pKatushka2S-B

Vector for expressing Katushka2S in bacteria.

Sequence Author: Evrogen

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No matches
AvaI - BsoBI - PaeR7I - XhoI (1) EcoO109I (4122) AatII (4068) ZraI (4066) XmnI (3745) PvuI (3516) FspI (3368) AseI (3318) AhdI (3146) AflIII - PciI (2253) BspQI - SapI (2137) BstAPI (2077) NdeI (2076) PsiI (49) MfeI (59) lac operator RBS BseRI (114) ATG BsaBI * (125) BamHI (126) AgeI (134) AleI (147) BstXI (149) BclI * (169) BsrGI (196) Bsu36I (412) SfiI (622) BsgI (651) PshAI (786) EagI - NotI (856) HindIII (863) NheI (983) BmtI (987) Bpu10I (1008) BspEI (1231) PflMI * (1467) XbaI (1840) PfoI (1897) PflFI - Tth111I (2000) BsaAI (2007) AccI (2025) BstZ17I (2026) pKatushka2S-B 4138 bp
AvaI  (1)
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  (1)
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.
PaeR7I  (1)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1)
1 site
C T C G A G G A G C T C
EcoO109I  (4122)
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.
AatII  (4068)
1 site
G A C G T C C T G C A G
ZraI  (4066)
1 site
G A C G T C C T G C A G
XmnI  (3745)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (3516)
1 site
C G A T C G G C T A G C
FspI  (3368)
1 site
T G C G C A A C G C G T
AseI  (3318)
1 site
A T T A A T T A A T T A
AhdI  (3146)
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.
AflIII  (2253)
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  (2253)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2137)
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  (2137)
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.
BstAPI  (2077)
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.
NdeI  (2076)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
PsiI  (49)
1 site
T T A T A A A A T A T T
MfeI  (59)
1 site
C A A T T G G T T A A C
BseRI  (114)
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.
BsaBI  (125)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
BamHI  (126)
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.
AgeI  (134)
1 site
A C C G G T T G G C C A
AleI  (147)
1 site
C A C N N N N G T G G T G N N N N C A C
BstXI  (149)
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.
BclI  (169)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BsrGI  (196)
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.
Bsu36I  (412)
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.
SfiI  (622)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
BsgI  (651)
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).
PshAI  (786)
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.
EagI  (856)
1 site
C G G C C G G C C G G C
NotI  (856)
1 site
G C G G C C G C C G C C G G C G
HindIII  (863)
1 site
A A G C T T T T C G A A
NheI  (983)
1 site
G C T A G C C G A T C G
BmtI  (987)
1 site
G C T A G C C G A T C G
Bpu10I  (1008)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
BspEI  (1231)
1 site
T C C G G A A G G C C T
PflMI  (1467)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
XbaI  (1840)
1 site
T C T A G A A G A T C T
PfoI  (1897)
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.
PflFI  (2000)
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  (2000)
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.
BsaAI  (2007)
1 site
Y A C G T R R T G C A Y
AccI  (2025)
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.
BstZ17I  (2026)
1 site
G T A T A C C A T A T G
AmpR
3073 .. 3933  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3073 .. 3864  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3073 .. 3933  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   3865 .. 3933  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3073 .. 3933  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ATG
114 .. 116  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
114 .. 116  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
Katushka2S
147 .. 854  =  708 bp
235 amino acids  =  26.4 kDa
Product: improved version of the far-red fluorescent protein TurboFP635/Katushka (Luker et al., 2015)
mammalian codon-optimized
Katushka2S
147 .. 854  =  708 bp
235 amino acids  =  26.4 kDa
Product: improved version of the far-red fluorescent protein TurboFP635/Katushka (Luker et al., 2015)
mammalian codon-optimized
CmR
1022 .. 1681  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1022 .. 1681  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
2314 .. 2902  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2314 .. 2902  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3934 .. 4038  =  105 bp
AmpR promoter
3934 .. 4038  =  105 bp
lambda t0 terminator
884 .. 978  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
884 .. 978  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
1746 .. 1832  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
1746 .. 1832  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 1:  
   10 .. 24  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 2:  -35  
   25 .. 30  =  6 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 3:  
   31 .. 47  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 4:  -10  
   48 .. 54  =  7 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
lac operator
62 .. 78  =  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
62 .. 78  =  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).
RBS
96 .. 107  =  12 bp
strong bacterial ribosome binding site (Elowitz and Leibler, 2000)
RBS
96 .. 107  =  12 bp
strong bacterial ribosome binding site (Elowitz and Leibler, 2000)
lac operator
30 .. 46  =  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
30 .. 46  =  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).
ORF:  1022 .. 1681  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3203 .. 3469  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  114 .. 854  =  741 bp
ORF:  246 amino acids  =  27.4 kDa
ORF:  260 .. 505  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  3073 .. 3933  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  132 .. 629  =  498 bp
ORF:  165 amino acids  =  17.0 kDa
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