pNIC-CTHF

Bacterial vector encoding a C-terminal TEV-6xHis-FLAG® cassette plus a SacB negative selection marker, for purification of recombinant proteins.

Sequence Author: Structural Genomics Consortium

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BglII (7241) SgrAI (7200) SphI (7052) BstAPI (6843) MluI (6519) BclI * (6505) BstEII (6337) NmeAIII (6319) ApaI (6316) PspOMI (6312) BssHII (6108) PshAI (5678) BglI (5462) FspI - FspAI (5441) PpuMI (5413) PflFI - Tth111I (4676) BspQI - SapI (4535) BssSI (4245) AlwNI (4009) NruI (3563) T7 promoter XbaI (47) AflII (69) RBS BfuAI (81) sacB promoter StuI * (770) MscI (792) AanI (1169) BsrGI (1209) SnaBI (1278) Acc65I (1586) KpnI (1590) SacII (1608) ZraI (1989) AatII (1991) PstI (2008) AarI - BfuAI (2011) TEV site 6xHis BamHI (2075) EcoRI (2081) Eco53kI (2089) SacI (2091) SalI (2094) HindIII (2100) EagI - NotI (2107) PaeR7I - PspXI - XhoI (2115) BlpI (2194) DraIII (2522) AanI (2647) AsiSI - PvuI (3222) TspMI - XmaI (3344) SmaI (3346) pNIC-CTHF 7260 bp
BglII  (7241)
1 site
A G A T C T T C T A G A
SgrAI  (7200)
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  (7052)
1 site
G C A T G C C G T A C G
BstAPI  (6843)
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  (6519)
1 site
A C G C G T T G C G C A
BclI  (6505)
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  (6337)
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  (6319)
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  (6316)
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  (6312)
1 site
G G G C C C C C C G G G
BssHII  (6108)
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.
PshAI  (5678)
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  (5462)
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  (5441)
1 site
T G C G C A A C G C G T
FspAI  (5441)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5413)
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  (4676)
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  (4676)
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  (4535)
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  (4535)
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  (4245)
1 site
C A C G A G G T G C T C
AlwNI  (4009)
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  (3563)
1 site
T C G C G A A G C G C T
XbaI  (47)
1 site
T C T A G A A G A T C T
AflII  (69)
1 site
C T T A A G G A A T T C
BfuAI  (81)
2 sites
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.
StuI  (770)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (792)
1 site
T G G C C A A C C G G T
AanI  (1169)
2 sites
T T A T A A A A T A T T
BsrGI  (1209)
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  (1278)
1 site
T A C G T A A T G C A T
Acc65I  (1586)
1 site
G G T A C C C C A T G G
KpnI  (1590)
1 site
G G T A C C C C A T G G
SacII  (1608)
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.
ZraI  (1989)
1 site
G A C G T C C T G C A G
AatII  (1991)
1 site
G A C G T C C T G C A G
PstI  (2008)
1 site
C T G C A G G A C G T C
AarI  (2011)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Cleavage may be enhanced when more than one copy of the AarI recognition sequence is present.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
BfuAI  (2011)
2 sites
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.
BamHI  (2075)
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  (2081)
1 site
G A A T T C C T T A A G
Eco53kI  (2089)
1 site
G A G C T C C T C G A G
SacI  (2091)
1 site
G A G C T C C T C G A G
SalI  (2094)
1 site
G T C G A C C A G C T G
HindIII  (2100)
1 site
A A G C T T T T C G A A
EagI  (2107)
1 site
C G G C C G G C C G G C
NotI  (2107)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2115)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2115)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2115)
1 site
C T C G A G G A G C T C
BlpI  (2194)
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  (2522)
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  (2647)
2 sites
T T A T A A A A T A T T
AsiSI  (3222)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3222)
1 site
C G A T C G G C T A G C
TspMI  (3344)
1 site
C C C G G G G G G C C C
XmaI  (3344)
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  (3346)
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
529 .. 1950  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   529 .. 615  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
529 .. 1950  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   616 .. 1950  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
529 .. 1950  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
5792 .. 6874  =  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
5792 .. 6874  =  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
2837 .. 3652  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
2837 .. 3652  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
3774 .. 4362  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3774 .. 4362  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replica