pBEN1-SGC

Bacterial vector encoding an N-terminal SET1-SBP-TEV cassette plus a SacB negative selection marker, for purification of recombinant proteins.

Sequence Author: Structural Genomics Consortium

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XbaI (7481) lac operator BglII (7415) SgrAI (7374) SphI (7226) BstAPI (7017) MluI (6693) BclI * (6679) BstEII (6511) NmeAIII (6493) ApaI (6490) PspOMI (6486) BssHII (6282) FspI - FspAI (5615) PpuMI (5587) PflFI - Tth111I (4850) BspQI - SapI (4709) BssSI (4419) AlwNI (4183) NruI (3737) NdeI (6) ATG SpeI (137) PmlI (175) Bsu36I (234) BsaI (302) NcoI (303) ZraI (316) AatII (318) BfuAI - BspMI (336) sacB promoter StuI * (1007) MscI (1029) AanI (1406) BsrGI (1446) SnaBI (1515) SacII (1845) BsaI (2233) BamHI (2249) EcoRI (2255) Eco53kI (2263) SacI (2265) SalI (2268) HindIII (2274) EagI - NotI (2281) PaeR7I - PspXI - XhoI (2289) BlpI (2368) AanI (2821) AsiSI - PvuI (3396) TspMI - XmaI (3518) SmaI (3520) pBEN1-SGC 7515 bp
XbaI  (7481)
1 site
T C T A G A A G A T C T
BglII  (7415)
1 site
A G A T C T T C T A G A
SgrAI  (7374)
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  (7226)
1 site
G C A T G C C G T A C G
BstAPI  (7017)
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  (6693)
1 site
A C G C G T T G C G C A
BclI  (6679)
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  (6511)
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  (6493)
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  (6490)
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  (6486)
1 site
G G G C C C C C C G G G
BssHII  (6282)
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.
FspI  (5615)
1 site
T G C G C A A C G C G T
FspAI  (5615)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (5587)
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  (4850)
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  (4850)
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  (4709)
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  (4709)
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  (4419)
1 site
C A C G A G G T G C T C
AlwNI  (4183)
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  (3737)
1 site
T C G C G A A G C G C T
NdeI  (6)
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.
SpeI  (137)
1 site
A C T A G T T G A T C A
PmlI  (175)
1 site
C A C G T G G T G C A C
Bsu36I  (234)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BsaI  (302)
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  (303)
1 site
C C A T G G G G T A C C
ZraI  (316)
1 site
G A C G T C C T G C A G
AatII  (318)
1 site
G A C G T C C T G C A G
BfuAI  (336)
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  (336)
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  (1007)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
MscI  (1029)
1 site
T G G C C A A C C G G T
AanI  (1406)
2 sites
T T A T A A A A T A T T
BsrGI  (1446)
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  (1515)
1 site
T A C G T A A T G C A T
SacII  (1845)
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  (2233)
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  (2249)
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  (2255)
1 site
G A A T T C C T T A A G
Eco53kI  (2263)
1 site
G A G C T C C T C G A G
SacI  (2265)
1 site
G A G C T C C T C G A G
SalI  (2268)
1 site
G T C G A C C A G C T G
HindIII  (2274)
1 site
A A G C T T T T C G A A
EagI  (2281)
1 site
C G G C C G G C C G G C
NotI  (2281)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (2289)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2289)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2289)
1 site
C T C G A G G A G C T C
BlpI  (2368)
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.
AanI  (2821)
2 sites
T T A T A A A A T A T T
AsiSI  (3396)
1 site
G C G A T C G C C G C T A G C G
PvuI  (3396)
1 site
C G A T C G G C T A G C
TspMI  (3518)
1 site
C C C G G G G G G C C C
XmaI  (3518)
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  (3520)
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
766 .. 2187  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   766 .. 852  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
766 .. 2187  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   853 .. 2187  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
766 .. 2187  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
5966 .. 7048  =  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
5966 .. 7048  =  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
3011 .. 3826  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
3011 .. 3826  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
3948 .. 4536  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3948 .. 4536  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2463 .. 2918  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2463 .. 2918  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
sacB promoter
320 .. 765  =  446 bp
sacB promoter and control region
sacB promoter
320 .. 765  =  446 bp
sacB promoter and control region
ATG
8 .. 10  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
8 .. 10  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SET1
11 .. 136  =  126 bp
42 amino acids  =  4.4 kDa
Product: solubility-enhancement tag derived from T7 phage gene 10B
SET1
11 .. 136  =  126 bp
42 amino acids  =  4.4 kDa
Product: solubility-enhancement tag derived from T7 phage gene 10B
SBP
143 .. 253  =  111 bp
37 amino acids  =  4.2 kDa
Product: streptavidin-binding peptide
selected from a peptide library; binds streptavidin with nanomolar affinity
SBP
143 .. 253  =  111 bp
37 amino acids  =  4.2 kDa
Product: streptavidin-binding peptide
selected from a peptide library; binds streptavidin with nanomolar affinity
TEV site
284 .. 304  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
284 .. 304  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
rop
4966 .. 5157  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4966 .. 5157  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
7049 .. 7126  =  78 bp
lacI promoter
7049 .. 7126  =  78 bp
T7 terminator
2379 .. 2426  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2379 .. 2426  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
7454 .. 7478  =  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
7454 .. 7478  =  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
7435 .. 7453  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
7435 .. 7453  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2295 .. 2312  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2295 .. 2312  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ORF:  766 .. 2187  =  1422 bp
ORF:  473 amino acids  =  53.0 kDa
ORF:  5932 .. 6183  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  7087 .. 7350  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  8 .. 349  =  342 bp
ORF:  113 amino acids  =  12.3 kDa
ORF:  5189 .. 5557  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  7224 .. 7463  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  4966 .. 5190  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  5554 .. 5910  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5946 .. 6209  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6798 .. 7298  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  7295 .. 7  =  228 bp
ORF:  75 amino acids  =  7.9 kDa
ORF:  3011 .. 3826  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5966 .. 6925  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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