pMCSG19C (linearized)

Linearized bacterial expression vector for ligation-independent cloning (LIC), with an MBP-TVMV-6xHis-TEV leader  and inducible TVMV protease. See also pMCSG19B.

Sequence Author: Midwest Center for Structural Genomics

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6xHis ScaI (6554) AhdI (6074) PciI (5181) BspQI - SapI (5065) BstZ17I (4952) PflFI - Tth111I (4926) Bpu10I (4287) PpuMI (4187) FspAI (4162) PshAI (3925) HpaI (3586) EcoRV (3530) ApaI (3291) EagI - NotI (7415) SalI (7428) Eco53kI (7437) SacI (7439) BamHI (7447) End (7453) Start (0) Acc65I (25) KpnI (29) 6xHis BsrGI (102) BsmI (605) BmgBI (681) BglII (860) PsiI (871) BsiWI (929) RBS lac operator T7 promoter BspDI * - ClaI * (1330) SgrAI (1372) SphI (1528) ZraI (1532) AatII (1534) BseRI (1770) NsiI (1892) AgeI (1935) DraIII (2103) SpeI (2172) SacII (2291) SphI (2555) EcoNI (2615) PflMI (2662) BstEII (3261) PspOMI (3287) pMCSG19C 7453 bp
ScaI  (6554)
1 site
A G T A C T T C A T G A
AhdI  (6074)
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.
PciI  (5181)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5065)
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  (5065)
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  (4952)
1 site
G T A T A C C A T A T G
PflFI  (4926)
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  (4926)
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  (4287)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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  (4187)
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  (4162)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (3925)
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.
HpaI  (3586)
1 site
G T T A A C C A A T T G
EcoRV  (3530)
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  (3291)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EagI  (7415)
1 site
C G G C C G G C C G G C
NotI  (7415)
1 site
G C G G C C G C C G C C G G C G
SalI  (7428)
1 site
G T C G A C C A G C T G
Eco53kI  (7437)
1 site
G A G C T C C T C G A G
SacI  (7439)
1 site
G A G C T C C T C G A G
BamHI  (7447)
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.
End  (7453)
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
BsrGI  (102)
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.
BsmI  (605)
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  (681)
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  (860)
1 site
A G A T C T T C T A G A
PsiI  (871)
1 site
T T A T A A A A T A T T
BsiWI  (929)
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.
BspDI  (1330)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1330)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1372)
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  (1528)
2 sites
G C A T G C C G T A C G
ZraI  (1532)
1 site
G A C G T C C T G C A G
AatII  (1534)
1 site
G A C G T C C T G C A G
BseRI  (1770)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
NsiI  (1892)
1 site
A T G C A T T A C G T A
AgeI  (1935)
1 site
A C C G G T T G G C C A
DraIII  (2103)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SpeI  (2172)
1 site
A C T A G T T G A T C A
SacII  (2291)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
SphI  (2555)
2 sites
G C A T G C C G T A C G
EcoNI  (2615)
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  (2662)
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.
BstEII  (3261)
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  (3287)
1 site
G G G C C C C C C G G G
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
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
MBP
124 .. 1224  =  1101 bp
367 amino acids  =  40.3 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
124 .. 1224  =  1101 bp
367 amino acids  =  40.3 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
2730 .. 3812  =  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
2730 .. 3812  =  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).
bla(M)
6001 .. 6789  =  789 bp
262 amino acids  =  28.8 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
bla(M)
6001 .. 6789  =  789 bp
262 amino acids  =  28.8 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
TVMV protease
1708 .. 2418  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TVMV protease
1708 .. 2418  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
ori
5242 .. 5830  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5242 .. 5830  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
4621 .. 4812  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4621 .. 4812  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6862 .. 6965  =  104 bp
AmpR promoter
6862 .. 6965  =  104 bp
rrnB T1 terminator
2451 .. 2537  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
2451 .. 2537  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacI promoter
2652 .. 2729  =  78 bp
lacI promoter
2652 .. 2729  =  78 bp
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
   Segment 1:  
   1611 .. 1629  =  19 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
   Segment 2:  -35  
   1630 .. 1635  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
   Segment 3:  
   1636 .. 1652  =  17 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
   Segment 4:  -10  
   1653 .. 1658  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
   Segment 5:  
   1659 .. 1684  =  26 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1611 .. 1684  =  74 bp
5 segments
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
T7 terminator
7275 .. 7322  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
7275 .. 7322  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1269 .. 1293  =  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
1269 .. 1293  =  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
1294 .. 1312  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1294 .. 1312  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
7389 .. 7406  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
7389 .. 7406  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1233 .. 1238  =  6 bp
ribosome binding site
RBS
1233 .. 1238  =  6 bp
ribosome binding site
tet operator
1611 .. 1629  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1611 .. 1629  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1636 .. 1654  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1636 .. 1654  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  1 .. 885  =  885 bp
ORF:  294 amino acids  =  34.1 kDa  (no start codon)
ORF:  1105 .. 1557  =  453 bp
ORF:  150 amino acids  =  15.9 kDa
ORF:  1708 .. 2418  =  711 bp
ORF:  236 amino acids  =  26.6 kDa
ORF:  3868 .. 4224  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4588 .. 4812  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2285 .. 2530  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  2615 .. 2980  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3569 .. 3832  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6131 .. 6397  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2853 .. 3812  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  1 .. 1224  =  1224 bp
ORF:  408 amino acids  =  45.0 kDa
ORF:  1276 .. 1527  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1693 .. 2109  =  417 bp
ORF:  138 amino acids  =  16.2 kDa
ORF:  3595 .. 3846  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  6001 .. 6861  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  4221 .. 4589  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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