pOSIP-KP

Prokaryotic one-step cloning and chromosomal integration vector encoding a kanamycin resistance marker and the phage φ80 integrase.
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EcoRI (1) rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator BsgI (6123) PflFI - Tth111I (6109) PfoI (6006) λ tL3 terminator BbvCI (5773) BseRI (5761) BmtI (5562) NheI (5558) DraI (4967) SnaBI (4729) SalI (4644) PpuMI * (4437) PvuI (4188) EcoNI (4100) NruI (3845) BspDI - ClaI (3507) Eco53kI (9) SacI (11) Acc65I (13) KpnI (17) SacII (29) MCS 1 BssHII (79) SrfI (234) BsaI (372) BglII (478) BciVI (932) PstI (1153) MCS 2 KasI (1167) NarI (1168) SfoI (1169) PluTI (1171) rrnB T2 terminator BstBI (1237) MfeI (1242) PacI (1343) AgeI (1346) ZraI (1382) AatII (1384) AvrII (1421) BsmI (1721) BstAPI (1968) PshAI (2093) AflIII - MluI (2243) BfuAI - BspMI - PaqCI (2629) PspXI (2939) FspI (3014) BsiWI (3095) pOSIP-KP 6938 bp
EcoRI  (1)
1 site
G A A T T C C T T A A G
BsgI  (6123)
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  (6109)
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  (6109)
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  (6006)
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  (5773)
1 site
C C T C A G C G G A G T C G
BseRI  (5761)
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.
BmtI  (5562)
1 site
G C T A G C C G A T C G
NheI  (5558)
1 site
G C T A G C C G A T C G
DraI  (4967)
1 site
T T T A A A A A A T T T
SnaBI  (4729)
1 site
T A C G T A A T G C A T
SalI  (4644)
1 site
G T C G A C C A G C T G
PpuMI  (4437)
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  (4188)
1 site
C G A T C G G C T A G C
EcoNI  (4100)
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.
NruI  (3845)
1 site
T C G C G A A G C G C T
BspDI  (3507)
1 site
A T C G A T T A G C T A
ClaI  (3507)
1 site
A T C G A T T A G C T A
Eco53kI  (9)
1 site
G A G C T C C T C G A G
SacI  (11)
1 site
G A G C T C C T C G A G
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
SacII  (29)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
BssHII  (79)
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  (234)
1 site
G C C C G G G C C G G G C C C G
BsaI  (372)
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  (478)
1 site
A G A T C T T C T A G A
BciVI  (932)
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.
PstI  (1153)
1 site
C T G C A G G A C G T C
KasI  (1167)
1 site
G G C G C C C C G C G G
NarI  (1168)
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  (1169)
1 site
G G C G C C C C G C G G
PluTI  (1171)
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  (1237)
1 site
T T C G A A A A G C T T
MfeI  (1242)
1 site
C A A T T G G T T A A C
PacI  (1343)
1 site
T T A A T T A A A A T T A A T T
AgeI  (1346)
1 site
A C C G G T T G G C C A
ZraI  (1382)
1 site
G A C G T C C T G C A G
AatII  (1384)
1 site
G A C G T C C T G C A G
AvrII  (1421)
1 site
C C T A G G G G A T C C
BsmI  (1721)
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  (1968)
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  (2093)
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  (2243)
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  (2243)
1 site
A C G C G T T G C G C A
BfuAI  (2629)
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  (2629)
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.
PaqCI  (2629)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PspXI  (2939)
1 site
V C T C G A G B B G A G C T C V
FspI  (3014)
1 site
T G C G C A A C G C G T
BsiWI  (3095)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
phage φ80 integrase
2372 .. 3580  =  1209 bp
402 amino acids  =  46.7 kDa
Product: integrase from phage φ80
phage φ80 integrase
2372 .. 3580  =  1209 bp
402 amino acids  =  46.7 kDa
Product: integrase from phage φ80
KanR
3757 .. 4572  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
3757 .. 4572  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
λ repressor (ts)
1427 .. 2140  =  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)
1427 .. 2140  =  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
486 .. 1074  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
486 .. 1074  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
R6K γ ori
4694 .. 5082  =  389 bp
γ replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication
R6K γ ori
4694 .. 5082  =  389 bp
γ replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication
phage Φ80 attP
5247 .. 5564  =  318 bp
attachment site of phage φ80
phage Φ80 attP
5247 .. 5564  =  318 bp
attachment site of phage φ80
ccdB
151 .. 456  =  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
151 .. 456  =  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
5653 .. 5899  =  247 bp
transcription terminator tL3 from phage λ
λ tL3 terminator
5653 .. 5899  =  247 bp
transcription terminator tL3 from phage λ
rrnB T1 terminator
6219 .. 6305  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6219 .. 6305  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6400 .. 6486  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6400 .. 6486  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6581 .. 6667  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6581 .. 6667  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6762 .. 6848  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
6762 .. 6848  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
MCS 1
1 .. 84  =  84 bp
multiple cloning site, part 1
MCS 1
1 .. 84  =  84 bp
multiple cloning site, part 1
FRT
5093 .. 5140  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
FRT
5093 .. 5140  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
FRT (minimal)
1387 .. 1420  =  34 bp
supports FLP-mediated excision but not integration (Turan and Bode, 2011)
FRT (minimal)
1387 .. 1420  =  34 bp
supports FLP-mediated excision but not integration (Turan and Bode, 2011)
tonB terminator
1279 .. 1310  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
tonB terminator
1279 .. 1310  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
rrnB T2 terminator
1203 .. 1230  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
1203 .. 1230  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
MCS 2
1149 .. 1172  =  24 bp
multiple cloning site, part 2
MCS 2
1149 .. 1172  =  24 bp
multiple cloning site, part 2
ORF:  151 .. 456  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  2119 .. 2436  =  318 bp
ORF:  105 amino acids  =  11.9 kDa
ORF:  3757 .. 4572  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5149 .. 5373  =  225 bp
ORF:  74 amino acids  =  8.3 kDa
ORF:  2372 .. 3580  =  1209 bp
ORF:  402 amino acids  =  46.7 kDa
ORF:  6135 .. 6374  =  240 bp
ORF:  79 amino acids  =  9.2 kDa
ORF:  1427 .. 2140  =  714 bp
ORF:  237 amino acids  =  26.2 kDa
ORF:  4367 .. 4591  =  225 bp
ORF:  74 amino acids  =  8.7 kDa
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