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Plasmid Files

pMCSG23 (linearized)

Linearized bacterial coexpression vector for ligation-independent cloning (LIC), with a 6xHis-MBP-TEV leader and a CloDF13 origin.

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pMCSG23 (linearized) Sequence and MappMCSG23 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 T7 promoter EcoNI (3457) AclI (3329) MluI (2989) ApaI (2786) PspOMI (2782) HpaI (2487) BsmBI (2374) PluTI (2354) SfoI (2352) NarI * (2351) KasI (2350) XbaI (2069) SgrAI (1396) NspI (1373) PciI (1369) BmtI (1273) NheI (1269) BspHI (1222) RBS ATG 6xHis AscI (3609) PstI - SbfI (3619) AflII (3647) BsrGI (3674) T7 promoter RBS NdeI (3782) ATG 6xHis BsaI (3889) BspEI (3945) BsiWI (4116) PsiI (4178) BmgBI (4368) BsmI (4446) AlwNI (4471) BsaBI (4501) AfeI (4736) EcoRV (4935) End (4956) Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) BmeT110I (58) PacI (132) AvrII (136) EcoO109I (181) Bsu36I (220) AgeI (269) DrdI - PflFI - Tth111I (329) NgoMIV (522) NaeI (524) BglI (546) pMCSG23 4956 bp
EcoNI  (3457)
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.
AclI  (3329)
1 site
A A C G T T T T G C A A
MluI  (2989)
1 site
A C G C G T T G C G C A
ApaI  (2786)
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  (2782)
1 site
G G G C C C C C C G G G
HpaI  (2487)
1 site
G T T A A C C A A T T G
BsmBI  (2374)
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.
PluTI  (2354)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (2352)
1 site
G G C G C C C C G C G G
NarI  (2351)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (2350)
1 site
G G C G C C C C G C G G
XbaI  (2069)
1 site
T C T A G A A G A T C T
SgrAI  (1396)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
NspI  (1373)
1 site
R C A T G Y Y G T A C R
PciI  (1369)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BmtI  (1273)
1 site
G C T A G C C G A T C G
NheI  (1269)
1 site
G C T A G C C G A T C G
BspHI  (1222)
1 site
T C A T G A A G T A C T
AscI  (3609)
1 site
G G C G C G C C C C G C G C G G
PstI  (3619)
1 site
C T G C A G G A C G T C
SbfI  (3619)
1 site
C C T G C A G G G G A C G T C C
AflII  (3647)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BsrGI  (3674)
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.
NdeI  (3782)
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.
BsaI  (3889)
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.
BspEI  (3945)
1 site
T C C G G A A G G C C T
BsiWI  (4116)
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.
PsiI  (4178)
1 site
T T A T A A A A T A T T
BmgBI  (4368)
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.
BsmI  (4446)
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.
AlwNI  (4471)
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.
BsaBI  (4501)
1 site
G A T N N N N A T C C T A N N N N T A G
AfeI  (4736)
1 site
A G C G C T T C G C G A
EcoRV  (4935)
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.
End  (4956)
0 sites
Start  (0)
0 sites
AvaI  (57)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (57)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PaeR7I  (57)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (57)
1 site
V C T C G A G B B G A G C T C V
XhoI  (57)
1 site
C T C G A G G A G C T C
BmeT110I  (58)
1 site
C Y C G R G G R G C Y C
PacI  (132)
1 site
T T A A T T A A A A T T A A T T
AvrII  (136)
1 site
C C T A G G G G A T C C
EcoO109I  (181)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
Bsu36I  (220)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
AgeI  (269)
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.
DrdI  (329)
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.
PflFI  (329)
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  (329)
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.
NgoMIV  (522)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (524)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
BglI  (546)
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.
MBP
3829 .. 4926  =  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
3829 .. 4926  =  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
2262 .. 3344  =  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
2262 .. 3344  =  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
383 .. 1174  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
383 .. 1174  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
CloDF13 ori
1314 .. 2052  =  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
1314 .. 2052  =  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
1175 .. 1266  =  92 bp
AmpR promoter
1175 .. 1266  =  92 bp
lacI promoter
3345 .. 3422  =  78 bp
lacI promoter
3345 .. 3422  =  78 bp
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
3487 .. 3511  =  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
3487 .. 3511  =  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
3717 .. 3741  =  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
3717 .. 3741  =  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
4948 .. 4968  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
4948 .. 4968  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
3468 .. 3486  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3468 .. 3486  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3698 .. 3716  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3698 .. 3716  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3567 .. 3584  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3567 .. 3584  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3787 .. 3804  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3787 .. 3804  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
3542 .. 3547  =  6 bp
ribosome binding site
RBS
3542 .. 3547  =  6 bp
ribosome binding site
RBS
3770 .. 3775  =  6 bp
ribosome binding site
RBS
3770 .. 3775  =  6 bp
ribosome binding site
ATG
3555 .. 3557  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3555 .. 3557  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3784 .. 3786  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3784 .. 3786  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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