pMCSG41 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a C-terminal TEV-6xHis cassette plus TVMV protease.

Sequence Author: Midwest Center for Structural Genomics

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T7 terminator ScaI (5330) PvuI (5220) BsaI (4911) AhdI (4850) PciI (3957) BspQI - SapI (3841) BstZ17I (3728) BsaAI (3709) PflFI - Tth111I (3702) Bpu10I (3063) PpuMI (2963) FspAI (2938) BlpI (6105) 6xHis EagI - NotI (6191) SalI (6204) Eco53kI (6213) SacI (6215) BamHI (6223) 6xHis End (6270) Start (0) RBS lac operator T7 promoter BspDI * - ClaI * (106) SgrAI (148) SphI (304) ZraI (308) AatII (310) NdeI (482) BseRI (546) NsiI (668) AgeI (711) DraIII (879) SpeI (948) SacII (1067) NcoI (1203) SphI (1331) EcoNI (1391) PflMI (1438) BstAPI (1539) BclI * (1870) BstEII (2037) PspOMI (2063) ApaI (2067) BssHII (2267) EcoRV (2306) HpaI (2362) PshAI (2701) pMCSG41 6270 bp
ScaI  (5330)
1 site
A G T A C T T C A T G A
PvuI  (5220)
1 site
C G A T C G G C T A G C
BsaI  (4911)
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  (4850)
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  (3957)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3841)
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  (3841)
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  (3728)
1 site
G T A T A C C A T A T G
BsaAI  (3709)
1 site
Y A C G T R R T G C A Y
PflFI  (3702)
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  (3702)
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  (3063)
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  (2963)
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  (2938)
1 site
R T G C G C A Y Y A C G C G T R
BlpI  (6105)
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.
EagI  (6191)
1 site
C G G C C G G C C G G C
NotI  (6191)
1 site
G C G G C C G C C G C C G G C G
SalI  (6204)
1 site
G T C G A C C A G C T G
Eco53kI  (6213)
1 site
G A G C T C C T C G A G
SacI  (6215)
1 site
G A G C T C C T C G A G
BamHI  (6223)
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  (6270)
0 sites
Start  (0)
0 sites
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.
SgrAI  (148)
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  (304)
2 sites
G C A T G C C G T A C G
ZraI  (308)
1 site
G A C G T C C T G C A G
AatII  (310)
1 site
G A C G T C C T G C A G
NdeI  (482)
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.
BseRI  (546)
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  (668)
1 site
A T G C A T T A C G T A
AgeI  (711)
1 site
A C C G G T T G G C C A
DraIII  (879)
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  (948)
1 site
A C T A G T T G A T C A
SacII  (1067)
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.
NcoI  (1203)
1 site
C C A T G G G G T A C C
SphI  (1331)
2 sites
G C A T G C C G T A C G
EcoNI  (1391)
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  (1438)
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  (1539)
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.
BclI  (1870)
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  (2037)
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  (2063)
1 site
G G G C C C C C C G G G
ApaI  (2067)
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  (2267)
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  (2306)
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  (2362)
1 site
G T T A A C C A A T T G
PshAI  (2701)
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.
lacI
1506 .. 2588  =  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
1506 .. 2588  =  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
4777 .. 5637  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4777 .. 5568  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4777 .. 5637  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5569 .. 5637  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4777 .. 5637  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TVMV protease
484 .. 1194  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TVMV protease
484 .. 1194  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
ori
4018 .. 4606  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4018 .. 4606  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
3397 .. 3588  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
3397 .. 3588  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
5638 .. 5741  =  104 bp
AmpR promoter
5638 .. 5741  =  104 bp
rrnB T1 terminator
1227 .. 1313  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
1227 .. 1313  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacI promoter
1428 .. 1505  =  78 bp
lacI promoter
1428 .. 1505  =  78 bp
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
   Segment 1:  
   387 .. 405  =  19 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
   Segment 2:  -35  
   406 .. 411  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
   Segment 3:  
   412 .. 428  =  17 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
   Segment 4:  -10  
   429 .. 434  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
   Segment 5:  
   435 .. 460  =  26 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
387 .. 460  =  74 bp
5 segments
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
T7 terminator
6051 .. 6098  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
6051 .. 6098  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
6xHis
6233 .. 6250  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6233 .. 6250  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
6257 .. 6277  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
6257 .. 6277  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
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).
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
6165 .. 6182  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6165 .. 6182  =  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
tet operator
387 .. 405  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
387 .. 405  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
412 .. 430  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
412 .. 430  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  1 .. 333  =  333 bp
ORF:  110 amino acids  =  11.2 kDa  (no start codon)
ORF:  484 .. 1194  =  711 bp
ORF:  236 amino acids  =  26.6 kDa
ORF:  2644 .. 3000  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3364 .. 3588  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  1061 .. 1306  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  1391 .. 1756  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  2345 .. 2608  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4907 .. 5173  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1629 .. 2588  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  52 .. 303  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  469 .. 885  =  417 bp
ORF:  138 amino acids  =  16.2 kDa
ORF:  2371 .. 2622  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4777 .. 5637  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2997 .. 3365  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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