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pMCSG58

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

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pMCSG58.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (4053) PvuI (3943) FspI (3795) BglI (3693) BsaI (3634) AhdI (3573) AlwNI (3096) DrdI (2788) PciI (2680) BspQI - SapI (2564) BstZ17I (2451) BsaAI (2432) 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 (226) SmaI (228) XbaI (265) BspDI * - ClaI * (334) AgeI (376) AscI (593) SbfI (604) BstBI (609) BfuAI - BspMI (622) MreI (741) AfeI (827) NcoI (1011) PsiI (1026) EcoNI (1115) PfoI (1147) PflMI (1162) BstAPI (1263) MluI (1580) BclI * (1594) BstEII (1761) PspOMI (1787) ApaI (1791) EcoRV (2030) HpaI (2086) BsmBI (2195) PshAI (2425) pMCSG58 4748 bp
ScaI  (4053)
1 site
A G T A C T T C A T G A
PvuI  (3943)
1 site
C G A T C G G C T A G C
FspI  (3795)
1 site
T G C G C A A C G C G T
BglI  (3693)
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  (3634)
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  (3573)
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  (3096)
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  (2788)
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  (2680)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2564)
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  (2564)
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  (2451)
1 site
G T A T A C C A T A T G
BsaAI  (2432)
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  (226)
1 site
C C C G G G G G G C C C
XmaI  (226)
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  (228)
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  (265)
1 site
T C T A G A A G A T C T
BspDI  (334)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (334)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (376)
1 site
A C C G G T T G G C C A
AscI  (593)
1 site
G G C G C G C C C C G C G C G G
SbfI  (604)
1 site
C C T G C A G G G G A C G T C C
BstBI  (609)
1 site
T T C G A A A A G C T T
BfuAI  (622)
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  (622)
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  (741)
1 site
C G C C G G C G G C G G C C G C
AfeI  (827)
1 site
A G C G C T T C G C G A
NcoI  (1011)
1 site
C C A T G G G G T A C C
PsiI  (1026)
1 site
T T A T A A A A T A T T
EcoNI  (1115)
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  (1147)
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  (1162)
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  (1263)
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  (1580)
1 site
A C G C G T T G C G C A
BclI  (1594)
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  (1761)
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  (1787)
1 site
G G G C C C C C C G G G
ApaI  (1791)
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  (2030)
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  (2086)
1 site
G T T A A C C A A T T G
BsmBI  (2195)
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  (2425)
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
1230 .. 2312  =  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
1230 .. 2312  =  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
3500 .. 4360  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3500 .. 4291  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3500 .. 4360  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4292 .. 4360  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3500 .. 4360  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2741 .. 3329  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2741 .. 3329  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4361 .. 4464  =  104 bp
AmpR promoter
4361 .. 4464  =  104 bp
lacI promoter
1152 .. 1229  =  78 bp
lacI promoter
1152 .. 1229  =  78 bp
argU
591 .. 667  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
591 .. 667  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
937 .. 1012  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
937 .. 1012  =  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
273 .. 297  =  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
273 .. 297  =  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
298 .. 316  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
298 .. 316  =  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
237 .. 242  =  6 bp
ribosome binding site
RBS
237 .. 242  =  6 bp
ribosome binding site
ORF:  208 .. 441  =  234 bp
ORF:  77 amino acids  =  8.4 kDa
ORF:  1013 .. 1480  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  2069 .. 2332  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  726 .. 980  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1353 .. 2312  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3630 .. 3896  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3500 .. 4360  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2095 .. 2346  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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