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pMCSG70

Compact bacterial vector encoding tRNA genes for rare Arg and Ile codons, with a C-terminal TEV-10xHis-TVMV-MBP cassette.

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pMCSG70.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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PaeR7I - PspXI - XhoI (158) ScaI (5216) FspI (4958) BglI (4856) AhdI (4736) DrdI (3951) PciI (3843) BspQI - SapI (3727) BstZ17I (3614) PshAI (3588) BsmBI (3358) HpaI (3249) EcoRV (3193) ApaI (2954) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) BsmI (694) BmgBI (770) BglII (949) BsiWI (1018) TspMI - XmaI (1389) SmaI (1391) XbaI (1428) BspDI * - ClaI * (1497) AgeI (1539) AscI (1756) SbfI (1767) BstBI (1772) MreI (1904) EcoNI (2278) PflMI (2325) MluI (2743) BstEII (2924) PspOMI (2950) pMCSG70 5911 bp
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  (5216)
1 site
A G T A C T T C A T G A
FspI  (4958)
1 site
T G C G C A A C G C G T
BglI  (4856)
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.
AhdI  (4736)
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.
DrdI  (3951)
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  (3843)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3727)
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  (3727)
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  (3614)
1 site
G T A T A C C A T A T G
PshAI  (3588)
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.
BsmBI  (3358)
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.
HpaI  (3249)
1 site
G T T A A C C A A T T G
EcoRV  (3193)
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  (2954)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 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
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.
BsmI  (694)
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  (770)
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.
BglII  (949)
1 site
A G A T C T T C T A G A
BsiWI  (1018)
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.
TspMI  (1389)
1 site
C C C G G G G G G C C C
XmaI  (1389)
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  (1391)
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  (1428)
1 site
T C T A G A A G A T C T
BspDI  (1497)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1497)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (1539)
1 site
A C C G G T T G G C C A
AscI  (1756)
1 site
G G C G C G C C C C G C G C G G
SbfI  (1767)
1 site
C C T G C A G G G G A C G T C C
BstBI  (1772)
1 site
T T C G A A A A G C T T
MreI  (1904)
1 site
C G C C G G C G G C G G C C G C
EcoNI  (2278)
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  (2325)
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  (2743)
1 site
A C G C G T T G C G C A
BstEII  (2924)
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  (2950)
1 site
G G G C C C C C C G G G
MBP
213 .. 1310  =  1098 bp
366 amino acids  =  40.2 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
213 .. 1310  =  1098 bp
366 amino acids  =  40.2 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.
lacI
2393 .. 3475  =  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
2393 .. 3475  =  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
4663 .. 5523  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4663 .. 5454  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4663 .. 5523  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5455 .. 5523  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4663 .. 5523  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3904 .. 4492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3904 .. 4492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
5524 .. 5627  =  104 bp
AmpR promoter
5524 .. 5627  =  104 bp
lacI promoter
2315 .. 2392  =  78 bp
lacI promoter
2315 .. 2392  =  78 bp
argU
1754 .. 1830  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1754 .. 1830  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
2100 .. 2175  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
2100 .. 2175  =  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
10xHis
1332 .. 1361  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
1332 .. 1361  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
1436 .. 1460  =  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
1436 .. 1460  =  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).
TVMV site
1311 .. 1331  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
1311 .. 1331  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TEV site
1368 .. 1388  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1368 .. 1388  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
1461 .. 1479  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1461 .. 1479  =  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
1400 .. 1405  =  6 bp
ribosome binding site
RBS
1400 .. 1405  =  6 bp
ribosome binding site
ORF:  2176 .. 2643  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  3232 .. 3495  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  1889 .. 2143  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  2516 .. 3475  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4793 .. 5059  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1194 .. 1604  =  411 bp
ORF:  136 amino acids  =  15.1 kDa
ORF:  4663 .. 5523  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  204 .. 869  =  666 bp
ORF:  221 amino acids  =  24.0 kDa
ORF:  3258 .. 3509  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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