Resources
Plasmid Files

pMCSG66 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a 6xHis-2xHA-TEV leader plus a second LIC site for expressing an untagged protein.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pMCSG66 (linearized) Sequence and MappMCSG66 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
Download Free Trial Get SnapGene Viewer

 PaeR7I - PspXI - XhoI (5279) BlpI (5201) StyI (5178) T7 terminator ScaI (4426) PvuI (4316) PstI (4191) BsaI (4007) AhdI (3946) AlwNI (3469) PciI (3053) BspQI - SapI (2937) BstZ17I (2824) BsaAI (2805) PflFI - Tth111I (2798) Bpu10I (2159) EagI - NotI (5287) HindIII (5294) SalI (5300) Eco53kI (5309) SacI (5311) EcoRI (5313) BamHI (5319) TspMI - XmaI (5353) SmaI (5355) End (5381) Start (0) TEV site Acc65I (25) KpnI (29) BfuAI - BspMI (37) HA BsiWI (48) HA BglII (86) 6xHis ATG NdeI (122) RBS XbaI (160) lac operator T7 promoter BspDI * - ClaI * (229) SgrAI (271) SphI (427) EcoNI (487) PflMI (534) BstAPI (635) MluI (952) BclI * (966) BstEII (1133) PspOMI (1159) ApaI (1163) BssHII (1363) EcoRV (1402) HpaI (1458) PshAI (1797) FspAI (2034) PpuMI (2059) pMCSG66 5381 bp
PaeR7I  (5279)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5279)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5279)
1 site
C T C G A G G A G C T C
BlpI  (5201)
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.
StyI  (5178)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
ScaI  (4426)
1 site
A G T A C T T C A T G A
PvuI  (4316)
1 site
C G A T C G G C T A G C
PstI  (4191)
1 site
C T G C A G G A C G T C
BsaI  (4007)
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.
AhdI  (3946)
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.
AlwNI  (3469)
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.
PciI  (3053)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2937)
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  (2937)
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  (2824)
1 site
G T A T A C C A T A T G
BsaAI  (2805)
1 site
Y A C G T R R T G C A Y
PflFI  (2798)
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  (2798)
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.
Bpu10I  (2159)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
EagI  (5287)
1 site
C G G C C G G C C G G C
NotI  (5287)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5294)
1 site
A A G C T T T T C G A A
SalI  (5300)
1 site
G T C G A C C A G C T G
Eco53kI  (5309)
1 site
G A G C T C C T C G A G
SacI  (5311)
1 site
G A G C T C C T C G A G
EcoRI  (5313)
1 site
G A A T T C C T T A A G
BamHI  (5319)
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.
TspMI  (5353)
1 site
C C C G G G G G G C C C
XmaI  (5353)
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  (5355)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
End  (5381)
0 sites
Start  (0)
0 sites
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
BfuAI  (37)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (37)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsiWI  (48)
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.
BglII  (86)
1 site
A G A T C T T C T A G A
NdeI  (122)
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  (160)
1 site
T C T A G A A G A T C T
BspDI  (229)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (229)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (271)
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.
SphI  (427)
1 site
G C A T G C C G T A C G
EcoNI  (487)
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  (534)
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.
BstAPI  (635)
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  (952)
1 site
A C G C G T T G C G C A
BclI  (966)
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.
BstEII  (1133)
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  (1159)
1 site
G G G C C C C C C G G G
ApaI  (1163)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (1363)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
EcoRV  (1402)
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.
HpaI  (1458)
1 site
G T T A A C C A A T T G
PshAI  (1797)
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.
FspAI  (2034)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2059)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
602 .. 1684  =  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
602 .. 1684  =  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
3873 .. 4733  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3873 .. 4664  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3873 .. 4733  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4665 .. 4733  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3873 .. 4733  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3114 .. 3702  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3114 .. 3702  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2493 .. 2684  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2493 .. 2684  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
4734 .. 4837  =  104 bp
AmpR promoter
4734 .. 4837  =  104 bp
lacI promoter
524 .. 601  =  78 bp
lacI promoter
524 .. 601  =  78 bp
T7 terminator
5147 .. 5194  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5147 .. 5194  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
HA
31 .. 57  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
31 .. 57  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
58 .. 84  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
58 .. 84  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
lac operator
168 .. 192  =  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
168 .. 192  =  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
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
193 .. 211  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
193 .. 211  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
103 .. 120  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
103 .. 120  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5261 .. 5278  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5261 .. 5278  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
132 .. 137  =  6 bp
ribosome binding site
RBS
132 .. 137  =  6 bp
ribosome binding site
RBS
5364 .. 5369  =  6 bp
ribosome binding site
RBS
5364 .. 5369  =  6 bp
ribosome binding site
ATG
121 .. 123  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
121 .. 123  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2016 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter