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Plasmid Files

pCFD2-dU6:2gRNA

Drosophila expression vector in which a guide RNA (gRNA) is expressed from the U6-2 promoter.

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pCFD2-dU6-2gRNA Sequence and MappCFD2-dU6-2gRNA.dna
Map and Sequence File   
Sequence Author:  Bullock Lab / Addgene #49409
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 BglII (6222) EcoRI (1) BbsI (402) BbsI (424) polIII terminator XbaI (735) pCFD2-dU6:2gRNA 6227 bp
BglII  (6222)
1 site
A G A T C T T C T A G A
EcoRI  (1)
1 site
G A A T T C C T T A A G
BbsI  (402)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (424)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
XbaI  (735)
1 site
T C T A G A A G A T C T
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 6:  
   3832 .. 3949  =  118 bp
   38 amino acids  =  4.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 5:  
   4026 .. 4119  =  94 bp
   32 amino acids  =  3.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 4:  
   4211 .. 4817  =  607 bp
   202 amino acids  =  24.0 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 3:  
   4872 .. 5005  =  134 bp
   45 amino acids  =  5.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 2:  
   5066 .. 5226  =  161 bp
   53 amino acids  =  6.1 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 1:  
   5300 .. 5325  =  26 bp
   9 amino acids  =  899.0 Da
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
3832 .. 5325  =  1494 bp
379 amino acids  =  44.4 kDa
6 segments
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
AmpR
2011 .. 2871  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2011 .. 2802  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2011 .. 2871  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2803 .. 2871  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2011 .. 2871  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1252 .. 1840  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1252 .. 1840  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3002 .. 3457  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3002 .. 3457  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
dU6-2 promoter
7 .. 406  =  400 bp
RNA polymerase III promoter for Drosophila U6-2
snRNA (Das et al., 1987)
dU6-2 promoter
7 .. 406  =  400 bp
RNA polymerase III promoter for Drosophila U6-2
snRNA (Das et al., 1987)
AmpR promoter
2872 .. 2976  =  105 bp
AmpR promoter
2872 .. 2976  =  105 bp
gRNA scaffold
425 .. 500  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
425 .. 500  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
attB
5640 .. 5709  =  70 bp
attB site for the φC31 integrase (Groth et al., 2000)
attB
5640 .. 5709  =  70 bp
attB site for the φC31 integrase (Groth et al., 2000)
polIII terminator
501 .. 506  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
501 .. 506  =  6 bp
RNA polymerase III transcription terminator
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