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Plasmid Files

pMCSG73 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC), with a NusA-TVMV-6xHis-Strep-Tag® II-TEV leader plus tRNA genes for rare Arg and Ile codons.

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pMCSG73 (linearized) Sequence and MappMCSG73 (linearized).dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 BspEI (6078) ScaI (5381) FspI (5123) BglI (5021) AhdI (4901) AlwNI (4424) DrdI (4116) PciI (4008) BspQI - SapI (3892) BstZ17I (3779) BsaAI (3760) PshAI (3753) BsmBI (3523) HpaI (3414) ApaI (3119) PspOMI (3115) BstEII (3089) MluI (2908) End (6209) Start (0) TEV site 6xHis TVMV site BglII (182) BsmI (226) MscI (314) StuI (367) ZraI (1425) AatII (1427) NdeI (1556) XbaI (1593) T7 promoter AgeI (1704) AscI (1921) SbfI (1932) MreI (2069) AfeI (2155) PsiI (2354) EcoNI (2443) PfoI (2475) PflMI (2490) pMCSG73 6209 bp
BspEI  (6078)
1 site
T C C G G A A G G C C T
ScaI  (5381)
1 site
A G T A C T T C A T G A
FspI  (5123)
1 site
T G C G C A A C G C G T
BglI  (5021)
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  (4901)
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.
AlwNI  (4424)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
DrdI  (4116)
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  (4008)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3892)
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  (3892)
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  (3779)
1 site
G T A T A C C A T A T G
BsaAI  (3760)
1 site
Y A C G T R R T G C A Y
PshAI  (3753)
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  (3523)
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.
HpaI  (3414)
1 site
G T T A A C C A A T T G
ApaI  (3119)
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  (3115)
1 site
G G G C C C C C C G G G
BstEII  (3089)
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.
MluI  (2908)
1 site
A C G C G T T G C G C A
End  (6209)
0 sites
Start  (0)
0 sites
BglII  (182)
1 site
A G A T C T T C T A G A
BsmI  (226)
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.
MscI  (314)
1 site
T G G C C A A C C G G T
StuI  (367)
1 site
A G G C C T T C C G G A
ZraI  (1425)
1 site
G A C G T C C T G C A G
AatII  (1427)
1 site
G A C G T C C T G C A G
NdeI  (1556)
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  (1593)
1 site
T C T A G A A G A T C T
AgeI  (1704)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
AscI  (1921)
1 site
G G C G C G C C C C G C G C G G
SbfI  (1932)
1 site
C C T G C A G G G G A C G T C C
MreI  (2069)
1 site
C G C C G G C G G C G G C C G C
AfeI  (2155)
1 site
A G C G C T T C G C G A
PsiI  (2354)
1 site
T T A T A A A A T A T T
EcoNI  (2443)
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.
PfoI  (2475)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflMI  (2490)
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.
NusA
97 .. 1557  =  1461 bp
487 amino acids  =  54.0 kDa
Product: transcription elongation protein (N
utilization substance protein A)
highly soluble in E. coli
NusA
97 .. 1557  =  1461 bp
487 amino acids  =  54.0 kDa
Product: transcription elongation protein (N
utilization substance protein A)
highly soluble in E. coli
lacI
2558 .. 3640  =  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
2558 .. 3640  =  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
4828 .. 5688  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4828 .. 5619  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4828 .. 5688  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5620 .. 5688  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4828 .. 5688  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4069 .. 4657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4069 .. 4657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
5689 .. 5792  =  104 bp
AmpR promoter
5689 .. 5792  =  104 bp
lacI promoter
2480 .. 2557  =  78 bp
lacI promoter
2480 .. 2557  =  78 bp
argU
1919 .. 1995  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1919 .. 1995  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
2265 .. 2340  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
2265 .. 2340  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
6102 .. 6149  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
6102 .. 6149  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
1601 .. 1625  =  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
1601 .. 1625  =  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).
Strep-Tag II
25 .. 48  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
Strep-Tag II
25 .. 48  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin®, an
engineered form of streptavidin
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
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
TVMV site
67 .. 87  =  21 bp
7 amino acids  =  866.0 Da
Product:
tobacco vein mottling virus (TVMV) NIa
protease recognition and cleavage site
T7 promoter
1626 .. 1644  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1626 .. 1644  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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
RBS
1566 .. 1571  =  6 bp
ribosome binding site
RBS
1566 .. 1571  =  6 bp
ribosome binding site
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