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Plasmid Files

pJG4-5

Yeast two-hybrid vector with a GAL1 promoter, for fusing the B42 transcriptional activator to a partner protein. Also known as pB42AD.

 
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 SacI (6444) Eco53kI (6442) PvuII (6255) BspQI - SapI (6192) AflIII - PciI (6075) DrdI (5973) AlwNI (5666) AhdI (5187) BsaI (5121) ScaI (4706) TatI (4704) AatII (4268) ZraI (4266) PmlI (4130) Bsu36I (3845) BsgI (3679) AarI (3653) BstBI (3347) Acc65I (6446) KpnI (1) AgeI (150) BtgZI (376) ATG EcoRI (849) MCS PaeR7I - XhoI (861) PsiI (1046) SphI (1195) BamHI (1330) SalI (1342) NotI (1351) BtgI (1630) MscI * (1635) BclI * (1673) SpeI (1688) NdeI (1709) XcmI (1765) SnaBI (2413) PfoI * (3152) pJG4-5 6449 bp
SacI  (6444)
1 site
G A G C T C C T C G A G
Eco53kI  (6442)
1 site
G A G C T C C T C G A G
PvuII  (6255)
1 site
C A G C T G G T C G A C
BspQI  (6192)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (6192)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
AflIII  (6075)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (6075)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (5973)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AlwNI  (5666)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
AhdI  (5187)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BsaI  (5121)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
ScaI  (4706)
1 site
A G T A C T T C A T G A
TatI  (4704)
1 site
W G T A C W W C A T G W
AatII  (4268)
1 site
G A C G T C C T G C A G
ZraI  (4266)
1 site
G A C G T C C T G C A G
PmlI  (4130)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Bsu36I  (3845)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BsgI  (3679)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AarI  (3653)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BstBI  (3347)
1 site
T T C G A A A A G C T T
Acc65I  (6446)
1 site
G G T A C C C C A T G G
KpnI  (1)
1 site
G G T A C C C C A T G G
AgeI  (150)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BtgZI  (376)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
EcoRI  (849)
1 site
G A A T T C C T T A A G
PaeR7I  (861)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (861)
1 site
C T C G A G G A G C T C
PsiI  (1046)
1 site
T T A T A A A A T A T T
SphI  (1195)
1 site
G C A T G C C G T A C G
BamHI  (1330)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
SalI  (1342)
1 site
G T C G A C C A G C T G
NotI  (1351)
1 site
G C G G C C G C C G C C G G C G
BtgI  (1630)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
MscI  (1635)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
BclI  (1673)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
SpeI  (1688)
1 site
A C T A G T T G A T C A
NdeI  (1709)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
XcmI  (1765)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SnaBI  (2413)
1 site
T A C G T A A T G C A T
PfoI  (3152)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
2μ ori
1713 .. 3055  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
1713 .. 3055  =  1343 bp
yeast 2μ plasmid origin of replication
AmpR
4403 .. 5263  =  861 bp
287 codons
   Segment 1:  signal sequence  
   4403 .. 4471  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4403 .. 5263  =  861 bp
287 codons
   Segment 2:  
   4472 .. 5263  =  792 bp
   264 codons
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4403 .. 5263  =  861 bp
287 codons
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TRP1
3416 .. 4090  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
3416 .. 4090  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
ori
5431 .. 6019  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5431 .. 6019  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
GAL1 promoter
81 .. 522  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
81 .. 522  =  442 bp
inducible promoter, regulated by Gal4
B42 transcriptional activator
573 .. 809  =  237 bp
79 amino acids  =  8.8 kDa
Product: yeast transcriptional activator cretaed from
E. coli genomic DNA fragments (Ma and Ptashne,
1987)
B42 transcriptional activator
573 .. 809  =  237 bp
79 amino acids  =  8.8 kDa
Product: yeast transcriptional activator cretaed from
E. coli genomic DNA fragments (Ma and Ptashne,
1987)
ADH1 terminator
1004 .. 1191  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
1004 .. 1191  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
4295 .. 4399  =  105 bp
AmpR promoter
4295 .. 4399  =  105 bp
TRP1 promoter
4091 .. 4192  =  102 bp
TRP1 promoter
4091 .. 4192  =  102 bp
HA
816 .. 842  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
816 .. 842  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
546 .. 566  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
546 .. 566  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
MCS
849 .. 866  =  18 bp
multiple cloning site
MCS
849 .. 866  =  18 bp
multiple cloning site
ATG
534 .. 536  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
534 .. 536  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
UAS
81 .. 198  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
81 .. 198  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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