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pMCSG62

Bacterial vector encoding tRNA genes for rare Arg and Ile codons, for expressing a protein with a 6xHis-AviTag™-TEV leader plus the BirA biotin ligase.

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pMCSG62 Sequence and MappMCSG62.dna
Map and Sequence File   
Sequence Author:  Midwest Center for Structural Genomics
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 BspEI (2) ScaI (5174) FspI (4916) BglI (4814) BsaI (4755) AhdI (4694) DrdI (3909) PciI (3801) BspQI - SapI (3685) BstZ17I (3572) PshAI (3546) BsmBI (3316) HpaI (3207) EcoRV (3151) ApaI (2912) BlpI (80) 6xHis AvaI - BsoBI - PaeR7I - PspXI - XhoI (158) BmeT110I (159) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) BsmI (651) DraIII (1079) SspI (1247) TEV site Acc65I (1272) KpnI (1276) 6xHis ATG NdeI (1348) RBS XbaI (1386) lac operator T7 promoter BspDI * - ClaI * (1455) AgeI (1497) AscI (1714) SbfI (1725) BfuAI - BspMI (1743) MreI (1862) AfeI (1948) NcoI (2132) PsiI (2147) EcoNI (2236) PfoI (2268) PflMI (2283) BstAPI (2384) MluI (2701) BclI * (2715) BstEII (2882) PspOMI (2908) pMCSG62 5869 bp
BspEI  (2)
1 site
T C C G G A A G G C C T
ScaI  (5174)
1 site
A G T A C T T C A T G A
FspI  (4916)
1 site
T G C G C A A C G C G T
BglI  (4814)
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  (4755)
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  (4694)
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  (3909)
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  (3801)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3685)
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  (3685)
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  (3572)
1 site
G T A T A C C A T A T G
PshAI  (3546)
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  (3316)
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  (3207)
1 site
G T T A A C C A A T T G
EcoRV  (3151)
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  (2912)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°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.
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.
BsmI  (651)
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.
DraIII  (1079)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SspI  (1247)
1 site
A A T A T T T T A T A A
Acc65I  (1272)
1 site
G G T A C C C C A T G G
KpnI  (1276)
1 site
G G T A C C C C A T G G
NdeI  (1348)
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  (1386)
1 site
T C T A G A A G A T C T
BspDI  (1455)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1455)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (1497)
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  (1714)
1 site
G G C G C G C C C C G C G C G G
SbfI  (1725)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (1743)
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  (1743)
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  (1862)
1 site
C G C C G G C G G C G G C C G C
AfeI  (1948)
1 site
A G C G C T T C G C G A
NcoI  (2132)
1 site
C C A T G G G G T A C C
PsiI  (2147)
1 site
T T A T A A A A T A T T
EcoNI  (2236)
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  (2268)
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  (2283)
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  (2384)
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  (2701)
1 site
A C G C G T T G C G C A
BclI  (2715)
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  (2882)
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  (2908)
1 site
G G G C C C C C C G G G
lacI
2351 .. 3433  =  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
2351 .. 3433  =  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).
BirA
241 .. 1206  =  966 bp
321 amino acids  =  35.3 kDa
Product: E. coli biotin ligase
BirA
241 .. 1206  =  966 bp
321 amino acids  =  35.3 kDa
Product: E. coli biotin ligase
AmpR
4621 .. 5481  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4621 .. 5412  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4621 .. 5481  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5413 .. 5481  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4621 .. 5481  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3862 .. 4450  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3862 .. 4450  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
5482 .. 5585  =  104 bp
AmpR promoter
5482 .. 5585  =  104 bp
lacI promoter
2273 .. 2350  =  78 bp
lacI promoter
2273 .. 2350  =  78 bp
argU
1712 .. 1788  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
1712 .. 1788  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
2058 .. 2133  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
2058 .. 2133  =  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
AviTag™
1278 .. 1322  =  45 bp
15 amino acids  =  1.8 kDa
Product: peptide tag that allows for enzymatic
biotinylation
AviTag™
1278 .. 1322  =  45 bp
15 amino acids  =  1.8 kDa
Product: peptide tag that allows for enzymatic
biotinylation
lac operator
1394 .. 1418  =  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
1394 .. 1418  =  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
1251 .. 1271  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
1251 .. 1271  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
1419 .. 1437  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1419 .. 1437  =  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
1329 .. 1346  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1329 .. 1346  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
1215 .. 1220  =  6 bp
ribosome binding site
RBS
1215 .. 1220  =  6 bp
ribosome binding site
RBS
1358 .. 1363  =  6 bp
ribosome binding site
RBS
1358 .. 1363  =  6 bp
ribosome binding site
ATG
1347 .. 1349  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1347 .. 1349  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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