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.

Sequence Author: MilliporeSigma (Novagen)

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HomePlasmidspET & Duet Vectors (Novagen)pRSF-2 Ek_LIC
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) RBS 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) BssSI - BssSαI (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
Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
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
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
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
Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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.
BssSI  (1724)
1 site		 C A C G A G G T G C T C
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.
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
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
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
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
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
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
ORF:  2353 .. 2604  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  519 .. 1334  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  2367 .. 2630  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3219 .. 3584  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  2387 .. 3346  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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Download pRSF-2 Ek_LIC.dna file

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Individual Sequences & Maps

pACYCDuet-1
pET-24a(+)
pET-3a
pET-51b(+) Ek_LIC (linearized)
pCDF-1b
pET-24b(+)
pET-3b
pET-52b(+)
pCDF-2 Ek_LIC
pET-24c(+)
pET-3c
pET-52b(+) 3C_LIC
pCDF-2 Ek_LIC (linearized)
pET-24d(+)
pET-3d
pET-52b(+) 3C_LIC (linearized)
pCDFDuet-1
pET-25b(+)
pET-40b(+)
pET-53-DEST
pCOLA-2-DEST
pET-26b(+)
pET-41 Ek_LIC
pET-54-DEST
pCOLA-3-DEST
pET-27b(+)
pET-41 Ek_LIC (linearized)
pET-55-DEST
pCOLADuet-1
pET-28a(+)
pET-41a(+)
pET-56-DEST
pET-11a
pET-28b(+)
pET-41b(+)
pET-57-DEST
pET-11b
pET-28c(+)
pET-41c(+)
pET-58-DEST
pET-11c
pET-29a(+)
pET-42a(+)
pET-59-DEST
pET-11d
pET-29b(+)
pET-42b(+)
pET-60-DEST
pET-14b
pET-29c(+)
pET-42c(+)
pET-61-DEST
pET-15b
pET-30 Ek_LIC
pET-43.1 Ek_LIC
pET-62-DEST
pET-16b
pET-30 Ek_LIC (linearized)
pET-43.1 Ek_LIC (linearized)
pET-9a
pET-17b
pET-30 Xa_LIC
pET-43.1a(+)
pET-9b
pET-19b
pET-30 Xa_LIC (linearized)
pET-43.1b(+)
pET-9c
pET-20b(+)
pET-30a(+)
pET-43.1c(+)
pET-9d
pET-21(+)
pET-30b(+)
pET-44a(+)
pETBlue-1
pET-21a(+)
pET-30c(+)
pET-44b(+)
pETBlue-2
pET-21b(+)
pET-31b(+)
pET-44c(+)
pETcoco-1
pET-21c(+)
pET-32 Ek_LIC
pET-45b(+)
pETcoco-2
pET-21d(+)
pET-32 Ek_LIC (linearized)
pET-46 Ek_LIC
pETDuet-1
pET-22b(+)
pET-32 Xa_LIC
pET-46 Ek_LIC (linearized)
pLacI
pET-23(+)
pET-32 Xa_LIC (linearized)
pET-47b(+)
pLysE
pET-23a(+)
pET-32a(+)
pET-48b(+)
pLysS
pET-23b(+)
pET-32b(+)
pET-49b(+)
pRSF-1b
pET-23c(+)
pET-32c(+)
pET-50b(+)
pRSF-2 Ek_LIC
pET-23d(+)
pET-33b(+)
pET-51b(+)
pRSF-2 Ek_LIC (linearized)
pET-24(+)
pET-39b(+)
pET-51b(+) Ek_LIC
pRSFDuet-1

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