pMCSG34 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), encoding N-terminal MBP-TVMV and C-terminal TEV-6xHis plus TVMV protease. See also pMCSG34B.

Sequence Author: Midwest Center for Structural Genomics

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EagI - NotI (7356) ScaI (6495) AhdI (6015) PciI (5122) BspQI - SapI (5006) BstZ17I (4893) PflFI - Tth111I (4867) Bpu10I (4228) PpuMI (4128) FspAI (4103) PshAI (3866) HpaI (3527) EcoRV (3471) ApaI (3232) SalI (7369) Eco53kI (7378) SacI (7380) BamHI (7388) TEV site End (7435) Start (0) BsrGI (43) BsmI (546) BmgBI (622) BglII (801) PsiI (812) BsiWI (870) RBS T7 promoter BspDI * - ClaI * (1271) SgrAI (1313) SphI (1469) ZraI (1473) AatII (1475) BseRI (1711) NsiI (1833) AgeI (1876) DraIII (2044) SpeI (2113) SacII (2232) SphI (2496) EcoNI (2556) PflMI (2603) BstEII (3202) PspOMI (3228) pMCSG34 7435 bp
EagI  (7356)
1 site
C G G C C G G C C G G C
NotI  (7356)
1 site
G C G G C C G C C G C C G G C G
ScaI  (6495)
1 site
A G T A C T T C A T G A
AhdI  (6015)
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  (5122)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5006)
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  (5006)
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  (4893)
1 site
G T A T A C C A T A T G
PflFI  (4867)
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  (4867)
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  (4228)
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  (4128)
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  (4103)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (3866)
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  (3527)
1 site
G T T A A C C A A T T G
EcoRV  (3471)
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  (3232)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SalI  (7369)
1 site
G T C G A C C A G C T G
Eco53kI  (7378)
1 site
G A G C T C C T C G A G
SacI  (7380)
1 site
G A G C T C C T C G A G
BamHI  (7388)
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  (7435)
0 sites
Start  (0)
0 sites
BsrGI  (43)
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  (546)
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  (622)
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  (801)
1 site
A G A T C T T C T A G A
PsiI  (812)
1 site
T T A T A A A A T A T T
BsiWI  (870)
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  (1271)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1271)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (1313)
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  (1469)
2 sites
G C A T G C C G T A C G
ZraI  (1473)
1 site
G A C G T C C T G C A G
AatII  (1475)
1 site
G A C G T C C T G C A G
BseRI  (1711)
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  (1833)
1 site
A T G C A T T A C G T A
AgeI  (1876)
1 site
A C C G G T T G G C C A
DraIII  (2044)
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  (2113)
1 site
A C T A G T T G A T C A
SacII  (2232)
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  (2496)
2 sites
G C A T G C C G T A C G
EcoNI  (2556)
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  (2603)
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  (3202)
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  (3228)
1 site
G G G C C C C C C G G G
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
TVMV site
8 .. 28  =  21 bp
7 amino acids  =  865.9 Da
Product:
tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site
MBP
65 .. 1165  =  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
65 .. 1165  =  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
2671 .. 3753  =  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
2671 .. 3753  =  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
5942 .. 6802  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   5942 .. 6733  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5942 .. 6802  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   6734 .. 6802  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5942 .. 6802  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TVMV protease
1649 .. 2359  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
TVMV protease
1649 .. 2359  =  711 bp
236 amino acids  =  26.6 kDa
Product: tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)
ori
5183 .. 5771  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5183 .. 5771  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
4562 .. 4753  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4562 .. 4753  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
6803 .. 6906  =  104 bp
AmpR promoter
6803 .. 6906  =  104 bp
rrnB T1 terminator
2392 .. 2478  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
2392 .. 2478  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacI promoter
2593 .. 2670  =  78 bp
lacI promoter
2593 .. 2670  =  78 bp
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
   Segment 1:  
   1552 .. 1570  =  19 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
   Segment 2:  -35  
   1571 .. 1576  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
   Segment 3:  
   1577 .. 1593  =  17 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
   Segment 4:  -10  
   1594 .. 1599  =  6 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
   Segment 5:  
   1600 .. 1625  =  26 bp
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
PLtetO-1 promoter
1552 .. 1625  =  74 bp
5 segments
modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)
T7 terminator
7216 .. 7263  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
7216 .. 7263  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
6xHis
7398 .. 7415  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
7398 .. 7415  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
7422 .. 7442  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
7422 .. 7442  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
lac operator
1210 .. 1234  =  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
1210 .. 1234  =  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
1235 .. 1253  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1235 .. 1253  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
7330 .. 7347  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
7330 .. 7347  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1174 .. 1179  =  6 bp
ribosome binding site
RBS
1174 .. 1179  =  6 bp
ribosome binding site
tet operator
1552 .. 1570  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1552 .. 1570  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1577 .. 1595  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
1577 .. 1595  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  2794 .. 3753  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2 .. 826  =  825 bp
ORF:  274 amino acids  =  31.8 kDa  (no start codon)
ORF:  1046 .. 1498  =  453 bp
ORF:  150 amino acids  =  15.9 kDa
ORF:  1649 .. 2359  =  711 bp
ORF:  236 amino acids  =  26.6 kDa
ORF:  3809 .. 4165  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4529 .. 4753  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2226 .. 2471  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  2556 .. 2921  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3510 .. 3773  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6072 .. 6338  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2 .. 1165  =  1164 bp
ORF:  388 amino acids  =  42.6 kDa
ORF:  1217 .. 1468  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1634 .. 2050  =  417 bp
ORF:  138 amino acids  =  16.2 kDa
ORF:  3536 .. 3787  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5942 .. 6802  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  4162 .. 4530  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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