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.

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) CsiI - 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) ATG 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.
CsiI  (3666)
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  (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
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

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.
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.
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
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
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™
AmpR
4783 .. 5643  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   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
2 segments
   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
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).
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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