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Plasmid Files

pDB-HisGST

Bacterial expression vector with a kanamycin resistance marker, for appending an N-terminal 6xHis-GST tag followed by a TEV site.

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pDB-HisGST Sequence and MappDB-HisGST.dna
Map and Sequence File   
Sequence Author:  Berkeley Structural Genomics Center
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 6xHis PaeR7I - PspXI - XhoI (5891) EagI - NotI (5883) HindIII (5876) SalI (5870) SacI (5867) Eco53kI (5865) EcoRI (5857) BamHI (5851) T7 tag (gene 10 leader) BmtI (5822) NheI (5818) NdeI (5813) ScaI (5677) SwaI (5531) BstBI (5501) MscI (5311) 6xHis ATG NcoI (5069) RBS XbaI (5030) T7 promoter BglII (4964) SgrAI (4923) SphI (4775) BstAPI (4566) MluI (4242) BstEII (4060) NmeAIII (4042) ApaI (4039) PspOMI (4035) BssHII (3831) HpaI (3740) PshAI (3401) BglI (3185) FspI - FspAI (3164) BlpI (5970) DraIII (245) PsiI (370) AsiSI - PvuI (945) TspMI - XmaI (1067) SmaI (1069) BspDI - ClaI (1250) NruI (1286) AcuI (1599) AlwNI (1732) BssS α I (1968) BstZ17I (2374) PflFI - Tth111I (2399) PpuMI (3136) pDB-HisGST 6053 bp
PaeR7I  (5891)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5891)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5891)
1 site
C T C G A G G A G C T C
EagI  (5883)
1 site
C G G C C G G C C G G C
NotI  (5883)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5876)
1 site
A A G C T T T T C G A A
SalI  (5870)
1 site
G T C G A C C A G C T G
SacI  (5867)
1 site
G A G C T C C T C G A G
Eco53kI  (5865)
1 site
G A G C T C C T C G A G
EcoRI  (5857)
1 site
G A A T T C C T T A A G
BamHI  (5851)
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.
BmtI  (5822)
1 site
G C T A G C C G A T C G
NheI  (5818)
1 site
G C T A G C C G A T C G
NdeI  (5813)
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.
ScaI  (5677)
1 site
A G T A C T T C A T G A
SwaI  (5531)
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.
BstBI  (5501)
1 site
T T C G A A A A G C T T
MscI  (5311)
1 site
T G G C C A A C C G G T
NcoI  (5069)
1 site
C C A T G G G G T A C C
XbaI  (5030)
1 site
T C T A G A A G A T C T
BglII  (4964)
1 site
A G A T C T T C T A G A
SgrAI  (4923)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
SphI  (4775)
1 site
G C A T G C C G T A C G
BstAPI  (4566)
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.
MluI  (4242)
1 site
A C G C G T T G C G C A
BstEII  (4060)
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.
NmeAIII  (4042)
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).
ApaI  (4039)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4035)
1 site
G G G C C C C C C G G G
BssHII  (3831)
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.
HpaI  (3740)
1 site
G T T A A C C A A T T G
PshAI  (3401)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BglI  (3185)
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.
FspI  (3164)
1 site
T G C G C A A C G C G T
FspAI  (3164)
1 site
R T G C G C A Y Y A C G C G T R
BlpI  (5970)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
DraIII  (245)
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.
PsiI  (370)
1 site
T T A T A A A A T A T T
AsiSI  (945)
1 site
G C G A T C G C C G C T A G C G
PvuI  (945)
1 site
C G A T C G G C T A G C
TspMI  (1067)
1 site
C C C G G G G G G C C C
XmaI  (1067)
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  (1069)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BspDI  (1250)
1 site
A T C G A T T A G C T A
ClaI  (1250)
1 site
A T C G A T T A G C T A
NruI  (1286)
1 site
T C G C G A A G C G C T
AcuI  (1599)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AlwNI  (1732)
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.
BssSαI  (1968)
1 site
C A C G A G G T G C T C
BstZ17I  (2374)
1 site
G T A T A C C A T A T G
PflFI  (2399)
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  (2399)
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.
PpuMI  (3136)
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.
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
GST
5107 .. 5757  =  651 bp
217 amino acids  =  25.4 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
5107 .. 5757  =  651 bp
217 amino acids  =  25.4 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4598 .. 4675  =  78 bp
lacI promoter
4598 .. 4675  =  78 bp
T7 terminator
5981 .. 6028  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5981 .. 6028  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 tag (gene 10 leader)
5815 .. 5847  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
T7 tag (gene 10 leader)
5815 .. 5847  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
lac operator
5003 .. 5027  =  25 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
5003 .. 5027  =  25 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).
TEV Site
5791 .. 5811  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV Site
5791 .. 5811  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5083 .. 5100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5083 .. 5100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5897 .. 5914  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5897 .. 5914  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
5058 .. 5063  =  6 bp
ribosome binding site
RBS
5058 .. 5063  =  6 bp
ribosome binding site
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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