pMCSG30 (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a 6xHis-TEV-MBP leader and a SacB negative selection marker that is lost during cloning.

Sequence Author: Midwest Center for Structural Genomics

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AvaI - BmeT110I - BsoBI - PaeR7I - PspXI - XhoI (6339) ScaI (5486) PstI (5251) AhdI (5006) BspQI - SapI (3997) PflFI - Tth111I (3858) PpuMI (3119) FspAI (3094) PshAI (2857) EagI - NotI (6347) SalI (6360) Eco53kI (6369) SacI (6371) BamHI (6379) End (6411) Start (0) NcoI (148) BsmI (495) BmgBI (571) AanI (761) BsiWI (819) TEV site 6xHis ATG NdeI (1182) RBS XbaI (1220) T7 promoter SgrAI (1331) SphI (1487) EcoNI (1547) PflMI (1594) MluI (2012) BstEII (2193) PspOMI (2219) ApaI (2223) EcoRV (2462) pMCSG30 6411 bp
AvaI  (6339)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BmeT110I  (6339)
1 site
C Y C G R G G R G C Y C
BsoBI  (6339)
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  (6339)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6339)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6339)
1 site
C T C G A G G A G C T C
ScaI  (5486)
1 site
A G T A C T T C A T G A
PstI  (5251)
1 site
C T G C A G G A C G T C
AhdI  (5006)
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.
BspQI  (3997)
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  (3997)
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.
PflFI  (3858)
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  (3858)
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.
PpuMI  (3119)
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  (3094)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (2857)
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.
EagI  (6347)
1 site
C G G C C G G C C G G C
NotI  (6347)
1 site
G C G G C C G C C G C C G G C G
SalI  (6360)
1 site
G T C G A C C A G C T G
Eco53kI  (6369)
1 site
G A G C T C C T C G A G
SacI  (6371)
1 site
G A G C T C C T C G A G
BamHI  (6379)
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  (6411)
0 sites
Start  (0)
0 sites
NcoI  (148)
1 site
C C A T G G G G T A C C
BsmI  (495)
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  (571)
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.
AanI  (761)
1 site
T T A T A A A A T A T T
BsiWI  (819)
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.
NdeI  (1182)
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  (1220)
1 site
T C T A G A A G A T C T
SgrAI  (1331)
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  (1487)
1 site
G C A T G C C G T A C G
EcoNI  (1547)
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  (1594)
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  (2012)
1 site
A C G C G T T G C G C A
BstEII  (2193)
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  (2219)
1 site
G G G C C C C C C G G G
ApaI  (2223)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (2462)
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.
MBP
38 .. 1111  =  1074 bp
358 amino acids  =  39.4 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
38 .. 1111  =  1074 bp
358 amino acids  =  39.4 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.
TEV site
1118 .. 1138  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1118 .. 1138  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
1163 .. 1180  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1163 .. 1180  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
1181 .. 1183  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1181 .. 1183  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lacI
1662 .. 2744  =  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
1662 .. 2744  =  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
4933 .. 5793  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4933 .. 5724  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4933 .. 5793  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5725 .. 5793  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4933 .. 5793  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
4174 .. 4762  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4174 .. 4762  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
3553 .. 3744  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
3553 .. 3744  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
5794 .. 5897  =  104 bp
AmpR promoter
5794 .. 5897  =  104 bp
lacI promoter
1584 .. 1661  =  78 bp
lacI promoter
1584 .. 1661  =  78 bp
T7 terminator
6207 .. 6254  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
6207 .. 6254  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1228 .. 1252  =  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
1228 .. 1252  =  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
1253 .. 1271  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1253 .. 1271  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
6321 .. 6338  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6321 .. 6338  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1192 .. 1197  =  6 bp
ribosome binding site
RBS
1192 .. 1197  =  6 bp
ribosome binding site
ORF:  2800 .. 3156  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3520 .. 3744  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2 .. 775  =  774 bp
ORF:  257 amino acids  =  29.6 kDa  (no start codon)
ORF:  995 .. 1912  =  918 bp
ORF:  305 amino acids  =  32.8 kDa
ORF:  2501 .. 2764  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5063 .. 5329  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1785 .. 2744  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2 .. 1183  =  1182 bp
ORF:  394 amino acids  =  43.1 kDa
ORF:  1235 .. 1486  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  1360 .. 1623  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  2527 .. 2778  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4933 .. 5793  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3153 .. 3521  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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