pMCSG21 (linearized)

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

Sequence Author: Midwest Center for Structural Genomics

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T7 promoter EcoNI (3457) PfoI (3421) AclI (3329) BstAPI (3313) MluI (2989) ApaI (2786) PspOMI (2782) HpaI (2487) BsmBI - Esp3I (2374) PluTI (2354) SfoI (2352) NarI * (2351) KasI (2350) XbaI (2069) RBS NcoI (3553) ATG 6xHis AscI (3609) PstI - SbfI (3619) AflII (3647) BsrGI - TatI (3674) T7 promoter RBS NdeI (3782) ATG 6xHis BglII (3816) Acc65I (3823) KpnI (3827) End (3837) TEV site Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) PacI (132) AvrII (136) EcoO109I (181) Bsu36I (220) AgeI (269) DrdI - PflFI - Tth111I (329) BsaAI (418) NgoMIV (522) NaeI (524) BglI (546) PvuI (796) BspHI (1222) NheI (1269) BmtI (1273) PciI (1369) NspI (1373) SgrAI (1396) pMCSG21 3837 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.
PfoI  (3421)
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.
AclI  (3329)
1 site
A A C G T T T T G C A A
BstAPI  (3313)
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  (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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (2374)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I 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
NcoI  (3553)
1 site
C C A T G G G G T A C C
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
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.
TatI  (3674)
1 site
W G T A C W W C A T G W
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.
BglII  (3816)
1 site
A G A T C T T C T A G A
Acc65I  (3823)
1 site
G G T A C C C C A T G G
KpnI  (3827)
1 site
G G T A C C C C A T G G
End  (3837)
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
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
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.
BsaAI  (418)
1 site
Y A C G T R R T G C A Y
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.
PvuI  (796)
1 site
C G A T C G G C T A G C
BspHI  (1222)
1 site
T C A T G A A G T A C T
NheI  (1269)
1 site
G C T A G C C G A T C G
BmtI  (1273)
1 site
G C T A G C C G A T C G
PciI  (1369)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1373)
1 site
R C A T G Y Y G T A C R
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.
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
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
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
TEV site
3829 .. 3849  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
3829 .. 3849  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
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
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
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
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).
T7 promoter
3698 .. 3716  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3698 .. 3716  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3468 .. 3486  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3468 .. 3486  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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
ORF:  752 .. 1135  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  2228 .. 2479  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  597 .. 875  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  885 .. 1142  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  2242 .. 2505  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3094 .. 3642  =  549 bp
ORF:  182 amino acids  =  19.6 kDa
ORF:  2262 .. 3221  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  383 .. 1174  =  792 bp
ORF:  263 amino acids  =  29.3 kDa
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