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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HomePlasmidsStructural Genomics VectorspNIC28-Bsa4
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
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
ori
3798 .. 4386  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3798 .. 4386  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2313 .. 2768  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2313 .. 2768  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
170 .. 615  =  446 bp
sacB promoter and control region
sacB promoter
170 .. 615  =  446 bp
sacB promoter and control region
rop
4816 .. 5007  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4816 .. 5007  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
6899 .. 6976  =  78 bp
lacI promoter
6899 .. 6976  =  78 bp
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
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
TEV site
134 .. 154  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
134 .. 154  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 terminator
2229 .. 2276  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2229 .. 2276  =  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
2145 .. 2162  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2145 .. 2162  =  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:  616 .. 2037  =  1422 bp
ORF:  473 amino acids  =  53.0 kDa
ORF:  5782 .. 6033  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6937 .. 7200  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  5039 .. 5407  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  7074 .. 29  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  4816 .. 5040  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  5404 .. 5760  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5796 .. 6059  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6648 .. 7148  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  7145 .. 109  =  249 bp
ORF:  82 amino acids  =  8.8 kDa
ORF:  2861 .. 3676  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5816 .. 6775  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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Individual Sequences & Maps

p11
pMCSG19B (linearized)
pMCSG41
pMCSG68 (linearized)
p15Tv-L
pMCSG19C
pMCSG41 (linearized)
pMCSG69
p283
pMCSG19C (linearized)
pMCSG42
pMCSG69 (linearized)
pBEN1-SGC
pMCSG20
pMCSG42 (linearized)
pMCSG7
pBEN1-SGC (linearized)
pMCSG20 (linearized)
pMCSG43
pMCSG7 (linearized)
pDB-HisGST
pMCSG21
pMCSG43 (linearized)
pMCSG70