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Plasmid Files

pNIC-CH

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

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pNIC-CH Sequence and MappNIC-CH.dna
Map and Sequence File   
Sequence Author:  Structural Genomics Consortium
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 SphI (44) BstAPI (7053) MluI (6729) BclI * (6715) BstEII (6547) NmeAIII (6529) ApaI (6526) PspOMI (6522) BssHII (6318) PshAI (5888) BglI (5672) FspI - FspAI (5651) PpuMI (5623) PflFI - Tth111I (4886) BspQI - SapI (4745) AlwNI (4219) NruI (3773) SmaI (3556) SgrAI (192) BglII (233) T7 promoter XbaI (299) AflII (321) RBS BfuAI (333) StuI * (1022) MscI (1044) AanI - AanI (1421) BsrGI (1461) SnaBI (1530) Acc65I (1838) KpnI (1842) SacII (1860) ZraI (2241) AatII (2243) PstI (2260) AarI - BfuAI (2263) BamHI (2285) EcoRI (2291) Eco53kI (2299) SacI (2301) SalI (2304) HindIII (2310) EagI - NotI (2317) PaeR7I - PspXI - XhoI (2325) BlpI (2404) DraIII (2732) AanI - AanI (2857) AsiSI - PvuI (3432) TspMI - XmaI (3554) pNIC-CH 7218 bp
SphI  (44)
1 site
G C A T G C C G T A C G
BstAPI  (7053)
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  (6729)
1 site
A C G C G T T G C G C A
BclI  (6715)
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  (6547)
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  (6529)
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  (6526)
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  (6522)
1 site
G G G C C C C C C G G G
BssHII  (6318)
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  (5888)
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  (5672)
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  (5651)
1 site
T G C G C A A C G C G T
FspAI  (5651)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5623)
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  (4886)
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  (4886)
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  (4745)
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  (4745)
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.
AlwNI  (4219)
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  (3773)
1 site
T C G C G A A G C G C T
SmaI  (3556)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SgrAI  (192)
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.
BglII  (233)
1 site
A G A T C T T C T A G A
XbaI  (299)
1 site
T C T A G A A G A T C T
AflII  (321)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BfuAI  (333)
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  (1022)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (1044)
1 site
T G G C C A A C C G G T
AanI  (1421)
2 sites
T T A T A A A A T A T T
AanI  (1421)
2 sites
T T A T A A A A T A T T
BsrGI  (1461)
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  (1530)
1 site
T A C G T A A T G C A T
Acc65I  (1838)
1 site
G G T A C C C C A T G G
KpnI  (1842)
1 site
G G T A C C C C A T G G
SacII  (1860)
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  (2241)
1 site
G A C G T C C T G C A G
AatII  (2243)
1 site
G A C G T C C T G C A G
PstI  (2260)
1 site
C T G C A G G A C G T C
AarI  (2263)
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.
BfuAI  (2263)
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  (2285)
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  (2291)
1 site
G A A T T C C T T A A G
Eco53kI  (2299)
1 site
G A G C T C C T C G A G
SacI  (2301)
1 site
G A G C T C C T C G A G
SalI  (2304)
1 site
G T C G A C C A G C T G
HindIII  (2310)
1 site
A A G C T T T T C G A A
EagI  (2317)
1 site
C G G C C G G C C G G C
NotI  (2317)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2325)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2325)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2325)
1 site
C T C G A G G A G C T C
BlpI  (2404)
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  (2732)
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  (2857)
2 sites
T T A T A A A A T A T T
AanI  (2857)
2 sites
T T A T A A A A T A T T
AsiSI  (3432)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3432)
1 site
C G A T C G G C T A G C
TspMI  (3554)
1 site
C C C G G G G G G C C C
XmaI  (3554)
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.
SacB
781 .. 2202  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 1:  signal peptide  
   781 .. 867  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
781 .. 2202  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 2:  
   868 .. 2202  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
781 .. 2202  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
lacI
6002 .. 7084  =  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
6002 .. 7084  =  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
3047 .. 3862  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
3047 .. 3862  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
3984 .. 4572  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3984 .. 4572  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2499 .. 2954  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2499 .. 2954  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
sacB promoter
348 .. 780  =  433 bp
sacB promoter and control region
sacB promoter
348 .. 780  =  433 bp
sacB promoter and control region
rop
5002 .. 5193  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
5002 .. 5193  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
7085 .. 7162  =  78 bp
lacI promoter
7085 .. 7162  =  78 bp
T7 terminator
2415 .. 2462  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
2415 .. 2462  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
272 .. 296  =  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
272 .. 296  =  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).
T7 promoter
253 .. 271  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
253 .. 271  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2264 .. 2281  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2264 .. 2281  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2331 .. 2348  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2331 .. 2348  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
327 .. 332  =  6 bp
ribosome binding site
RBS
327 .. 332  =  6 bp
ribosome binding site
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