pMCSG30

Bacterial expression vector with a 6xHis-TEV-MBP leader and a SacB negative selection marker, for high-throughput purification of recombinant proteins.

Sequence Author: Midwest Center for Structural Genomics

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AvaI - BmeT110I - BsoBI - PaeR7I - PspXI - XhoI (158) ScaI (7619) PstI (7384) AhdI (7139) BspQI - SapI (6130) PflFI - Tth111I (5991) PpuMI (5252) FspAI (5227) PshAI (4990) EcoRV (4595) ApaI (4356) PspOMI (4352) BstEII (4326) EagI - NotI (166) SalI (179) Eco53kI (188) SacI (190) BamHI (198) SfiI (249) SacII (598) SnaBI (926) BsrGI (991) AanI (1035) MscI (1412) StuI * (1434) SfiI (2133) NcoI (2281) BsmI (2628) BmgBI (2704) AanI (2894) BsiWI (2952) TEV site 6xHis ATG NdeI (3315) RBS XbaI (3353) T7 promoter SgrAI (3464) SphI (3620) EcoNI (3680) PflMI (3727) MluI (4145) pMCSG30 8314 bp
AvaI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BmeT110I  (158)
1 site
C Y C G R G G R G C Y C
BsoBI  (158)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
ScaI  (7619)
1 site
A G T A C T T C A T G A
PstI  (7384)
1 site
C T G C A G G A C G T C
AhdI  (7139)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BspQI  (6130)
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  (6130)
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.
PflFI  (5991)
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  (5991)
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.
PpuMI  (5252)
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.
FspAI  (5227)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (4990)
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.
EcoRV  (4595)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
ApaI  (4356)
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  (4352)
1 site
G G G C C C C C C G G G
BstEII  (4326)
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.
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
BamHI  (198)
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.
SfiI  (249)
2 sites
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
SacII  (598)
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.
SnaBI  (926)
1 site
T A C G T A A T G C A T
BsrGI  (991)
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.
AanI  (1035)
2 sites
T T A T A A A A T A T T
MscI  (1412)
1 site
T G G C C A A C C G G T
StuI  (1434)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
SfiI  (2133)
2 sites
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
NcoI  (2281)
1 site
C C A T G G G G T A C C
BsmI  (2628)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BmgBI  (2704)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
AanI  (2894)
2 sites
T T A T A A A A T A T T
BsiWI  (2952)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
NdeI  (3315)
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.
XbaI  (3353)
1 site
T C T A G A A G A T C T
SgrAI  (3464)
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  (3620)
1 site
G C A T G C C G T A C G
EcoNI  (3680)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PflMI  (3727)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
MluI  (4145)
1 site
A C G C G T T G C G C A
SacB
255 .. 1676  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   255 .. 1589  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
255 .. 1676  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   1590 .. 1676  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
255 .. 1676  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
MBP
2171 .. 3244  =  1074 bp
358 amino acids  =  39.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
2171 .. 3244  =  1074 bp
358 amino acids  =  39.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
TEV site
3251 .. 3271  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
3251 .. 3271  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
3296 .. 3313  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3296 .. 3313  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
3314 .. 3316  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3314 .. 3316  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lacI
3795 .. 4877  =  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
3795 .. 4877  =  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).
AmpR
7066 .. 7926  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   7066 .. 7857  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7066 .. 7926  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   7858 .. 7926  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7066 .. 7926  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
6307 .. 6895  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6307 .. 6895  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
sacB promoter
1677 .. 2122  =  446 bp
sacB promoter and control region
sacB promoter
1677 .. 2122  =  446 bp
sacB promoter and control region
rop
5686 .. 5877  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
5686 .. 5877  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
7927 .. 8030  =  104 bp
AmpR promoter
7927 .. 8030  =  104 bp
lacI promoter
3717 .. 3794  =  78 bp
lacI promoter
3717 .. 3794  =  78 bp
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
3361 .. 3385  =  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
3361 .. 3385  =  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
3386 .. 3404  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3386 .. 3404  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
3325 .. 3330  =  6 bp
ribosome binding site
RBS
3325 .. 3330  =  6 bp
ribosome binding site
ORF:  4933 .. 5289  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5653 .. 5877  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  3128 .. 4045  =  918 bp
ORF:  305 amino acids  =  32.8 kDa
ORF:  4634 .. 4897  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  7196 .. 7462  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3918 .. 4877  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2123 .. 3316  =  1194 bp
ORF:  397 amino acids  =  43.4 kDa
ORF:  3368 .. 3619  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  3493 .. 3756  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  4660 .. 4911  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  7066 .. 7926  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  255 .. 1676  =  1422 bp
ORF:  473 amino acids  =  53.0 kDa
ORF:  5286 .. 5654  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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