| Plasmid
name |
Selection | Comment/description | Contact info | |
| in yeast | in E.coli | |||
| pMW101 | HIS3 | CmR | Basic plasmids to clone
Bait as fusion with LexA.
Expression is driven by the ADH1 promoter |
RB |
| pMW103 | KmR | |||
| pEG202 | ApR | Origene, Clontech, MoBiTec, Display |
||
| pJK202 | pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate Bait to nucleus) | Origene | ||
| pNLexA | Polylinker is upstream of LexA; allows fusion of LexA to C-terminus of Bait, leaving amino-terminal residues of Bait unblocked | Origene | ||
| pEG202I | pEG202 derivative, which can be integrated into yeast HIS3 gene after digestion with KpnI; ensures lower levels of Bait expression | RB | ||
| 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 | Origene, Clontech |
||
| pDD |
KmR |
R.Hopkins |
||
| 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. | Invitrogen | |
| 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 | Contact info | |
| in yeast | in E.coli | |||
| pEG202-Ras | HIS3 |
ApR |
a negative control for activation and positive control for interaction with Raf1 and RalGDS | I.Serebriiskii |
| pEG202-hsRPB7 | a weak positive control for activation | I.Serebriiskii | ||
| pEG202-Krit1 | a moderate positive control for activation | I.Serebriiskii | ||
| 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. | Origene | ||
| pSH17-4
(control) |
GAL4 activation domain cloned into pEG202 backbone is recommended as a positive control for transcriptional activation. | Origene | ||
| Plasmid
name |
Selection | Comment/description | Contact info | |
| 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 | Origene, Clontech, MoBiTec, Display |
| pJLo |
A derivative of pJG4-5 that has lower copy number
(CEN/ARS ori ) |
R.Hopkins |
||
| 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) | RB | ||
| 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 | Invitrogen | ||
| 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 | M. Brown | ||
| pMW102 | KmR | Same as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available | RB | |
| 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 | Contact info | |
| in yeast | in E.coli | |||
| pJG4-5-Raf | TRP1 | ApR |
a positive control for interaction with Ras | I.Serebriiskii |
| pYesTrp-RalGDS | a positive control for interaction with Ras and Krev | I.Serebriiskii | ||
| pJG4-5-Krit1 | a positive control for interaction with Krev | I.Serebriiskii | ||
| Plasmid
name |
Selection | Comment/description | Contact info | ||
| 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 |
RB |
| pMW109 | 2 | ||||
| pMW111 | 1 | ||||
| pMW107 | CmR | 8 | |||
| pMW108 | 2 | ||||
| pMW110 | 1 | ||||
| pSH18-34 | ApR | 8 | Origene, Invitrogen, Clontech |
||
| pJK103 | 2 | Origene | |||
| pRB1840 | 1 | Origene | |||
| 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 ) | Origene | ||
| pGNG1 | 8? | lacZ gene is replaced by GFP | MoBiTec, Display |
||
| pLexAop -lucU |
8 | lacZ gene is replaced by luciferase | A.Fujita | ||
| Strain name | Genotype | Comment/description | Contact info | |
| # of ope- rators |
. | |||
| EGY48 | MATalpha, trp1,
his3, ura3, lexAops-LEU2 |
6 | Transcription of the LEU2 gene is under control of lexA operators; EGY48 is a basic strain used to select for interacting clones from a cDNA library, while EGY191 provides a more stringent selection, producing lower background with Baits with intrinsic ability to activate transcription. | Origene,
Invitrogen, Clontech, MoBiTec, Display |
| EGY191 | 2 | |||
| L40 | MATalpha, trp1, leu2, ade2, GAL4,
lexAops-HIS3, lexAops-lacZ . |
4 8 . |
Expression driven from GAL1 promoter is constitutive in L40 (inducible in EGY strains). Selection is for his prototrophy, and integrated lacZ reported is considerably less sensitive than pSH18-34 in EGY strains. | Invitrogen |
| RFY206 | MATa, trp1, his3, ura3, leu2, lys2 | . | Used in mating assay | Origene |
| Item | Comment/description | Contact info |
| 5´ bait primer | for sequencing bait-fusion protein junction | Origene,
Invitrogen,
Clontech, MoBiTec, Display |
| 3´ bait primer | for PCR | Invitrogen |
| 5´ target primer | for PCR and sequencing | Origene,
Invitrogen,
Clontech, MoBiTec, Display |
| 3´ target primer | for PCR and sequencing (cannot be used to sequence PCR fragments) | Origene,
Invitrogen,
MoBiTec, Display |
| additional plasmids related to basic bait/prey plasmids | for expression in yeast | Origene |
| additional yeast reporter strains | contain intermediate sensitivity LEU2 or LacZ reporters | Origene, Clontech,
MoBiTec, Display |
| KC8 (E. coli) | trp- strain used for rescuing target plasmids from yeast | Origene, Clontech |
| antibodies against LexA | can be used for characterizing hybrid proteins generated with bait plasmids | Invitrogen, Clontech, Santa Cruz |
By red font are shown items recommended as basic set.
For basic plasmids, maps are
available
Plasmids marked commercially available from Clontech have different commercial
names
Credits:
Interaction Trap reagents represent the work of many contributors: the
original basic reagents were developed in the Brent laboratory. Plasmids
with altered antibiotic resistance markers (all pMW plasmids) were constructed
at Glaxo in Research Triangle Park. Plasmids and strains for specialized
applications have been developed by: E. Golemis, Fox Chase Cancer Center,
Philadelphia - pEG202, EG48, EG191; J. Gyuris, Mitotix, Cambridge - pJG4-5;
J. Kamens, BASF, Worcester - pJK101, pJK202; cumulative efforts of I. York,
Dana-Farber Cancer Center, Boston and M.Sainz and S.Nottwehr, U. Oregon
- pNLexA; D.A. Shaywitz, MIT Center for Cancer Research, Cambridge - pGilda;
R. Buckholz, Glaxo Research Triangle Park - pEG202I, pJG4-5I; S. Hanes,
Wadsworth Institute, Albany - pSH18-34, pSH17-4; R. Brent, Dept. of Molecular
Biology, MGH, Boston - pRB1840; R. Finley, Wayne State University, Detroit
- pRFHM1, RFY206; Vojtek A.B., and Hollenberg, S.M, Fred Hatchinson Cancer
Research Center, Seattle - L40.
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