pMCSG63

Compact bacterial vector encoding tRNA genes for rare Arg and Ile codons, for expressing a protein with a 6xHis-TEV leader plus a second untagged protein.

Sequence Author: Midwest Center for Structural Genomics

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PaeR7I - PspXI - XhoI (158) BlpI (80) T7 terminator BspEI (2) ScaI (4169) PvuI (4059) FspI (3911) BglI (3809) BsaI (3750) AhdI (3689) AlwNI (3212) DrdI (2904) PciI (2796) BspQI - SapI (2680) BstZ17I (2567) BsaAI (2548) PshAI (2541) BsmBI - Esp3I (2311) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) TspMI - XmaI (232) SmaI (234) RBS SspI (275) TEV site Acc65I (300) KpnI (304) BglII (307) ATG NdeI (343) RBS XbaI (381) lac operator T7 promoter BspDI * - ClaI * (450) AgeI (492) AscI (709) SbfI (720) BstBI (725) BfuAI - BspMI (738) MreI (857) AfeI (943) NcoI (1127) PsiI (1142) EcoNI (1231) PfoI (1263) PflMI (1278) BstAPI (1379) MluI (1696) BclI * (1710) BstEII (1877) PspOMI (1903) ApaI (1907) EcoRV (2146) HpaI (2202) pMCSG63 4864 bp
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
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.
BspEI  (2)
1 site
T C C G G A A G G C C T
ScaI  (4169)
1 site
A G T A C T T C A T G A
PvuI  (4059)
1 site
C G A T C G G C T A G C
FspI  (3911)
1 site
T G C G C A A C G C G T
BglI  (3809)
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.
BsaI  (3750)
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  (3689)
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  (3212)
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  (2904)
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  (2796)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2680)
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  (2680)
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  (2567)
1 site
G T A T A C C A T A T G
BsaAI  (2548)
1 site
Y A C G T R R T G C A Y
PshAI  (2541)
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  (2311)
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  (2311)
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.
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.
TspMI  (232)
1 site
C C C G G G G G G C C C
XmaI  (232)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (234)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SspI  (275)
1 site
A A T A T T T T A T A A
Acc65I  (300)
1 site
G G T A C C C C A T G G
KpnI  (304)
1 site
G G T A C C C C A T G G
BglII  (307)
1 site
A G A T C T T C T A G A
NdeI  (343)
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  (381)
1 site
T C T A G A A G A T C T
BspDI  (450)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (450)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (492)
1 site
A C C G G T T G G C C A
AscI  (709)
1 site
G G C G C G C C C C G C G C G G
SbfI  (720)
1 site
C C T G C A G G G G A C G T C C
BstBI  (725)
1 site
T T C G A A A A G C T T
BfuAI  (738)
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  (738)
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  (857)
1 site
C G C C G G C G G C G G C C G C
AfeI  (943)
1 site
A G C G C T T C G C G A
NcoI  (1127)
1 site
C C A T G G G G T A C C
PsiI  (1142)
1 site
T T A T A A A A T A T T
EcoNI  (1231)
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  (1263)
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  (1278)
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  (1379)
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  (1696)
1 site
A C G C G T T G C G C A
BclI  (1710)
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  (1877)
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  (1903)
1 site
G G G C C C C C C G G G
ApaI  (1907)
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  (2146)
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  (2202)
1 site
G T T A A C C A A T T G
lacI
1346 .. 2428  =  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
1346 .. 2428  =  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
3616 .. 4476  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3616 .. 4407  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3616 .. 4476  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4408 .. 4476  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3616 .. 4476  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2857 .. 3445  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2857 .. 3445  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4477 .. 4580  =  104 bp
AmpR promoter
4477 .. 4580  =  104 bp
lacI promoter
1268 .. 1345  =  78 bp
lacI promoter
1268 .. 1345  =  78 bp
argU
707 .. 783  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
707 .. 783  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
1053 .. 1128  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
1053 .. 1128  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
TEV site
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
279 .. 299  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
324 .. 341  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
324 .. 341  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
342 .. 344  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
342 .. 344  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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
389 .. 413  =  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
389 .. 413  =  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
414 .. 432  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
414 .. 432  =  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
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
243 .. 248  =  6 bp
ribosome binding site
RBS
353 .. 358  =  6 bp
ribosome binding site
RBS
353 .. 358  =  6 bp
ribosome binding site
ORF:  1129 .. 1596  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  2185 .. 2448  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  842 .. 1096  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1469 .. 2428  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3746 .. 4012  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  324 .. 557  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
ORF:  3616 .. 4476  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2211 .. 2462  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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