To download sequence files, please right-click the click button and choose "save" or "download".
All files are created using a free software "ApE".
Although you can open these files in a simple text format, we recommend to use ApE.
Attention!
When you use a series of vectors for chromosomal integration, please note that not all the transformants are proper integrants.
According to the previous paper [Genetics. 1994;136(3):849-56.], this phenomenon occurs due to gene conversion.
Although it was reported that gene conversion occurs in nearly 30% of the transformants, we estimate that this frequency is less than 10% in our integration systems.
But we recommend to check expression of the tagged proteins before you use in your experiment.
It is enough to check two transformants in most cases.
Additional information
And also, please note that the LR reaction should be carried out using low concentration of entry clones and destination vectors.
Although we do not know the reason, it is very difficult to obtain correct expression plasmids when high concentration of vectors and entry clones are used in the reaction.
We usually use about 10 ng (or less) of each plasmid in the LR reaction.
The reaction volume can be reduced to at least 3 microliter for cost saving.
| Gateway-compatible vectors for chromosomal integration |
|
Plasmid name
|
Backbone
|
Promoter
|
Tag
|
Tag position
|
Multicopy marker
|
Targeted locus
(negative) selection
|
Positive selection marker
|
Sequence
|
| pLYS1U-CFH1c |
pUC119
|
nmt1
|
CFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-CFH21c |
pUC119
|
tub1
|
CFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-CFH61c |
pUC119
|
ef1a-c
|
CFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-FFH1c |
pUC119
|
nmt1
|
FLAG2-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-GFH21c |
pUC119
|
tub1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-GFH61c |
pUC119
|
ef1a-c
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-YFH1c |
pUC119
|
nmt1
|
YFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-YFH21c |
pUC119
|
tub1
|
YFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-YFH61c |
pUC119
|
ef1a-c
|
YFP-FLAG-His6
|
3'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFC1c |
pUC119
|
nmt1
|
His6-FLAG-CFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFC21c |
pUC119
|
tub1
|
His6-FLAG-CFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFC61c |
pUC119
|
ef1a-c
|
His6-FLAG-CFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFY1c |
pUC119
|
nmt1
|
His6-FLAG-YFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFY21c |
pUC119
|
tub1
|
His6-FLAG-YFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1U-HFY61c |
pUC119
|
ef1a-c
|
His6-FLAG-YFP
|
5'
|
-
|
lys1+
|
ura4+
|
|
| pLYS1K-FFH1c |
pUC119
|
nmt1
|
FLAG2-His6
|
3'
|
-
|
lys1+
|
kanr
|
|
| pLYS1K-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
lys1+
|
kanr
|
|
| pLYS1K-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
lys1+
|
kanr
|
|
| pLYS1K-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
lys1+
|
kanr
|
|
| pHIS3U-CFH1c |
pUC119
|
nmt1
|
CFP-FLAG-His6
|
3'
|
-
|
his3+
|
ura4+
|
|
| pHIS3U-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
his3+
|
ura4+
|
|
| pHIS3U-YFH1c |
pUC119
|
nmt1
|
YFP-FLAG-His6
|
3'
|
-
|
his3+
|
ura4+
|
|
| pHIS3U-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
his3+
|
ura4+
|
|
| pHIS3U-HFC1c |
pUC119
|
nmt1
|
His6-FLAG-CFP
|
5'
|
-
|
his3+
|
ura4+
|
|
| pHIS3U-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
his3+
|
ura4+
|
|
| pHIS3K-CFH1c |
pUC119
|
nmt1
|
CFP-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-CFH21c |
pUC119
|
tub1
|
CFP-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-CFH61c |
pUC119
|
ef1a-c
|
CFP-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-GFH21c |
pUC119
|
tub1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-GFH61c |
pUC119
|
ef1a-c
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-HFC1c |
pUC119
|
nmt1
|
His6-FLAG-CFP
|
5'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-HFC21c |
pUC119
|
tub1
|
His6-FLAG-CFP
|
5'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
his3+
|
kanr
|
|
| pHIS3K-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
his3+
|
kanr
|
|
| pARG1U-FFH1c |
pUC119
|
nmt1
|
FLAG2-His6
|
3'
|
-
|
arg1+
|
ura4+
|
|
| pARG1U-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
arg1+
|
ura4+
|
|
| pARG1U-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
arg1+
|
ura4+
|
|
| pARG1U-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
arg1+
|
ura4+
|
|
| pARG1K-FFH1c |
pUC119
|
nmt1
|
FLAG2-His6
|
3'
|
-
|
arg1+
|
kanr
|
|
| pARG1K-GFH1c |
pUC119
|
nmt1
|
GFP(S65C)-FLAG-His6
|
3'
|
-
|
arg1+
|
kanr
|
|
| pARG1K-HFF1c |
pUC119
|
nmt1
|
His6-FLAG2
|
5'
|
-
|
arg1+
|
kanr
|
|
| pARG1K-HFG1c |
pUC119
|
nmt1
|
His6-FLAG-GFP(S65C)
|
5'
|
-
|
arg1+
|
kanr
|
|
| pARG1KF-CFH1c |
pUC119
|
nmt1
|
CFP-FLAG-His6
|
3'
|
ura4+
|
arg1+
|
kanr
|
|
|
|
* Medium strength nmt1 promoter, ** Low strength nmt1 promoter
|
 |
 |
|
