pET200 D-TOPO

Bacterial directional TOPO® cloning vector with a kanamycin resistance marker, for inducible expression of an N-terminally 6xHis-tagged protein.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsTOPO Cloning VectorspET200 D-TOPO
EcoNI (5272) MluI (4804) BclI * (4790) BstEII (4622) ApaI (4601) PspOMI (4597) ApoI (4529) HincII - HpaI (4302) PshAI (3963) NruI (3705) BsmI (3322) AvaI - BsoBI (3250) FspAI (3223) Bpu10I (3095) BstZ17I (2433) AccI (2432) TatI (2396) SgrAI (5485) BglII (5526) lac operator XbaI (5592) RBS NdeI (5632) ATG 6xHis NheI (5670) BmtI (5674) T7 tag (gene 10 leader) Xpress™ tag Eco53kI (9) SacI (11) BlpI (68) BseRI (129) HindIII (305) BspDI - ClaI (312) ZraI (417) AatII (419) SspI (533) PstI (732) RsrII (1195) BstBI (1361) AlwNI (1794) PciI (2203) pET200/D-TOPO® 5737 bp
EcoNI  (5272)
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.
MluI  (4804)
1 site		 A C G C G T T G C G C A
BclI  (4790)
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  (4622)
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  (4601)
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  (4597)
1 site		 G G G C C C C C C G G G
ApoI  (4529)
1 site		 R A A T T Y Y T T A A R
ApoI is typically used at 50°C, but is 50% active at 37°C.
HincII  (4302)
1 site		 G T Y R A C C A R Y T G
HpaI  (4302)
1 site		 G T T A A C C A A T T G
PshAI  (3963)
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.
NruI  (3705)
1 site		 T C G C G A A G C G C T
BsmI  (3322)
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  (3250)
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  (3250)
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.
FspAI  (3223)
1 site		 R T G C G C A Y Y A C G C G T R
Bpu10I  (3095)
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.
BstZ17I  (2433)
1 site		 G T A T A C C A T A T G
AccI  (2432)
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.
TatI  (2396)
1 site		 W G T A C W W C A T G W
SgrAI  (5485)
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  (5526)
1 site		 A G A T C T T C T A G A
XbaI  (5592)
1 site		 T C T A G A A G A T C T
NdeI  (5632)
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.
NheI  (5670)
1 site		 G C T A G C C G A T C G
BmtI  (5674)
1 site		 G C T A G C C G A T C 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
BlpI  (68)
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.
BseRI  (129)
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.
HindIII  (305)
1 site		 A A G C T T T T C G A A
BspDI  (312)
1 site		 A T C G A T T A G C T A
ClaI  (312)
1 site		 A T C G A T T A G C T A
ZraI  (417)
1 site		 G A C G T C C T G C A G
AatII  (419)
1 site		 G A C G T C C T G C A G
SspI  (533)
1 site		 A A T A T T T T A T A A
PstI  (732)
1 site		 C T G C A G G A C G T C
RsrII  (1195)
1 site		 C G G W C C G G C C W G G C
Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BstBI  (1361)
1 site		 T T C G A A A A G C T T
AlwNI  (1794)
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  (2203)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
lacI
4077 .. 5159  =  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
4077 .. 5159  =  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).
NeoR/KanR
551 .. 1345  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
551 .. 1345  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
1559 .. 2147  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1559 .. 2147  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
2574 .. 2765  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
2574 .. 2765  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
446 .. 550  =  105 bp
AmpR promoter
446 .. 550  =  105 bp
ATG
5634 .. 5636  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5634 .. 5636  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
5646 .. 5663  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5646 .. 5663  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
T7 tag (gene 10 leader)
5667 .. 5699  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene 10
promotes efficient translation in E. coli
T7 tag (gene 10 leader)
5667 .. 5699  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene 10
promotes efficient translation in E. coli
Xpress™ tag
5703 .. 5726  =  24 bp
8 amino acids  =  998.0 Da
2 segments
   Segment 1:  
   5703 .. 5711  =  9 bp
   3 amino acids  =  409.4 Da
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
Xpress™ tag
5703 .. 5726  =  24 bp
8 amino acids  =  998.0 Da
2 segments
   Segment 2:  enterokinase site  
   5712 .. 5726  =  15 bp
   5 amino acids  =  606.5 Da
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
Xpress™ tag
5703 .. 5726  =  24 bp
8 amino acids  =  998.0 Da
2 segments
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
lacI promoter
5160 .. 5237  =  78 bp
lacI promoter
5160 .. 5237  =  78 bp
T7 terminator
79 .. 126  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
79 .. 126  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
5565 .. 5589  =  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
5565 .. 5589  =  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
5546 .. 5564  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5546 .. 5564  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
5619 .. 5626  =  8 bp
ribosome binding site
RBS
5619 .. 5626  =  8 bp
ribosome binding site
ORF:  2797 .. 3165  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  5335 .. 5574  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  551 .. 1345  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  3599 .. 4294  =  696 bp
ORF:  231 amino acids  =  25.4 kDa
ORF:  5198 .. 5461  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  723 .. 1109  =  387 bp
ORF:  128 amino acids  =  14.5 kDa
ORF:  2574 .. 2798  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  3162 .. 3500  =  339 bp
ORF:  112 amino acids  =  12.6 kDa
ORF:  4077 .. 5036  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  5406 .. 5741  =  336 bp
ORF:  111 amino acids  =  11.9 kDa  (no start codon)
ORF:  860 .. 1114  =  255 bp
ORF:  84 amino acids  =  9.7 kDa
ORF:  3404 .. 4021  =  618 bp
ORF:  205 amino acids  =  21.8 kDa
ORF:  4057 .. 4320  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4909 .. 5409  =  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)

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