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Plasmid Files

pSP64 Poly(A)

Cloning vector for in vitro transcription from the SP6 promoter with the addition of a synthetic 30-base poly(A) tail.

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pSP64 Poly(A) Sequence and MappSP64 Poly(A).dna
Map and Sequence File   
Sequence Author:  Promega
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 HindIII (7) BsrGI (2950) BtgZI (2808) BmtI (2793) NheI (2789) NaeI (2758) NgoMIV (2756) SgrAI (2747) AfeI (2665) BtgI (2629) SphI (2599) EcoNI (2534) PfoI (2367) AatII (2256) ZraI (2254) SspI (2138) XmnI (1933) ScaI (1814) PvuI (1704) FspI (1556) NmeAIII (1482) BfuAI - BspMI (9) PstI (20) SalI (22) AccI (23) HincII (24) XbaI (28) BamHI (34) AvaI - BsoBI - TspMI - XmaI (39) BmeT110I (40) SmaI (41) Eco53kI (48) SacI (50) ApoI - EcoRI (86) lac operator PvuII (265) BspQI - SapI (325) AflIII - PciI (441) BseYI (745) PspFI (749) AlwNI (857) AhdI (1334) BsaI (1395) BpmI (1404) BglI (1454) pSP64 Poly(A) 3030 bp
HindIII  (7)
1 site
A A G C T T T T C G A A
BsrGI  (2950)
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.
BtgZI  (2808)
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.
BmtI  (2793)
1 site
G C T A G C C G A T C G
NheI  (2789)
1 site
G C T A G C C G A T C G
NaeI  (2758)
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  (2756)
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.
SgrAI  (2747)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
AfeI  (2665)
1 site
A G C G C T T C G C G A
BtgI  (2629)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SphI  (2599)
1 site
G C A T G C C G T A C G
EcoNI  (2534)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PfoI  (2367)
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.
AatII  (2256)
1 site
G A C G T C C T G C A G
ZraI  (2254)
1 site
G A C G T C C T G C A G
SspI  (2138)
1 site
A A T A T T T T A T A A
XmnI  (1933)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (1814)
1 site
A G T A C T T C A T G A
PvuI  (1704)
1 site
C G A T C G G C T A G C
FspI  (1556)
1 site
T G C G C A A C G C G T
NmeAIII  (1482)
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).
BfuAI  (9)
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  (9)
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.
PstI  (20)
1 site
C T G C A G G A C G T C
SalI  (22)
1 site
G T C G A C C A G C T G
AccI  (23)
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  (24)
1 site
G T Y R A C C A R Y T G
XbaI  (28)
1 site
T C T A G A A G A T C T
BamHI  (34)
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.
AvaI  (39)
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  (39)
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.
TspMI  (39)
1 site
C C C G G G G G G C C C
XmaI  (39)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
BmeT110I  (40)
1 site
C Y C G R G G R G C Y C
SmaI  (41)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Eco53kI  (48)
1 site
G A G C T C C T C G A G
SacI  (50)
1 site
G A G C T C C T C G A G
ApoI  (86)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (86)
1 site
G A A T T C C T T A A G
PvuII  (265)
1 site
C A G C T G G T C G A C
BspQI  (325)
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  (325)
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  (441)
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  (441)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (745)
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.
PspFI  (749)
1 site
C C C A G C G G G T C G
AlwNI  (857)
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  (1334)
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.
BsaI  (1395)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BpmI  (1404)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BglI  (1454)
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.
MCS
7 .. 91  =  85 bp
multiple cloning site
MCS
7 .. 91  =  85 bp
multiple cloning site
AmpR
1261 .. 2121  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1261 .. 2052  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1261 .. 2121  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2053 .. 2121  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1261 .. 2121  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
502 .. 1090  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
502 .. 1090  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
2122 .. 2226  =  105 bp
AmpR promoter
2122 .. 2226  =  105 bp
lac promoter
148 .. 178  =  31 bp
   Segment 3:  -10  
   148 .. 154  =  7 bp
promoter for the E. coli lac operon
lac promoter
148 .. 178  =  31 bp
   Segment 2:  
   155 .. 172  =  18 bp
promoter for the E. coli lac operon
lac promoter
148 .. 178  =  31 bp
   Segment 1:  -35  
   173 .. 178  =  6 bp
promoter for the E. coli lac operon
lac promoter
148 .. 178  =  31 bp
3 segments
promoter for the E. coli lac operon
SP6 promoter
3014 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3014 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
M13 rev
100 .. 116  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
100 .. 116  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
124 .. 140  =  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
124 .. 140  =  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).
poly(A)
54 .. 83  =  30 bp
poly(A)
54 .. 83  =  30 bp
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