pSCREEN-iT lacZ-DEST

Gateway® destination vector that allows assessment of the potency of an RNAi knockdown reagent using a β-gal reporter assay.

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (8700) AhdI (7779) BspQI - SapI (6770) PsiI (6363) BstAPI (5959) EcoO109I (5956) PmeI (5693) PstI (5541) BmgBI (5320) SmaI - SrfI (5286) TspMI - XmaI (5284) BbvCI (5140) BspEI (4411) MfeI (161) NruI (208) SpeI (249) SnaBI (590) Bsu36I (1114) BspDI - ClaI (1714) EcoRV (2003) BclI * (2236) MauBI (2388) AfeI (2725) BsiWI (3661) PfoI * (3870) BlpI (3903) PaeR7I - PspXI - XhoI (3955) pSCREEN-iT™/lacZ-DEST 8702 bp
SgrDI  (8700)
1 site
C G T C G A C G G C A G C T G C
AhdI  (7779)
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.
BspQI  (6770)
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  (6770)
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.
PsiI  (6363)
1 site
T T A T A A A A T A T T
BstAPI  (5959)
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.
EcoO109I  (5956)
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.
PmeI  (5693)
1 site
G T T T A A A C C A A A T T T G
PstI  (5541)
1 site
C T G C A G G A C G T C
BmgBI  (5320)
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.
SmaI  (5286)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (5286)
1 site
G C C C G G G C C G G G C C C G
TspMI  (5284)
1 site
C C C G G G G G G C C C
XmaI  (5284)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
BbvCI  (5140)
1 site
C C T C A G C G G A G T C G
BspEI  (4411)
1 site
T C C G G A A G G C C T
MfeI  (161)
1 site
C A A T T G G T T A A C
NruI  (208)
1 site
T C G C G A A G C G C T
SpeI  (249)
1 site
A C T A G T T G A T C A
SnaBI  (590)
1 site
T A C G T A A T G C A T
Bsu36I  (1114)
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.
BspDI  (1714)
1 site
A T C G A T T A G C T A
ClaI  (1714)
1 site
A T C G A T T A G C T A
EcoRV  (2003)
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.
BclI  (2236)
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.
MauBI  (2388)
1 site
C G C G C G C G G C G C G C G C
AfeI  (2725)
1 site
A G C G C T T C G C G A
BsiWI  (3661)
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.
PfoI  (3870)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BlpI  (3903)
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.
PaeR7I  (3955)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3955)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3955)
1 site
C T C G A G G A G C T C
lacZ
903 .. 3947  =  3045 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
lacZ
903 .. 3947  =  3045 bp
1015 amino acids  =  115.5 kDa
Product: β-galactosidase
AmpR
7706 .. 8566  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7706 .. 8497  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7706 .. 8566  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   8498 .. 8566  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7706 .. 8566  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
4202 .. 4861  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
4202 .. 4861  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
6947 .. 7535  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6947 .. 7535  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6005 .. 6433  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6005 .. 6433  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
ccdB
5203 .. 5508  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
ccdB
5203 .. 5508  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA gyrase
Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains.
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
attR1
3969 .. 4093  =  125 bp
recombination site for the Gateway® LR reaction
attR1
3969 .. 4093  =  125 bp
recombination site for the Gateway® LR reaction
attR2
5549 .. 5673  =  125 bp
recombination site for the Gateway® LR reaction
attR2
5549 .. 5673  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
8567 .. 8671  =  105 bp
AmpR promoter
8567 .. 8671  =  105 bp
HSV TK poly(A) signal
5755 .. 5803  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
5755 .. 5803  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
lac UV5 promoter
4118 .. 4148  =  31 bp
3 segments
   Segment 1:  -35  
   4118 .. 4123  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
4118 .. 4148  =  31 bp
3 segments
   Segment 2:  
   4124 .. 4141  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
4118 .. 4148  =  31 bp
3 segments
   Segment 3:  -10  
   4142 .. 4148  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
4118 .. 4148  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
ORF:  4202 .. 4861  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  774 .. 4004  =  3231 bp
ORF:  1076 amino acids  =  122.7 kDa
ORF:  7706 .. 8566  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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