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Plasmid Files

pMCSG70 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with a a C-terminal TEV-10xHis-TVMV-MBP cassette plus tRNA genes for rare Arg and Ile codons.

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pMCSG70 (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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AvaI - BsoBI - PaeR7I - PspXI - XhoI (4678) ScaI (3825) FspI (3567) BglI (3465) AhdI (3345) DrdI (2560) PciI (2452) BspQI - SapI (2336) BstZ17I (2223) PshAI (2197) BsmBI (1967) HpaI (1858) EcoRV (1802) ApaI (1563) PspOMI (1559) BstEII (1533) BmeT110I (4679) EagI - NotI (4686) HindIII (4693) SalI (4699) Eco53kI (4708) SacI (4710) EcoRI (4712) BamHI (4718) BsmI (5214) BmgBI (5290) BglII (5469) BsiWI (5538) End (5901) Start (0) XbaI (37) BspDI * - ClaI * (106) AgeI (148) AscI (365) SbfI (376) BstBI (381) MreI (513) EcoNI (887) PflMI (934) MluI (1352) pMCSG70 5901 bp
AvaI  (4678)
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  (4678)
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  (4678)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4678)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4678)
1 site
C T C G A G G A G C T C
ScaI  (3825)
1 site
A G T A C T T C A T G A
FspI  (3567)
1 site
T G C G C A A C G C G T
BglI  (3465)
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.
AhdI  (3345)
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.
DrdI  (2560)
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.
PciI  (2452)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2336)
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  (2336)
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  (2223)
1 site
G T A T A C C A T A T G
PshAI  (2197)
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.
BsmBI  (1967)
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.
HpaI  (1858)
1 site
G T T A A C C A A T T G
EcoRV  (1802)
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.
ApaI  (1563)
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  (1559)
1 site
G G G C C C C C C G G G
BstEII  (1533)
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.
BmeT110I  (4679)
1 site
C Y C G R G G R G C Y C
EagI  (4686)
1 site
C G G C C G G C C G G C
NotI  (4686)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4693)
1 site
A A G C T T T T C G A A
SalI  (4699)
1 site
G T C G A C C A G C T G
Eco53kI  (4708)
1 site
G A G C T C C T C G A G
SacI  (4710)
1 site
G A G C T C C T C G A G
EcoRI  (4712)
1 site
G A A T T C C T T A A G
BamHI  (4718)
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.
BsmI  (5214)
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.
BmgBI  (5290)
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.
BglII  (5469)
1 site
A G A T C T T C T A G A
BsiWI  (5538)
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.
End  (5901)
0 sites
Start  (0)
0 sites
XbaI  (37)
1 site
T C T A G A A G A T C T
BspDI  (106)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (106)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (148)
1 site
A C C G G T T G G C C A
AscI  (365)
1 site
G G C G C G C C C C G C G C G G
SbfI  (376)
1 site
C C T G C A G G G G A C G T C C
BstBI  (381)
1 site
T T C G A A A A G C T T
MreI  (513)
1 site
C G C C G G C G G C G G C C G C
EcoNI  (887)
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  (934)
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.
MluI  (1352)
1 site
A C G C G T T G C G C A
MBP
4733 .. 5830  =  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
4733 .. 5830  =  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
1002 .. 2084  =  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
1002 .. 2084  =  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
3272 .. 4132  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3272 .. 4063  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3272 .. 4132  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4064 .. 4132  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3272 .. 4132  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2513 .. 3101  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2513 .. 3101  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4133 .. 4236  =  104 bp
AmpR promoter
4133 .. 4236  =  104 bp
lacI promoter
924 .. 1001  =  78 bp
lacI promoter
924 .. 1001  =  78 bp
argU
363 .. 439  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
363 .. 439  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
709 .. 784  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
709 .. 784  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
4546 .. 4593  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
4546 .. 4593  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
10xHis
5852 .. 5881  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
5852 .. 5881  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
45 .. 69  =  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
45 .. 69  =  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).
TVMV site
5831 .. 5851  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
5831 .. 5851  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TEV site
5888 .. 5908  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
5888 .. 5908  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
70 .. 88  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
70 .. 88  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
4660 .. 4677  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4660 .. 4677  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
9 .. 14  =  6 bp
ribosome binding site
RBS
9 .. 14  =  6 bp
ribosome binding site
ORF:  785 .. 1252  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  1841 .. 2104  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  498 .. 752  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1125 .. 2084  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3402 .. 3668  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3272 .. 4132  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  4724 .. 5911  =  1188 bp
ORF:  395 amino acids  =  43.7 kDa  (no start codon)
ORF:  1867 .. 2118  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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