pET101 D-TOPO

Bacterial directional TOPO® cloning vector, for inducible expression of a C-terminally V5- and 6xHis-tagged protein.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsTOPO Cloning VectorspET101 D-TOPO
EcoNI (5383) BclI * (4901) BstEII (4733) ApaI (4712) PspOMI (4708) BssHII (4504) HpaI (4413) PshAI (4074) EagI (3847) NruI (3816) BfuAI - BspMI (3732) BsmI (3433) AvaI - BsoBI (3361) MscI * (3344) FspAI (3334) Bpu10I (3206) PflFI - Tth111I (2569) BsaAI (2563) BstZ17I (2544) AccI (2543) BspQI - SapI (2431) SphI (5448) SgrAI (5596) BglII (5637) T7 promoter XbaI (5703) RBS EcoRI (5734) RBS Eco53kI (9) SacI (11) BstBI (16) BseRI (40) AgeI (73) 6xHis BlpI (156) T7 terminator BspDI - ClaI (400) ZraI (505) AatII (507) SspI (621) ScaI (945) PvuI (1057) PstI (1184) AhdI (1426) AlwNI (1905) PciI (2314) pET101/D-TOPO® 5749 bp
EcoNI  (5383)
1 site		 C C T N N N N N A G G G G A N N N N N T C C
The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BclI  (4901)
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  (4733)
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.
ApaI  (4712)
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  (4708)
1 site		 G G G C C C C C C G G G
BssHII  (4504)
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  (4413)
1 site		 G T T A A C C A A T T G
PshAI  (4074)
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  (3847)
1 site		 C G G C C G G C C G G C
NruI  (3816)
1 site		 T C G C G A A G C G C T
BfuAI  (3732)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (3732)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsmI  (3433)
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.
AvaI  (3361)
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  (3361)
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.
MscI  (3344)
1 site		 T G G C C A A C C G G T
* Blocked by Dcm methylation.
FspAI  (3334)
1 site		 R T G C G C A Y Y A C G C G T R
Bpu10I  (3206)
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.
PflFI  (2569)
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  (2569)
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.
BsaAI  (2563)
1 site		 Y A C G T R R T G C A Y
BstZ17I  (2544)
1 site		 G T A T A C C A T A T G
AccI  (2543)
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.
BspQI  (2431)
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  (2431)
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.
SphI  (5448)
1 site		 G C A T G C C G T A C G
SgrAI  (5596)
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.
BglII  (5637)
1 site		 A G A T C T T C T A G A
XbaI  (5703)
1 site		 T C T A G A A G A T C T
EcoRI  (5734)
1 site		 G A A T T C C T T A A G
Eco53kI  (9)
1 site		 G A G C T C C T C G A G
SacI  (11)
1 site		 G A G C T C C T C G A G
BstBI  (16)
1 site		 T T C G A A A A G C T T
BseRI  (40)
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.
AgeI  (73)
1 site		 A C C G G T T G G C C A
BlpI  (156)
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.
BspDI  (400)
1 site		 A T C G A T T A G C T A
ClaI  (400)
1 site		 A T C G A T T A G C T A
ZraI  (505)
1 site		 G A C G T C C T G C A G
AatII  (507)
1 site		 G A C G T C C T G C A G
SspI  (621)
1 site		 A A T A T T T T A T A A
ScaI  (945)
1 site		 A G T A C T T C A T G A
PvuI  (1057)
1 site		 C G A T C G G C T A G C
PstI  (1184)
1 site		 C T G C A G G A C G T C
AhdI  (1426)
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.
AlwNI  (1905)
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  (2314)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
lacI
4188 .. 5270  =  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
4188 .. 5270  =  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
639 .. 1499  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   639 .. 707  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
639 .. 1499  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   708 .. 1499  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
639 .. 1499  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1670 .. 2258  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1670 .. 2258  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
2685 .. 2876  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
2685 .. 2876  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
534 .. 638  =  105 bp
AmpR promoter
534 .. 638  =  105 bp
lacI promoter
5271 .. 5348  =  78 bp
lacI promoter
5271 .. 5348  =  78 bp
V5 tag
28 .. 69  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
28 .. 69  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
79 .. 96  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
79 .. 96  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
T7 terminator
167 .. 214  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
167 .. 214  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
5676 .. 5700  =  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
5676 .. 5700  =  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
5657 .. 5675  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5657 .. 5675  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
5730 .. 5736  =  7 bp
ribosome binding site
RBS
5730 .. 5736  =  7 bp
ribosome binding site
RBS
5740 .. 5744  =  5 bp
ribosome binding site
RBS
5740 .. 5744  =  5 bp
ribosome binding site
ORF:  2908 .. 3276  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  5446 .. 5685  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  3710 .. 4405  =  696 bp
ORF:  231 amino acids  =  25.4 kDa
ORF:  5309 .. 5572  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  639 .. 1499  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2685 .. 2909  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  3273 .. 3611  =  339 bp
ORF:  112 amino acids  =  12.6 kDa
ORF:  4188 .. 5147  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  5517 .. 5753  =  237 bp
ORF:  78 amino acids  =  8.1 kDa  (no start codon)
ORF:  1103 .. 1369  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3515 .. 4132  =  618 bp
ORF:  205 amino acids  =  21.8 kDa
ORF:  4168 .. 4431  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5020 .. 5520  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
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Individual Sequences & Maps

pBAD-TOPO (linearized)
pcDNA5 FRT TO-TOPO (linearized)
pEF6 V5-His-TOPO (linearized)
pGlow-TOPO (linearized)
pBAD Thio-TOPO (linearized)
pcDNA5 FRT V5-His-TOPO (linearized)
pENTR D-TOPO
pIB V5-His-TOPO (linearized)
pBAD202 D-TOPO
pcDNA6.2 C-EmGFP-GW TOPO (linearized)
pENTR SD D-TOPO
pLenti6.3 V5-TOPO (linearized)
pBlue-TOPO (linearized)
pcDNA6.2 C-YFP-GW TOPO (linearized)
pENTR TEV D-TOPO
pLenti6 V5-D-TOPO
pcDNA3.1D V5-His-TOPO
pcDNA6.2 N-EmGFP-GW TOPO (linearized)
pENTR5'-TOPO (linearized)
pLenti7.3 V5-TOPO (linearized)
pcDNA3.2 V5 GW D-TOPO
pCR-XL-2-TOPO (linearized)
pET100 D-TOPO
pLPS-B (linearized)
pcDNA6.2 cGeneBLAzer-GW D-TOPO
pCR-XL-TOPO (linearized)
pET101 D-TOPO
pMT V5-His-TOPO (linearized)
pcDNA6.2 V5 GW D-TOPO
pCR2.1-TOPO (linearized)
pET102 D-TOPO
pOptiVEC-TOPO (linearized)
pcDNA3.1 CT-GFP-TOPO (linearized)
pCR4-TOPO (linearized)
pET151 D-TOPO
pSecTag FRT V5-His-TOPO (linearized)
pcDNA3.1 NT-GFP-TOPO (linearized)
pCR4Blunt-TOPO (linearized)
pET160 GW D-TOPO
pTOP Blunt V2 (linearized)
pcDNA3.1 V5-His-TOPO (linearized)
pCR8 GW TOPO (linearized)
pET161 GW D-TOPO
pTrcHis-TOPO (linearized)
pcDNA3.3-TOPO (linearized)
pCR-Blunt II-TOPO (linearized)
pET200 D-TOPO
pTrcHis2-TOPO (linearized)
pcDNA3.4-TOPO (linearized)
pCRII-TOPO (linearized)
pEXP5-CT TOPO (linearized)
pYES2.1 V5-His-TOPO (linearized)
pcDNA4 HisMax-TOPO (linearized)
pEF5 FRT V5-D-TOPO
pEXP5-NT TOPO (linearized)

Plasmid Sets

Basic Cloning Vectors
I.M.A.G.E. Consortium Plasmids
pET & Duet Vectors (Novagen)
TA and GC Cloning Vectors
Coronavirus Resources
Insect Cell Vectors
pGEX Vectors (GE Healthcare)
TOPO Cloning Vectors
CRISPR Plasmids
Luciferase Vectors
Plant Vectors
Viral Expression & Packaging Vectors
Fluorescent Protein Genes & Plasmids
Lucigen Vectors
Qiagen Vectors
Yeast Plasmids
Gateway® Cloning Vectors
Mammalian Expression Vectors
Structural Genomics Vectors

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