pTrcHis2-TOPO (linearized)

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

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsTA and GC Cloning VectorspTrcHis2-TOPO (linearized)
PluTI (3832) SfoI (3830) NarI * (3829) KasI (3828) EcoRV (3639) ApaI - BanII (3400) PspOMI (3396) BstEII (3370) BclI * (3203) MluI (3189) PflMI (2771) NsiI (2752) SphI (2750) MauBI (2728) PfoI (2660) PflFI - Tth111I (2561) BstZ17I (2536) NdeI (2484) BspQI - SapI (2422) PciI (2305) AlwNI (1896) BfuAI - BspMI (4032) trc promoter lac operator RBS BtgI - NcoI - StyI (4371) End (4381) Start (1) EcoRI (7) BstBI (11) HindIII (14) SnaBI (21) Myc SalI (64) 6xHis PmeI (94) ScaI (936) TsoI (1019) PvuI (1048) FspI (1194) BglI (1299) AhdI (1417) pTrcHis2-TOPO® 4380 bp
PluTI  (3832)
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  (3830)
1 site		 G G C G C C C C G C G G
NarI  (3829)
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  (3828)
1 site		 G G C G C C C C G C G G
EcoRV  (3639)
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  (3400)
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  (3400)
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  (3396)
1 site		 G G G C C C C C C G G G
BstEII  (3370)
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  (3203)
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  (3189)
1 site		 A C G C G T T G C G C A
PflMI  (2771)
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  (2752)
1 site		 A T G C A T T A C G T A
SphI  (2750)
1 site		 G C A T G C C G T A C G
MauBI  (2728)
1 site		 C G C G C G C G G C G C G C G C
PfoI  (2660)
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  (2561)
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  (2561)
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.
BstZ17I  (2536)
1 site		 G T A T A C C A T A T G
NdeI  (2484)
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  (2422)
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  (2422)
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  (2305)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
AlwNI  (1896)
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  (4032)
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  (4032)
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  (4371)
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  (4371)
1 site		 C C A T G G G G T A C C
StyI  (4371)
1 site		 C C W W G G G G W W C C
Sticky ends from different StyI sites may not be compatible.
End  (4381)
0 sites
Start  (1)
0 sites
EcoRI  (7)
1 site		 G A A T T C C T T A A G
BstBI  (11)
1 site		 T T C G A A A A G C T T
HindIII  (14)
1 site		 A A G C T T T T C G A A
SnaBI  (21)
1 site		 T A C G T A A T G C A T
SalI  (64)
1 site		 G T C G A C C A G C T G
PmeI  (94)
1 site		 G T T T A A A C C A A A T T T G
ScaI  (936)
1 site		 A G T A C T T C A T G A
TsoI  (1019)
1 site		 T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9
Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (1048)
1 site		 C G A T C G G C T A G C
FspI  (1194)
1 site		 T G C G C A A C G C G T
BglI  (1299)
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  (1417)
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
2839 .. 3921  =  1083 bp
360 amino acids  =  38.5 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
2839 .. 3921  =  1083 bp
360 amino acids  =  38.5 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
630 .. 1490  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   630 .. 698  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
630 .. 1490  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   699 .. 1490  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
630 .. 1490  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1661 .. 2249  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1661 .. 2249  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
538 .. 629  =  92 bp
AmpR promoter
538 .. 629  =  92 bp
rrnB T1 terminator
313 .. 399  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
313 .. 399  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacIq promoter
2761 .. 2838  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
2761 .. 2838  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
rrnG antiterminator
4223 .. 4292  =  70 bp
antiterminator from the E. coli rrnG leader region (Berg et al., 1989)
rrnG antiterminator
4223 .. 4292  =  70 bp
antiterminator from the E. coli rrnG leader region (Berg et al., 1989)
Myc
25 .. 54  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
25 .. 54  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
6xHis
70 .. 87  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
70 .. 87  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
trc promoter
4153 .. 4182  =  30 bp
3 segments
   Segment 1:  -35  
   4153 .. 4158  =  6 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4153 .. 4182  =  30 bp
3 segments
   Segment 2:  
   4159 .. 4175  =  17 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4153 .. 4182  =  30 bp
3 segments
   Segment 3:  -10  
   4176 .. 4182  =  7 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
trc promoter
4153 .. 4182  =  30 bp
3 segments
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
rrnB T2 terminator
491 .. 518  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
491 .. 518  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
minicistron
4343 .. 4369  =  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
4343 .. 4369  =  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
4190 .. 4206  =  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
4190 .. 4206  =  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
4306 .. 4314  =  9 bp
translational enhancer from gene 10 of bacteriophage T7 (Olins and Rangwala, 1989)
gene 10 translational enhancer
4306 .. 4314  =  9 bp
translational enhancer from gene 10 of bacteriophage T7 (Olins and Rangwala, 1989)
RBS
4329 .. 4333  =  5 bp
ribosome binding site
RBS
4329 .. 4333  =  5 bp
ribosome binding site
ORF:  2962 .. 3921  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  509 .. 733  =  225 bp
ORF:  74 amino acids  =  8.7 kDa
ORF:  630 .. 1490  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2772 .. 3089  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  3678 .. 3941  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  1094 .. 1360  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  154 .. 480  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
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