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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.dna
Map and Sequence File:    Download    Open   
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
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
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
Function: regulator; Degradation of small molecules: Carbon compounds
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
ORF:  169 .. 534  =  366 bp
ORF:  121 amino acids  =  14.0 kDa
ORF:  2119 .. 2550  =  432 bp
ORF:  143 amino acids  =  16.2 kDa
ORF:  2656 .. 2994  =  339 bp
ORF:  112 amino acids  =  12.6 kDa
ORF:  3358 .. 3582  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2060 .. 2752  =  693 bp
ORF:  230 amino acids  =  24.8 kDa
ORF:  4899 .. 5165  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2991 .. 3359  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  959 .. 1795  =  837 bp
ORF:  278 amino acids  =  32.3 kDa
ORF:  2084 .. 2557  =  474 bp
ORF:  157 amino acids  =  17.2 kDa
ORF:  4769 .. 5629  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  37 .. 975  =  939 bp
ORF:  312 amino acids  =  35.7 kDa
ORF:  1231 .. 1476  =  246 bp
ORF:  81 amino acids  =  9.4 kDa
ORF:  2035 .. 2265  =  231 bp
ORF:  76 amino acids  =  8.1 kDa
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