pNIC-Zb

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

Sequence Author: Structural Genomics Consortium

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BglII (7427) SgrAI (7386) SphI (7238) BstAPI (7029) MluI (6705) BclI * (6691) BstEII (6523) NmeAIII (6505) ApaI (6502) PspOMI (6498) BssHII (6294) BglI (5648) FspAI (5627) PpuMI (5599) PflFI - Tth111I (4862) BssSI (4431) AlwNI (4195) NruI (3749) T7 promoter XbaI (47) RBS NdeI (87) ATG 6xHis TEV site BsaI (314) NcoI (315) ZraI (328) AatII (330) sacB promoter StuI * (1019) MscI (1041) AanI (1418) BsrGI (1458) SnaBI (1527) SacII (1857) BsaI (2245) BamHI (2261) EcoRI (2267) SalI (2280) HindIII (2286) EagI - NotI (2293) PaeR7I - PspXI - XhoI (2301) BlpI (2380) DraIII (2708) AanI (2833) AsiSI - PvuI (3408) TspMI - XmaI (3530) SmaI (3532) pNIC-Zb 7446 bp
BglII  (7427)
1 site
A G A T C T T C T A G A
SgrAI  (7386)
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  (7238)
1 site
G C A T G C C G T A C G
BstAPI  (7029)
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  (6705)
1 site
A C G C G T T G C G C A
BclI  (6691)
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  (6523)
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  (6505)
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  (6502)
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  (6498)
1 site
G G G C C C C C C G G G
BssHII  (6294)
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  (5648)
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.
FspAI  (5627)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5599)
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  (4862)
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  (4862)
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.
BssSI  (4431)
1 site
C A C G A G G T G C T C
AlwNI  (4195)
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  (3749)
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
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  (314)
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  (315)
1 site
C C A T G G G G T A C C
ZraI  (328)
1 site
G A C G T C C T G C A G
AatII  (330)
1 site
G A C G T C C T G C A G
StuI  (1019)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (1041)
1 site
T G G C C A A C C G G T
AanI  (1418)
2 sites
T T A T A A A A T A T T
BsrGI  (1458)
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  (1527)
1 site
T A C G T A A T G C A T
SacII  (1857)
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  (2245)
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  (2261)
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  (2267)
1 site
G A A T T C C T T A A G
SalI  (2280)
1 site
G T C G A C C A G C T G
HindIII  (2286)
1 site
A A G C T T T T C G A A
EagI  (2293)
1 site
C G G C C G G C C G G C
NotI  (2293)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2301)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2301)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2301)
1 site
C T C G A G G A G C T C
BlpI  (2380)
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  (2708)
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  (2833)
2 sites
T T A T A A A A T A T T
AsiSI  (3408)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3408)
1 site
C G A T C G G C T A G C
TspMI  (3530)
1 site
C C C G G G G G G C C C
XmaI  (3530)
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  (3532)
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
778 .. 2199  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   778 .. 864  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
778 .. 2199  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   865 .. 2199  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
778 .. 2199  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
5978 .. 7060  =  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
5978 .. 7060  =  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
3023 .. 3838  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
3023 .. 3838  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
3960 .. 4548  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3960 .. 4548  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2475 .. 2930  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2475 .. 2930  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
332 .. 777  =  446 bp
sacB promoter and control region
sacB promoter
332 .. 777  =  446 bp
sacB promoter and control region
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
92 .. 109  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
92 .. 109  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
Z-basic tag
119 .. 289  =  171 bp
57 amino acids  =  6.8 kDa
Product: Z domain of staphylococcal protein A, engineered to contain multiple positive charges (Hedhammar and Hober, 2007)
useful for purifying tagged proteins by cation-exchange chromatography
Z-basic tag
119 .. 289  =  171 bp
57 amino acids  =  6.8 kDa
Product: Z domain of staphylococcal protein A, engineered to contain multiple positive charges (Hedhammar and Hober, 2007)
useful for purifying tagged proteins by cation-exchange chromatography
TEV site
296 .. 316  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
296 .. 316  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
rop
4978 .. 5169  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4978 .. 5169  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
7061 .. 7138  =  78 bp
lacI promoter
7061 .. 7138  =  78 bp
T7 terminator
2391 .. 2438  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2391 .. 2438  =  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).
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
2307 .. 2324  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2307 .. 2324  =  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
ORF:  778 .. 2199  =  1422 bp
ORF:  473 amino acids  =  53.0 kDa
ORF:  5944 .. 6195  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  7099 .. 7362  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  89 .. 361  =  273 bp
ORF:  90 amino acids  =  10.7 kDa
ORF:  5201 .. 5569  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  7236 .. 29  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  106 .. 336  =  231 bp
ORF:  76 amino acids  =  9.1 kDa
ORF:  4978 .. 5202  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  5566 .. 5922  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5958 .. 6221  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6810 .. 7310  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  7307 .. 172  =  312 bp
ORF:  103 amino acids  =  11.1 kDa
ORF:  3023 .. 3838  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5978 .. 6937  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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