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Plasmid Files

pGBKT7

Yeast two-hybrid "bait" vector for expressing proteins fused to the GAL4 DNA-binding domain.

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pGBKT7 Sequence and MappGBKT7.dna
Map and Sequence File   
Sequence Author:  Clontech
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 DraIII (7031) AarI (6462) BstXI (6327) EcoRV (6314) PmlI (5989) BspEI * (5894) XcmI (5437) BmgBI (5226) NsiI (4436) PluTI (3887) SfoI (3885) NarI (3884) KasI (3883) PflFI - Tth111I (3767) PvuI (0) BsaBI (268) PaeR7I - XhoI (979) HpaI (1039) T7 promoter ATG Myc PpuMI (1274) NdeI (1282) BtgI - NcoI (1288) SfiI (1293) EcoRI (1299) TspMI - XmaI (1304) SmaI (1306) BamHI (1309) SalI (1315) PstI (1325) NotI (1327) BssHII (1385) M13 rev lac operator lac promoter AvrII (3045) BstBI (3202) RsrII (3367) NmeAIII (3664) pGBKT7 7303 bp
DraIII  (7031)
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.
AarI  (6462)
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.
BstXI  (6327)
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.
EcoRV  (6314)
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.
PmlI  (5989)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BspEI  (5894)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
XcmI  (5437)
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.
BmgBI  (5226)
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.
NsiI  (4436)
1 site
A T G C A T T A C G T A
PluTI  (3887)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (3885)
1 site
G G C G C C C C G C G G
NarI  (3884)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (3883)
1 site
G G C G C C C C G C G G
PflFI  (3767)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (3767)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PvuI  (0)
1 site
C G A T C G G C T A G C
BsaBI  (268)
1 site
G A T N N N N A T C C T A N N N N T A G
PaeR7I  (979)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (979)
1 site
C T C G A G G A G C T C
HpaI  (1039)
1 site
G T T A A C C A A T T G
PpuMI  (1274)
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.
NdeI  (1282)
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.
BtgI  (1288)
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  (1288)
1 site
C C A T G G G G T A C C
SfiI  (1293)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
EcoRI  (1299)
1 site
G A A T T C C T T A A G
TspMI  (1304)
1 site
C C C G G G G G G C C C
XmaI  (1304)
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  (1306)
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  (1309)
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  (1315)
1 site
G T C G A C C A G C T G
PstI  (1325)
1 site
C T G C A G G A C G T C
NotI  (1327)
1 site
G C G G C C G C C G C C G G C G
BssHII  (1385)
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.
AvrII  (3045)
1 site
C C T A G G G G A T C C
BstBI  (3202)
1 site
T T C G A A A A G C T T
RsrII  (3367)
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.
NmeAIII  (3664)
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).
2μ ori
4147 .. 5489  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
4147 .. 5489  =  1343 bp
yeast 2μ plasmid origin of replication
NeoR/KanR
3221 .. 4015  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
3221 .. 4015  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ADH1 promoter
30 .. 734  =  705 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
30 .. 734  =  705 bp
promoter for alcohol dehydrogenase 1
TRP1
6030 .. 6704  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
6030 .. 6704  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2025 .. 2613  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2025 .. 2613  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6807 .. 7262  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6807 .. 7262  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
GAL4 DNA binding domain
762 .. 1202  =  441 bp
147 amino acids  =  16.9 kDa
Product: DNA binding domain of the GAL4
transcriptional activator
GAL4 DNA binding domain
762 .. 1202  =  441 bp
147 amino acids  =  16.9 kDa
Product: DNA binding domain of the GAL4
transcriptional activator
TRP1 promoter
5748 .. 6029  =  282 bp
TRP1 promoter
5748 .. 6029  =  282 bp
ADH1 terminator
1412 .. 1599  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
1412 .. 1599  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
4016 .. 4120  =  105 bp
AmpR promoter
4016 .. 4120  =  105 bp
MCS
1281 .. 1333  =  53 bp
multiple cloning site
MCS
1281 .. 1333  =  53 bp
multiple cloning site
T7 terminator
1338 .. 1385  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
1338 .. 1385  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
HSV TK poly(A) signal
2942 .. 2989  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
2942 .. 2989  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
lac promoter
1671 .. 1701  =  31 bp
   Segment 3:  -10  
   1671 .. 1677  =  7 bp
promoter for the E. coli lac operon
lac promoter
1671 .. 1701  =  31 bp
   Segment 2:  
   1678 .. 1695  =  18 bp
promoter for the E. coli lac operon
lac promoter
1671 .. 1701  =  31 bp
   Segment 1:  -35  
   1696 .. 1701  =  6 bp
promoter for the E. coli lac operon
lac promoter
1671 .. 1701  =  31 bp
3 segments
promoter for the E. coli lac operon
Myc
1251 .. 1280  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1251 .. 1280  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
T7 promoter
1213 .. 1231  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1213 .. 1231  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 rev
1623 .. 1639  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
1623 .. 1639  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
1647 .. 1663  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1647 .. 1663  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
ATG
1245 .. 1247  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for in vitro
transcription/translation
ATG
1245 .. 1247  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for in vitro
transcription/translation
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