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Plasmid Files

pRSF-2 Ek/LIC

Bacterial vector with an RSF 1030 origin for ligation-independent cloning (LIC) to express 6xHis- and S-tagged proteins with an enterokinase site.

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pRSF-2 Ek_LIC Sequence and MappRSF-2 Ek_LIC.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 T7 promoter PfoI (3546) AclI (3454) BstAPI (3438) MluI (3114) BclI * (3100) BstEII (2932) NmeAIII (2914) ApaI (2911) PspOMI (2907) BssHII (2703) HpaI (2612) PluTI (2479) SfoI (2477) NarI * (2476) KasI (2475) EaeI (2440) AcuI (2254) XbaI (2194) BtgI - NcoI (69) ATG 6xHis BsaAI - PmlI (94) enterokinase site PshAI (111) BseRI (115) PaeR7I - XhoI (134) AccI (191) PacI (209) AvrII (213) EcoO109I (258) Bsu36I (297) AgeI (346) DrdI (417) BsaI (425) XmnI (513) Bpu10I (882) TspMI - XmaI (1026) SmaI (1028) BspDI - ClaI (1209) NruI (1245) SphI (1434) AfeI (1438) BspQI - SapI (1446) PciI (1562) BssS α I (1724) pRSF-2 Ek/LIC 3609 bp
PfoI  (3546)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AclI  (3454)
1 site
A A C G T T T T G C A A
BstAPI  (3438)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (3114)
1 site
A C G C G T T G C G C A
BclI  (3100)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BstEII  (2932)
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.
NmeAIII  (2914)
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).
ApaI  (2911)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (2907)
1 site
G G G C C C C C C G G G
BssHII  (2703)
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.
HpaI  (2612)
1 site
G T T A A C C A A T T G
PluTI  (2479)
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.
SfoI  (2477)
1 site
G G C G C C C C G C G G
NarI  (2476)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (2475)
1 site
G G C G C C C C G C G G
EaeI  (2440)
1 site
Y G G C C R R C C G G Y
AcuI  (2254)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
XbaI  (2194)
1 site
T C T A G A A G A T C T
BtgI  (69)
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  (69)
1 site
C C A T G G G G T A C C
BsaAI  (94)
1 site
Y A C G T R R T G C A Y
PmlI  (94)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PshAI  (111)
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.
BseRI  (115)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
PaeR7I  (134)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (134)
1 site
C T C G A G G A G C T C
AccI  (191)
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.
PacI  (209)
1 site
T T A A T T A A A A T T A A T T
AvrII  (213)
1 site
C C T A G G G G A T C C
EcoO109I  (258)
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.
Bsu36I  (297)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
AgeI  (346)
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.
DrdI  (417)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BsaI  (425)
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.
XmnI  (513)
1 site
G A A N N N N T T C C T T N N N N A A G
Bpu10I  (882)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
TspMI  (1026)
1 site
C C C G G G G G G C C C
XmaI  (1026)
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  (1028)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BspDI  (1209)
1 site
A T C G A T T A G C T A
ClaI  (1209)
1 site
A T C G A T T A G C T A
NruI  (1245)
1 site
T C G C G A A G C G C T
SphI  (1434)
1 site
G C A T G C C G T A C G
AfeI  (1438)
1 site
A G C G C T T C G C G A
BspQI  (1446)
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  (1446)
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.
PciI  (1562)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BssSαI  (1724)
1 site
C A C G A G G T G C T C
lacI
2387 .. 3469  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
lacI
2387 .. 3469  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
KanR
519 .. 1334  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
519 .. 1334  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
RSF ori
1442 .. 2191  =  750 bp
Plasmids containing the RSF 1030 origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
RSF ori
1442 .. 2191  =  750 bp
Plasmids containing the RSF 1030 origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
AmpR promoter
1335 .. 1426  =  92 bp
AmpR promoter
1335 .. 1426  =  92 bp
lacI promoter
3470 .. 3547  =  78 bp
lacI promoter
3470 .. 3547  =  78 bp
T7 terminator
242 .. 289  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
242 .. 289  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
146 .. 190  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
146 .. 190  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
3 .. 27  =  25 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
3 .. 27  =  25 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).
T7 promoter
3593 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3593 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
77 .. 94  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
77 .. 94  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
98 .. 112  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
98 .. 112  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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