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Plasmid Files

pMCS5

E. coli cloning vector with an extensive multiple cloning site containing 59 unique restriction sites.

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pMCS5 Sequence and MappMCS5.dna
Map and Sequence File   
Sequence Author:  MoBiTec
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 PstI - SbfI (211) SphI (205) BfuAI - BspMI (200) HindIII (195) RsrII (189) AvrII (182) I-SceI (180) PsiI (2973) DraIII (2848) BtgZI (2840) XmnI (2295) ScaI (2176) TatI (2174) NmeAIII (1844) BpmI (1766) BsaI (1757) AhdI (1696) SalI (213) AccI (214) XbaI (219) BamHI (225) PmlI (233) TspMI - XmaI (237) SmaI - SrfI (239) PmeI (247) MfeI (252) BstBI (259) BsiWI (264) NruI (272) PaeR7I - PspXI - XhoI (276) SnaBI (284) Acc65I (288) KpnI (292) Eco53kI (296) SacI (298) EcoRI (300) EagI - NotI (307) SacII (313) PspOMI (314) ApaI (318) NdeI (320) SfiI (322) AgeI - SgrAI (329) FseI (341) BglII (344) NheI (350) BmtI (354) SpeI (356) KasI (362) NarI (363) SfoI (364) PluTI (366) MluI (368) EcoRV (376) NsiI (384) HpaI (388) BspDI * - ClaI * (393) NcoI (398) AscI - BssHII (404) PacI (415) SwaI (422) lac operator BspQI - SapI (687) PciI (803) BseYI (1107) PspFI (1111) AlwNI (1219) pMCS5 3081 bp
PstI  (211)
1 site
C T G C A G G A C G T C
SbfI  (211)
1 site
C C T G C A G G G G A C G T C C
SphI  (205)
1 site
G C A T G C C G T A C G
BfuAI  (200)
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  (200)
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.
HindIII  (195)
1 site
A A G C T T T T C G A A
RsrII  (189)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
AvrII  (182)
1 site
C C T A G G G G A T C C
I-SceI  (180)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its
electrophoretic mobility.
PsiI  (2973)
1 site
T T A T A A A A T A T T
DraIII  (2848)
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.
BtgZI  (2840)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
XmnI  (2295)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2176)
1 site
A G T A C T T C A T G A
TatI  (2174)
1 site
W G T A C W W C A T G W
NmeAIII  (1844)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BpmI  (1766)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BsaI  (1757)
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  (1696)
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.
SalI  (213)
1 site
G T C G A C C A G C T G
AccI  (214)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
XbaI  (219)
1 site
T C T A G A A G A T C T
BamHI  (225)
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.
PmlI  (233)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
TspMI  (237)
1 site
C C C G G G G G G C C C
XmaI  (237)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (239)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (239)
1 site
G C C C G G G C C G G G C C C G
PmeI  (247)
1 site
G T T T A A A C C A A A T T T G
MfeI  (252)
1 site
C A A T T G G T T A A C
BstBI  (259)
1 site
T T C G A A A A G C T T
BsiWI  (264)
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.
NruI  (272)
1 site
T C G C G A A G C G C T
PaeR7I  (276)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (276)
1 site
V C T C G A G B B G A G C T C V
XhoI  (276)
1 site
C T C G A G G A G C T C
SnaBI  (284)
1 site
T A C G T A A T G C A T
Acc65I  (288)
1 site
G G T A C C C C A T G G
KpnI  (292)
1 site
G G T A C C C C A T G G
Eco53kI  (296)
1 site
G A G C T C C T C G A G
SacI  (298)
1 site
G A G C T C C T C G A G
EcoRI  (300)
1 site
G A A T T C C T T A A G
EagI  (307)
1 site
C G G C C G G C C G G C
NotI  (307)
1 site
G C G G C C G C C G C C G G C G
SacII  (313)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
PspOMI  (314)
1 site
G G G C C C C C C G G G
ApaI  (318)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
NdeI  (320)
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.
SfiI  (322)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
AgeI  (329)
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.
SgrAI  (329)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
FseI  (341)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BglII  (344)
1 site
A G A T C T T C T A G A
NheI  (350)
1 site
G C T A G C C G A T C G
BmtI  (354)
1 site
G C T A G C C G A T C G
SpeI  (356)
1 site
A C T A G T T G A T C A
KasI  (362)
1 site
G G C G C C C C G C G G
NarI  (363)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (364)
1 site
G G C G C C C C G C G G
PluTI  (366)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
MluI  (368)
1 site
A C G C G T T G C G C A
EcoRV  (376)
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.
NsiI  (384)
1 site
A T G C A T T A C G T A
HpaI  (388)
1 site
G T T A A C C A A T T G
BspDI  (393)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (393)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
NcoI  (398)
1 site
C C A T G G G G T A C C
AscI  (404)
1 site
G G C G C G C C C C G C G C G G
BssHII  (404)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
PacI  (415)
1 site
T T A A T T A A A A T T A A T T
SwaI  (422)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BspQI  (687)
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  (687)
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.
PciI  (803)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1107)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
PspFI  (1111)
1 site
C C C A G C G G G T C G
AlwNI  (1219)
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.
AmpR
1623 .. 2483  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1623 .. 2414  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1623 .. 2483  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2415 .. 2483  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1623 .. 2483  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
864 .. 1452  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
864 .. 1452  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
lacZα
3044 .. 466  =  504 bp
167 amino acids  =  18.7 kDa
Product: LacZα fragment of β-galactosidase
lacZα
3044 .. 466  =  504 bp
167 amino acids  =  18.7 kDa
Product: LacZα fragment of β-galactosidase
f1 ori
2615 .. 3043  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2615 .. 3043  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
AmpR promoter
2484 .. 2588  =  105 bp
AmpR promoter
2484 .. 2588  =  105 bp
lac promoter
510 .. 540  =  31 bp
   Segment 3:  -10  
   510 .. 516  =  7 bp
promoter for the E. coli lac operon
lac promoter
510 .. 540  =  31 bp
   Segment 2:  
   517 .. 534  =  18 bp
promoter for the E. coli lac operon
lac promoter
510 .. 540  =  31 bp
   Segment 1:  -35  
   535 .. 540  =  6 bp
promoter for the E. coli lac operon
lac promoter
510 .. 540  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
486 .. 502  =  17 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
486 .. 502  =  17 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).
MCS
168 .. 426  =  259 bp
multiple cloning site
MCS
168 .. 426  =  259 bp
multiple cloning site
T7 promoter
429 .. 447  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
429 .. 447  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 fwd
142 .. 158  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
142 .. 158  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
462 .. 478  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
462 .. 478  =  17 bp
common sequencing primer, one of multiple similar
variants
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