Resources
Plasmid Files

pGAD-C1

Yeast two-hybrid "prey" vector for fusing a gene to the GAL4 activation domain. For other reading frames, use pGAD‑C2 or pGAD‑C3.

 
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 NsiI (6381) BsaAI - SnaBI (6025) BmgBI (5583) PfoI (5362) AatII (5251) ZraI (5249) ScaI (4809) PvuI (4699) NmeAIII (4477) BglI (4449) BsaI (4390) BmrI (4369) AhdI (4329) AlwNI (3852) PspFI (3744) BseYI (3740) BsgI (352) Acc65I (479) KpnI (483) RsrII (557) SexAI * (697) MluI (726) EcoRI (833) TspMI - XmaI (839) SmaI (841) BamHI (845) BspDI - ClaI (852) SalI (857) PstI (867) BglII (869) MscI (1005) BsrGI (1523) BtgI (1783) EcoRV (1898) AflII (2329) BstEII (2609) PflMI (2738) PpuMI (2754) PvuII (3260) BspQI - SapI (3320) pGAD-C1 6667 bp
NsiI  (6381)
1 site
A T G C A T T A C G T A
BsaAI  (6025)
1 site
Y A C G T R R T G C A Y
SnaBI  (6025)
1 site
T A C G T A A T G C A T
BmgBI  (5583)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
PfoI  (5362)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AatII  (5251)
1 site
G A C G T C C T G C A G
ZraI  (5249)
1 site
G A C G T C C T G C A G
ScaI  (4809)
1 site
A G T A C T T C A T G A
PvuI  (4699)
1 site
C G A T C G G C T A G C
NmeAIII  (4477)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BglI  (4449)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
BsaI  (4390)
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.
BmrI  (4369)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the
absence of magnesium.
AhdI  (4329)
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.
AlwNI  (3852)
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.
PspFI  (3744)
1 site
C C C A G C G G G T C G
BseYI  (3740)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
BsgI  (352)
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).
Acc65I  (479)
1 site
G G T A C C C C A T G G
KpnI  (483)
1 site
G G T A C C C C A T G G
RsrII  (557)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
SexAI  (697)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
MluI  (726)
1 site
A C G C G T T G C G C A
EcoRI  (833)
1 site
G A A T T C C T T A A G
TspMI  (839)
1 site
C C C G G G G G G C C C
XmaI  (839)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (841)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (845)
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.
BspDI  (852)
1 site
A T C G A T T A G C T A
ClaI  (852)
1 site
A T C G A T T A G C T A
SalI  (857)
1 site
G T C G A C C A G C T G
PstI  (867)
1 site
C T G C A G G A C G T C
BglII  (869)
1 site
A G A T C T T C T A G A
MscI  (1005)
1 site
T G G C C A A C C G G T
BsrGI  (1523)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BtgI  (1783)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
EcoRV  (1898)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
AflII  (2329)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BstEII  (2609)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
PflMI  (2738)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
PpuMI  (2754)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
PvuII  (3260)
1 site
C A G C T G G T C G A C
BspQI  (3320)
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  (3320)
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.
2μ ori
5503 .. 6667  =  1165 bp
yeast 2μ plasmid origin of replication
2μ ori
5503 .. 6667  =  1165 bp
yeast 2μ plasmid origin of replication
LEU2
1554 .. 2648  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
LEU2
1554 .. 2648  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required
for leucine biosynthesis
yeast auxotrophic marker
AmpR
4256 .. 5116  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4256 .. 5047  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4256 .. 5116  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5048 .. 5116  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4256 .. 5116  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3497 .. 4085  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
3497 .. 4085  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
GAL4 activation domain
422 .. 829  =  408 bp
136 amino acids  =  14.9 kDa
Product: activation domain of the GAL4
transcriptional activator
GAL4 activation domain
422 .. 829  =  408 bp
136 amino acids  =  14.9 kDa
Product: activation domain of the GAL4
transcriptional activator
LEU2 promoter
2649 .. 3053  =  405 bp
LEU2 promoter
2649 .. 3053  =  405 bp
ADH1 promoter
5 .. 406  =  402 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
5 .. 406  =  402 bp
promoter for alcohol dehydrogenase 1
ADH1 terminator
1249 .. 1436  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
1249 .. 1436  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
5117 .. 5221  =  105 bp
AmpR promoter
5117 .. 5221  =  105 bp
MCS
833 .. 874  =  42 bp
multiple cloning site
MCS
833 .. 874  =  42 bp
multiple cloning site
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