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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.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Xbrane Bioscience
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AatII (4680) ZraI (4678) XmnI (4357) ScaI (4238) BsaI (3819) PciI (2865) BspQI - SapI (2749) EcoRI (2509) BanII - SacI (2507) Eco53kI (2505) KpnI (2501) Acc65I (2497) BamHI (2459) XbaI (2453) AccI (2448) SalI (2447) EcoO109I (4734) 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
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
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.
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
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
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
ORF:  539 .. 904  =  366 bp
ORF:  121 amino acids  =  14.0 kDa
ORF:  3815 .. 4081  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  186 .. 410  =  225 bp
ORF:  74 amino acids  =  8.7 kDa
ORF:  1329 .. 2165  =  837 bp
ORF:  278 amino acids  =  32.3 kDa
ORF:  407 .. 1345  =  939 bp
ORF:  312 amino acids  =  35.7 kDa
ORF:  1601 .. 1846  =  246 bp
ORF:  81 amino acids  =  9.4 kDa
ORF:  3685 .. 4545  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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