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Plasmid Files

pPrime (linearized)

Linearized vector with 3'-U overhangs for UA cloning of PCR products produced by non-proofreading DNA polymerases.

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pPrime (linearized) Sequence and MappPrime (linearized).dna
Map and Sequence File   
Sequence Author:  5 Prime
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 BspQI - SapI (3527) PciI (3410) PspFI (3110) BseYI (3106) AlwNI (3001) PflMI (2560) Bpu10I (2314) BsmBI (2313) AsiSI (2297) EcoNI (2209) SmaI (2171) TspMI - XmaI (2169) BspDI - ClaI (1988) NruI (1954) AhdI (1647) BsaI (1581) lac operator Acc65I (3802) KpnI (3806) SphI (3814) PstI (3819) MluI (3821) SnaBI (3829) BamHI (3835) EcoRI (3842) End (3853) Start (1) EcoRI (6) SalI (12) AccI (13) HincII (14) HindIII (18) PaeR7I - XhoI (24) AvrII - StyI (30) NheI (35) BmtI (39) XbaI (41) AleI - PmlI (52) BstXI (54) EcoO109I - PspOMI (59) ApaI (63) Eco53kI (67) SacI (69) EagI - NotI (72) SP6 promoter AanI - PsiI (369) DraIII (497) BtgZI (498) NgoMIV (598) NaeI (600) BsaHI (1107) TatI (1164) ScaI (1166) NmeAIII (1500) BpmI (1578) pPrime 3852 bp
BspQI  (3527)
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  (3527)
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.
PciI  (3410)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (3110)
1 site
C C C A G C G G G T C G
BseYI  (3106)
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  (3001)
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.
PflMI  (2560)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
Bpu10I  (2314)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BsmBI  (2313)
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.
AsiSI  (2297)
1 site
G C G A T C G C C G C T A G C G
EcoNI  (2209)
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.
SmaI  (2171)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (2169)
1 site
C C C G G G G G G C C C
XmaI  (2169)
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.
BspDI  (1988)
1 site
A T C G A T T A G C T A
ClaI  (1988)
1 site
A T C G A T T A G C T A
NruI  (1954)
1 site
T C G C G A A G C G C T
AhdI  (1647)
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  (1581)
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.
Acc65I  (3802)
1 site
G G T A C C C C A T G G
KpnI  (3806)
1 site
G G T A C C C C A T G G
SphI  (3814)
1 site
G C A T G C C G T A C G
PstI  (3819)
1 site
C T G C A G G A C G T C
MluI  (3821)
1 site
A C G C G T T G C G C A
SnaBI  (3829)
1 site
T A C G T A A T G C A T
BamHI  (3835)
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  (3842)
2 sites
G A A T T C C T T A A G
End  (3853)
0 sites
Start  (1)
0 sites
EcoRI  (6)
2 sites
G A A T T C C T T A A G
SalI  (12)
1 site
G T C G A C C A G C T G
AccI  (13)
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  (14)
1 site
G T Y R A C C A R Y T G
HindIII  (18)
1 site
A A G C T T T T C G A A
PaeR7I  (24)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (24)
1 site
C T C G A G G A G C T C
AvrII  (30)
1 site
C C T A G G G G A T C C
StyI  (30)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
NheI  (35)
1 site
G C T A G C C G A T C G
BmtI  (39)
1 site
G C T A G C C G A T C G
XbaI  (41)
1 site
T C T A G A A G A T C T
AleI  (52)
1 site
C A C N N N N G T G G T G N N N N C A C
PmlI  (52)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstXI  (54)
1 site
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.
EcoO109I  (59)
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.
PspOMI  (59)
1 site
G G G C C C C C C G G G
ApaI  (63)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
Eco53kI  (67)
1 site
G A G C T C C T C G A G
SacI  (69)
1 site
G A G C T C C T C G A G
EagI  (72)
1 site
C G G C C G G C C G G C
NotI  (72)
1 site
G C G G C C G C C G C C G G C G
AanI  (369)
1 site
T T A T A A A A T A T T
PsiI  (369)
1 site
T T A T A A A A T A T T
DraIII  (497)
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.
BtgZI  (498)
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.
NgoMIV  (598)
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.
NaeI  (600)
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.
BsaHI  (1107)
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.
TatI  (1164)
1 site
W G T A C W W C A T G W
ScaI  (1166)
1 site
A G T A C T T C A T G A
NmeAIII  (1500)
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).
BpmI  (1578)
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.
AmpR
860 .. 1720  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   860 .. 928  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
860 .. 1720  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   929 .. 1720  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
860 .. 1720  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
KanR
1866 .. 2681  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
1866 .. 2681  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
2766 .. 3354  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2766 .. 3354  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
273 .. 728  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
273 .. 728  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
AmpR promoter
755 .. 859  =  105 bp
AmpR promoter
755 .. 859  =  105 bp
LacZα
3752 .. 3852  =  101 bp
33 amino acids  =  3.9 kDa
Product: LacZα fragment of β-galactosidase
LacZα
3752 .. 3852  =  101 bp
33 amino acids  =  3.9 kDa
Product: LacZα fragment of β-galactosidase
MCS
2 .. 78  =  77 bp
multiple cloning site
MCS
2 .. 78  =  77 bp
multiple cloning site
lac promoter
3678 .. 3708  =  31 bp
   Segment 1:  -35  
   3678 .. 3683  =  6 bp
promoter for the E. coli lac operon
lac promoter
3678 .. 3708  =  31 bp
   Segment 2:  
   3684 .. 3701  =  18 bp
promoter for the E. coli lac operon
lac promoter
3678 .. 3708  =  31 bp
   Segment 3:  -10  
   3702 .. 3708  =  7 bp
promoter for the E. coli lac operon
lac promoter
3678 .. 3708  =  31 bp
3 segments
promoter for the E. coli lac operon
SP6 promoter
84 .. 102  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
84 .. 102  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
M13 fwd
116 .. 132  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
116 .. 132  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
3716 .. 3732  =  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
3716 .. 3732  =  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).
LacZα
2 .. 281  =  280 bp
92 amino acids  =  10.5 kDa
Product: LacZα fragment of β-galactosidase
LacZα
2 .. 281  =  280 bp
92 amino acids  =  10.5 kDa
Product: LacZα fragment of β-galactosidase
MCS
3802 .. 3852  =  51 bp
multiple cloning site
MCS
3802 .. 3852  =  51 bp
multiple cloning site
T7 promoter
3775 .. 3793  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3775 .. 3793  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 rev
3740 .. 3756  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3740 .. 3756  =  17 bp
common sequencing primer, one of multiple similar
variants
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