pNIC28-Bsa4

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

Sequence Author: Structural Genomics Consortium

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SgrAI (7224) SphI (7076) BstAPI (6867) MluI (6543) BclI * (6529) BstEII (6361) NmeAIII (6343) ApaI (6340) PspOMI (6336) BssHII (6132) BglI (5486) FspI - FspAI (5465) PpuMI (5437) PflFI - Tth111I (4700) BspQI - SapI (4559) BssSI (4269) AlwNI (4033) T7 promoter XbaI (47) RBS NdeI (87) ATG 6xHis TEV site BsaI (152) NcoI (153) ZraI (166) AatII (168) BfuAI - BspMI (186) StuI * (857) MscI (879) AanI (1256) BsrGI (1296) SnaBI (1365) SacII (1695) BsaI (2083) BamHI (2099) EcoRI (2105) Eco53kI (2113) SacI (2115) SalI (2118) HindIII (2124) EagI - NotI (2131) PaeR7I - PspXI - XhoI (2139) BlpI (2218) DraIII (2546) AanI (2671) AsiSI - PvuI (3246) TspMI - XmaI (3368) SmaI (3370) NruI (3587) pNIC28-Bsa4 7284 bp
SgrAI  (7224)
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  (7076)
1 site
G C A T G C C G T A C G
BstAPI  (6867)
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  (6543)
1 site
A C G C G T T G C G C A
BclI  (6529)
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  (6361)
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  (6343)
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  (6340)
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  (6336)
1 site
G G G C C C C C C G G G
BssHII  (6132)
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  (5486)
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  (5465)
1 site
T G C G C A A C G C G T
FspAI  (5465)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5437)
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  (4700)
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  (4700)
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  (4559)
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  (4559)
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  (4269)
1 site
C A C G A G G T G C T C
AlwNI  (4033)
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.
XbaI  (47)
1 site
T C T A G A A G A T C T
NdeI  (87)
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.
BsaI  (152)
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  (153)
1 site
C C A T G G G G T A C C
ZraI  (166)
1 site
G A C G T C C T G C A G
AatII  (168)
1 site
G A C G T C C T G C A G
BfuAI  (186)
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  (186)
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  (857)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (879)
1 site
T G G C C A A C C G G T
AanI  (1256)
2 sites
T T A T A A A A T A T T
BsrGI  (1296)
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  (1365)
1 site
T A C G T A A T G C A T
SacII  (1695)
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  (2083)
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  (2099)
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  (2105)
1 site
G A A T T C C T T A A G
Eco53kI  (2113)
1 site
G A G C T C C T C G A G
SacI  (2115)
1 site
G A G C T C C T C G A G
SalI  (2118)
1 site
G T C G A C C A G C T G
HindIII  (2124)
1 site
A A G C T T T T C G A A
EagI  (2131)
1 site
C G G C C G G C C G G C
NotI  (2131)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2139)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2139)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2139)
1 site
C T C G A G G A G C T C
BlpI  (2218)
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  (2546)
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  (2671)
2 sites
T T A T A A A A T A T T
AsiSI  (3246)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3246)
1 site
C G A T C G G C T A G C
TspMI  (3368)
1 site
C C C G G G G G G C C C
XmaI  (3368)
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  (3370)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
NruI  (3587)
1 site
T C G C G A A G C G C T
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   616 .. 702  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   703 .. 2037  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
616 .. 2037  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
5816 .. 6898  =  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
5816 .. 6898  =  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
2861 .. 3676  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes