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MATCHMAKER GAL4 Two-HybridVectors Handbook
(PT3062-1)
Information Supplement for:
Catalog # Product
K1605-1 MATCHMAKER Two-Hybrid System
K1604-1 MATCHMAKER Two-Hybrid System 2
(many) MATCHMAKER Libraries
FOR RESEARCH USE ONLY
(PR6X890)
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-12 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
I. Introduction 3
II. GAL4 Activation Domain (AD) Cloning Vectors 5
pACT 5
λACT 6pACT2 7
pGAD10 8
pGAD424 9
pGAD GH 10
pGAD GL 11
III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors 12
pAS2 12
pAS2-1 13
pGBT9 14
IV. Control Plasmids 15
pCL1 15
pVA3 16
pVA3-1 16
pTD1 17
pTD1-1 17
pLAM5' 18
pLAM5'-1 18
V. References 19
VI. Plasmid and System Ordering Information 20
APPENDIX. Additional MATCHMAKER Plasmid Information 21
List of Tables
Table I. Yeast Promoter Constructs in the MATCHMAKER GAL4 Two-Hybrid Cloning Vectors 4
Table II. List of Abbreviations 4
Table III. MATCHMAKER GAL4 Two-Hybrid System Cloning Vectors 21
Table IV. MATCHMAKER GAL4 Two-Hybrid System Reporter Plamids 22
Table of Contents
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 3
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
I. Introduction
This Handbook provides vector maps, MCSs, transformation markers, references, GenBank accessionnumbers (where available), and other pertinent information for the cloning and control plasmids used inthe yeast GAL4-based MATCHMAKER Two-Hybrid Systems and Libraries. For ease of use andcomparison of the plasmids, much of this information is summarized in the Tables in the Appendix.Complete sequence information for most of the plasmids is available by request from CLONTECH'sTechnical Support Dept. Selected vector sequences are accessible through our worldwide web site(http://www.clontech.com).
All of the cloning and control plasmids are shuttle vectors that replicate autonomously in both E. coli(using the Col E1 or pBR322 origins of replication) and in S. cerevisiae (using the 2µ ori). AllMATCHMAKER plasmids carry the β-lactamase (bla) gene, which confers ampicillin resistance (Ampr)in E. coli, and a nutritional gene (TRP1 or LEU2 ) that allows yeast auxotrophs transformed with theplasmid to grow on synthetic defined medium lacking tryptophan or leucine, respectively. The nutritionalmarkers also allow selection in certain auxotrophic strains of E. coli on M9 minimal medium.
The cloning plasmids generate fusion proteinsAll of the cloning plasmids provided in the MATCHMAKER Two-Hybrid Systems are used for theconstruction and expression of hybrid (fusion) proteins. The GAL4 AD plasmids generate a hybrid thatcontains the sequences for the yeast GAL4 activation domain (a.a. 768–881) and a cloned protein orcDNA library insert. The GAL4 DNA-BD plasmids generate a hybrid that contains the sequences for theGAL4 DNA-binding domain (a.a. 1–147) and a cloned protein, most often used as the bait protein in atwo-hybrid library screening. These vectors have unique restriction sites located in the MCS region atthe 3' end of the open reading frame for either the DNA-BD or the AD sequence.
For the construction of a specific hybrid, the gene encoding the protein of interest is inserted into the MCSin the correct orientation and reading frame such that a hybrid protein is generated. In the case ofMATCHMAKER Libraries, the inserts may be cloned into the MCS either directionally or nondirectionally(check the Product Analysis Certificate and the User Manual for detailed information on how the librarywas constructed). The fusion proteins are expressed in yeast cells from the full-length ADH1 promoteror a modified form of it. See Table I for the relative expression levels observed for the different plasmids;for further information on the promoters, see the Yeast Protocols Handbook (YPH), Chapter II. In allplasmids, transcription is terminated at the ADH1 transcription termination signal.
The hybrid proteins are targeted to the yeast nucleus by nuclear localization sequences. In the GAL4DNA-BD cloning plasmids, the nuclear localization sequence is an intrinsic part of the DNA-BD (Silveret al., 1984) and is not specifically indicated on the vector maps. In the GAL4 AD cloning plasmids, thenuclear localization sequence from SV40 T-antigen has been cloned into the vector between the ADH1promoter and the AD sequence. (For detailed information regarding the construction of this sequence,see Chien et al., 1991.)
Some of the cloning vectors also encode the influenza hemagglutinin (HA) epitope in the junctionbetween the GAL4 sequence and the MCS; if present, this element is indicated on the vector map. TheHA epitope tag is useful for detecting expressed fusion proteins on Western blots using an anti-HAantibody. GAL4 AD and DNA-BD Monoclonal Antibodies (#5398-1 and 5399-1, respectively) areavailable from CLONTECH for probing Western blots when using medium- to high-expression levelcloning vectors (see Table I). The DNA-BD plasmids pAS2 and pAS2-1 (Harper et al., 1993) carry thewild-type yeast CYHS2 gene, which confers sensitivity to cycloheximide in transformed cells. CYHS2 canbe used to isolate AD/library plasmids in yeast segregants after a two-hybrid library screening, and thusfacilitates the elimination of false positives.
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-14 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
I. Introduction continued
TABLE I. YEAST PROMOTER CONSTRUCTS IN THE MATCHMAKER GAL4 CLONING VECTORS
Regulation/ SignalRelative Protein Strength on
Vectors Promoter Expression Level Western blot a
pGAD GH, pAS2, pAS2-1 ADH1 (full-length) Ethanol-repressedb/High +++
pACT2, pACT ADH1 (truncated+)c Constitutive/medium ++
pGAD GL ADH1 (truncated) Constitutive/low +/– (weak)
pGAD424, pGAD10, pGBT9 Constitutive/ very low (not detectable)
a Soluble protein extracts were prepared from CG-1945 transformed with the indicated plasmid. Samples equivalent to ~1 OD600unit of cells were electrophoresed and then blotted to nitrocellulose filters. The blots were probed with either GAL4 DNA-BDmAb (0.5 µg/ml; #5398-1) or GAL4 AD mAb (0.4 µg/ml; #5399-1), followed by HRP-conjugated polyclonal goat anti-mouse IgG(Jackson Immunological Research; diluted 1:15,000 in TBST). Signals were detected using a chemiluminescent detectionassay and a 2.5-min exposure of x-ray film. Signal intensities were compared to that of known amounts of purified GAL4 DNA-BD or GAL4 AD.
b Transcription is repressed in late lag phase by the ethanol that accumulates in the medium as a by-product of yeast metabolism.c The truncated ADH1 promoter in pACT2 is adjacent to a section of pBR322 which acts as a transcriptional enhancer in yeast.
TABLE II. LIST OF ABBREVIATIONS
AD GAL4 activation domain (a.a. 768–881)
DNA-BD (or BD) GAL4 DNA-binding domain (a.a. 1–147)
P promoter
T transcription termination sequence
HA hemagglutinin sequence
AD/library plasmid Plasmid encoding a fusion of theGAL4 AD and a library insert
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 5
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
II. GAL4 Activation Domain (AD) Cloning Vectors
Figure 1. pACT map and MCS. Unique sites are in bold. pACT (Durfee et al., 1993) is used to generatea hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a protein encodedby a cDNA in a fusion library). The hybrid protein is expressed at medium levels in yeast host cells fromthe enhanced, truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigennuclear localization sequence (▲ ; Chien et al., 1991). pACT contains the LEU2 gene for selection in Leu–auxotrophic yeast strains. The Bgl II (*) sites can be used as a unique cloning site. pACT Libraries fromCLONTECH are constructed in λACT; recombinant plasmids are released by Cre-lox recombination(see Figure 2). No further pACT sequence information is available.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA
CCA AAC CCA AAA AAA GAG ATC TGG AAT TCG GAT CCT CGA GAG ATC TAT GA
A TCG TAG ATA CTG AAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC
Xho IBamH IEcoR I Bgl II*Bgl II*
GAL4 AD Sequencing Primer
STOP(ORF 1)
STOP(ORF 3)
STOP(ORF 2)
MATCHMAKER 3' AD LD-Insert-Screening Amplimer
MATCHMAKER 5' AD LD-InsertScreening Amplimer
GAL4 AD
MCSBgl II*EcoR I BamH IXho IBgl II*
EcoR I(1)
Xba I(0.7)
Hpa I(1.7)
Cla I(2.7)
EcoR I(3.2)
EcoR V(3.3)
Aat II(7.5)
pACT7.65 kb
Col E1ori
Ampr
PADH1
2 µori
TADH1
LEU2∆
GAL4 AD
♦
Not I Not I
loxP
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-16 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 2. Conversion of a recombinant λACT to the corresponding pACT. The λACT MCS is locatedwithin an embedded plasmid, which is flanked by loxP sites at the λ junctions. Transduction of a λACTlysate into E. coli strain BNN132 promotes Cre recombinase-mediated release and recircularization ofpACT at the loxP sites. Note that the resulting plasmid contains only one loxP site.
λ Left Arm (27.4 kb) λ Right Arm (15.2 kb)
pACT
λACT (42.6 kb)
Clone cDNA into MCS of λACT
High-efficiency, single-step conversionvia site-specific recombination at loxP sites (in E. coli BNN132)
loxP loxP
MCS
MCS with insert
loxP loxP
pACT7.65 kb
Col E1ori
Ampr
PADH1
2 µori
GAL4AD
TADH1
LEU2
∆
insert
♦loxP
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 7
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 3. pACT2 map and MCS. Unique sites are in bold. pACT2 (Li et al., 1994) is used to generatea hybrid containing the GAL4 AD (a.a. 768–881), an HA epitope tag (Durfee et al., 1993), and anotherprotein of interest (or a protein encoded by a cDNA in a fusion library). The hybrid protein is expressedat medium levels in yeast host cells from an enhanced, truncated ADH1 promoter and is targeted to theyeast nucleus by the SV40 T-antigen nuclear localization sequence (▲ ; Chien et al., 1991). pACT2contains the LEU2 gene for selection in Leu– auxotrophic yeast strains. The Bgl II (*) sites can be usedas a unique cloning site, but this will remove the HA epitope. pACT2 libraries from CLONTECH areconstructed in λACT2; recombinant plasmids are released by Cre-lox recombination (see Figure 2).GenBank Accession: #U29899.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA
AAC CCA AAA AAA GAG ATC TGT ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC TTG
GGT GGT CAT ATG GCC ATG GAG GCC CCG GGG ATC CGA ATT CGA GCT CGA GAG ATC TAT GA
A TCG TAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC
Bgl II*
STOP (ORF 1)
STOP(ORF 3)
STOP (ORF 2)
5095•
5035
•
4976
•
5155•
Sma I/Xma I
Nco I BamH I EcoR INde I
Sfi I
Xho I
Tyr Pro Tyr Asp Val Pro Asp Tyr A la
HA epitope
Sac I
Bgl II*
GAL4AD
GAL4 AD MATCHMAKER 5' AD LD-Insert
Screening AmplimerGAL4 AD Sequencing Primer
MATCHMAKER 3' AD LD-Insert-Screening Amplimer
pACT28.1 kb
MCS
(5082–4981) * Bgl II
HA epitope Nde I Sfi I
Nco ISma I Xma I
BamH IEcoR I
Sac IXho I
* Bgl II
Nde I(235)
Nde I(6131)
Not I (4163)
Cla I(2626)
Hpa I(1581)
Xba I(598)
2 µ ori
GAL4 AD
PADH1
TADH1
LEU2
Col E1ori
Ampr
Not I (4345)
loxP
HA
∆
Hind III(4739)
Hind III(5497)
♦
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-18 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 4. pGAD10 map and MCS. Unique sites are in bold. pGAD10 (Bartel et al., 1993) is used togenerate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD10 contains the LEU2 gene for selection in Leu–auxotrophic yeast strains.GenBank Accession: #U13188.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA
CCA AAC CCA AAA AAA GAG ATC TCT CGA GGA TCC GAA TTC CAG ATC TAT GA
A TCG TAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC
Xho I BamH I EcoR I Bgl II*Bgl II*
GAL4 AD
STOP(ORF 1)
STOP(ORF 3)
STOP(ORF 2)
MATCHMAKER 3' AD LD-Insert-Screening Amplimer
MATCHMAKER 5' AD LD-InsertScreening Amplimer755
•
812•
862•
GAL4 AD Sequencing Primer
2 µori
GAL4AD
pGAD106.65 kb
PADH1
TADH1
LEU2
Col E1ori
Ampr
Sph I(10)
Hind III(410)
Kpn I (484) Mlu I
(727)
Hind III(1096)
Sph I (1424)
EcoR V(1882)
Cla I (2478)Bgl I(4433)
Pvu I(4683)
Sca I (4793)
Aat II(5235)
SnaB I(6009)
MCS(828-857)Bgl II* Xho I BamH I EcoR I Bgl II*
Pvu II (3243)
∆
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 9
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 5. pGAD424 map and MCS. Unique sites are in bold. pGAD424 (Bartel et al., 1993) is used togenerate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD424 contains the LEU2 gene for selection in Leu–auxotrophic yeast strains.GenBank Accession: #U07647.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA
CCA AAC CCA AAA AAA GAG ATC GAA TTC CCG GGG ATC CGT CGA CCT GCA GAG ATC TAT GA
A TCG TAG ATA CTG AAA AAC CCC GCA AGT TCA CTT CAA CTG TGC ATC GTG CAC CAT CTC
Sma I BamH I Sal I Pst IEcoR I Bgl II
GAL4 AD
STOP(ORF 1)
STOP
(ORF 3)
STOP(ORF 2)
MATCHMAKER 3' AD LD-Insert-Screening Amplimer
MATCHMAKER 5'AD LD-Insert-Screening Amplimer
812•
755•
871•
GAL4 AD Sequencing Primer
2 µori
GAL4AD
pGAD4246.6 kb
PADH1
TADH1
LEU2
Col E1ori
Ampr
Sph I(10)
Hind III(410)
Kpn I (484) Mlu I
(727)
Hind III(1105)
Sph I (1433)
EcoR V(1891)
Cla I (2487)Bgl I(4442)
Pvu I(4692)
Sca I (4802)
Aat II(5244)
SnaB I(6018)
MCS(833-866)EcoR I Sma I BamH ISal IPst IBgl II
Pvu II (3253)
∆
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-110 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 6. pGAD GH map and MCS. Unique sites are in bold. pGAD GH (van Aelst et al., 1993) is usedto generate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at high levels in yeast host cellsfrom the full-length ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD GH contains the LEU2 gene for selection in Leu–auxotrophic yeast strains. pGAD GH was derived from pGAD GL (Figure 7) and is nearly identical to it,except in the promoter region. Also, an Sph I site (at bp 1600) in pGAD GL was mutagenized whenpGAD GH was constructed. Indicated bp's are approximate. No further pGAD GH sequence informationis available.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA
AAC CCA AAA AAA GAG ATC CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AAT TCG ATA TC
A AGC TTA TCG ATA CCG TCG ACC TCG AGG GGG GGC CCG GTA CCC AAT TCG CCC TAT AG
T GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT
GAT CTA TGA ATC GTA GAT ACT GAA AAA CCC CGC AAG TTC ACT TCA ACT GTG CAT CGT GCA
SK primer
KS primer
M13 –20 forward primer
T7 primer
STOP (ORF 3)
STOP(ORF 2)
STOP(ORF 1)
Hind III Xho I
BamH I
Sal I
Spe I Sma I Pst I EcoR I EcoR V
Cla I Apa I Kpn I
GAL4 AD GAL4 AD Sequencing Primer
MATCHMAKER 3' AD LD-InsertScreening Amplimer
MATCHMAKER 5' AD LD-InsertScreening Amplimer
2 µori
GAL4AD
PADH1
TADH1
LEU2
Col E1ori
Ampr
Sph I (6.9)
Hind III (0.4)
Hind III(1.2)
Aat II(5.5)
SnaB I(6.4)
Not I (3.45)
pGAD GH7.8 kb
Sph I(0)
MCS(0.8–0.9)Spe I BamH I Sma I EcoR I Hind III Sal I Xho IApa I
∆
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 11
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
II. GAL4 Activation Domain (AD) Cloning Vectors continued
Figure 7. pGAD GL map and MCS. Unique sites are in bold. pGAD GL (van Aelst et al., 1993) is usedto generate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD GL contains the LEU2 gene for selection in Leu–auxotrophic yeast strains. Indicated bp's are approximate. No further pGAD GL sequence informationis available.
AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA
AAC CCA AAA AAA GAG ATC CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AAT TCG ATA TC
A AGC TTA TCG ATA CCG TCG ACC TCG AGG GGG GGC CCG GTA CCC AAT TCG CCC TAT AG
T GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT
GAT CTA TGA ATC GTA GAT ACT GAA AAA CCC CGC AAG TTC ACT TCA ACT GTG CAT CGT GCA
SK primer
KS primer
M13 –20 forward primer
T7 primer
STOP (ORF 3)
STOP(ORF 2)
STOP(ORF 1)
Hind III Xho I
BamH I
Sal I
Spe I Sma I Pst I EcoR I EcoR V
Cla I Apa I Kpn I
GAL4 AD GAL4 AD Sequencing Primer
MATCHMAKER 3' AD LD-InsertScreening Amplimer
MATCHMAKER 5' AD LD-InsertScreening Amplimer
2 µori
GAL4AD
PADH1
TADH1
LEU2
Col E1ori
Ampr
Sph I(0)
Hind III(0.4) Mlu I
(0.7)
Hind III(1.25)
Sph I (1.6)
EcoR V (2.0)
Cla I (2.6)
Bgl I(4.7)
Pvu I(5.0)
Sca I (5.1)
Aat II(5.5)
SnaB I(6.3)
∆
Not I (3.45)
pGAD GL6.9 kb
MCS(0.8–0.9)Spe IBamH I Sma IEcoR I EcoR VHind III Cla I Sal I Xho I Apa IKpn I
Pvu II (3.4)
Pvu II (3.5)
Kpn I (0.5)
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-112 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors
Figure 8. pAS2 map and MCS. Unique sites are in bold. pAS2 is a cloning vector used to generatefusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). pAS2 is synonomous with pAS1CHY2 (Harperet al., 1993) and carries the wild-type yeast CYHS2 gene, which confers sensitivity to cycloheximide intransformed yeast cells. The hybrid protein is expressed at high levels in yeast host cells from the full-length ADH1 promoter. The hybrid protein is targeted to the yeast nucleus by nuclear localizationsequences (Silver et al., 1984). pAS2 contains the TRP1 gene for selection in Trp– auxotropic yeaststrains. The Xba I site at bp 4763 (†) is methylation sensitive. The acidic amino acids of the HA epitopetag fused to the GAL4 DNA-BD causes high backgrounds in the two-hybrid assay when pAS2 is usedas a negative control. The HA epitope also leads to high backgrounds in some plasmid constructs basedon pAS2. pAS2 has been discontinued and replaced by its derivative pAS2-1 (Figure 9), which does notcontain an HA epitope.GenBank Accession: #U30496.
f1ori
TRP1
CYH2
MCS (5981–6036)
Pst I Sal I
BamH I Sma I
Sfi INco I Nde I Nhe I
HA epitope
pAS28.4 kb
Col E1ori
2 µoriAmpr
PADH1
GAL4 BD
TADH1
Bgl II (3968)
EcoR I (3950)
EcoR I (5946)
EcoR V (4769)
Xba I† (4763)
EcoR V (3562)
Sac I (3297)
Nae I (2988)
EcoR V (2168)
Xba I (1969)
Xba I (982)
Hind III (3284)
Hind III (2397)
Hind III(5478)
Hind III(6246)
6094
•
GGT GGT CAT ATG GCC ATG GAG GCC CCG GGG ATC CGT CGA CCT GCA GCC AAG
CCG GAA TTC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC TTG
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
CTA ATT CCG GGC GAA TTT CTT ATG ATT TAT GAT TTT TAT TAT TAAATAAGTT
Nde I Sal ISma I/Xma I
Nco I
EcoR I
Pst I
pAS2-specific sequence
pAS2-specific sequence
HA epitope
Nhe I
BamH I
STOP(ORF 3)
STOP(ORF 2)
STOP(ORF 1)
5943
•
5991
•
6042
•
TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG
5895
•
ATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACG
MATCHMAKER 5' BD Insert
Screening Amplimer & GAL4 DNA-BD Sequencing Primer
MATCHMAKER 3' BD InsertScreening Amplimer
GAL4 DNA-BD
Sfi I
a.a.147
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MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors continued
Figure 9. pAS2-1 map and MCS. Unique sites are in bold. pAS2-1 is a cloning vector used to generatefusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). pAS2-1 is derived from pAS2 (Figure 8)and hence from pAS1CHY2 (Harper et al., 1993) and carries the CYH
S2 gene for cycloheximide sensitivity.The hybrid protein is expressed at high levels in yeast host cells from the full-length ADH1 promoter(PADH1). The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (Silver etal., 1984). The Xba I site at bp 4763 (†) is methylation sensitive. pAS2-1 contains the TRP1 gene forselection in Trp– auxotrophic yeast strains. Plasmid modification was performed at CLONTECH.GenBank Accession: #U30497.Compared with pAS2, pAS2-1 contains a neutral, short peptide instead of an HA epitope tag. Removingthe HA epitope tag and converting a.a.149 from Glu to Val completely eliminates the autonomousactivation activity of pAS2 (assayed in Y187 using the lacZ reporter; Holtz & Zhu, 1995). pAS2-1 has adifferent MCS than pAS2; however, the cloning sites that they have in common are in the same readingframe. Furthermore, the EcoR I, Xma I/Sma I, BamH I, Sal I, and Pst I sites in the pAS2-1 MCS are inthe same reading frames as those in pGBT9. Thus, inserts from pAS2 or pGBT9 can be excised andmoved to pAS2-1 without changing the reading frame. The EcoR I site in the pAS2-1 MCS is uniquebecause the EcoR I site in the CYHS2 gene in pAS2 was eliminated by site-directed mutagenesis.
STOP(ORF 3)
STOP(ORF 1)
5957
•
STOP (ORF 2)
Sal ISma I/Xma I
Pst IBamH IEcoR I
6020
•
6088
•
pAS2-1 specific sequence
TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG GTA TTG CAA TAC
CCA GCT TTG ACT CAT ATG GCC ATG GAG GCC GAA TTC CCG GGG ATC CGT CGA CCT GCA GCC AAG
CTA ATT CCG GGC GAA TTT CTT ATG ATT TAT GAT TTT TAT TAT TAA ATAAGTTATAAAAAAAATAAGTG
TATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACG
MATCHMAKER 3' BD InsertScreening Amplimer
GAL4 DNA-BD
a.a.147
5895
•
MATCHMAKER 5' BD Insert Screening Amplimer & GAL4 DNA-BD Sequencing Primer
Nde I Nco ISfi I
MCS (5969–6014)
Pst ISal I
BamH I Sma IXma I
EcoR ISfi I
Nco INde I
pAS2-18.4 kb
Col E1ori
f1ori
2 µoriAmp
r
TRP1
CYH2
PADH1
GAL4 BD
TADH1
Bgl II (3969)
Xba I† (4763)
Sac I (3298)
Nae I (2989)
Xba I (1968)
Xba I (981)
Hind III (2397)
Hind III (3284)
Hind III(5477)
Hind III(6224)
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-114 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors continued
Figure 10. pGBT9 map and MCS. Unique sites are in bold. pGBT9 (Bartel et al., 1993) is a cloning vectorused to generate fusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). The hybrid protein isexpressed at low levels in yeast host cells from a truncated ADH1 promoter. The hybrid protein istargeted to the yeast nucleus by nuclear localization sequences (Silver et al., 1984). pGBT9 contains theTRP1 gene for selection in Trp– auxotropic yeast strains.GenBank Accession: #U07646.
TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG
GAA TTC CCG GGG ATC CGT CGA CCT GCA GCC AAG CTA ATT CCG GGC GAA TTT
CTT ATG ATT TAT GAT TTT TAT TAT TAA ATA AGT TAT AAA AAA AAT AAG TGT ATA
CAA ATT TTA AAG TGA CTC TTA GGT TTT AAA ACG
EcoR I Sma I
878 •
Pst ISal IBamH I
929 •
827 •
STOP(ORF 2)
GAL4 DNA-BD
STOP(ORF 1)
STOP(ORF 3)
MATCHMAKER 5' BD Insert Screening Amplimer &
GAL4 DNA-BD Sequencing Primer
983 •
MATCHMAKER 3'BD Insert
Screening Amplimer
pGBT95.5 kb
GAL4BD
Ampr
PADH1
2 µori
TADH1
TRP1
Col E1ori
Hind III (410)
Hind III (1319)
Nae I (1115)
Pvu II(2118)
Xba I(1748)
Aat II(4109)
SnaB I(4883)
MCS(1004–1098)EcoR ISma IBamH ISal IPst I
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MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
Figure 11. pCL1 map. pCL1 (Fields & Song, 1989) is a positive control plasmid that encodes the full-length, wild-type GAL4 protein. The GAL4 protein activates reporter genes under the control of aGAL4-responsive element (UAS
G). Thus, pCL1 is used as a positive control for the transcription assay
in GAL4-based MATCHMAKER Two-Hybrid Systems. This vector is a derivative of YCp50 referencedin Fields & Song, 1989. pCL1 has not been sequenced.
IV. Contol Plasmids
pCL115.3 kb
Ampr PADH1
GAL4
TADH1
LEU2
CEN4
Hind III
Hind III
Hind III
pMB1ori
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-116 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
Figure 12. pVA3 map. pVA3 (Iwabuchi et al., 1993) is a positive control plasmid that encodes a fusionof the murine p53 protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA(GenBank Accession #K01700) was cloned into pGBT9 (Figure 10). pVA3 has not been sequenced andit is not known whether any of the sites are unique.
Figure 13. pVA3-1 map. pVA3-1 is a positive control plasmid that encodes a fusion of the murine p53protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA (GenBank Accession#K01700) was cloned into pAS1CYH2 (a precursor of pAS2-1 [Figure 9]; Harper et al., 1993). The p53insert was derived from the plasmid described in Iwabuchi et al. (1993); plasmid modification wasperformed at CLONTECH. The Xba I site at bp 4763 (†) is methylation sensitive. pVA3-1 has not beensequenced and it is not known whether any of the sites are unique.
IV. Contol Plasmids continued
MCSPst I Sal I BamH I EcoR I
pVA3-19.4 kb
Col E1ori
f1ori
2 µoriAmp
r
TRP1
CYH2
PADH1
GAL4 BD
TADH1
Bgl II (3969)
Xba I† (4763)
Sac I (3298)
Nae I (2989)
Xba I (1968)
Xba I (981)
Murine p53 insert
bp 371(a.a. 72)
stop codonbp 1328
(a.a. 390)
bp 1345
Hind III (2397)
Hind III(3284)
Hind III(5477)
Hind III
pVA36.4 kb
Col E1ori
2 µori
Amp r
TRP1
PADH1
TADH1
GAL4 BD
SnaB I
MCSEcoR IBamH ISal IPst I
Hind III
Nae IHind III
Xba I
Pvu II
Aat II
Murine p53 insert
bp 371(a.a. 72)
stop codon
bp 1345
bp 1328 (a.a. 390)
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MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
BamH I Hind III
Pvu I
Pvu I
Xba I, Pvu I
Xba I
Xba I*
EcoR I
EcoR I
EcoR I
Hind III
Sph I
Sph I
Sal I
Sph ISal I
pTD1~15 kb
Ampr
PADH1
2 µori
GAL4AD
TADH1
LEU2
pBR322ori
∆
Pvu II
SV40 large T-antigen
bp 4559(a.a. 87)
Hind IIIbp 4002
Nde Ibp 3808
Hind IIIbp 3477
bp 2539
Stop codonbp 2693(a.a. 708)
Pvu IIbp 3509
IV. Contol Plasmids continued
Figure 15. pTD1-1 map. pTD1-1 is a positive control plasmid that encodes a fusion of the SV40 largeT-antigen (a.a. 87–708) and the GAL4 AD (a.a. 768–881). The SV40 large T-antigen cDNA (GenBankLocus SV4CG) was cloned into pACT2 (Figure 3). The SV40 T-antigen insert was derived from theplasmid referenced in Li & Fields (1993); plasmid modification was performed at CLONTECH. pTD1-1has not been sequenced and it is not known whether any of the sites are unique.
Figure 14. pTD1 map. pTD1 (Li & Fields, 1993) is a positive control plasmid that encodes a fusion ofthe SV40 large T-antigen (a.a. 86–708) and the GAL4 AD (a.a. 768–881). The SV40 large T-antigencDNA was cloned into pGAD3F (Chien et al., 1991). pTD1 has not been sequenced and it is not knownwhether any of the sites are unique.
pTD1-110.1 kb
MCSBgl II
HA epitopeNde I
Sfi INco ISma IXma I
BamH IEcoR IXho I Bgl II
Not I (4163)
Cla I(2626)
Hpa I(1581)
Xba I(598)
2 µ ori
GAL4 AD
PADH1
TADH1
LEU2
Col E1ori
Ampr
Not I (4345)
loxP
HA
SV40 large T-antigen
bp 4559(a.a. 87)
Hind IIIbp 4002
Nde I
bp 3808
Hind IIIbp 3477
Stop codon bp 2693 (a.a. 708)
bp 2539
Hind III(4739)
Hind III
♦
∆
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-118 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
Figure 16. pLAM 5' map. pLAM5' is a false-positive detection plasmid that encodes a fusion of thehuman lamin C protein (a.a. 66–230) and the GAL4 DNA-BD (a.a. 1–147). The lamin C cDNA insert(GenBank Accession #M13451) was derived from the plasmid referenced in Bartel et al. (1993), and wascloned into pGBT9 (Figure 10). Plasmid modification was performed at CLONTECH. pLam5' has notbeen sequenced and it is not known whether any of the sites are unique.
Figure 17. pLAM 5'-1 map. pLAM5'-1 is a false-positive detection plasmid that encodes a fusion of thehuman lamin C protein (a.a. 66–230) and the GAL4 DNA-BD (a.a. 1–147). The lamin C cDNA insert(GenBank Accession #M13451) was derived from the plasmid referenced in Bartel et al. (1993), and wascloned into pAS2-1 (Figure 9). Plasmid modification was performed at CLONTECH.pLAM5'-1 has not been sequenced and it is not known whether any of the sites are unique.
IV. Contol Plasmids continued
pLAM5'6.0 kb
Col E1ori
2 µori
Amp r
TRP1
PADH1
TADH1
GAL4 BD
SnaB IMCS EcoR ISma IBamH ISal IPst I
Hind III
Nae I
Hind III
Xba I
Pvu II
Aat II
Human lamin C
bp 330 (a.a. 66)
bp 825(a.a. 230)
Hind IIIbp 645
MCSPst ISal I BamH ISma IEcoR ISfi I Nco I Nde I
pLAM 5'-19.1 kb
Col E1ori
f1ori
2 µoriAmp
r
TRP1
CYH2
PADH1
GAL4 BD
TADH1
Bgl II (3969)
Xba I†(4763)
Sac I (3298)
Nae I (2989)
Xba I (1968)
Xba I (981)
Human lamin C
bp 330 (a.a. 66)
bp 825 (a.a. 230)
Hind III(5477)
Hind III
Hind III (2397)
Hind III (3284)
Hind IIIbp 645
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 19
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
V. References
Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. (1993) Using the two-hybrid system to detect protein-protein interactions.In Cellular Interactions in Development: A Practical Approach., ed. Hartley, D.A. (Oxford University Press, Oxford) pp. 153–179.
Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) The two-hybrid system: A method to identify and clone genes forproteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA 88:9578–9582.
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilbburn, A. E., Lee, W. H. & Elledge, S. J. (1993) The retinoblastomaprotein associates with the protein phosphatase type 1 catalytic subunit. Genes Devel. 7:555–569.
Fields, S. & Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–247.
Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potentinhibitor of G1 cyclin-dependent kinases. Cell 75:805–816.
Iwabuchi, K., Li, B., Bartel, P. & Fields, S. (1993) Use of the two-hybrid system to identify the domain of p53 involved inoligomerization. Oncogene 8:1693–1696.
Li, B. & Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybridsystem. FASEB J. 7:957–963.
Li, L., Elledge, S. J., Peterson, C.A., Bales, E. S. & Legerski, R. J. (1994) Specific association between the human DNA repairproteins XPA and ERCC1. Proc. Natl. Acad. Sci. USA 91:5012–5016.
Silver, P. A., Keegan, L. P. & Ptashne, M. (1984) Amino terminus of the yeast GAL4 gene product is sufficient for nuclearlocalization. Prod. Natl. Acad. Sci. USA 81:5951–5955.
van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. (1993) Complex formation between RAS and RAF and other proteinkinases. Proc. Natl. Acad. Sci. USA 90:6213–6217.
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-120 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
VI. Plasmid and System Ordering Information
Product Size Cat.#
MATCHMAKER Two-Hybrid System each K1605-1
MATCHMAKER Two-Hybrid System 2 each K1604-1
Two-Hybrid cDNA Library Construction Kit each K1607-1
MATCHMAKER cDNA Libraries many
MATCHMAKER DNA-BD Insert Screening Amplimer Set 100 rxn 5417-1
MATCHMAKER AD LD-Insert Screening Amplimer Set 100 rxn 9103-1
GAL4 Activation Domain Sequencing Primer 2.5 µg 6473-1
GAL4 Binding Domain Sequencing Primer 2.5 µg 6474-1
GAL4 AD mAb 20 µg 5398-1
GAL4 DNA-BD mAb 20 µg 5399-1
Vectors available separately
pACT2 40 µg K1604-A
pGAD10 40 µg 6180-1
pGAD424 40 µg K1605-B
pGAD GH 40 µg 6182-1
pGAD GL 40 µg 6181-1
pAS2-1 40 µg K1604-B
pGBT9 40 µg K1605-A
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 21
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
APPENDIX. Additional MATCHMAKER Plasmid Information
TA
BLE
III.
MA
TC
HM
AK
ER
GA
L4
TW
O-H
YB
RID
SY
ST
EM C
LON
ING
VE
CT
OR
S
Sel
ectio
n on
Siz
eD
iagn
ostic
Gen
Ban
kV
ecto
rS
yste
ma
Des
crip
tion
SD
Med
ium
(kb)
R.E
. Site
s (k
b)A
cces
sion
#R
efer
ence
s
pAC
TM
M L
ibra
ries
GA
L4 A
D,
–Leu
7.65
Eco
R I
not a
vaila
ble
Dur
fee
et a
l., 1
993;
LEU
2, A
mpr
,(3
.0, 3
.05,
1.6
)E
lledg
e, p
ers.
com
m.
HA
epi
tope
tag
pAC
T2
GA
L4 2
H-2
GA
L4 A
D,
–Leu
8.1
Hin
d III
U29
899
Li e
t al.,
199
4;(#
K16
04-1
) &
LEU
2, A
mpr
,(7
.3, 0
.8)
Elle
dge,
per
s. c
omm
.M
M L
ibra
ries
HA
epi
tope
tag
pAS
2(#
K16
05-D
)bG
AL4
DN
A-B
D,
–Trp
8.4
Hin
d III
U30
496
Dur
fee
et a
l., 1
993;
TR
P1,
Am
pr(4
.6, 2
.2, 0
.9, 0
.7)d
Har
per
et a
l., 1
993
pAS
2-1
cG
AL4
2H
-2G
AL4
DN
A-B
D,
–Trp
8.4
Hin
d III
U30
497
Har
per
et a
l., 1
993
(#K
1604
-1)
TR
P1,
Am
pr, C
YH
s 2(4
.6, 2
.2, 0
.9, 0
.7)d
pGA
D10
MM
Lib
rarie
sG
AL4
AD
,–L
eu6.
6H
ind
IIIU
1318
8B
arte
l et a
l., 1
993
LEU
2, A
mpr
(5.9
, 0.7
)e
pGA
D42
4G
AL4
2H
GA
L4 A
D,
–Leu
6.6
Hin
d III
U07
647
Bar
tel e
t al.,
199
3(#
K16
05-1
)LE
U2,
Am
pr(5
.9, 0
.7)e
pGA
D G
HM
M L
ibra
ries
GA
L4 A
D,
–Leu
7.9
Hin
d III
not a
vaila
ble
van
Ael
st e
t al .,
199
3LE
U2,
Am
pr(7
.1, 0
.5, 0
.3)
pGA
D G
LM
M L
ibra
ryG
AL4
AD
,–L
eu6.
9H
ind
IIIno
t ava
ilabl
eva
n A
elst
et a
l ., 1
993
LEU
2, A
mpr
(6.1
, 0.5
, 0.3
)
pGB
T9
GA
L4 2
HG
AL4
DN
A-B
D,
–Trp
5.5
Hin
d III
U07
646
Bar
tel e
t al.,
199
3(#
K16
05-1
)T
RP
1, A
mpr
(4.6
, 0.9
)
aK
ey t
o sy
stem
abb
revi
atio
ns:
GA
L4 2
H-2
= M
AT
CH
MA
KE
R T
wo-
Hyb
rid S
yste
m 2
(#K
1604
-1);
GA
L4 2
H =
MA
TC
HM
AK
ER
Tw
o-H
ybrid
Sys
tem
(#K
1605
-1).
Som
e pl
asm
ids
are
also
avai
labl
e se
para
tely
(se
e S
ectio
n V
I).
bT
he M
AT
CH
MA
KE
R S
uppl
emen
t Kit
(#K
1605
-D)
was
rep
lace
d by
the
MA
TC
HM
AK
ER
Tw
o-H
ybrid
Sys
tem
2 (
#K16
04-1
).c
pAS
2-1
is a
der
ivat
ive
of th
e pl
asm
id d
escr
ibed
in th
is r
efer
ence
; the
pla
smid
was
mod
ified
at C
LON
TE
CH
.d
The
Eco
R I
site
is u
niqu
e in
pA
S2-
1, b
ut n
ot in
pA
S2.
epG
AD
424
is li
near
ized
by
dige
stio
n w
ith S
al I;
pG
AD
10 d
oes
not c
onta
in a
Sal
I si
te.
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-122 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890
APPENDIX. Additional MATCHMAKER Plasmid Information continued
TA
BLE
IV.
MA
TC
HM
AK
ER
GA
L4
TW
O-H
YB
RID
SY
ST
EM
CO
NT
RO
L P
LAS
MID
S
Sel
ectio
n on
Siz
eV
ecto
raS
yste
mD
escr
iptio
nS
D M
ediu
m(k
b)D
iagn
ostic
R.E
. Site
s (k
b)R
efer
ence
s
pCL1
GA
L4 2
H &
2H
-2w
ild-t
ype
full-
leng
th G
AL4
–Leu
~15
.3H
ind
IIIF
ield
s &
Son
g, 1
989
(#K
1605
-1 &
gene
in a
YC
p50
deriv
ativ
e,(~
11.2
, 2.8
, 1.8
)#K
1604
-1)
LEU
2 , A
mpr
pLA
M5'
GA
L4 2
HH
uman
lam
in C
(66–
230)
–Trp
6.0
Hin
d III
Bar
tel e
t al.,
199
3(#
K16
05-1
)in
pG
BT
9, T
RP
1, A
mpr
(4.6
, 0.8
, 0.6
)
pLA
M5'
-1G
AL4
2H
-2H
uman
lam
in C
(66–
230)
–Trp
~9.
0H
ind
IIIB
arte
l et a
l., 1
993
(#K
1604
-1)
in p
AS
2-1
TR
P1,
Am
pr(4
.6, 2
.2, 0
.9, 0
.85,
0.4
)
pTD
1G
AL4
2H
SV
40 la
rge
T-a
ntig
en (8
4–70
8)–L
eu~
15.0
Hin
d III
Li &
Fie
lds,
199
3;(#
K16
05-1
)in
pG
AD
3F, L
EU
2, A
mpr
(12,
1.3
, 1.2
, 0.5
)C
hien
et a
l., 1
991
pTD
1-1
GA
L4 2
H-2
SV
40 la
rge
T-a
ntig
en (8
4–70
8)–L
eu~
10.0
Hin
d III
Li &
Fie
lds,
199
3;(#
K16
04-1
)in
pA
CT
2, L
EU
2, A
mpr
(7.3
, 1.2
, 1.0
, 0.5
)
pVA
3G
AL4
2H
mur
ine
p53
(72–
390)
in p
GB
T9
–Trp
6.4
Hin
d III
Iwab
uchi
et a
l., 1
993
(#K
1605
-1)
TR
P1 ,
Am
pr(4
.6, 1
.8)
pVA
3-1
GA
L4 2
H-2
mur
ine
p53
(72–
390)
in p
AS
2-1
–Trp
9.4
Hin
d III
Iwab
uchi
et a
l., 1
993
(#K
1604
-1)
TR
P1 ,
Am
pr(4
.6, 2
.2, 1
.7, 0
.9)
Chi
en e
t al.,
199
1
apL
AM
5', p
LAM
5'-1
, pT
D1-
1, a
nd p
VA
3-1
are
deriv
ativ
es o
f the
pla
smid
s de
scrib
ed in
the
indi
cate
d re
fere
nces
; pla
smid
s w
ere
mod
ified
at C
LON
TE
CH
.
Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 23
MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.
Notice to PurchaserPractice of the two-hybrid system is covered by US Patents #5,283,173 and #5,468,614 assigned to the Research Foundationof the State University of New York. Purchase of any CLONTECH two-hybrid reagent does not imply or convey a license topractice the two-hybrid system covered by these patents. Commercial entities purchasing these reagents must obtain a licensefrom the Research Foundation of the State University of New York before using them. CLONTECH is required by its licensingagreement to submit a report of all purchasers of two-hybrid reagents to SUNY Stony Brook. Please contact Carol Dempster,Ph.D., at the Long Island Research Institute for license information (Tel: 516-361-6800; Fax: 516-361-6840).
All plasmids (except for pACT2 and pAS2-1) are licensed from The Research Foundation of the State University of New York.pACT2 and pAS2-1 are licensed from Baylor University. CLONTECH encourages researchers not to redistribute the plasmidsor yeast strains without prior written consent.
1997, CLONTECH Laboratories, Inc. All rights reserved.