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Plasmid Files

pMCSG46

Bacterial vector with a 6xHN-NusA-TEV leader for high-throughput purification of recombinant proteins.

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pMCSG46.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) ScaI (6064) PstI (5829) BsaI (5645) AhdI (5584) AlwNI (5107) PciI (4691) BspQI - SapI (4575) BstZ17I (4462) BsaAI (4443) PflFI - Tth111I (4436) Bpu10I (3797) PpuMI (3697) FspAI (3672) PshAI (3435) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) BglII (344) BsmI (388) MscI (476) StuI (529) BfuAI - BspMI (678) NcoI (804) ZraI (1587) AatII (1589) BstBI (1646) NdeI (1760) XbaI (1798) SphI (2065) EcoNI (2125) PflMI (2172) MluI (2590) BstEII (2771) PspOMI (2797) ApaI (2801) HpaI (3096) pMCSG46 6759 bp
AvaI  (158)
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  (158)
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  (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
ScaI  (6064)
1 site
A G T A C T T C A T G A
PstI  (5829)
1 site
C T G C A G G A C G T C
BsaI  (5645)
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  (5584)
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  (5107)
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  (4691)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4575)
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  (4575)
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  (4462)
1 site
G T A T A C C A T A T G
BsaAI  (4443)
1 site
Y A C G T R R T G C A Y
PflFI  (4436)
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  (4436)
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  (3797)
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.
PpuMI  (3697)
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.
FspAI  (3672)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (3435)
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.
BmeT110I  (159)
1 site
C Y C G R G G R G C Y 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
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
BamHI  (198)
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.
SspI  (219)
1 site
A A T A T T T T A T A A
BglII  (344)
1 site
A G A T C T T C T A G A
BsmI  (388)
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.
MscI  (476)
1 site
T G G C C A A C C G G T
StuI  (529)
1 site
A G G C C T T C C G G A
BfuAI  (678)
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  (678)
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.
NcoI  (804)
1 site
C C A T G G G G T A C C
ZraI  (1587)
1 site
G A C G T C C T G C A G
AatII  (1589)
1 site
G A C G T C C T G C A G
BstBI  (1646)
1 site
T T C G A A A A G C T T
NdeI  (1760)
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  (1798)
1 site
T C T A G A A G A T C T
SphI  (2065)
1 site
G C A T G C C G T A C G
EcoNI  (2125)
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  (2172)
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  (2590)
1 site
A C G C G T T G C G C A
BstEII  (2771)
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  (2797)
1 site
G G G C C C C C C G G G
ApaI  (2801)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HpaI  (3096)
1 site
G T T A A C C A A T T G
NusA
259 .. 1716  =  1458 bp
486 amino acids  =  53.8 kDa
Product: transcription elongation protein (N utilization substance protein A)
highly soluble in E. coli
NusA
259 .. 1716  =  1458 bp
486 amino acids  =  53.8 kDa
Product: transcription elongation protein (N utilization substance protein A)
highly soluble in E. coli
lacI
2240 .. 3322  =  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
2240 .. 3322  =  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
5511 .. 6371  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5511 .. 6302  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5511 .. 6371  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   6303 .. 6371  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5511 .. 6371  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
4752 .. 5340  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4752 .. 5340  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
4131 .. 4322  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4131 .. 4322  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6372 .. 6475  =  104 bp
AmpR promoter
6372 .. 6475  =  104 bp
lacI promoter
2162 .. 2239  =  78 bp
lacI promoter
2162 .. 2239  =  78 bp
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
6xHN
1717 .. 1752  =  36 bp
12 amino acids  =  1.5 kDa
Product: 6x(His-Asn) affinity tag
6xHN
1717 .. 1752  =  36 bp
12 amino acids  =  1.5 kDa
Product: 6x(His-Asn) affinity tag
lac operator
1806 .. 1830  =  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
1806 .. 1830  =  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
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
1831 .. 1849  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1831 .. 1849  =  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
RBS
1770 .. 1775  =  6 bp
ribosome binding site
RBS
1770 .. 1775  =  6 bp
ribosome binding site
ATG
1759 .. 1761  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1759 .. 1761  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  676 .. 1533  =  858 bp
ORF:  285 amino acids  =  33.2 kDa
ORF:  1726 .. 2490  =  765 bp
ORF:  254 amino acids  =  26.9 kDa
ORF:  3079 .. 3342  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5641 .. 5907  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  329 .. 565  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  2363 .. 3322  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3378 .. 3734  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4098 .. 4322  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  205 .. 1761  =  1557 bp
ORF:  518 amino acids  =  57.6 kDa
ORF:  1813 .. 2064  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1938 .. 2201  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  3105 .. 3356  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5511 .. 6371  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3731 .. 4099  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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