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Plasmid Files

pMCSG76

Bacterial vector with a CloDF13 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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pMCSG76 Sequence and MappMCSG76.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 6xHis BspEI * (2) EcoNI (4304) PfoI (4268) PflMI (4261) AclI (4176) BstAPI (4160) MluI (3836) ApaI (3633) PspOMI (3629) HpaI (3334) BsmBI (3221) PciI (2216) BmtI (2120) PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) AccI (180) Eco53kI (188) SacI (190) EcoRI (192) TspMI - XmaI (232) SmaI (234) RBS SspI (275) TatI (287) Acc65I (300) KpnI (304) BglII (307) 6xHis ATG NdeI (343) RBS lac operator T7 promoter BspDI * - ClaI * (450) AgeI (492) XmnI (561) AscI (709) PstI - SbfI (720) BstBI (725) MreI (857) AfeI (943) NcoI (1127) PsiI (1142) PflFI - Tth111I (1176) BsaAI (1265) BglI (1393) PvuI (1643) NheI (2116) pMCSG76 4310 bp
BspEI  (2)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
EcoNI  (4304)
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  (4268)
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  (4261)
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.
AclI  (4176)
1 site
A A C G T T T T G C A A
BstAPI  (4160)
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  (3836)
1 site
A C G C G T T G C G C A
ApaI  (3633)
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  (3629)
1 site
G G G C C C C C C G G G
HpaI  (3334)
1 site
G T T A A C C A A T T G
BsmBI  (3221)
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.
PciI  (2216)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BmtI  (2120)
1 site
G C T A G C C G A T C G
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
AccI  (180)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
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
TatI  (287)
1 site
W G T A C W W C A T G W
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.
BspDI  (450)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (450)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (492)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
XmnI  (561)
1 site
G A A N N N N T T C C T T N N N N A A G
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
BstBI  (725)
1 site
T T C G A A A A G C T T
MreI  (857)
1 site
C G C C G G C G G C G G C C G C
AfeI  (943)
1 site
A G C G C T T C G C G A
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
PflFI  (1176)
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  (1176)
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.
BsaAI  (1265)
1 site
Y A C G T R R T G C A Y
BglI  (1393)
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.
PvuI  (1643)
1 site
C G A T C G G C T A G C
NheI  (2116)
1 site
G C T A G C C G A T C G
lacI
3109 .. 4191  =  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
3109 .. 4191  =  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).
SmR
1230 .. 2021  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
1230 .. 2021  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
CloDF13 ori
2161 .. 2899  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
CloDF13 ori
2161 .. 2899  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
AmpR promoter
2022 .. 2113  =  92 bp
AmpR promoter
2022 .. 2113  =  92 bp
lacI promoter
4192 .. 4269  =  78 bp
lacI promoter
4192 .. 4269  =  78 bp
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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