pSpot4

Bacterial plasmid for inducible expression of fusion proteins with an N-terminal Spot-Tag®.

Sequence Author: ChromoTek

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lac operator MfeI (2900) PvuII (2813) BspQI - SapI (2750) AflIII - PciI (2633) PspFI (2333) BseYI (2329) AlwNI (2224) AhdI (1745) BsrFI (1660) BglI (1627) NmeAIII (1598) FspI (1522) T7 promoter XbaI (47) RBS BssHII (102) Spot-Tag BamHI (127) EcoRI (133) Eco53kI (141) BanII - SacI (143) SalI (146) AccI (147) HincII (148) HindIII (152) EagI - NotI (159) AvaI - BsoBI - PaeR7I - PspXI - XhoI (167) BlpI (235) StyI (257) NdeI (571) BstAPI (575) PfoI (706) EcoO109I (765) ZraI (824) AatII (826) ScaI (1264) TsoI (1347) PvuI (1376) pSpot4 2972 bp
MfeI  (2900)
1 site
C A A T T G G T T A A C
PvuII  (2813)
1 site
C A G C T G G T C G A C
BspQI  (2750)
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  (2750)
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.
AflIII  (2633)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2633)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2333)
1 site
C C C A G C G G G T C G
BseYI  (2329)
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  (2224)
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  (1745)
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.
BsrFI  (1660)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
BglI  (1627)
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.
NmeAIII  (1598)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
FspI  (1522)
1 site
T G C G C A A C G C G T
XbaI  (47)
1 site
T C T A G A A G A T C T
BssHII  (102)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BamHI  (127)
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.
EcoRI  (133)
1 site
G A A T T C C T T A A G
Eco53kI  (141)
1 site
G A G C T C C T C G A G
BanII  (143)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (143)
1 site
G A G C T C C T C G A G
SalI  (146)
1 site
G T C G A C C A G C T G
AccI  (147)
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.
HincII  (148)
1 site
G T Y R A C C A R Y T G
HindIII  (152)
1 site
A A G C T T T T C G A A
EagI  (159)
1 site
C G G C C G G C C G G C
NotI  (159)
1 site
G C G G C C G C C G C C G G C G
AvaI  (167)
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  (167)
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.
PaeR7I  (167)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (167)
1 site
V C T C G A G B B G A G C T C V
XhoI  (167)
1 site
C T C G A G G A G C T C
BlpI  (235)
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.
StyI  (257)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
NdeI  (571)
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.
BstAPI  (575)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
PfoI  (706)
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.
EcoO109I  (765)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
ZraI  (824)
1 site
G A C G T C C T G C A G
AatII  (826)
1 site
G A C G T C C T G C A G
ScaI  (1264)
1 site
A G T A C T T C A T G A
TsoI  (1347)
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  (1376)
1 site
C G A T C G G C T A G C
AmpR
958 .. 1818  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   958 .. 1026  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
958 .. 1818  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1027 .. 1818  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
958 .. 1818  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1989 .. 2577  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1989 .. 2577  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
853 .. 957  =  105 bp
AmpR promoter
853 .. 957  =  105 bp
MCS
127 .. 173  =  47 bp
multiple cloning site
MCS
127 .. 173  =  47 bp
multiple cloning site
ATG
88 .. 90  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
88 .. 90  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Spot-Tag
91 .. 126  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
Spot-Tag
91 .. 126  =  36 bp
12 amino acids  =  1.4 kDa
Product: engineered Spot-Tag® peptide that binds with high affinity to a nanobody
lac operator
20 .. 44  =  25 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
20 .. 44  =  25 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).
RBS
59 .. 81  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
RBS
59 .. 81  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
lac operator
2903 .. 2919  =  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
2903 .. 2919  =  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).
ORF:  958 .. 1818  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1422 .. 1688  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2737 .. 403  =  639 bp
ORF:  212 amino acids  =  23.6 kDa
ORF:  325 .. 549  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
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Download pSpot4.dna file

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