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Plasmid Files

pYES2.1/V5-His-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and expression of tagged proteins in yeast cells.

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pYES2.1 V5-His-TOPO (linearized) Sequence and MappYES2.1 V5-His-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 NaeI (5229) NgoMIV (5227) SwaI (4901) SnaBI (4597) BmeT110I (4248) AvaI - BsoBI (4247) HpaI (3952) BmgBI (3892) PfoI * (3850) NruI (3546) BlpI (3510) BspDI - ClaI (3366) BmtI (3361) NheI (3357) NdeI (3217) BsgI (3188) SbfI (3154) BfuAI - BspMI (3143) XcmI (3005) EcoRV (2944) NcoI (2923) BsmI (2772) ApaI * (2758) PspOMI * (2754) BsmBI (2506) SpeI (5369) PvuII (5845) End (5886) Start (1) BstEII (16) XbaI (106) BsrGI (285) BciVI (757) BspHI (1274) AhdI (1447) BglI (1567) BsaHI (1984) pYES2.1/V5-His-TOPO® 5885 bp
NaeI  (5229)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
NgoMIV  (5227)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
SwaI  (4901)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
SnaBI  (4597)
1 site
T A C G T A A T G C A T
BmeT110I  (4248)
1 site
C Y C G R G G R G C Y C
AvaI  (4247)
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  (4247)
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.
HpaI  (3952)
1 site
G T T A A C C A A T T G
BmgBI  (3892)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
PfoI  (3850)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
NruI  (3546)
1 site
T C G C G A A G C G C T
BlpI  (3510)
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  (3366)
1 site
A T C G A T T A G C T A
ClaI  (3366)
1 site
A T C G A T T A G C T A
BmtI  (3361)
1 site
G C T A G C C G A T C G
NheI  (3357)
1 site
G C T A G C C G A T C G
NdeI  (3217)
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.
BsgI  (3188)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
SbfI  (3154)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (3143)
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  (3143)
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.
XcmI  (3005)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
EcoRV  (2944)
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.
NcoI  (2923)
1 site
C C A T G G G G T A C C
BsmI  (2772)
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.
ApaI  (2758)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
PspOMI  (2754)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
BsmBI  (2506)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
SpeI  (5369)
1 site
A C T A G T T G A T C A
PvuII  (5845)
1 site
C A G C T G G T C G A C
End  (5886)
0 sites
Start  (1)
0 sites
BstEII  (16)
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.
XbaI  (106)
1 site
T C T A G A A G A T C T
BsrGI  (285)
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.
BciVI  (757)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
BspHI  (1274)
1 site
T C A T G A A G T A C T
AhdI  (1447)
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.
BglI  (1567)
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.
BsaHI  (1984)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
2μ ori
3953 .. 4833  =  881 bp
yeast 2μ plasmid origin of replication
2μ ori
3953 .. 4833  =  881 bp
yeast 2μ plasmid origin of replication
AmpR
1374 .. 2234  =  861 bp
287 codons
   Segment 2:  
   1374 .. 2165  =  792 bp
   264 codons
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1374 .. 2234  =  861 bp
287 codons
   Segment 1:  signal sequence  
   2166 .. 2234  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1374 .. 2234  =  861 bp
287 codons
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
URA3
2330 .. 3133  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
URA3
2330 .. 3133  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
ori
615 .. 1203  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
615 .. 1203  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4902 .. 5357  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4902 .. 5357  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
GAL1 promoter
5376 .. 5817  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
5376 .. 5817  =  442 bp
inducible promoter, regulated by Gal4
CYC1 terminator
120 .. 367  =  248 bp
transcription terminator for CYC1
CYC1 terminator
120 .. 367  =  248 bp
transcription terminator for CYC1
URA3 promoter
3134 .. 3349  =  216 bp
URA3 promoter
3134 .. 3349  =  216 bp
V5 tag
31 .. 72  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
31 .. 72  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
5849 .. 5867  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5849 .. 5867  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
82 .. 99  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
82 .. 99  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
UAS
5376 .. 5493  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
5376 .. 5493  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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