pSpot5

Yeast centromeric plasmid for expression of fusion proteins with an N-terminal Spot-Tag®.

Sequence Author: ChromoTek

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SphI (7602) PacI (7535) Bsu36I (7186) AatII (6823) ZraI (6821) FseI (6132) EagI (6130) PspFI (5423) BseYI (5419) AlwNI (5314) AhdI (4835) PmlI (3901) EcoRI (1) PstI (51) BamHI (53) AvrII (58) PspOMI (65) ApaI (69) HindIII (71) EcoRV (80) PaeR7I - PspXI - XhoI (86) TspMI - XmaI (93) SmaI (95) NheI (100) BmtI (104) Eco53kI (109) SacI (111) M13 fwd BtgZI (637) DraIII (645) KasI (1134) NarI (1135) SfoI (1136) PluTI (1138) HpaI (1167) BstEII (1609) BspDI - ClaI (1727) AflII (1890) BtgI (2436) BfuAI - BspMI (2569) BsrGI (2696) AccI (2769) PfoI (3282) CEN/ARS SwaI (3877) pSpot5 7813 bp
SphI  (7602)
1 site
G C A T G C C G T A C G
PacI  (7535)
1 site
T T A A T T A A A A T T A A T T
Bsu36I  (7186)
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.
AatII  (6823)
1 site
G A C G T C C T G C A G
ZraI  (6821)
1 site
G A C G T C C T G C A G
FseI  (6132)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
EagI  (6130)
1 site
C G G C C G G C C G G C
PspFI  (5423)
1 site
C C C A G C G G G T C G
BseYI  (5419)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
AlwNI  (5314)
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.
AhdI  (4835)
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.
PmlI  (3901)
1 site
C A C G T G G T G C A C
EcoRI  (1)
1 site
G A A T T C C T T A A G
PstI  (51)
1 site
C T G C A G G A C G T C
BamHI  (53)
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.
AvrII  (58)
1 site
C C T A G G G G A T C C
PspOMI  (65)
1 site
G G G C C C C C C G G G
ApaI  (69)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HindIII  (71)
1 site
A A G C T T T T C G A A
EcoRV  (80)
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.
PaeR7I  (86)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (86)
1 site
V C T C G A G B B G A G C T C V
XhoI  (86)
1 site
C T C G A G G A G C T C
TspMI  (93)
1 site
C C C G G G G G G C C C
XmaI  (93)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (95)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
NheI  (100)
1 site
G C T A G C C G A T C G
BmtI  (104)
1 site
G C T A G C C G A T C G
Eco53kI  (109)
1 site
G A G C T C C T C G A G
SacI  (111)
1 site
G A G C T C C T C G A G
BtgZI  (637)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
DraIII  (645)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
KasI  (1134)
1 site
G G C G C C C C G C G G
NarI  (1135)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (1136)
1 site
G G C G C C C C G C G G
PluTI  (1138)
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.
HpaI  (1167)
1 site
G T T A A C C A A T T G
BstEII  (1609)
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.
BspDI  (1727)
1 site
A T C G A T T A G C T A
ClaI  (1727)
1 site
A T C G A T T A G C T A
AflII  (1890)
1 site
C T T A A G G A A T T C
BtgI  (2436)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
BfuAI  (2569)
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  (2569)
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.
BsrGI  (2696)
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.
AccI  (2769)
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.
PfoI  (3282)
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.
SwaI  (3877)
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.
LEU2
1576 .. 2670  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
LEU2
1576 .. 2670  =  1095 bp
364 amino acids  =  39.0 kDa
Product: 3-isopropylmalate dehydrogenase, required for leucine biosynthesis
yeast auxotrophic marker
AmpR
4048 .. 4908  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4048 .. 4116  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4048 .. 4908  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4117 .. 4908  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4048 .. 4908  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
5079 .. 5667  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5079 .. 5667  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
TPI1 promoter
7303 .. 7812  =  510 bp
strong constitutive promoter for yeast triose phosphate isomerase
TPI1 promoter
7303 .. 7812  =  510 bp
strong constitutive promoter for yeast triose phosphate isomerase
CEN/ARS
3403 .. 3906  =  504 bp
S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence
CEN/ARS
3403 .. 3906  =  504 bp
S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence
f1 ori
412 .. 867  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
412 .. 867  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
LEU2 promoter
1168 .. 1575  =  408 bp
LEU2 promoter
1168 .. 1575  =  408 bp
AmpR promoter
3943 .. 4047  =  105 bp
AmpR promoter
3943 .. 4047  =  105 bp
AmpR promoter
6689 .. 6793  =  105 bp
AmpR promoter
6689 .. 6793  =  105 bp
MCS
47 .. 112  =  66 bp
multiple cloning site
MCS
47 .. 112  =  66 bp
multiple cloning site
T7 promoter
230 .. 248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
230 .. 248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 fwd
255 .. 271  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
255 .. 271  =  17 bp
common sequencing primer, one of multiple similar variants
ATG
10 .. 12  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
10 .. 12  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Spot-Tag
13 .. 48  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
Spot-Tag
13 .. 48  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
ORF:  1576 .. 2670  =  1095 bp
ORF:  364 amino acids  =  39.0 kDa
ORF:  4048 .. 4908  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  176 .. 400  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  6092 .. 6688  =  597 bp
ORF:  198 amino acids  =  21.9 kDa
ORF:  7754 .. 192  =  252 bp
ORF:  83 amino acids  =  9.7 kDa
ORF:  2238 .. 2549  =  312 bp
ORF:  103 amino acids  =  11.2 kDa
ORF:  4512 .. 4778  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  7434 .. 7667  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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Download pSpot5.dna file

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