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Plasmid Files

pRHA-67

pUC18-derived plasmid encoding the rhaB promoter plus rhaR and rhaS genes, for rhamnose-inducible protein expression in E. coli.

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pRHA-67 Sequence and MappRHA-67.dna
Map and Sequence File   
Sequence Author:  Xbrane Bioscience
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 EcoO109I (4734) AatII (4680) ZraI (4678) XmnI (4357) ScaI (4238) BsaI (3819) PciI (2865) BspQI - SapI (2749) lac operator M13 rev EcoRI (2509) BanII - SacI (2507) Eco53kI (2505) KpnI (2501) Acc65I (2497) BamHI (2459) XbaI (2453) AccI (2448) SalI (2447) RBS NdeI (184) KasI (235) NarI (236) SfoI (237) PluTI (239) HindIII (399) PacI (407) PflMI (440) AvaI - BsoBI - TspMI - XmaI (460) BmeT110I (461) SmaI (462) BstXI (610) BsgI (627) NheI (692) BmtI (696) XcmI (1349) BstEII - DraIII (1369) BtgZI (1453) BbvCI (1515) AflII (1539) BmgBI (1651) BglII (1721) AarI (1804) BspDI * - ClaI * (1893) AgeI - SgrAI (1989) MluI (2120) BsaAI (2247) EcoRI (2401) pRHA-67 4745 bp
EcoO109I  (4734)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
AatII  (4680)
1 site
G A C G T C C T G C A G
ZraI  (4678)
1 site
G A C G T C C T G C A G
XmnI  (4357)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (4238)
1 site
A G T A C T T C A T G A
BsaI  (3819)
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  (2865)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2749)
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  (2749)
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.
EcoRI  (2509)
2 sites
G A A T T C C T T A A G
BanII  (2507)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (2507)
1 site
G A G C T C C T C G A G
Eco53kI  (2505)
1 site
G A G C T C C T C G A G
KpnI  (2501)
1 site
G G T A C C C C A T G G
Acc65I  (2497)
1 site
G G T A C C C C A T G G
BamHI  (2459)
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.
XbaI  (2453)
1 site
T C T A G A A G A T C T
AccI  (2448)
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.
SalI  (2447)
1 site
G T C G A C C A G C T G
NdeI  (184)
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.
KasI  (235)
1 site
G G C G C C C C G C G G
NarI  (236)
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  (237)
1 site
G G C G C C C C G C G G
PluTI  (239)
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.
HindIII  (399)
1 site
A A G C T T T T C G A A
PacI  (407)
1 site
T T A A T T A A A A T T A A T T
PflMI  (440)
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.
AvaI  (460)
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  (460)
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.
TspMI  (460)
1 site
C C C G G G G G G C C C
XmaI  (460)
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.
BmeT110I  (461)
1 site
C Y C G R G G R G C Y C
SmaI  (462)
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  (610)
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.
BsgI  (627)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
NheI  (692)
1 site
G C T A G C C G A T C G
BmtI  (696)
1 site
G C T A G C C G A T C G
XcmI  (1349)
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  (1369)
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  (1369)
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  (1453)
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.
BbvCI  (1515)
1 site
C C T C A G C G G A G T C G
AflII  (1539)
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  (1651)
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  (1721)
1 site
A G A T C T T C T A G A
AarI  (1804)
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.
BspDI  (1893)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (1893)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (1989)
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  (1989)
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  (2120)
1 site
A C G C G T T G C G C A
BsaAI  (2247)
1 site
Y A C G T R R T G C A Y
EcoRI  (2401)
2 sites
G A A T T C C T T A A G
AmpR
3685 .. 4545  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3685 .. 4476  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3685 .. 4545  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4477 .. 4545  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3685 .. 4545  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
rhaR
407 .. 1255  =  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
407 .. 1255  =  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
1329 .. 2165  =  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
1329 .. 2165  =  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
2926 .. 3514  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2926 .. 3514  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rhaB promoter
2311 .. 2429  =  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
2311 .. 2429  =  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
4546 .. 4650  =  105 bp
AmpR promoter
4546 .. 4650  =  105 bp
MCS
2447 .. 2508  =  62 bp
pUC19 multiple cloning site
MCS
2447 .. 2508  =  62 bp
pUC19 multiple cloning site
lac promoter
2572 .. 2602  =  31 bp
   Segment 3:  -10  
   2572 .. 2578  =  7 bp
promoter for the E. coli lac operon
lac promoter
2572 .. 2602  =  31 bp
   Segment 2:  
   2579 .. 2596  =  18 bp
promoter for the E. coli lac operon
lac promoter
2572 .. 2602  =  31 bp
   Segment 1:  -35  
   2597 .. 2602  =  6 bp
promoter for the E. coli lac operon
lac promoter
2572 .. 2602  =  31 bp
3 segments
promoter for the E. coli lac operon
M13 rev
2524 .. 2540  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2524 .. 2540  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
2548 .. 2564  =  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
2548 .. 2564  =  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).
RBS
2439 .. 2444  =  6 bp
ribosome binding site
RBS
2439 .. 2444  =  6 bp
ribosome binding site
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