pRetroQ-mCherry-N1

Self-inactivating retroviral vector for expressing a protein fused to the N-terminus of mCherry.

Sequence Author: Clontech (TaKaRa)

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SspI (7252) XmnI (7047) ScaI (6928) FspI (6670) AflIII - PciI (5555) BspQI - SapI (5439) AvrII (5308) SfiI (5261) EcoRV (4184) DraIII (4083) RsrII (3628) CMV enhancer AscI (542) PshAI (676) CMV enhancer NheI (2172) BmtI (2176) BglII (2190) PaeR7I - XhoI (2194) BstBI (2208) EcoRI (2210) SalI (2220) AccI (2221) PspOMI (2234) ApaI (2238) BamHI (2241) SbfI (2609) PflMI (2693) BbvCI (2792) AleI (2896) SgrAI (2935) XcmI (2940) NotI (2966) XbaI (2976) BlpI (3265) BsmI (3423) BfuAI - BspMI (3501) BsiWI (3568) pRetroQ-mCherry-N1 7597 bp
SspI  (7252)
1 site
A A T A T T T T A T A A
XmnI  (7047)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (6928)
1 site
A G T A C T T C A T G A
FspI  (6670)
1 site
T G C G C A A C G C G T
AflIII  (5555)
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  (5555)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5439)
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  (5439)
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.
AvrII  (5308)
1 site
C C T A G G G G A T C C
SfiI  (5261)
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.
EcoRV  (4184)
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.
DraIII  (4083)
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.
RsrII  (3628)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
AscI  (542)
1 site
G G C G C G C C C C G C G C G G
PshAI  (676)
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.
NheI  (2172)
1 site
G C T A G C C G A T C G
BmtI  (2176)
1 site
G C T A G C C G A T C G
BglII  (2190)
1 site
A G A T C T T C T A G A
PaeR7I  (2194)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2194)
1 site
C T C G A G G A G C T C
BstBI  (2208)
1 site
T T C G A A A A G C T T
EcoRI  (2210)
1 site
G A A T T C C T T A A G
SalI  (2220)
1 site
G T C G A C C A G C T G
AccI  (2221)
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.
PspOMI  (2234)
1 site
G G G C C C C C C G G G
ApaI  (2238)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BamHI  (2241)
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.
SbfI  (2609)
1 site
C C T G C A G G G G A C G T C C
PflMI  (2693)
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.
BbvCI  (2792)
1 site
C C T C A G C G G A G T C G
AleI  (2896)
1 site
C A C N N N N G T G G T G N N N N C A C
SgrAI  (2935)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
XcmI  (2940)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
NotI  (2966)
1 site
G C G G C C G C C G C C G G C G
XbaI  (2976)
1 site
T C T A G A A G A T C T
BlpI  (3265)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BsmI  (3423)
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.
BfuAI  (3501)
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  (3501)
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.
BsiWI  (3568)
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.
AmpR
6375 .. 7235  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   6375 .. 7166  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6375 .. 7235  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   7167 .. 7235  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6375 .. 7235  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
mCherry
2254 .. 2964  =  711 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent protein
mammalian codon-optimized
mCherry
2254 .. 2964  =  711 bp
236 amino acids  =  26.7 kDa
Product: monomeric derivative of DsRed fluorescent protein
mammalian codon-optimized
PuroR
3512 .. 4111  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3512 .. 4111  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
5616 .. 6204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5616 .. 6204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
PGK promoter
2992 .. 3491  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
2992 .. 3491  =  500 bp
mouse phosphoglycerate kinase 1 promoter
3' LTR (ΔU3)
4296 .. 4728  =  433 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
3' LTR (ΔU3)
4296 .. 4728  =  433 bp
self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1151 .. 1567  =  417 bp
truncated MMLV gag gene lacking the start codon
SV40 promoter
4994 .. 5323  =  330 bp
SV40 enhancer and early promoter
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SV40 promoter
4994 .. 5323  =  330 bp
SV40 enhancer and early promoter