pTrcHis-TOPO (linearized)

Linearized bacterial vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning and inducible expression of N-terminally 6xHis-tagged proteins.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsTOPO Cloning VectorspTrcHis-TOPO (linearized)
PluTI (3745) SfoI (3743) NarI * (3742) KasI (3741) HpaI (3608) EcoRV (3552) ApaI - BanII (3313) PspOMI (3309) BstEII (3283) BclI * (3116) MluI (3102) PflMI (2684) NsiI (2665) SphI (2663) MauBI (2641) PfoI (2573) PflFI - Tth111I (2474) BsaAI (2468) BstZ17I (2449) AccI (2448) NdeI (2397) BspQI - SapI (2335) PciI (2218) AlwNI (1809) BfuAI - BspMI (3945) trc promoter lac operator gene 10 translational enhancer minicistron BtgI - NcoI - StyI (4284) ATG NheI (4322) BmtI (4326) T7 tag (gene 10 leader) Xpress™ tag BsaBI * (4377) BamHI (4378) End (4390) Start (1) EcoRI (7) BstBI (16) HindIII (19) rrnB T1 terminator rrnB T2 terminator ScaI (849) PvuI (961) FspI (1107) BglI (1212) AhdI (1330) pTrcHis-TOPO® 4389 bp
PluTI  (3745)
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  (3743)
1 site		 G G C G C C C C G C G G
NarI  (3742)
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  (3741)
1 site		 G G C G C C C C G C G G
HpaI  (3608)
1 site		 G T T A A C C A A T T G
EcoRV  (3552)
1 site		 G A T A T C C T A T A G
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
ApaI  (3313)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
BanII  (3313)
1 site		 G R G C Y C C Y C G R G
Sticky ends from different BanII sites may not be compatible.
PspOMI  (3309)
1 site		 G G G C C C C C C G G G
BstEII  (3283)
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.
BclI  (3116)
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.
MluI  (3102)
1 site		 A C G C G T T G C G C A
PflMI  (2684)
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.
NsiI  (2665)
1 site		 A T G C A T T A C G T A
SphI  (2663)
1 site		 G C A T G C C G T A C G
MauBI  (2641)
1 site		 C G C G C G C G G C G C G C G C
PfoI  (2573)
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.
PflFI  (2474)
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  (2474)
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  (2468)
1 site		 Y A C G T R R T G C A Y
BstZ17I  (2449)
1 site		 G T A T A C C A T A T G
AccI  (2448)
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.
NdeI  (2397)
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.
BspQI  (2335)
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  (2335)
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  (2218)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
AlwNI  (1809)
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.
BfuAI  (3945)
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  (3945)
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.
BtgI  (4284)
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  (4284)
1 site		 C C A T G G G G T A C C
StyI  (4284)
1 site		 C C W W G G G G W W C C
Sticky ends from different StyI sites may not be compatible.
NheI  (4322)
1 site		 G C T A G C C G A T C G
BmtI  (4326)
1 site		 G C T A G C C G A T C G
BsaBI  (4377)
1 site		 G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
BamHI  (4378)
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.
End  (4390)
0 sites
Start  (1)
0 sites
EcoRI  (7)
1 site		 G A A T T C C T T A A G
BstBI  (16)
1 site		 T T C G A A A A G C T T
HindIII  (19)
1 site		 A A G C T T T T C G A A
ScaI  (849)
1 site		 A G T A C T T C A T G A
PvuI  (961)
1 site		 C G A T C G G C T A G C
FspI  (1107)
1 site		 T G C G C A A C G C G T
BglI  (1212)
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.
AhdI  (1330)
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.
lacI
2752 .. 3834  =  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
2752 .. 3834  =  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
543 .. 1403  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   543 .. 611  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
543 .. 1403  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   612 .. 1403  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
543 .. 1403  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1574 .. 2162  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1574 .. 2162  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ATG
4286 .. 4288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4286 .. 4288  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
4298 .. 4315  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4298 .. 4315  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
T7 tag (gene 10 leader)
4319 .. 4351  =  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)
4319 .. 4351  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene 10
promotes efficient translation in E. coli
Xpress™ tag
4355 .. 4378  =  24 bp
8 amino acids  =  998.0 Da
2 segments
   Segment 1:  
   4355 .. 4363  =  9 bp
   3 amino acids  =  409.4 Da
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
Xpress™ tag
4355 .. 4378  =  24 bp
8 amino acids  =  998.0 Da
2 segments
   Segment 2:  enterokinase site  
   4364 .. 4378  =  15 bp
   5 amino acids  =  606.5 Da
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
Xpress™ tag
4355 .. 4378  =  24 bp
8 amino acids  =  998.0 Da
2 segments
Product: Xpress™ epitope tag, including an enterokinase recognition and cleavage site
AmpR promoter
451 .. 542  =  92 bp
AmpR promoter
451 .. 542  =  92 bp
rrnB T1 terminator
227 .. 313  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
227 .. 313  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacIq promoter
2674 .. 2751  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
2674 .. 2751  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
rrnG antiterminator
4136 .. 4205  =  70 bp
antiterminator from the E. coli rrnG leader region (Berg et al., 1989)
rrnG antiterminator
4136 .. 4205  =  70 bp
antiterminator from the E. coli rrnG leader region (Berg et al., 1989)
trc promoter
4066 .. 4095  =  30 bp
3 segments
   Segment 1:  -35  
   4066 .. 4071  =  6 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4066 .. 4095  =  30 bp
3 segments
   Segment 2:  
   4072 .. 4088  =  17 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4066 .. 4095  =  30 bp
3 segments
   Segment 3:  -10  
   4089 .. 4095  =  7 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4066 .. 4095  =  30 bp
3 segments
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
rrnB T2 terminator
405 .. 432  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
405 .. 432  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
minicistron
4256 .. 4282  =  27 bp
8 amino acids  =  1.1 kDa
Product: synthetic cistron containing a ribosome binding site (Shine-Dalgarno sequence), for enhancing the bacterial expression of a downstream cistron (Schoner, 1997)
This first cistron should terminate 3 bp upstream of the ATG for the second cistron.
minicistron
4256 .. 4282  =  27 bp
8 amino acids  =  1.1 kDa
Product: synthetic cistron containing a ribosome binding site (Shine-Dalgarno sequence), for enhancing the bacterial expression of a downstream cistron (Schoner, 1997)
This first cistron should terminate 3 bp upstream of the ATG for the second cistron.
lac operator
4103 .. 4119  =  17 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
4103 .. 4119  =  17 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).
gene 10 translational enhancer
4219 .. 4227  =  9 bp
translational enhancer from gene 10 of bacteriophage T7 (Olins and Rangwala, 1989)
gene 10 translational enhancer
4219 .. 4227  =  9 bp
translational enhancer from gene 10 of bacteriophage T7 (Olins and Rangwala, 1989)
RBS
4242 .. 4246  =  5 bp
ribosome binding site
RBS
4242 .. 4246  =  5 bp
ribosome binding site
ORF:  2875 .. 3834  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  543 .. 1403  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2685 .. 3002  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  3591 .. 3854  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  68 .. 394  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  1007 .. 1273  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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Individual Sequences & Maps

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pEF5 FRT V5-D-TOPO
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