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Plasmid Files

pYES2/NT B

High-copy episomal vector for galactose-inducible expression of N-terminally tagged proteins in S. cerevisiae. For other reading frames, use pYES2/NT A or pYES2/NT C.

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pYES2 NT B Sequence and MappYES2 NT B.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SpeI (6034) NaeI (5894) NgoMIV (5892) AanI (5663) SwaI (5566) SnaBI (5262) HpaI (4617) BmgBI (4557) PfoI * (4515) NruI (4211) BlpI (4175) BspDI - ClaI (4031) NdeI (3882) BsgI (3853) SbfI (3819) BfuAI - BspMI (3808) XcmI (3670) BsmI (3437) BsmBI (3171) GAL1 promoter PvuII (471) HindIII (501) ATG 6xHis Acc65I (602) KpnI (606) Bsu36I (607) BamHI (611) BstXI (622) EcoRI (626) BstXI (648) EagI - NotI (653) PaeR7I - PspXI - XhoI (659) XbaI (665) V5 tag 6xHis PmeI (758) BsrGI (950) BciVI (1422) BspHI (1939) AhdI (2112) BglI (2232) BsaHI (2649) AanI (2982) pYES2/NT B 6039 bp
SpeI  (6034)
1 site
A C T A G T T G A T C A
NaeI  (5894)
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  (5892)
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  (5663)
2 sites
T T A T A A A A T A T T
SwaI  (5566)
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  (5262)
1 site
T A C G T A A T G C A T
HpaI  (4617)
1 site
G T T A A C C A A T T G
BmgBI  (4557)
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  (4515)
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  (4211)
1 site
T C G C G A A G C G C T
BlpI  (4175)
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  (4031)
1 site
A T C G A T T A G C T A
ClaI  (4031)
1 site
A T C G A T T A G C T A
NdeI  (3882)
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  (3853)
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  (3819)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (3808)
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  (3808)
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  (3670)
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.
BsmI  (3437)
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  (3171)
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  (602)
1 site
G G T A C C C C A T G G
KpnI  (606)
1 site
G G T A C C C C A T G G
Bsu36I  (607)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BamHI  (611)
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  (622)
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  (626)
1 site
G A A T T C C T T A A G
BstXI  (648)
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.
EagI  (653)
1 site
C G G C C G G C C G G C
NotI  (653)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (659)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (659)
1 site
V C T C G A G B B G A G C T C V
XhoI  (659)
1 site
C T C G A G G A G C T C
XbaI  (665)
1 site
T C T A G A A G A T C T
PmeI  (758)
1 site
G T T T A A A C C A A A T T T G
BsrGI  (950)
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  (1422)
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  (1939)
1 site
T C A T G A A G T A C T
AhdI  (2112)
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  (2232)
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  (2649)
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  (2982)
2 sites
T T A T A A A A T A T T
2μ ori
4618 .. 5498  =  881 bp
yeast 2μ plasmid origin of replication
2μ ori
4618 .. 5498  =  881 bp
yeast 2μ plasmid origin of replication
AmpR
2039 .. 2899  =  861 bp
286 amino acids  =  31.7 kDa
   Segment 2:  
   2039 .. 2830  =  792 bp
   263 amino acids  =  29.1 kDa
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
AmpR
2039 .. 2899  =  861 bp
286 amino acids  =  31.7 kDa
   Segment 1:  signal sequence  
   2831 .. 2899  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
AmpR
2039 .. 2899  =  861 bp
286 amino acids  =  31.7 kDa
2 segments
Product: β-lactamase
confers resistance fo ampicillin, carbenicillin, and
related antibiotics
URA3
2995 .. 3798  =  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
2995 .. 3798  =  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
1280 .. 1868  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1280 .. 1868  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5567 .. 6022  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5567 .. 6022  =  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
785 .. 1032  =  248 bp
transcription terminator for CYC1
CYC1 terminator
785 .. 1032  =  248 bp
transcription terminator for CYC1
URA3 promoter
3799 .. 4014  =  216 bp
URA3 promoter
3799 .. 4014  =  216 bp
MCS
602 .. 670  =  69 bp
multiple cloning site
MCS
602 .. 670  =  69 bp
multiple cloning site
V5 tag
683 .. 724  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
683 .. 724  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 tag (gene 10 leader)
543 .. 575  =  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)
543 .. 575  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
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
522 .. 539  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
522 .. 539  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
734 .. 751  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
734 .. 751  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
510 .. 512  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
510 .. 512  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
UAS
2 .. 119  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
2 .. 119  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
Xpress™ tag
579 .. 602  =  24 bp
8 amino acids  =  998.0 Da
   Segment 1:  
   579 .. 587  =  9 bp
   3 amino acids  =  409.5 Da
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
Xpress™ tag
579 .. 602  =  24 bp
8 amino acids  =  998.0 Da
   Segment 2:  enterokinase site  
   588 .. 602  =  15 bp
   5 amino acids  =  606.5 Da
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
Xpress™ tag
579 .. 602  =  24 bp
8 amino acids  =  998.0 Da
2 segments
Product: Xpress™ epitope tag, including an
enterokinase recognition and cleavage site
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