| Name |
|
|
|
| SKY48 | MATatrp1, his3, ura3, lexAop-LEU2, cIop-Lys2 |
|
Provides a more stringent selection for interaction partners of cI-fused Baits, and more sensitive lexA-responsive LEU2 reporter than the one in SKY191 |
| SKY191 |
|
Provides a more stringent lexA-responsive LEU2 reporter, and more sensitive cI-responsive LYS2 reporter than the one in SKY48. | |
| SKY473 | MATahis3, leu2, trp1, ura3, lexAop-LEU2 cIop-LYS2 |
3 cI |
Mating partner for SKY strains; can be also used as a reporter strain itself. Sensitivity of LEU2 reporter is intermediate between sensitivity of LEU2 in SKY48 and SKY191. Sensitivity of LYS2 reporter is the same as sensitivity of LYS2 in SKY191. |
| Plasmid
name |
Selection | Comment/description | |
| in yeast | in E.coli | ||
| pMW101 | HIS3 | CmR | Basic plasmids to clone
Bait as fusion with LexA.
Expression is driven by the ADH1 promoter |
| pMW103 | KmR | ||
| pEG202 | ApR | ||
| pJK202 | pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate Bait to nucleus) | ||
| pNLexA | Polylinker is upstream of LexA; allows fusion of LexA to C-terminus of Bait, leaving amino-terminal residues of Bait unblocked | ||
| pEG202I | pEG202 derivative, which can be integrated into yeast HIS3 gene after digestion with KpnI; ensures lower levels of Bait expression | ||
| pGilda | GAL1 promoter and CEN-ARS backbone facilitate a tightly controlled, galactose-inducible bait expression; should be used if continuous presence of the Bait is toxic to yeast | ||
| pDD |
KmR |
||
| pHybLex/Zeo | Zeo R | Bait cloning vector, compatible with IT and all other two-hybrid systems. Minimal ADH1 promoter expresses LexA followed by extended polylinker. | |
| pCGLex/ p2GLex |
Gal-inducible Bait vector, compatible with IT and all other two-hybrid systems. GAL1 promoter promoter expresses LexA followed by extended polylinker; both high- and low copy number versions available. | ||
| Plasmid
name |
Selection | Comment/description | |
| in yeast | in E.coli | ||
| pEG202-Ras | HIS3 |
ApR |
a negative control for activation and positive control for interaction with Raf1 and RalGDS |
| pEG202-hsRPB7 | a weak positive control for activation | ||
| pEG202-Krit1 | a moderate positive control for activation | ||
| pRFHM1
(control) |
The homeodomain of bicoid cloned into pEG202 backbone; the resulting non-activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay. | ||
| pSH17-4
(control) |
GAL4 activation domain cloned into pEG202 backbone is recommended as a positive control for transcriptional activation. | ||
| Plasmid
name |
Selection | Comment/description | |
| in yeast | in E.coli | ||
| pJG4-5 | TRP1 | ApR | Library construction plasmid; GAL1 promoter provides efficient expression of a gene fused to a cassette consisting of nuclear localization sequence, transcriptional activation domain, and HA epitope tag |
| pJLo |
A derivative of pJG4-5 that has lower copy number
(CEN/ARS ori ) |
||
| pJG4-5I | A derivative of pJG4-5 that can be integrated into yeast TRP1 gene after digestion with Bsu36I; designed to study interactions that occur physiologically at low protein concentrations ( in combination with pEE202I) | ||
| pYESTrp | GAL1 promoter expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site. Used to express cDNA libraries | ||
| pNB42 series |
Allow fusion to the N-terminus of an AD, leaving N-terminal residues of Prey unblocked; various multiple cloning sites. No libraries yet available | ||
| pMW102 | KmR | Same as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available | |
| pMW104 | CmR | ||
| pCGB42/ p2GB42 |
|
|
The same Tn903-encoded gene confers kanamycin resistance in E.coliand geneticin resistance in yeast; both high- and low copy number versions available. Multiple cloning site. |
| Plasmid
name |
Selection | Comment/description | |
| in yeast | in E.coli | ||
| pJG4-5-Raf | TRP1 | ApR |
a positive control for interaction with Ras |
| pYesTrp-RalGDS | a positive control for interaction with Ras and Krev | ||
| pJG4-5-Krit1 | a positive control for interaction with Krev | ||
| Plasmid
name |
Selection | Comment/description | ||
| in yeast | in E.coli | # of ope- rators |
. | |
| pMW112 | URA3 | KmR | 8 | Transcription of the lacZ gene is directed by
lexA operators: the most sensitive indicator plasmids for transcriptional activation have 8 operators, intermediate reporters 2, and the most stringent reporters 1 operator |
| pMW109 | 2 | |||
| pMW111 | 1 | |||
| pMW107 | CmR | 8 | ||
| pMW108 | 2 | |||
| pMW110 | 1 | |||
| pSH18-34 | ApR | 8 | ||
| pJK103 | 2 | |||
| pRB1840 | 1 | |||
| pJK101 (control) |
(2) | The basal activity of lacZ gene is under control of 2 lexA operators; used to monitor Bait binding to operator sequences (in repression assay ) | ||
| pGNG1 | 8? | lacZ gene is replaced by GFP | ||
| pLexAop -lucU |
8 | lacZ gene is replaced by luciferase | ||
| Item | Comment/description |
| 5´ bait primer | for sequencing bait-fusion protein junction |
| 3´ bait primer | for PCR |
| 5´ target primer | for PCR and sequencing |
| 3´ target primer | for PCR and sequencing (cannot be used to sequence PCR fragments) |
| additional plasmids related to basic bait/prey plasmids | for expression in yeast |
| additional yeast reporter strains | contain intermediate sensitivity LEU2 or LacZ reporters |
| KC8 (E. coli) | trp- strain used for rescuing target plasmids from yeast |
| antibodies against LexA | can be used for characterizing hybrid proteins generated with bait plasmids |