pGilda

Centromeric yeast two-hybrid vector for galactose-regulated expression of LexA fused to the N-terminus of a protein.

Sequence Author: Clontech (TaKaRa)

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PspOMI (6552) PspFI (5849) BseYI (5845) AlwNI (5740) AhdI (5261) BsaI (5195) ScaI (4780) BsaHI (4721) PpuMI (4338) PmlI (4327) SwaI (4303) PfoI (3708) AfeI (3548) ApaI (6556) AgeI (293) BstAPI (545) HpaI (702) MluI (770) EcoRV (1057) SnaBI (1109) PmeI (1213) XcmI (1258) EcoRI (1298) TspMI - XmaI (1303) SmaI (1305) BamHI (1308) BtgI - NcoI - StyI (1319) EagI - NotI (1325) PaeR7I - PspXI - XhoI (1331) BfuAI - BspMI (1349) SphI (1554) Eco53kI (1684) SacI (1686) M13 fwd NgoMIV (2007) NaeI (2009) DraIII (2115) BssHII (2444) NsiI (2481) AleI (2697) NheI (2748) BmtI (2752) BsiWI (2840) BstXI (2871) MscI (3049) BsaBI * (3199) pGilda 6570 bp
PspOMI  (6552)
1 site
G G G C C C C C C G G G
PspFI  (5849)
1 site
C C C A G C G G G T C G
BseYI  (5845)
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.
AlwNI  (5740)
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  (5261)
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  (5195)
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  (4780)
1 site
A G T A C T T C A T G A
BsaHI  (4721)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
PpuMI  (4338)
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.
PmlI  (4327)
1 site
C A C G T G G T G C A C
SwaI  (4303)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
PfoI  (3708)
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.
AfeI  (3548)
1 site
A G C G C T T C G C G A
ApaI  (6556)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AgeI  (293)
1 site
A C C G G T T G G C C A
BstAPI  (545)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
HpaI  (702)
1 site
G T T A A C C A A T T G
MluI  (770)
1 site
A C G C G T T G C G C A
EcoRV  (1057)
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.
SnaBI  (1109)
1 site
T A C G T A A T G C A T
PmeI  (1213)
1 site
G T T T A A A C C A A A T T T G
XcmI  (1258)
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.
EcoRI  (1298)
1 site
G A A T T C C T T A A G
TspMI  (1303)
1 site
C C C G G G G G G C C C
XmaI  (1303)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (1305)
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  (1308)
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.
BtgI  (1319)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (1319)
1 site
C C A T G G G G T A C C
StyI  (1319)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
EagI  (1325)
1 site
C G G C C G G C C G G C
NotI  (1325)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1331)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1331)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1331)
1 site
C T C G A G G A G C T C
BfuAI  (1349)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1349)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
SphI  (1554)
1 site
G C A T G C C G T A C G
Eco53kI  (1684)
1 site
G A G C T C C T C G A G
SacI  (1686)
1 site
G A G C T C C T C G A G
NgoMIV  (2007)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (2009)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
DraIII  (2115)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BssHII  (2444)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
NsiI  (2481)
1 site
A T G C A T T A C G T A
AleI  (2697)
1 site
C A C N N N N G T G G T G N N N N C A C
NheI  (2748)
1 site
G C T A G C C G A T C G
BmtI  (2752)
1 site
G C T A G C C G A T C G
BsiWI  (2840)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BstXI  (2871)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
MscI  (3049)
1 site
T G G C C A A C C G G T
BsaBI  (3199)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4474 .. 4542  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4543 .. 5334  =  792 bp
   263 amino acids  =  29.0 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4474 .. 5334  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GAL1,10 promoter
8 .. 672  =  665 bp
divergent inducible promoter, regulated by Gal4
GAL1,10 promoter
8 .. 672  =  665 bp
divergent inducible promoter, regulated by Gal4
HIS3
2598 .. 3257  =  660 bp
219 amino acids  =  23.8 kDa
Product: imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis
yeast auxotrophic marker
HIS3
2598 .. 3257  =  660 bp
219 amino acids  =  23.8 kDa
Product: imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis
yeast auxotrophic marker
LexA
692 .. 1297  =  606 bp
202 amino acids  =  22.4 kDa
2 segments
   Segment 1:  DNA-binding domain  
   692 .. 943  =  252 bp
   84 amino acids  =  9.2 kDa
Product: E. coli DNA-binding protein that represses genes involved in the SOS response to DNA damage
The N-terminal DNA-binding domain recognizes the 16-bp palindromic sequence 5'-CTGTATATATATACAG-3'.
LexA
692 .. 1297  =  606 bp
202 amino acids  =  22.4 kDa
2 segments
   Segment 2:  
   944 .. 1297  =  354 bp
   118 amino acids  =  13.2 kDa
Product: E. coli DNA-binding protein that represses genes involved in the SOS response to DNA damage
The N-terminal DNA-binding domain recognizes the 16-bp palindromic sequence 5'-CTGTATATATATACAG-3'.
LexA
692 .. 1297  =  606 bp
202 amino acids  =  22.4 kDa
2 segments
Product: E. coli DNA-binding protein that represses genes involved in the SOS response to DNA damage
The N-terminal DNA-binding domain recognizes the 16-bp palindromic sequence 5'-CTGTATATATATACAG-3'.
ori
5505 .. 6093  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5505 .. 6093  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CEN/ARS
3829 .. 4332  =  504 bp
S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence
CEN/ARS
3829 .. 4332  =  504 bp
S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence
f1 ori
1882 .. 2337  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1882 .. 2337  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
ADH1 terminator
1363 .. 1550  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
1363 .. 1550  =  188 bp
transcription terminator for alcohol dehydrogenase 1
HIS3 promoter
3258 .. 3445  =  188 bp
HIS3 promoter
3258 .. 3445  =  188 bp
AmpR promoter
4369 .. 4473  =  105 bp
AmpR promoter
4369 .. 4473  =  105 bp
MCS
1298 .. 1336  =  39 bp
multiple cloning site
MCS
1298 .. 1336  =  39 bp
multiple cloning site
M13 fwd
1721 .. 1737  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
1721 .. 1737  =  17 bp
common sequencing primer, one of multiple similar variants
UAS
224 .. 341  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
UAS
224 .. 341  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
ORF:  4474 .. 5334  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  692 .. 1384  =  693 bp
ORF:  230 amino acids  =  25.6 kDa
ORF:  2598 .. 3257  =  660 bp
ORF:  219 amino acids  =  23.8 kDa
ORF:  4938 .. 5204  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2378 .. 2641  =  264 bp
ORF:  87 amino acids  =  10.4 kDa
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