pMCSG69 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with an MBP-TVMV-6xHis-TEV leader plus tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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AvaI - BsoBI - PaeR7I - PspXI - XhoI (5902) ScaI (5049) FspI (4791) BglI (4689) AhdI (4569) DrdI (3784) PciI (3676) BspQI - SapI (3560) BstZ17I (3447) PshAI (3421) BsmBI - Esp3I (3191) HpaI (3082) EcoRV (3026) ApaI (2787) PspOMI (2783) BstEII (2757) EagI - NotI (5910) HindIII (5917) SalI (5923) Eco53kI (5932) SacI (5934) EcoRI (5936) BamHI (5942) End (5948) Start (0) Acc65I (25) KpnI (29) 6xHis TVMV site BsrGI (102) BsmI (605) BmgBI (681) BglII (860) BsiWI (929) NdeI (1223) RBS XbaI (1261) T7 promoter BspDI * - ClaI * (1330) AgeI (1372) AscI (1589) SbfI (1600) BstBI (1605) MreI (1737) EcoNI (2111) PflMI (2158) MluI (2576) pMCSG69 5948 bp
AvaI  (5902)
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  (5902)
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  (5902)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5902)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5902)
1 site
C T C G A G G A G C T C
ScaI  (5049)
1 site
A G T A C T T C A T G A
FspI  (4791)
1 site
T G C G C A A C G C G T
BglI  (4689)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
AhdI  (4569)
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.
DrdI  (3784)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PciI  (3676)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3560)
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  (3560)
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  (3447)
1 site
G T A T A C C A T A T G
PshAI  (3421)
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.
BsmBI  (3191)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (3191)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
HpaI  (3082)
1 site
G T T A A C C A A T T G
EcoRV  (3026)
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  (2787)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (2783)
1 site
G G G C C C C C C G G G
BstEII  (2757)
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.
EagI  (5910)
1 site
C G G C C G G C C G G C
NotI  (5910)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5917)
1 site
A A G C T T T T C G A A
SalI  (5923)
1 site
G T C G A C C A G C T G
Eco53kI  (5932)
1 site
G A G C T C C T C G A G
SacI  (5934)
1 site
G A G C T C C T C G A G
EcoRI  (5936)
1 site
G A A T T C C T T A A G
BamHI  (5942)
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  (5948)
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
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.
NdeI  (1223)
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  (1261)
1 site
T C T A G A A G A T C T
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.
AgeI  (1372)
1 site
A C C G G T T G G C C A
AscI  (1589)
1 site
G G C G C G C C C C G C G C G G
SbfI  (1600)
1 site
C C T G C A G G G G A C G T C C
BstBI  (1605)
1 site
T T C G A A A A G C T T
MreI  (1737)
1 site
C G C C G G C G G C G G C C G C
EcoNI  (2111)
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  (2158)
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  (2576)
1 site
A C G C G T T G C G C A
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
2226 .. 3308  =  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
2226 .. 3308  =  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
4496 .. 5356  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4496 .. 5287  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4496 .. 5356  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5288 .. 5356  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4496 .. 5356  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3737 .. 4325  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3737 .. 4325  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
5357 .. 5460  =  104 bp
AmpR promoter
5357 .. 5460  =  104 bp
lacI promoter
2148 .. 2225  =  78 bp
lacI promoter
2148 .. 2225  =  78 bp
argU
1587 .. 1663  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1587 .. 1663  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
1933 .. 2008  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
1933 .. 2008  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
5770 .. 5817  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5770 .. 5817  =  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
5884 .. 5901  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5884 .. 5901  =  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
ORF:  1 .. 885  =  885 bp
ORF:  294 amino acids  =  34.1 kDa  (no start codon)
ORF:  1105 .. 1437  =  333 bp
ORF:  110 amino acids  =  12.3 kDa
ORF:  2009 .. 2476  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  3065 .. 3328  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  1722 .. 1976  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  2349 .. 3308  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4626 .. 4892  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  4496 .. 5356  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  1 .. 1224  =  1224 bp
ORF:  408 amino acids  =  45.0 kDa
ORF:  3091 .. 3342  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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