A- M- P- E- GTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTC
Y- V- Q- E- R- T- I- F- F- K- D- D- G- N- Y- K- T- R- A- E- V- K- FGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTG
E- G- D- T- L- V- N- R- I- E- L- K- G- I- D- F- K- E- D- G- N- I- LGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC
G- H- K- L- E- Y- N- Y- N- S- H- N- V- Y- I- M- A- D- K- Q- K- N- GATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG
I- K- V- N- F- K- I- R- H- N- I- E- D- G- S- V- Q- L- A- D- H- Y- QCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCC
Q- N- T- P- I- G- D- G- P- V- L- L- P- D- N- H- Y- L- S- T- Q- S- ACTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC
L- S- K- D- P- N- E- K- R- D- H- M- V- L- L- E- F- V- T- A- A- G- I
ACTCACGGCATGGACGAGCTGTACAAGTAA
T- H- G- M- D- E- L- Y- K- *

pGWB7, 8, 9

(6xHis)

18bp

CATCATCATCATCATCAT
H- H- H- H- H- H-

pGWB10, 11, 12(FLAG) 24bp

GACTACAAGGATGACGATGACAAG
D- Y- K- D- D- D- D- K-

pGWB13, 14, 15
Please consult(3xHA)

120bp

http://pingu.salk.edu/users/forsburg/ for further information

GGGTTAATTAACATCTTTTACCCATACGATGTTCCTGACTATGCGGGCTATCCCTATGACGTCCCGGAC
G- L- I- N- I- F- Y- P- Y- D- V- P- D- Y- A- G- Y- P- Y- D- V- P- DTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCTGCTCAGTGCAGC
Y- A- G- S- Y- P- Y- D- V- P- D- Y- A- A- Q- C- S-(1) The three HA units are underlined.
(2) The 54th nucleotide is a G in the reported sequence, but it is a C in our vectors. This does not affect theidentity of the amino acid, however [CCG (P) to CCC (P)].

pGWB16, 17, 18
Please consult(4xMyc) 171bp

http://pingu.salk.edu/users/forsburg/ for further information

GGGTTAATTAACGGTGAACAAAAGCTAATCTCCGAGGAAGACTTGAACGGTGAACAAAAATTAATCTCA
G- L- I- N- G- E- Q- K- L- I- S- E- E- D- L- N- G- E- Q- K- L- I- SGAAGAAGACTTGAACGGACTCGACGGTGAACAAAAGTTGATTTCTGAAGAAGATTTGAACGGTGAACAA
E- E- D- L- N- G- L- D- G- E- Q- K- L- I- S- E- E- D- L- N- G- E- QAAGCTAATCTCCGAGGAAGACTTGAACGGTAGC
K- L- I- S- E- E- D- L- N- G –S

The four Myc units aare underlined.

pGWB19, 20, 21(10xMyc) 402bp

GGGTTAATTAACGGTGAACAAAAGCTAATCTCCGAGGAAGACTTGAACGGTGAACAAAAATTAATCTCA
G- L- I- N- G- E- Q- K- L- I- S- E- E- D- L- N- G- E- Q- K- L- I- SGAAGAAGACTTGAACGGACTCGACGGTGAACAAAAGTTGATTTCTGAAGAAGATTTGAACGGTGAACAA
E- E- D- L- N- G- L- D- G- E- Q- K- L- I- S- E- E- D- L- N- G- E- QAAGCTAATCTCCGAGGAAGACTTGAACGGTGAACAAAAATTAATCTCAGAAGAAGACTTGAACGGACTC
K- L- I- S- E- E- D- L- N- G- E- Q- K- L- I- S- E- E- D- L- N- G- LGACGGTGAACAAAAGTTGATTTCTGAAGAAGATTTGAACGGTGAACAAAAGCTAATCTCCGAGGAAGAC
D- G- E- Q- K- L- I- S- E- E- D- L- N- G- E- Q- K- L- I- S- E- E- DTTGAACGGTGAACAAAAATTAATCTCAGAAGAAGACTTGAACGGACTCGACGGTGAACAAAAGTTGATT
L- N- G- E- Q- K- L- I- S- E- E- D- L- N- G- L- D- G- E- Q- K- L- ITCTGAAGAAGATTTGAACGGTGAACAAAAGCTAATCTCCGAGGAAGACTTGAACGGTAGC
S- E- E- D- L- N- G- E- Q- K- L- I- S- E- E- D- L- N- G- S-(1) The ten Myc units are underlined.
(2) The last codon AGC (Ser) is not included in pGWB19 and 20.

pGWB22,23,24 (GST)

678bp

The full-length GST sequence was derived from pGEX2T

ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATAT
M- S- P- I- L- G- Y- W- K- I- K- G- L- V- Q- P- T- R- L- L- L- E- YCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTT
L- E- E- K- Y- E- E- H- L- Y- E- R- D- E- G- D- K- W- R- N- K- K- FGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATG
E- L- G- L- E- F- P- N- L- P- Y- Y- I- D- G- D- V- K- L- T- Q- S- MGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATT
A- I- I- R- Y- I- A- D- K- H- N- M- L- G- G- C- P- K- E- R- A- E- ITCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTT
S- M- L- E- G- A- V- L- D- I- R- Y- G- V- S- R- I- A- Y- S- K- D- FGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGT
E- T- L- K- V- D- F- L- S- K- L- P- E- M- L- K- M- F- E- D- R- L- CCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTT
H- K- T- Y- L- N- G- D- H- V- T- H- P- D- F- M- L- Y- D- A- L- D- VGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAA
V- L- Y- M- D- P- M- C- L- D- A- F- P- K- L- V- C- F- K- K- R- I- EGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAA
A- I- P- Q- I- D- K- Y- L- K- S- S- K- Y- I- A- W- P- L- Q- G- W- QGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTGGATCC
A- T- F- G- G- G- D- H- P- P- K- S- D- L- V- P- R- G- S-

pGWB25, 26, 27

(T7)

33bp

ATGGCTAGCATGACTGGTGGACAGCAAATGGGT
M- A- S- M- T- G- G- Q- Q- M- G

PGWB28, 29

(TAP)

546bp

AAGAGAAGATGGAAAAAGAATTTCATAGCCGTCTCAGCAGCCAACCGCTTTAAGAAAATCTCATCCTCC
K- R- R- W- K- K- N- F- I- A- V- S- A- A- N- R- F- K- K- I- S- S- SGGGGCACTTGATTATGATATTCCAACTACTGCTAGCGAGAATTTGTATTTTCAGGGTGAGCTCAAAACC
G- A- L- D- Y- D- I- P- T- T- A- S- E- N- L- Y- F- Q- G- E- L- K- TGCGGCTCTTGCGCAACACGATGAAGCCGTGGACAACAAATTCAACAAAGAACAACAAAACGCGTTCTAT
A- A- L- A- Q- H- D- E- A- V- D- N- K- F- N- K- E- Q- Q- N- A- F- Y
GAGATCTTACATTTACCTAACTTAAACGAAGAACAACGAAACGCCTTCATCCAAAGTTTAAAAGATGAC
E- I- L- H- L- P- N- L- N- E- E- Q- R- N- A- F- I- Q- S- L- K- D- DCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCTCAGGCGCCGAAAGTAGAC
P- S- Q- S- A- N- L- L- A- E- A- K- K- L- N- D- A- Q- A- P- K- V- DAACAAATTCAACAAAGAACAACAAAACGCGTTCTATGAGATCTTACATTTACCTAACTTAAACGAAGAA
N- K- F- N- K- E- Q- Q- N- A- F- Y- E- I- L- H- L- P- N- L- N- E- ECAACGAAACGCCTTCATCCAAAGTTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCT
Q- R- N- A- F- I- Q- S- L- K- D- D- P- S- Q- S- A- N- L- L- A- E- AAAAAAGCTAAATGGTGCTCAGGCGCCGAAAGTAGACGCGAATTCCGCGGGGAAGTCAACCTGA
K- K- L- N- G- A- Q- A- P- K- V- D- A- N- S- A- G- K- S- T- *(1) The CBP sequence is underlined.
(2) TEV site is underlined in bold.
(3) The protein A sequence is double-underlined.

Acknowledgements
I would like to thank Lien Lai (Ohio State University) for critical reading of themanual, Takayuki Kurose and Mamoru Watanabe (Shimane University) for technicalassistance.