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Plasmid Files

LITMUS28

High copy-number E. coli vector with a multiple cloning site flanked by mutant T7 promoters. For wild-type T7 promoters, use LITMUS28i.

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LITMUS28 Sequence and MapLITMUS28.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 PvuII (2710) AflII (2603) StuI (2601) KpnI (2588) Acc65I (2584) SacI (2581) Eco53kI (2579) AvrII (2570) XbaI (2562) AgeI (2556) AatII (2554) ZraI (2552) BtgI - NcoI (2543) HindIII (2536) BamHI (2527) EcoRV (2522) PstI - SbfI (2518) EcoRI (2508) BsmBI (2505) PaeR7I - XhoI (2502) BsiWI (2495) BssHII (2487) BsmI - NsiI (2484) BstBI (2476) BglII (2470) SpeI (2460) SnaBI (2447) NspI (2313) AflIII - PciI (2309) PspFI (2009) BseYI (2005) AlwNI (1900) HpaI (3) TatI (447) ScaI (449) AseI (755) NmeAIII (783) BpmI (861) BsaI (864) SwaI (1102) PsiI (1199) DraIII (1327) LITMUS28 2823 bp
PvuII  (2710)
1 site
C A G C T G G T C G A C
AflII  (2603)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
StuI  (2601)
1 site
A G G C C T T C C G G A
KpnI  (2588)
1 site
G G T A C C C C A T G G
Acc65I  (2584)
1 site
G G T A C C C C A T G G
SacI  (2581)
1 site
G A G C T C C T C G A G
Eco53kI  (2579)
1 site
G A G C T C C T C G A G
AvrII  (2570)
1 site
C C T A G G G G A T C C
XbaI  (2562)
1 site
T C T A G A A G A T C T
AgeI  (2556)
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.
AatII  (2554)
1 site
G A C G T C C T G C A G
ZraI  (2552)
1 site
G A C G T C C T G C A G
BtgI  (2543)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (2543)
1 site
C C A T G G G G T A C C
HindIII  (2536)
1 site
A A G C T T T T C G A A
BamHI  (2527)
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.
EcoRV  (2522)
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.
PstI  (2518)
1 site
C T G C A G G A C G T C
SbfI  (2518)
1 site
C C T G C A G G G G A C G T C C
EcoRI  (2508)
1 site
G A A T T C C T T A A G
BsmBI  (2505)
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.
PaeR7I  (2502)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2502)
1 site
C T C G A G G A G C T C
BsiWI  (2495)
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.
BssHII  (2487)
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.
BsmI  (2484)
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.
NsiI  (2484)
1 site
A T G C A T T A C G T A
BstBI  (2476)
1 site
T T C G A A A A G C T T
BglII  (2470)
1 site
A G A T C T T C T A G A
SpeI  (2460)
1 site
A C T A G T T G A T C A
SnaBI  (2447)
1 site
T A C G T A A T G C A T
NspI  (2313)
1 site
R C A T G Y Y G T A C R
AflIII  (2309)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2309)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2009)
1 site
C C C A G C G G G T C G
BseYI  (2005)
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.
AlwNI  (1900)
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.
HpaI  (3)
1 site
G T T A A C C A A T T G
TatI  (447)
1 site
W G T A C W W C A T G W
ScaI  (449)
1 site
A G T A C T T C A T G A
AseI  (755)
1 site
A T T A A T T A A T T A
NmeAIII  (783)
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  (861)
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  (864)
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.
SwaI  (1102)
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.
PsiI  (1199)
1 site
T T A T A A A A T A T T
DraIII  (1327)
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.
AmpR
143 .. 1003  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   143 .. 211  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
143 .. 1003  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   212 .. 1003  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
143 .. 1003  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1665 .. 2253  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1665 .. 2253  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
M13 ori
1045 .. 1554  =  510 bp
M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
M13 ori
1045 .. 1554  =  510 bp
M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
lacZα
2424 .. 2795  =  372 bp
123 amino acids  =  14.3 kDa
Product: LacZα fragment of β-galactosidase
lacZα
2424 .. 2795  =  372 bp
123 amino acids  =  14.3 kDa
Product: LacZα fragment of β-galactosidase
AmpR promoter
38 .. 142  =  105 bp
AmpR promoter
38 .. 142  =  105 bp
lac promoter
2350 .. 2380  =  31 bp
   Segment 1:  -35  
   2350 .. 2355  =  6 bp
promoter for the E. coli lac operon
lac promoter
2350 .. 2380  =  31 bp
   Segment 2:  
   2356 .. 2373  =  18 bp
promoter for the E. coli lac operon
lac promoter
2350 .. 2380  =  31 bp
   Segment 3:  -10  
   2374 .. 2380  =  7 bp
promoter for the E. coli lac operon
lac promoter
2350 .. 2380  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
2388 .. 2404  =  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
2388 .. 2404  =  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
2460 .. 2608  =  149 bp
multiple cloning site
MCS
2460 .. 2608  =  149 bp
multiple cloning site
M13 rev
2412 .. 2428  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2412 .. 2428  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
2624 .. 2640  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
2624 .. 2640  =  17 bp
common sequencing primer, one of multiple similar
variants
T7 promoter (mutant)
2449 .. 2466  =  18 bp
mutant version of the promoter for bacteriophage
T7 RNA polymerase
T7 promoter (mutant)
2449 .. 2466  =  18 bp
mutant version of the promoter for bacteriophage
T7 RNA polymerase
T7 promoter (mutant)
2603 .. 2620  =  18 bp
mutant version of the promoter for bacteriophage
T7 RNA polymerase
T7 promoter (mutant)
2603 .. 2620  =  18 bp
mutant version of the promoter for bacteriophage
T7 RNA polymerase
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