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Plasmid Files

pOSIP-TO

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

 
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 Acc65I (13) EcoRI (1) rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator BsgI (6104) PflFI - Tth111I (6090) PfoI (5987) BseRI (5742) SnaBI (4883) SalI (4798) StuI (4659) ScaI (4265) BglI (4222) PmlI (3988) EcoRV (3546) BspDI - ClaI (3366) KpnI (17) BamHI (22) BssHII (70) SrfI (225) BsaI (363) BglII (469) BciVI (923) SpeI (1126) MCS 2 PstI (1144) rrnB T2 terminator BstBI (1234) PacI (1340) PspOMI (1349) ApaI (1353) ZraI (1379) AatII (1381) AvrII (1418) EarI (1442) BsmI (1718) PshAI (2090) AfeI (2473) NruI (2921) FspAI (3213) DraIII (3360) pOSIP-TO 6919 bp
Acc65I  (13)
1 site
G G T A C C C C A T G G
EcoRI  (1)
1 site
G A A T T C C T T A A G
BsgI  (6104)
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  (6090)
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  (6090)
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  (5987)
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.
BseRI  (5742)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
SnaBI  (4883)
1 site
T A C G T A A T G C A T
SalI  (4798)
1 site
G T C G A C C A G C T G
StuI  (4659)
1 site
A G G C C T T C C G G A
ScaI  (4265)
1 site
A G T A C T T C A T G A
BglI  (4222)
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.
PmlI  (3988)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
EcoRV  (3546)
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.
BspDI  (3366)
1 site
A T C G A T T A G C T A
ClaI  (3366)
1 site
A T C G A T T A G C T A
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.
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
BstBI  (1234)
1 site
T T C G A A A A G C T T
PacI  (1340)
1 site
T T A A T T A A A A T T A A T T
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.
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.
AfeI  (2473)
1 site
A G C G C T T C G C G A
NruI  (2921)
1 site
T C G C G A A G C G C T
FspAI  (3213)
1 site
R T G C G C A Y Y A C G C G T R
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.
TcR
3433 .. 4638  =  1206 bp
401 amino acids  =  43.4 kDa
Product: tetracycline efflux protein
confers resistance to tetracycline
TcR
3433 .. 4638  =  1206 bp
401 amino acids  =  43.4 kDa
Product: tetracycline efflux protein
confers resistance to tetracycline
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
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
4848 .. 5236  =  389 bp
γ replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication
R6K γ ori
4848 .. 5236  =  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
5634 .. 5880  =  247 bp
transcription terminator tL3 from phage λ
λ tL3 terminator
5634 .. 5880  =  247 bp
transcription terminator tL3 from phage λ
phage 186 attP
5307 .. 5521  =  215 bp
attachment site of phage 186
phage 186 attP
5307 .. 5521  =  215 bp
attachment site of phage 186
lambda t0 terminator
4697 .. 4791  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
4697 .. 4791  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
6200 .. 6286  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6200 .. 6286  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6381 .. 6467  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6381 .. 6467  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6562 .. 6648  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6562 .. 6648  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6743 .. 6829  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
6743 .. 6829  =  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
tetR/tetA promoters
3371 .. 3425  =  55 bp
overlapping promoters for bacterial tetR and tetA
tetR/tetA promoters
3371 .. 3425  =  55 bp
overlapping promoters for bacterial tetR and tetA
FRT
5247 .. 5294  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
FRT
5247 .. 5294  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
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
MCS 2
1126 .. 1145  =  20 bp
multiple cloning site, part 2
MCS 2
1126 .. 1145  =  20 bp
multiple cloning site, part 2
tet operator
3406 .. 3424  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
3406 .. 3424  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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