pNH-TrxT

Bacterial vector encoding an N-terminal 6xHis-thioredoxin-TEV cassette plus a SacB negative selection marker, for purification of recombinant proteins.

Sequence Author: Structural Genomics Consortium

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T7 promoter BglII (7536) SgrAI (7495) SphI (7347) BstAPI (7138) MluI (6814) BclI * (6800) BstEII (6632) NmeAIII (6614) ApaI (6611) PspOMI (6607) BssHII (6403) BglI (5757) FspI - FspAI (5736) PpuMI (5708) PflFI - Tth111I (4971) BspQI - SapI (4830) BssSI (4540) AlwNI (4304) NruI (3858) XbaI (0) RBS NdeI (40) ATG 6xHis RsrII (171) BsaI (423) NcoI (424) ZraI (437) AatII (439) BfuAI - BspMI (457) sacB promoter StuI * (1128) MscI (1150) AanI (1527) BsrGI (1567) SnaBI (1636) SacII (1966) BsaI (2354) BamHI (2370) EcoRI (2376) Eco53kI (2384) SacI (2386) SalI (2389) HindIII (2395) EagI - NotI (2402) PaeR7I - PspXI - XhoI (2410) BlpI (2489) DraIII (2817) AanI (2942) AsiSI - PvuI (3517) TspMI - XmaI (3639) SmaI (3641) pNH-TrxT 7602 bp
BglII  (7536)
1 site
A G A T C T T C T A G A
SgrAI  (7495)
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  (7347)
1 site
G C A T G C C G T A C G
BstAPI  (7138)
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  (6814)
1 site
A C G C G T T G C G C A
BclI  (6800)
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.
BstEII  (6632)
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  (6614)
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  (6611)
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  (6607)
1 site
G G G C C C C C C G G G
BssHII  (6403)
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.
BglI  (5757)
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  (5736)
1 site
T G C G C A A C G C G T
FspAI  (5736)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5708)
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.
PflFI  (4971)
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  (4971)
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.
BspQI  (4830)
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  (4830)
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.
BssSI  (4540)
1 site
C A C G A G G T G C T C
AlwNI  (4304)
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.
NruI  (3858)
1 site
T C G C G A A G C G C T
XbaI  (0)
1 site
T C T A G A A G A T C T
NdeI  (40)
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.
RsrII  (171)
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.
BsaI  (423)
2 sites
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.
NcoI  (424)
1 site
C C A T G G G G T A C C
ZraI  (437)
1 site
G A C G T C C T G C A G
AatII  (439)
1 site
G A C G T C C T G C A G
BfuAI  (457)
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  (457)
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.
StuI  (1128)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (1150)
1 site
T G G C C A A C C G G T
AanI  (1527)
2 sites
T T A T A A A A T A T T
BsrGI  (1567)
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.
SnaBI  (1636)
1 site
T A C G T A A T G C A T
SacII  (1966)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
BsaI  (2354)
2 sites
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.
BamHI  (2370)
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.
EcoRI  (2376)
1 site
G A A T T C C T T A A G
Eco53kI  (2384)
1 site
G A G C T C C T C G A G
SacI  (2386)
1 site
G A G C T C C T C G A G
SalI  (2389)
1 site
G T C G A C C A G C T G
HindIII  (2395)
1 site
A A G C T T T T C G A A
EagI  (2402)
1 site
C G G C C G G C C G G C
NotI  (2402)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2410)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2410)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2410)
1 site
C T C G A G G A G C T C
BlpI  (2489)
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  (2817)
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.
AanI  (2942)
2 sites
T T A T A A A A T A T T
AsiSI  (3517)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3517)
1 site
C G A T C G G C T A G C
TspMI  (3639)
1 site
C C C G G G G G G C C C
XmaI  (3639)
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  (3641)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SacB
887 .. 2308  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   887 .. 973  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
887 .. 2308  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   974 .. 2308  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
887 .. 2308  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
6087 .. 7169  =  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
6087 .. 7169  =  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
3132 .. 3947  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
3132 .. 3947  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
4069 .. 4657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4069 .. 4657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2584 .. 3039  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2584 .. 3039  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
441 .. 886  =  446 bp
sacB promoter and control region
sacB promoter
441 .. 886  =  446 bp
sacB promoter and control region
ATG
42 .. 44  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
42 .. 44  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
45 .. 62  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
45 .. 62  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TrxA
72 .. 398  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
TrxA
72 .. 398  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
TEV site
405 .. 425  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
405 .. 425  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
rop
5087 .. 5278  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
5087 .. 5278  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
7170 .. 7247  =  78 bp
lacI promoter
7170 .. 7247  =  78 bp
T7 terminator
2500 .. 2547  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2500 .. 2547  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
7575 .. 7599  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inh