pLXIN

Bicistronic retroviral vector for co-expression of a gene together with a neomycin (G418) resistance marker.

Sequence Author: Clontech (TaKaRa)

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SacII (6078) SspI (5736) ScaI (5412) PvuI (5302) AseI (5104) NdeI (3862) BstZ17I (3812) AccI (3811) XbaI (3267) AscI (443) PshAI (577) AfeI (989) BstEII (1153) CsiI - SexAI * (1281) BsrGI (1348) HpaI (1478) PaeR7I - PspXI - XhoI (1481) BamHI (1486) PspOMI (1645) ApaI (1649) AvrII (1683) HindIII (1756) PmlI (1848) PaqCI (1871) DraIII (1895) PflMI (1985) BmgBI (2075) SphI (2649) NcoI (2676) NgoMIV (2744) NaeI (2746) RsrII (2760) pLXIN 6098 bp
SacII  (6078)
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.
SspI  (5736)
1 site
A A T A T T T T A T A A
ScaI  (5412)
1 site
A G T A C T T C A T G A
PvuI  (5302)
1 site
C G A T C G G C T A G C
AseI  (5104)
1 site
A T T A A T T A A T T A
NdeI  (3862)
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.
BstZ17I  (3812)
1 site
G T A T A C C A T A T G
AccI  (3811)
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.
XbaI  (3267)
1 site
T C T A G A A G A T C T
AscI  (443)
1 site
G G C G C G C C C C G C G C G G
PshAI  (577)
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  (989)
1 site
A G C G C T T C G C G A
BstEII  (1153)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
CsiI  (1281)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (1281)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BsrGI  (1348)
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.
HpaI  (1478)
1 site
G T T A A C C A A T T G
PaeR7I  (1481)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1481)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1481)
1 site
C T C G A G G A G C T C
BamHI  (1486)
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.
PspOMI  (1645)
1 site
G G G C C C C C C G G G
ApaI  (1649)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (1683)
1 site
C C T A G G G G A T C C
HindIII  (1756)
1 site
A A G C T T T T C G A A
PmlI  (1848)
1 site
C A C G T G G T G C A C
PaqCI  (1871)
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.
DraIII  (1895)
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.
PflMI  (1985)
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.
BmgBI  (2075)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
SphI  (2649)
1 site
G C A T G C C G T A C G
NcoI  (2676)
1 site
C C A T G G G G T A C C
NgoMIV  (2744)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (2746)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
RsrII  (2760)
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.
AmpR
4859 .. 5719  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4859 .. 5650  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4859 .. 5719  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5651 .. 5719  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4859 .. 5719  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2107 .. 2910  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
2107 .. 2910  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
3' LTR
2970 .. 3563  =  594 bp
long terminal repeat from Moloney murine leukemia virus
3' LTR
2970 .. 3563  =  594 bp
long terminal repeat from Moloney murine leukemia virus
5' LTR
1 .. 589  =  589 bp
long terminal repeat from Moloney murine sarcoma virus
5' LTR
1 .. 589  =  589 bp
long terminal repeat from Moloney murine sarcoma virus
ori
4100 .. 4688  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4100 .. 4688  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
1532 .. 2105  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
1532 .. 2105  =  574 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
gag (truncated)
1052 .. 1468  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1052 .. 1468  =  417 bp
truncated MMLV gag gene lacking the start codon
MMLV Ψ
652 .. 851  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
MMLV Ψ
652 .. 851  =  200 bp
packaging signal of Moloney murine leukemia virus (MMLV)
AmpR promoter
5720 .. 5824  =  105 bp
AmpR promoter
5720 .. 5824  =  105 bp
MCS
1476 .. 1491  =  16 bp
multiple cloning site
MCS
1476 .. 1491  =  16 bp
multiple cloning site
ORF:  2107 .. 2910  =  804 bp
ORF:  267 amino acids  =  29.2 kDa
ORF:  2015 .. 2287  =  273 bp
ORF:  90 amino acids  =  10.3 kDa
ORF:  2288 .. 2674  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  4989 .. 5255  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3387 .. 3677  =  291 bp
ORF:  96 amino acids  =  10.5 kDa
ORF:  4859 .. 5719  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  739 .. 999  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  2425 .. 2679  =  255 bp
ORF:  84 amino acids  =  9.6 kDa
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