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pFRT/lacZeo2

Flp recombination target site (FRT) vector, with an SV40 promoter but no enhancer, for generating a Flp-In™ host cell line. See also pFRT/lacZeo.

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pFRT lacZeo2.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Invitrogen)
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SfiI (6477) ApaI (6473) PspOMI (6469) AhdI (5570) BsaI (5504) AseI (5395) ScaI (5089) XmnI (4970) EcoO109I (4590) PluTI (4345) SfoI (4343) NarI (4342) KasI (4341) PstI - SbfI (4169) SalI (4159) BamHI (4147) EcoRI (4137) BsmI (4087) PsiI (4026) FseI (3779) SexAI * (3666) BmgBI (3637) SgrAI (3617) BstBI (3425) BlpI (3374) NdeI (3318) BglII (1) NotI (14) NheI (27) BmtI (31) PaeR7I - PspXI - XhoI (32) BstAPI (58) NsiI (63) NcoI (148) StuI (240) AvrII (241) BsaAI - PmlI (301) Bsu36I (585) Bpu10I (866) BspDI - ClaI (1185) BsaBI * (1692) BstZ17I (3126) BsiWI (3132) pFRT/lacZeo2 6482 bp
SfiI  (6477)
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.
ApaI  (6473)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (6469)
1 site
G G G C C C C C C G G G
AhdI  (5570)
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.
BsaI  (5504)
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.
AseI  (5395)
1 site
A T T A A T T A A T T A
ScaI  (5089)
1 site
A G T A C T T C A T G A
XmnI  (4970)
1 site
G A A N N N N T T C C T T N N N N A A G
EcoO109I  (4590)
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.
PluTI  (4345)
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.
SfoI  (4343)
1 site
G G C G C C C C G C G G
NarI  (4342)
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.
KasI  (4341)
1 site
G G C G C C C C G C G G
PstI  (4169)
1 site
C T G C A G G A C G T C
SbfI  (4169)
1 site
C C T G C A G G G G A C G T C C
SalI  (4159)
1 site
G T C G A C C A G C T G
BamHI  (4147)
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.
EcoRI  (4137)
1 site
G A A T T C C T T A A G
BsmI  (4087)
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.
PsiI  (4026)
1 site
T T A T A A A A T A T T
FseI  (3779)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
SexAI  (3666)
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.
BmgBI  (3637)
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.
SgrAI  (3617)
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.
BstBI  (3425)
1 site
T T C G A A A A G C T T
BlpI  (3374)
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.
NdeI  (3318)
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.
BglII  (1)
1 site
A G A T C T T C T A G A
NotI  (14)
1 site
G C G G C C G C C G C C G G C G
NheI  (27)
1 site
G C T A G C C G A T C G
BmtI  (31)
1 site
G C T A G C C G A T C G
PaeR7I  (32)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (32)
1 site
V C T C G A G B B G A G C T C V
XhoI  (32)
1 site
C T C G A G G A G C T C
BstAPI  (58)
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.
NsiI  (63)
1 site
A T G C A T T A C G T A
NcoI  (148)
1 site
C C A T G G G G T A C C
StuI  (240)
1 site
A G G C C T T C C G G A
AvrII  (241)
1 site
C C T A G G G G A T C C
BsaAI  (301)
1 site
Y A C G T R R T G C A Y
PmlI  (301)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Bsu36I  (585)
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.
Bpu10I  (866)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
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.
BspDI  (1185)
1 site
A T C G A T T A G C T A
ClaI  (1185)
1 site
A T C G A T T A G C T A
BsaBI  (1692)
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.
BstZ17I  (3126)
1 site
G T A T A C C A T A T G
BsiWI  (3132)
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.
lacZ
374 .. 3418  =  3045 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
lacZ
374 .. 3418  =  3045 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
AmpR
4783 .. 5643  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4783 .. 4851  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4783 .. 5643  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4852 .. 5643  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4783 .. 5643  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
5814 .. 6402  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5814 .. 6402  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
BleoR
3500 .. 3877  =  378 bp
125 amino acids  =  13.9 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
3500 .. 3877  =  378 bp
125 amino acids  =  13.9 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
SV40 promoter
38 .. 256  =  219 bp
SV40 early promoter
SV40 promoter
38 .. 256  =  219 bp
SV40 early promoter
SV40 poly(A) signal
4007 .. 4128  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4007 .. 4128  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4678 .. 4782  =  105 bp
AmpR promoter
4678 .. 4782  =  105 bp
FRT
310 .. 357  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
FRT
310 .. 357  =  48 bp
FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011).
ATG
305 .. 307  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
305 .. 307  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
107 .. 242  =  136 bp
SV40 origin of replication
SV40 ori
107 .. 242  =  136 bp
SV40 origin of replication
ORF:  4783 .. 5643  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  305 .. 3877  =  3573 bp
ORF:  1190 amino acids  =  134.8 kDa
ORF:  6372 .. 204  =  315 bp
ORF:  104 amino acids  =  11.5 kDa
ORF:  5247 .. 5513  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1220 .. 1534  =  315 bp
ORF:  104 amino acids  =  12.0 kDa
ORF:  2645 .. 2881  =  237 bp
ORF:  78 amino acids  =  9.0 kDa
ORF:  2284 .. 2595  =  312 bp
ORF:  103 amino acids  =  10.5 kDa
ORF:  3412 .. 3783  =  372 bp
ORF:  123 amino acids  =  13.2 kDa
ORF:  4129 .. 4395  =  267 bp
ORF:  88 amino acids  =  10.1 kDa
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