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pMCSG59

Compact bacterial vector encoding tRNA genes for rare Arg and Ile codons, with a C-terminal TEV-6xHis cassette for high-throughput purification of recombinant proteins.

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pMCSG59.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (4080) PvuI (3970) FspI (3822) BglI (3720) BsaI (3661) AhdI (3600) AlwNI (3123) DrdI (2815) PciI (2707) BspQI - SapI (2591) BstZ17I (2478) BsaAI (2459) BspEI (2) BlpI (80) PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) TspMI - XmaI (253) SmaI (255) XbaI (292) BspDI * - ClaI * (361) AgeI (403) AscI (620) SbfI (631) BstBI (636) BfuAI - BspMI (649) MreI (768) AfeI (854) NcoI (1038) PsiI (1053) EcoNI (1142) PfoI (1174) PflMI (1189) BstAPI (1290) MluI (1607) BclI * (1621) BstEII (1788) PspOMI (1814) ApaI (1818) EcoRV (2057) HpaI (2113) BsmBI (2222) PshAI (2452) pMCSG59 4775 bp
ScaI  (4080)
1 site
A G T A C T T C A T G A
PvuI  (3970)
1 site
C G A T C G G C T A G C
FspI  (3822)
1 site
T G C G C A A C G C G T
BglI  (3720)
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.
BsaI  (3661)
1 site
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.
AhdI  (3600)
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.
AlwNI  (3123)
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.
DrdI  (2815)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PciI  (2707)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2591)
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  (2591)
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.
BstZ17I  (2478)
1 site
G T A T A C C A T A T G
BsaAI  (2459)
1 site
Y A C G T R R T G C A Y
BspEI  (2)
1 site
T C C G G A A G G C C T
BlpI  (80)
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.
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
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
HindIII  (173)
1 site
A A G C T T T T C G A A
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
EcoRI  (192)
1 site
G A A T T C C T T A 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.
TspMI  (253)
1 site
C C C G G G G G G C C C
XmaI  (253)
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  (255)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
XbaI  (292)
1 site
T C T A G A A G A T C T
BspDI  (361)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (361)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (403)
1 site
A C C G G T T G G C C A
AscI  (620)
1 site
G G C G C G C C C C G C G C G G
SbfI  (631)
1 site
C C T G C A G G G G A C G T C C
BstBI  (636)
1 site
T T C G A A A A G C T T
BfuAI  (649)
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  (649)
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.
MreI  (768)
1 site
C G C C G G C G G C G G C C G C
AfeI  (854)
1 site
A G C G C T T C G C G A
NcoI  (1038)
1 site
C C A T G G G G T A C C
PsiI  (1053)
1 site
T T A T A A A A T A T T
EcoNI  (1142)
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.
PfoI  (1174)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflMI  (1189)
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.
BstAPI  (1290)
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  (1607)
1 site
A C G C G T T G C G C A
BclI  (1621)
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  (1788)
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.
PspOMI  (1814)
1 site
G G G C C C C C C G G G
ApaI  (1818)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (2057)
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.
HpaI  (2113)
1 site
G T T A A C C A A T T G
BsmBI  (2222)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PshAI  (2452)
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.
lacI
1257 .. 2339  =  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
1257 .. 2339  =  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
3527 .. 4387  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3527 .. 4318  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3527 .. 4387  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4319 .. 4387  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3527 .. 4387  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2768 .. 3356  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2768 .. 3356  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4388 .. 4491  =  104 bp
AmpR promoter
4388 .. 4491  =  104 bp
lacI promoter
1179 .. 1256  =  78 bp
lacI promoter
1179 .. 1256  =  78 bp
argU
618 .. 694  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
618 .. 694  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
964 .. 1039  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
964 .. 1039  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
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
300 .. 324  =  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
300 .. 324  =  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).
TEV site
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
232 .. 252  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
325 .. 343  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
325 .. 343  =  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
6xHis
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
208 .. 225  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
264 .. 269  =  6 bp
ribosome binding site
RBS
264 .. 269  =  6 bp
ribosome binding site
ORF:  208 .. 468  =  261 bp
ORF:  86 amino acids  =  9.3 kDa
ORF:  1040 .. 1507  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  2096 .. 2359  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  753 .. 1007  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1380 .. 2339  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3657 .. 3923  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3527 .. 4387  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2122 .. 2373  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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