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Plasmid Files

pNIC-CTHF

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

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pNIC-CTHF Sequence and MappNIC-CTHF.dna
Map and Sequence File   
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) AlwNI (4009) NruI (3563) T7 promoter XbaI (47) AflII (69) RBS BfuAI (81) StuI * (770) MscI (792) AanI - 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) 6xHis BlpI (2194) DraIII (2522) AanI - 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.
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

The sticky ends produced by AflII are hard to ligate.
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
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

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  (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
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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
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
   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
   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 replication
f1 ori
2289 .. 2744  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2289 .. 2744  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
sacB promoter
96 .. 528  =  433 bp
sacB promoter and control region
sacB promoter
96 .. 528  =  433 bp
sacB promoter and control region
rop
4792 .. 4983  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
4792 .. 4983  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
6875 .. 6952  =  78 bp
lacI promoter
6875 .. 6952  =  78 bp
T7 terminator
2205 .. 2252  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
2205 .. 2252  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
20 .. 44  =  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
20 .. 44  =  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).
FLAG
2048 .. 2071  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
2048 .. 2071  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
TEV site
2009 .. 2029  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
2009 .. 2029  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2030 .. 2047  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2030 .. 2047  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2121 .. 2138  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2121 .. 2138  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
75 .. 80  =  6 bp
ribosome binding site
RBS
75 .. 80  =  6 bp
ribosome binding site
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