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Plasmid Files

pCFD4-U6:1_U6:3tandemgRNAs

Drosophila expression vector for simultaneously expressing two guide RNA (gRNA) sequences from the U6-1 and U6-3 promoters.

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pCFD4-U6-1_U6-3tandemgRNAs Sequence and MappCFD4-U6-1_U6-3tandemgRNAs.dna
Map and Sequence File   
Sequence Author:  Bullock Lab / Addgene #49411
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 XbaI (4603) polIII terminator BbsI (4292) BbsI (4270) BbsI (3777) BglII (2951) BbsI (3755) pCFD4-U6:1_U6:3tandemgRNAs 7255 bp
XbaI  (4603)
1 site
T C T A G A A G A T C T
BbsI  (4292)
4 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  (4270)
4 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  (3777)
4 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.
BglII  (2951)
1 site
A G A T C T T C T A G A
BbsI  (3755)
4 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.
vermilion
445 .. 1938  =  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
445 .. 1938  =  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
445 .. 1938  =  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
445 .. 1938  =  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
445 .. 1938  =  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
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 6:  
   445 .. 562  =  118 bp
   38 amino acids  =  4.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 5:  
   639 .. 732  =  94 bp
   32 amino acids  =  3.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 4:  
   824 .. 1430  =  607 bp
   202 amino acids  =  24.0 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 3:  
   1485 .. 1618  =  134 bp
   45 amino acids  =  5.5 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 2:  
   1679 .. 1839  =  161 bp
   53 amino acids  =  6.1 kDa
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  1494 bp
379 amino acids  =  44.4 kDa
   Segment 1:  
   1913 .. 1938  =  26 bp
   9 amino acids  =  899.0 Da
Product: tryptophan oxygenase
selectable marker, required to synthesize the brown
eye pigment in Drosophila
vermilion
445 .. 1938  =  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
5879 .. 6739  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5879 .. 6670  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5879 .. 6739  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6671 .. 6739  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5879 .. 6739  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
dU6-1 promoter
2963 .. 3759  =  797 bp
RNA polymerase III promoter for Drosophila U6-1
snRNA (Port et al., 2014)
dU6-1 promoter
2963 .. 3759  =  797 bp
RNA polymerase III promoter for Drosophila U6-1
snRNA (Port et al., 2014)
ori
5120 .. 5708  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5120 .. 5708  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6870 .. 70  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6870 .. 70  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
dU6-3 promoter
3854 .. 4274  =  421 bp
RNA polymerase III promoter for Drosophila U6-3
snRNA (Port et al., 2014)
dU6-3 promoter
3854 .. 4274  =  421 bp
RNA polymerase III promoter for Drosophila U6-3
snRNA (Port et al., 2014)
gypsy insulator
2564 .. 2949  =  386 bp
chromatin insulator from Drosophila
gypsy insulator
2564 .. 2949  =  386 bp
chromatin insulator from Drosophila
AmpR promoter
6740 .. 6844  =  105 bp
AmpR promoter
6740 .. 6844  =  105 bp
gRNA scaffold
3778 .. 3853  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
3778 .. 3853  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
4293 .. 4368  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
4293 .. 4368  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
attB
2253 .. 2322  =  70 bp
attB site for the φC31 integrase (Groth et al., 2000)
attB
2253 .. 2322  =  70 bp
attB site for the φC31 integrase (Groth et al., 2000)
polIII terminator
4369 .. 4374  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
4369 .. 4374  =  6 bp
RNA polymerase III transcription terminator
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