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Plasmid Files

pYES2/CT

High-copy episomal vector with a URA3 marker for galactose-inducible expression of C-terminally tagged proteins in S. cerevisiae.

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pYES2 CT Sequence and MappYES2 CT.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 NaeI (5818) NgoMIV (5816) AanI (5587) SwaI (5490) SnaBI (5186) HpaI (4541) BmgBI (4481) PfoI * (4439) NruI (4135) BlpI (4099) BspDI - ClaI (3955) BmtI (3950) NheI (3946) NdeI (3806) BsgI (3777) SbfI (3743) BfuAI - BspMI (3732) XcmI (3594) NcoI (3512) BsmI (3361) BsmBI (3095) PvuII (471) T7 promoter HindIII (501) Acc65I (507) KpnI (511) Eco53kI (515) SacI (517) BamHI (519) BstXI (546) EcoRI (550) BstXI (572) NotI (577) PaeR7I - PspXI - XhoI (583) XbaI (589) PmeI (682) CYC1 terminator BsrGI (874) BciVI (1346) BspHI (1863) AhdI (2036) BglI (2156) BsaHI (2573) AanI (2906) pYES2/CT 5963 bp
NaeI  (5818)
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  (5816)
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.
AanI  (5587)
2 sites
T T A T A A A A T A T T
SwaI  (5490)
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  (5186)
1 site
T A C G T A A T G C A T
HpaI  (4541)
1 site
G T T A A C C A A T T G
BmgBI  (4481)
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  (4439)
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  (4135)
1 site
T C G C G A A G C G C T
BlpI  (4099)
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  (3955)
1 site
A T C G A T T A G C T A
ClaI  (3955)
1 site
A T C G A T T A G C T A
BmtI  (3950)
1 site
G C T A G C C G A T C G
NheI  (3946)
1 site
G C T A G C C G A T C G
NdeI  (3806)
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  (3777)
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  (3743)
1 site
C C T G C A G G G G A C G T C C
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.
XcmI  (3594)
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.
NcoI  (3512)
1 site
C C A T G G G G T A C C
BsmI  (3361)
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.
BsmBI  (3095)
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.
PvuII  (471)
1 site
C A G C T G G T C G A C
HindIII  (501)
1 site
A A G C T T T T C G A A
Acc65I  (507)
1 site
G G T A C C C C A T G G
KpnI  (511)
1 site
G G T A C C C C A T G G
Eco53kI  (515)
1 site
G A G C T C C T C G A G
SacI  (517)
1 site
G A G C T C C T C G A G
BamHI  (519)
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.
BstXI  (546)
2 sites
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoRI  (550)
1 site
G A A T T C C T T A A G
BstXI  (572)
2 sites
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NotI  (577)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (583)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (583)
1 site
V C T C G A G B B G A G C T C V
XhoI  (583)
1 site
C T C G A G G A G C T C
XbaI  (589)
1 site
T C T A G A A G A T C T
PmeI  (682)
1 site
G T T T A A A C C A A A T T T G
BsrGI  (874)
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  (1346)
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  (1863)
1 site
T C A T G A A G T A C T
AhdI  (2036)
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  (2156)
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  (2573)
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.
AanI  (2906)
2 sites
T T A T A A A A T A T T
2μ ori
4542 .. 5422  =  881 bp
yeast 2μ plasmid origin of replication
2μ ori
4542 .. 5422  =  881 bp
yeast 2μ plasmid origin of replication
AmpR
1963 .. 2823  =  861 bp
286 amino acids  =  31.7 kDa
   Segment 2:  
   1963 .. 2754  =  792 bp
   263 amino acids  =  29.1 kDa
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
AmpR
1963 .. 2823  =  861 bp
286 amino acids  =  31.7 kDa
   Segment 1:  signal sequence  
   2755 .. 2823  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
AmpR
1963 .. 2823  =  861 bp
286 amino acids  =  31.7 kDa
2 segments
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
URA3
2919 .. 3722  =  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
2919 .. 3722  =  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
1204 .. 1792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1204 .. 1792  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5491 .. 5946  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5491 .. 5946  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
GAL1 promoter
2 .. 443  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
2 .. 443  =  442 bp
inducible promoter, regulated by Gal4
CYC1 terminator
709 .. 956  =  248 bp
transcription terminator for CYC1
CYC1 terminator
709 .. 956  =  248 bp
transcription terminator for CYC1
URA3 promoter
3723 .. 3938  =  216 bp
URA3 promoter
3723 .. 3938  =  216 bp
MCS
501 .. 594  =  94 bp
multiple cloning site
MCS
501 .. 594  =  94 bp
multiple cloning site
V5 tag
607 .. 648  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
607 .. 648  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
475 .. 493  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
475 .. 493  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
658 .. 675  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
658 .. 675  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
UAS
2 .. 119  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
2 .. 119  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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