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Plasmid Files

pMCSG52

Bacterial vector encoding tRNA genes for rare Arg and Ile codons, with a 6xHis-TEV leader for high-throughput purification of recombinant proteins.

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pMCSG52.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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ScaI (5114) PvuI (5004) BsaI (4695) AhdI (4634) AlwNI (4157) PciI (3741) BspQI - SapI (3625) BstZ17I (3512) BsaAI (3493) PflFI - Tth111I (3486) BlpI (80) AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) SspI (219) Acc65I (244) KpnI (248) BglII (251) NdeI (287) XbaI (325) BspDI * - ClaI * (394) AgeI (436) AscI (653) SbfI (664) BstBI (669) BfuAI - BspMI (682) MreI (801) NcoI (1071) PsiI (1086) EcoNI (1175) PflMI (1222) BstAPI (1323) MluI (1640) BclI * (1654) BstEII (1821) PspOMI (1847) ApaI (1851) EcoRV (2090) HpaI (2146) PshAI (2485) FspAI (2722) PpuMI (2747) pMCSG52 5809 bp
ScaI  (5114)
1 site
A G T A C T T C A T G A
PvuI  (5004)
1 site
C G A T C G G C T A G C
BsaI  (4695)
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  (4634)
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  (4157)
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.
PciI  (3741)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3625)
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  (3625)
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  (3512)
1 site
G T A T A C C A T A T G
BsaAI  (3493)
1 site
Y A C G T R R T G C A Y
PflFI  (3486)
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  (3486)
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.
BlpI  (80)
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.
AvaI  (158)
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  (158)
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  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
BmeT110I  (159)
1 site
C Y C G R G G R G C Y C
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
HindIII  (173)
1 site
A A G C T T T T C G A A
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
EcoRI  (192)
1 site
G A A T T C C T T A A G
BamHI  (198)
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.
SspI  (219)
1 site
A A T A T T T T A T A A
Acc65I  (244)
1 site
G G T A C C C C A T G G
KpnI  (248)
1 site
G G T A C C C C A T G G
BglII  (251)
1 site
A G A T C T T C T A G A
NdeI  (287)
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  (325)
1 site
T C T A G A A G A T C T
BspDI  (394)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (394)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (436)
1 site
A C C G G T T G G C C A
AscI  (653)
1 site
G G C G C G C C C C G C G C G G
SbfI  (664)
1 site
C C T G C A G G G G A C G T C C
BstBI  (669)
1 site
T T C G A A A A G C T T
BfuAI  (682)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (682)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
MreI  (801)
1 site
C G C C G G C G G C G G C C G C
NcoI  (1071)
1 site
C C A T G G G G T A C C
PsiI  (1086)
1 site
T T A T A A A A T A T T
EcoNI  (1175)
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  (1222)
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  (1323)
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.
MluI  (1640)
1 site
A C G C G T T G C G C A
BclI  (1654)
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  (1821)
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  (1847)
1 site
G G G C C C C C C G G G
ApaI  (1851)
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  (2090)
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  (2146)
1 site
G T T A A C C A A T T G
PshAI  (2485)
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.
FspAI  (2722)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2747)
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.
lacI
1290 .. 2372  =  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
1290 .. 2372  =  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
4561 .. 5421  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4561 .. 5352  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4561 .. 5421  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5353 .. 5421  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4561 .. 5421  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3802 .. 4390  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3802 .. 4390  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
3181 .. 3372  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
3181 .. 3372  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
5422 .. 5525  =  104 bp
AmpR promoter
5422 .. 5525  =  104 bp
lacI promoter
1212 .. 1289  =  78 bp
lacI promoter
1212 .. 1289  =  78 bp
argU
651 .. 727  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
651 .. 727  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
997 .. 1072  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
997 .. 1072  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
333 .. 357  =  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
333 .. 357  =  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).
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
223 .. 243  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
358 .. 376  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
358 .. 376  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
268 .. 285  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
297 .. 302  =  6 bp
ribosome binding site
RBS
297 .. 302  =  6 bp
ribosome binding site
ATG
286 .. 288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
286 .. 288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  268 .. 501  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
ORF:  2428 .. 2784  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3148 .. 3372  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  1073 .. 1540  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  2129 .. 2392  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4691 .. 4957  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  786 .. 1040  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1413 .. 2372  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2155 .. 2406  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4561 .. 5421  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2781 .. 3149  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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