pET-52b(+) 3C_LIC

Bacterial vector with an HRV 3C site for ligation-independent cloning (LIC) to express proteins tagged with Strep-Tag II.

Sequence Author: MilliporeSigma (Novagen)

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HomePlasmidspET & Duet Vectors (Novagen)pET-52b(+) 3C_LIC
AvrII (97) BlpI (80) DraIII (4985) PsiI (4857) SspI (4777) AhdI (4573) BsaI (4507) BglI (4455) FspI (4350) PvuI (4204) ScaI (4092) AlwNI (3379) PciI (2963) BspQI - SapI (2847) BstZ17I (2734) PflFI - Tth111I (2708) thrombin site Eco53kI (169) SacI (171) EagI - NotI (175) SalI (182) BsrGI (189) BamHI (195) Acc65I (202) AvaI - BsoBI - KpnI - TspMI - XmaI (206) SmaI (208) KflI (210) HRV 3C site BstBI (243) Strep-Tag II NcoI (268) ATG XbaI (307) lac operator T7 promoter BspDI * - ClaI * (376) SgrAI (418) SphI (574) PflMI (681) BstAPI (782) MluI (1099) BclI * (1113) BstEII (1280) PspOMI (1306) ApaI (1310) BssHII (1510) HpaI (1605) Bpu10I (2069) pET-52b(+) 3C/LIC 5227 bp
AvrII  (97)
1 site		 C C T A G G G G A T C C
BlpI  (80)
1 site		 G C T N A G C C G A N T C G
Sticky ends from different BlpI sites may not be compatible.
DraIII  (4985)
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.
PsiI  (4857)
1 site		 T T A T A A A A T A T T
SspI  (4777)
1 site		 A A T A T T T T A T A A
AhdI  (4573)
1 site		 G A C N N N N N G T C C T G N N N N N C A G
The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BsaI  (4507)
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.
BglI  (4455)
1 site		 G C C N N N N N G G C C G G N N N N N C C G
Sticky ends from different BglI sites may not be compatible.
FspI  (4350)
1 site		 T G C G C A A C G C G T
PvuI  (4204)
1 site		 C G A T C G G C T A G C
ScaI  (4092)
1 site		 A G T A C T T C A T G A
AlwNI  (3379)
1 site		 C A G N N N C T G G T C N N N G A C
Sticky ends from different AlwNI sites may not be compatible.
PciI  (2963)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BspQI  (2847)
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  (2847)
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.
BstZ17I  (2734)
1 site		 G T A T A C C A T A T G
PflFI  (2708)
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  (2708)
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.
Eco53kI  (169)
1 site		 G A G C T C C T C G A G
SacI  (171)
1 site		 G A G C T C C T C G A G
EagI  (175)
1 site		 C G G C C G G C C G G C
NotI  (175)
1 site		 G C G G C C G C C G C C G G C G
SalI  (182)
1 site		 G T C G A C C A G C T G
BsrGI  (189)
1 site		 T G T A C A A C A T G T
BsrGI is typically used at 37°C, but is even more active at 60°C.
BamHI  (195)
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  (202)
1 site		 G G T A C C C C A T G G
AvaI  (206)
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  (206)
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.
KpnI  (206)
1 site		 G G T A C C C C A T G G
TspMI  (206)
1 site		 C C C G G G G G G C C C
XmaI  (206)
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  (208)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
KflI  (210)
1 site		 G G G W C C C C C C W G G G
Sticky ends from different KflI sites may not be compatible.
BstBI  (243)
1 site		 T T C G A A A A G C T T
NcoI  (268)
1 site		 C C A T G G G G T A C C
XbaI  (307)
1 site		 T C T A G A A G A T C T
BspDI  (376)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (376)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (418)
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.
SphI  (574)
1 site		 G C A T G C C G T A C G
PflMI  (681)
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.
BstAPI  (782)
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  (1099)
1 site		 A C G C G T T G C G C A
BclI  (1113)
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  (1280)
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.
PspOMI  (1306)
1 site		 G G G C C C C C C G G G
ApaI  (1310)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
BssHII  (1510)
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  (1605)
1 site		 G T T A A C C A A T T G
Bpu10I  (2069)
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.
lacI
749 .. 1831  =  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
749 .. 1831  =  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).
AmpR
3786 .. 4646  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3786 .. 3854  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3786 .. 4646  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3855 .. 4646  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3786 .. 4646  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3024 .. 3612  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3024 .. 3612  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4761 .. 5216  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4761 .. 5216  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
2403 .. 2594  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
2403 .. 2594  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
10xHis
110 .. 139  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
110 .. 139  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
thrombin site
146 .. 163  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
146 .. 163  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
HRV 3C site
209 .. 232  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C protease
HRV 3C site
209 .. 232  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C protease
Strep-Tag II
239 .. 262  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an engineered form of streptavidin
Strep-Tag II
239 .. 262  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an engineered form of streptavidin
ATG
269 .. 271  =  3 bp
1 amino acid  =  149.2 Da
Product: start
ATG
269 .. 271  =  3 bp
1 amino acid  =  149.2 Da
Product: start
lacI promoter
671 .. 748  =  78 bp
lacI promoter
671 .. 748  =  78 bp
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
315 .. 339  =  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
315 .. 339  =  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
340 .. 358  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
340 .. 358  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
279 .. 284  =  6 bp
ribosome binding site
RBS
279 .. 284  =  6 bp
ribosome binding site
ORF:  67 .. 999  =  933 bp
ORF:  310 amino acids  =  33.1 kDa
ORF:  1588 .. 1851  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  872 .. 1831  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2370 .. 2594  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  3786 .. 4646  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2003 .. 2371  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  4250 .. 4516  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  322 .. 573  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  447 .. 710  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1614 .. 1865  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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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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