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Plasmid Files

pMCSG77

Bacterial vector with a p15A origin encoding tRNA genes for rare Arg and Ile codons, for expressing a protein with a 6xHis-TEV leader plus a second untagged protein.

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pMCSG77 Sequence and MappMCSG77.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 BspEI (2) BsrGI (5440) AlwNI (5098) SacII (4747) PfoI * (4700) BstZ17I (4508) BmtI (4499) NheI (4495) DraIII (4267) NruI (4076) EcoNI (3820) AsiSI (3735) AarI (2762) BlpI (80) EagI - NotI (166) Eco53kI (188) SacI (190) EcoRI (192) TspMI - XmaI (232) SmaI (234) RBS SspI (275) TEV site Acc65I (300) KpnI (304) BglII (307) ATG NdeI (343) RBS XbaI (381) lac operator T7 promoter AcuI (512) AscI (709) PstI - SbfI (720) MreI (857) NcoI (1127) PsiI (1142) AclI (1335) BstAPI (1356) ApaLI (1653) AflIII - MluI (1673) BclI * (1687) NmeAIII (1879) PspOMI (1880) ApaI (1884) EcoRV (2123) BciVI (2130) HpaI (2179) MfeI (2512) pMCSG77 5445 bp
BspEI  (2)
1 site
T C C G G A A G G C C T
BsrGI  (5440)
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.
AlwNI  (5098)
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.
SacII  (4747)
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.
PfoI  (4700)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BstZ17I  (4508)
1 site
G T A T A C C A T A T G
BmtI  (4499)
1 site
G C T A G C C G A T C G
NheI  (4495)
1 site
G C T A G C C G A T C G
DraIII  (4267)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
NruI  (4076)
1 site
T C G C G A A G C G C T
EcoNI  (3820)
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.
AsiSI  (3735)
1 site
G C G A T C G C C G C T A G C G
AarI  (2762)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
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.
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
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
TspMI  (232)
1 site
C C C G G G G G G C C C
XmaI  (232)
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  (234)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SspI  (275)
1 site
A A T A T T T T A T A A
Acc65I  (300)
1 site
G G T A C C C C A T G G
KpnI  (304)
1 site
G G T A C C C C A T G G
BglII  (307)
1 site
A G A T C T T C T A G A
NdeI  (343)
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  (381)
1 site
T C T A G A A G A T C T
AcuI  (512)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AscI  (709)
1 site
G G C G C G C C C C G C G C G G
PstI  (720)
1 site
C T G C A G G A C G T C
SbfI  (720)
1 site
C C T G C A G G G G A C G T C C
MreI  (857)
1 site
C G C C G G C G G C G G C C G C
NcoI  (1127)
1 site
C C A T G G G G T A C C
PsiI  (1142)
1 site
T T A T A A A A T A T T
AclI  (1335)
1 site
A A C G T T T T G C A A
BstAPI  (1356)
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.
ApaLI  (1653)
1 site
G T G C A C C A C G T G
AflIII  (1673)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (1673)
1 site
A C G C G T T G C G C A
BclI  (1687)
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.
NmeAIII  (1879)
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).
PspOMI  (1880)
1 site
G G G C C C C C C G G G
ApaI  (1884)
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  (2123)
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.
BciVI  (2130)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
HpaI  (2179)
1 site
G T T A A C C A A T T G
MfeI  (2512)
1 site
C A A T T G G T T A A C
lacI
1323 .. 2405  =  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
1323 .. 2405  =  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
3350 .. 4165  =  816 bp
271 amino acids  =  31.1 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
3350 .. 4165  =  816 bp
271 amino acids  =  31.1 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
p15A ori
4760 .. 5305  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
p15A ori
4760 .. 5305  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
lacIq promoter
1245 .. 1322  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
lacIq promoter
1245 .. 1322  =  78 bp
In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about
10-fold.
argU
707 .. 783  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
707 .. 783  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
1053 .. 1128  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
1053 .. 1128  =  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
389 .. 413  =  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
389 .. 413  =  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
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
414 .. 432  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
414 .. 432  =  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
324 .. 341  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
324 .. 341  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
353 .. 358  =  6 bp
ribosome binding site
RBS
353 .. 358  =  6 bp
ribosome binding site
ATG
342 .. 344  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
342 .. 344  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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