pCFD4-U6-1_U6-3tandemgRNAs

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

Sequence Author: Bullock Lab / Addgene #49411

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HomePlasmidsCRISPR PlasmidspCFD4-U6-1_U6-3tandemgRNAs
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  =  1140 bp
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  =  1140 bp
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  =  1140 bp
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  =  1140 bp
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  =  1140 bp
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  =  1140 bp
   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
6 segments  =  1140 bp
   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
6 segments  =  1140 bp
   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
6 segments  =  1140 bp
   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
6 segments  =  1140 bp
   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
6 segments  =  1140 bp
   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  =  1140 bp
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
2 segments
   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
2 segments
   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
ORF:  3217 .. 3753  =  537 bp
ORF:  178 amino acids  =  19.9 kDa
ORF:  942 .. 1190  =  249 bp
ORF:  82 amino acids  =  9.0 kDa
ORF:  6009 .. 6275  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2555 .. 2935  =  381 bp
ORF:  126 amino acids  =  15.2 kDa
ORF:  5879 .. 6739  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  804 .. 1397  =  594 bp
ORF:  197 amino acids  =  23.5 kDa
ORF:  1671 .. 1925  =  255 bp
ORF:  84 amino acids  =  9.7 kDa
ORF:  2223 .. 2636  =  414 bp
ORF:  137 amino acids  =  15.2 kDa
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Individual Sequences & Maps

3xFLAG-dCas9 pCMV-7.1
pCas9_GFP
pCRISPRa-Enhancer
pME-Cas9-T2A-GFP
3xFLAG-dCas9 pMXs-neo
pCas9D10A_GFP
pCS2+hSpCas9
pMJ806
3xFLAG-dCas9 pMXs-puro
pCas-Guide
pCS2-Cas9
pMJ841
Cas9m4
pcDNA-dCas9
pCS2-nCas9n
pMJ920
Cas9m4-VP64
pcDNA-dCas9-p300 Core
pCW-Cas9
pMLM3705
CRISPRi library backbone
pcDNA-dCas9-p300 Core (D1399Y)
pdCas9
pMSCV-LTR-dCas9-p65AD-BFP
dCAS9-VP64_GFP
pcDNA-dCas9-VP64
pdCas9-bacteria
pMSCV-LTR-dCas9-VP64-BFP
eSpCas9(1.1)
pcDNA3.1-CibN-dCas9-CibN
pdCas9-humanized
pMZ374
GeneArt CRISPR Nuclease Vector - CD4 (linearized)
pCFD1-dU6-1gRNA
pdCas9 BFP-humanized
pNW3
GeneArt CRISPR Nuclease Vector - OFP (linearized)
pCFD2-dU6-2gRNA
pDCC6
pRGE31
gRNA_AAVS1-T1
pCFD3-dU6-3gRNA
pDD162 (Peft-3_Cas9 + Empty sgRNA)
pRGEB31
gRNA_AAVS1-T2
pCFD4-U6-1_U6-3tandemgRNAs
pDR274
pRGEB32
gRNA_Cloning Vector
pCLIP-All-EFS-Ametrine
Peft-3_cas9-SV40_NLS_tbb-2 3'UTR
pROSA26-Puro-DNR
gRNA_GFP-T1
pCLIP-All-EFS-Blast
pET-28b-Cas9-His
pRPR1_gRNA_handle_RPR1t
gRNA_GFP-T2
pCLIP-All-EFS-Puro
pET-Cas9-6xHis
pSECC
HBT-pcoCas9
pCLIP-All-EFS-tRFP
pET-Cas9-NLS-6xHis
pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)
hCas9
pCLIP-All-EFS-ZsGreen
pET-NLS-Cas9-6xHis
pSLQ1658-dCas9-EGFP
hCas9_D10A
pCLIP-All-hCMV-Blast
pET28a Cas9-Cys
pSpCas9(BB)-2A-GFP (PX458)
JDS246
pCLIP-All-hCMV-Puro
pFGC-pcoCas9
pSpCas9(BB)-2A-Puro (PX459) V2.0
lenti dCAS-VP64_Blast
pCLIP-All-hCMV-tRFP
pGL3-U6-sgRNA-PGK-puromycin
pSpCas9n(BB)-2A-GFP (PX461)
lenti MS2-P65-HSF1_Hygro
pCLIP-All-hCMV-ZsGreen
pgRNA-bacteria
pSpCas9n(BB)-2A-Puro (PX462) V2.0
lenti sgRNA(MS2)_zeo backbone
pCLIP-All-Nickase(D10A)-EFS-Blast
pgRNA-humanized
pSpCas9n(BB) (PX460)
lentiCas9-Blast
pCLIP-All-Nickase(D10A)-EFS-Puro
pGuide
pSpCas9 (PX165)
lentiCas9-EGFP
pCLIP-All-Nickase(D10A)-EFS-tRFP
pGuide-it-sgRNA1 Vector (Linear)
pSPgRNA
lentiCas9n(D10A)-Blast
pCLIP-All-Nickase(D10A)-EFS-ZsGreen
pGuide-it-tdTomato Vector (Linear)
pSQT834
lentiCRISPR v2
pCLIP-All-Nickase(D10A)-hCMV-Puro
pGuide-it-ZsGreen1 Vector (Linear)
pSQT1313
lentiGuide-Puro
pCLIP-All-Nickase(D10A)-hCMV-tRFP
pHAGE EF1-alpha dCas9-KRAB
pST1374-N-NLS-flag-linker-Cas9-D10A
LSL-Cas9-Rosa26TV
pCLIP-All-Nickase(D10A)-hCMV-ZsGreen
pHAGE EF1-alpha dCas9-VP64
pST1374-N-NLS-flag-linker-Cas9-H840A
M-SPcas
pCLIP-All-Nickase(D10A)-TRE3G-Blast
pHAGE TRE dCas9
pST1374-NLS-flag-linker-Cas9
M-ST1cas
pCLIP-All-Nickase(D10A)-TRE3G-Puro
pHAGE TRE dCas9-KRAB
pT3TS-nCas9n
MLM3613
pCLIP-All-Nickase(D10A)-TRE3G-tRFP
pHAGE TRE dCas9-VP64
pT7-Cas9
MLM3636
pCLIP-All-Nickase(D10A)-TRE3G-ZsGreen
pHAGE-TO-dCas9-3XGFP
pT7-Cas9-Nickase
MLM3639
pCLIP-All-TRE3G-Blast
pHAGE-TO-dCas9-3XmCherry
pT7-gRNA
MS2-P65-HSF1_GFP
pCLIP-All-TRE3G-Puro
pHAGE-TO-nls-st1dCas9-3nls-3XTagBFP2
pT7-Guide-IVT
MSP1101
pCLIP-All-TRE3G-tRFP
pHAGE-TO-nmdCas9-3XGFP
pTDH3-dCas9
MSP2133
pCLIP-All-TRE3G-ZsGreen
pHAGE-TO-nmdCas9-3XmCherry
pTDH3-dCas9-Mxi1
MSP2135
pCLIP-Cas9(D10a)-Nickase-EFS-Puro
pHR-SFFV-dCas9-BFP
pTOL-hCMV-TET3G-Hygro Transactivator
MSP469
pCLIP-Cas9(D10a)-Nickase-EFS-Blast
pHR-SFFV-dCas9-BFP-KRAB
pTPGI_dCas9_VP64
p3s-Cas9HC
pCLIP-Cas9(D10a)-Nickase-EFS-tRFP
pHR-SFFV-KRAB-dCas9-P2A-mCherry
pU6-BbsI-chiRNA
p414-TEF1p-Cas9-CYC1t
pCLIP-Cas9(D10a)-Nickase-EFS-ZsGreen
pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP
PU6_klp-12_sgRNA
p415-GalL-Cas9-CYC1t
pCLIP-Cas9(D10a)-Nickase-hCMV-Blast
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B
p426-SNR52p-gRNA.CAN1.Y-SUP4t
pCLIP-Cas9(D10a)-Nickase-hCMV-Puro
pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS
pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6
pAAV-Guide-it-1 Vector (Linear)
pCLIP-Cas9(D10a)-Nickase-hCMV-tRFP
pHSN401
pUC119-gRNA
pAAV-Guide-it-Down Vector (Linear)
pCLIP-Cas9(D10a)-Nickase-hCMV-ZsGreen
pHsp70-Cas9
pUC57-sgRNA expression vector
pAAV-Guide-it-Up Vector
pCLIP-Cas9(D10a)-Nickase-TRE3G-Blast
pICH41308_hCas9
Puro-Cas9 donor
pAAVS1-BSD-DNR
pCLIP-Cas9(D10a)-Nickase-TRE3G-Puro
pICH47742_2x35S-5'UTR-hCas9(STOP)-NOST
pWJ66
pAAVS1-Puro-DNR
pCLIP-Cas9(D10a)-Nickase-TRE3G-tRFP
piCRg Entry
pWJ68
pAC2-dual-dCas9VP48-sgExpression
pCLIP-Cas9(D10a)-Nickase-TRE3G-ZsGreen
pICSL01009_AtU6p
pwtCas9-bacteria
pAc-sgRNA-Cas9
pCLIP-Cas9-Nuclease-EFS-Blast
pK7WGF2_hCas9
pX260
pAC149-pCR8-dCas9VP160
pCLIP-Cas9-Nuclease-EFS-Puro
pKLV-U6gRNA(BbsI)-PGKpuro2ABFP
pX330
pAC152-dual-dCas9VP64-sgExpression
pCLIP-Cas9-Nuclease-EFS-tRFP
pL-CRISPR.EFS.GFP
pX330A-1x2
pAC153-dual-dCas9VP96-sgExpression
pCLIP-Cas9-Nuclease-EFS-ZsGreen
pL-CRISPR.EFS.tRFP
pX330S-2
pAC154-dual-dCas9VP160-sgExpression
pCLIP-Cas9-Nuclease-hCMV-Blast
pL-CRISPR.SFFV.GFP
pX334
pAC155-pCR8-sgExpression
pCLIP-Cas9-Nuclease-hCMV-Puro
pL-CRISPR.SFFV.tRFP
pX335
pAC84-pCR8-dCas9
pCLIP-Cas9-Nuclease-hCMV-tRFP
pLenti-Cas9
PX551
pAC91-pmax-dCas9VP64
pCLIP-Cas9-Nuclease-hCMV-ZsGreen
pLenti-Cas9-IRES-Puro
PX552
pAC93-pmax-dCas9VP160
pCLIP-Cas9-Nuclease-TRE3G-Blast
pLenti-Cas9-P2A-tGFP
pX600
pAC94-pmax-dCas9VP160-2A-puro
pCLIP-Cas9-Nuclease-TRE3G-Puro
pLenti-Cas-Guide
pX601
pAC95-pmax-dCas9VP160-2A-neo
pCLIP-Cas9-Nuclease-TRE3G-tRFP
pLenti-EF1a-Cas9-IRES-Puro
pX602
pACT2-CAS9c
pCLIP-Cas9-Nuclease-TRE3G-ZsGreen
pLenti-Guide-Puro
PX851
pBS-Hsp70-Cas9
pCLIP-gRNA-EFS-Blast
pLKO.1-puro U6 sgRNA BfuAI stuffer
PX852
pCAG-T3-hCAS-pA
pCLIP-gRNA-EFS-Puro
pLSC-5
PX853
pCAG-T3-hCasD10A-pA
pCLIP-gRNA-EFS-tRFP
pLV hUbC-Cas9-T2A-GFP
PX854
pCas
pCLIP-gRNA-EFS-ZsGreen
pLV hUbC-dCas9 VP64-T2A-GFP
PX855
pCas-Guide-AAVS1
pCLIP-gRNA-hCMV-Blast
pLV hUbC-dCas9-T2A-GFP
PX856
pCas-Guide-CRISPRa
pCLIP-gRNA-hCMV-Puro
pLVX-hyg-sgRNA1 Vector (Linear)
pY010 (pcDNA3.1-hAsCpf1)
pCas-Guide-CRISPRi
pCLIP-gRNA-hCMV-tRFP
pLVX-puro-Cas9
pY016 (pcDNA3.1-hLbCpf1)
pCas-Guide-EF1a-CD4
pCLIP-gRNA-hCMV-ZsGreen
pLX-sgRNA
sgRNA(MS2) cloning backbone
pCas-Guide-EF1a-GFP
pCRCT
pMB66
SP-Cas9
pCas-Guide-Nickase
pCRISPomyces-1
pMB70
SP-dCas9-VPR
pCas-Guide-ROSA26
pCRISPomyces-2
pME-Cas9
VP12
pCas9
pCRISPR

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