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Plasmid Files

pOSIP-KO

Prokaryotic one-step cloning and chromosomal integration vector encoding a kanamycin resistance marker and the phage 186 integrase.

 
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 EcoRI (1) rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator BsgI (5657) PflFI - Tth111I (5643) PfoI (5540) λ tL3 terminator BbvCI (5307) Bsu36I (5153) DraI (4670) SnaBI (4432) SalI (4347) PpuMI * (4140) PvuI (3891) EcoNI (3803) DraIII (3360) Acc65I (13) KpnI (17) BamHI (22) BssHII (70) BsrGI (198) SrfI (225) BsaI (363) BglII (469) BciVI (923) SpeI (1126) PstI (1144) SphI (1150) KasI (1164) NarI (1165) SfoI (1166) PluTI (1168) BstBI (1234) MfeI (1239) PacI (1340) AgeI (1343) PspOMI (1349) ApaI (1353) ZraI (1379) AatII (1381) AvrII (1418) EarI (1442) BsmI (1718) BstAPI (1965) PshAI (2090) AflIII - MluI (2240) AfeI (2473) FspAI (3213) pOSIP-KO 6472 bp
EcoRI  (1)
1 site
G A A T T C C T T A A G
BsgI  (5657)
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).
PflFI  (5643)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (5643)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PfoI  (5540)
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.
BbvCI  (5307)
1 site
C C T C A G C G G A G T C G
Bsu36I  (5153)
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.
DraI  (4670)
1 site
T T T A A A A A A T T T
SnaBI  (4432)
1 site
T A C G T A A T G C A T
SalI  (4347)
1 site
G T C G A C C A G C T G
PpuMI  (4140)
1 site
R G G W C C Y Y C C W G G R
* Blocked by Dcm methylation.
Sticky ends from different PpuMI sites may not be compatible.
PvuI  (3891)
1 site
C G A T C G G C T A G C
EcoNI  (3803)
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.
DraIII  (3360)
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.
Acc65I  (13)
1 site
G G T A C C C C A T G G
KpnI  (17)
1 site
G G T A C C C C A T G G
BamHI  (22)
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.
BssHII  (70)
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.
BsrGI  (198)
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.
SrfI  (225)
1 site
G C C C G G G C C G G G C C C G
BsaI  (363)
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.
BglII  (469)
1 site
A G A T C T T C T A G A
BciVI  (923)
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.
SpeI  (1126)
1 site
A C T A G T T G A T C A
PstI  (1144)
1 site
C T G C A G G A C G T C
SphI  (1150)
1 site
G C A T G C C G T A C G
KasI  (1164)
1 site
G G C G C C C C G C G G
NarI  (1165)
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  (1166)
1 site
G G C G C C C C G C G G
PluTI  (1168)
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.
BstBI  (1234)
1 site
T T C G A A A A G C T T
MfeI  (1239)
1 site
C A A T T G G T T A A C
PacI  (1340)
1 site
T T A A T T A A A A T T A A T T
AgeI  (1343)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PspOMI  (1349)
1 site
G G G C C C C C C G G G
ApaI  (1353)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
ZraI  (1379)
1 site
G A C G T C C T G C A G
AatII  (1381)
1 site
G A C G T C C T G C A G
AvrII  (1418)
1 site
C C T A G G G G A T C C
EarI  (1442)
1 site
C T C T T C N G A G A A G N N N N

Efficient cleavage requires at least two copies of the EarI
recognition sequence.
Sticky ends from different EarI sites may not be compatible.
BsmI  (1718)
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.
BstAPI  (1965)
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.
PshAI  (2090)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
AflIII  (2240)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (2240)
1 site
A C G C G T T G C G C A
AfeI  (2473)
1 site
A G C G C T T C G C G A
FspAI  (3213)
1 site
R T G C G C A Y Y A C G C G T R
phage 186 integrase
2276 .. 3286  =  1011 bp
336 amino acids  =  39.0 kDa
Product: integrase from phage 186
phage 186 integrase
2276 .. 3286  =  1011 bp
336 amino acids  =  39.0 kDa
Product: integrase from phage 186
KanR
3460 .. 4275  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
3460 .. 4275  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
λ repressor (ts)
1424 .. 2137  =  714 bp
237 amino acids  =  26.2 kDa
Product: temperature-sensitive variant of the phage
λ repressor
thermosensitivity is conferred by the A67T mutation
λ repressor (ts)
1424 .. 2137  =  714 bp
237 amino acids  =  26.2 kDa
Product: temperature-sensitive variant of the phage
λ repressor
thermosensitivity is conferred by the A67T mutation
ori
477 .. 1065  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
477 .. 1065  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
R6K γ ori
4397 .. 4785  =  389 bp
γ replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication
R6K γ ori
4397 .. 4785  =  389 bp
γ replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication
ccdB
142 .. 447  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
142 .. 447  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
λ tL3 terminator
5187 .. 5433  =  247 bp
transcription terminator tL3 from phage λ
λ tL3 terminator
5187 .. 5433  =  247 bp
transcription terminator tL3 from phage λ
phage 186 attP
4856 .. 5070  =  215 bp
attachment site of phage 186
phage 186 attP
4856 .. 5070  =  215 bp
attachment site of phage 186
rrnB T1 terminator
5753 .. 5839  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
5753 .. 5839  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
5934 .. 6020  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
5934 .. 6020  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6115 .. 6201  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6115 .. 6201  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6296 .. 6382  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6296 .. 6382  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
MCS 1
1 .. 75  =  75 bp
multiple cloning site, part 1
MCS 1
1 .. 75  =  75 bp
multiple cloning site, part 1
FRT
4796 .. 4843  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
FRT
4796 .. 4843  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
MCS 2
1126 .. 1169  =  44 bp
multiple cloning site, part 2
MCS 2
1126 .. 1169  =  44 bp
multiple cloning site, part 2
FRT (minimal)
1384 .. 1417  =  34 bp
supports FLP-mediated excision but not integration
(Turan and Bode, 2011)
FRT (minimal)
1384 .. 1417  =  34 bp
supports FLP-mediated excision but not integration
(Turan and Bode, 2011)
tonB terminator
1276 .. 1307  =  32 bp
bidirectional E. coli tonB-P14 transcription
terminator
tonB terminator
1276 .. 1307  =  32 bp
bidirectional E. coli tonB-P14 transcription
terminator
rrnB T2 terminator
1200 .. 1227  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
1200 .. 1227  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
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