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Plasmid Files

pRHA-113

pBR322-derived low copy number plasmid encoding the rhaB promoter plus rhaR and rhaS genes, for rhamnose-inducible protein expression in E. coli.

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pRHA-113 Sequence and MappRHA-113.dna
Map and Sequence File   
Sequence Author:  Xbrane Bioscience
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 AatII (5764) ZraI (5762) ScaI (5322) PstI (5087) BsaI (4903) PciI (3949) BspQI - SapI (3833) NdeI (3772) BstZ17I (3722) PflFI - Tth111I (3696) BspEI * (3140) FspAI (2932) MscI * (2922) HindIII (29) PacI (37) TspMI - XmaI (90) SmaI (92) BstXI (240) NheI (322) BmtI (326) XcmI (979) BstEII - DraIII (999) BbvCI (1145) AflII (1169) BmgBI (1281) BglII (1351) AarI (1434) AgeI - SgrAI (1619) MluI (1750) SalI (2077) XbaI (2083) BamHI (2089) Acc65I (2122) KpnI (2126) PshAI (2192) EagI (2415) KasI (2681) NarI (2682) SfoI (2683) PluTI (2685) BsmI (2835) StyI (2845) pRHA-113 5837 bp
AatII  (5764)
1 site
G A C G T C C T G C A G
ZraI  (5762)
1 site
G A C G T C C T G C A G
ScaI  (5322)
1 site
A G T A C T T C A T G A
PstI  (5087)
1 site
C T G C A G G A C G T C
BsaI  (4903)
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.
PciI  (3949)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3833)
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  (3833)
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.
NdeI  (3772)
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.
BstZ17I  (3722)
1 site
G T A T A C C A T A T G
PflFI  (3696)
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  (3696)
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.
BspEI  (3140)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
FspAI  (2932)
1 site
R T G C G C A Y Y A C G C G T R
MscI  (2922)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
HindIII  (29)
1 site
A A G C T T T T C G A A
PacI  (37)
1 site
T T A A T T A A A A T T A A T T
TspMI  (90)
1 site
C C C G G G G G G C C C
XmaI  (90)
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  (92)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BstXI  (240)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NheI  (322)
1 site
G C T A G C C G A T C G
BmtI  (326)
1 site
G C T A G C C G A T C G
XcmI  (979)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BstEII  (999)
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.
DraIII  (999)
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.
BbvCI  (1145)
1 site
C C T C A G C G G A G T C G
AflII  (1169)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BmgBI  (1281)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BglII  (1351)
1 site
A G A T C T T C T A G A
AarI  (1434)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
AgeI  (1619)
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  (1619)
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.
MluI  (1750)
1 site
A C G C G T T G C G C A
SalI  (2077)
1 site
G T C G A C C A G C T G
XbaI  (2083)
1 site
T C T A G A A G A T C T
BamHI  (2089)
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.
Acc65I  (2122)
1 site
G G T A C C C C A T G G
KpnI  (2126)
1 site
G G T A C C C C A T G G
PshAI  (2192)
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.
EagI  (2415)
1 site
C G G C C G G C C G G C
KasI  (2681)
1 site
G G C G C C C C G C G G
NarI  (2682)
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  (2683)
1 site
G G C G C C C C G C G G
PluTI  (2685)
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.
BsmI  (2835)
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.
StyI  (2845)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AmpR
4769 .. 5629  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4769 .. 5560  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4769 .. 5629  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5561 .. 5629  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4769 .. 5629  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
rhaR
37 .. 885  =  849 bp
282 amino acids  =  32.3 kDa
Genetic Code:  Bacterial, Archaeal and Plant
   Plastid
Product: transcriptional activator of rhaSR
positive regulator for rhaRS operon
rhaR
37 .. 885  =  849 bp
282 amino acids  =  32.3 kDa
Genetic Code:  Bacterial, Archaeal and Plant
   Plastid
Product: transcriptional activator of rhaSR
positive regulator for rhaRS operon
rhaS
959 .. 1795  =  837 bp
278 amino acids  =  32.3 kDa
Genetic Code:  Bacterial, Archaeal and Plant
   Plastid
Product: transcriptional activator of rhaBAD and
rhaT
positive regulator for rhaBAD operon
rhaS
959 .. 1795  =  837 bp
278 amino acids  =  32.3 kDa
Genetic Code:  Bacterial, Archaeal and Plant
   Plastid
Product: transcriptional activator of rhaBAD and
rhaT
positive regulator for rhaBAD operon
ori
4010 .. 4598  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4010 .. 4598  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3391 .. 3582  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3391 .. 3582  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rhaB promoter
1941 .. 2059  =  119 bp
promoter of the E. coli rhaBAD operon, conferring
tight induction with L-rhamnose and repression with
D-glucose in the presence of RhaR and RhaS
(Giacalone et al., 2006)
rhaB promoter
1941 .. 2059  =  119 bp
promoter of the E. coli rhaBAD operon, conferring
tight induction with L-rhamnose and repression with
D-glucose in the presence of RhaR and RhaS
(Giacalone et al., 2006)
AmpR promoter
5630 .. 5734  =  105 bp
AmpR promoter
5630 .. 5734  =  105 bp
MCS
2077 .. 2127  =  51 bp
multiple cloning site
MCS
2077 .. 2127  =  51 bp
multiple cloning site
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