pCFD4-U6:1_U6:3tandemgRNAs vector (V012275)

LOCUS       40924_10491        7255 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Drosophila expression vector for simultaneously expressing two guide
            RNA (gRNA) sequences from the U6-1 and U6-3 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Port F, Chen HM, Lee T, Bullock SL.
  TITLE     Optimized CRISPR/Cas tools for efficient germline and somatic genome
            engineering in Drosophila.
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 2014;111:E2967-76.
  PUBMED    25002478
REFERENCE   2  (bases 1 to 7255)
  AUTHORS   Bullock Lab / Addgene #49411
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7255)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A."; date: "2014"; volume: "111"; pages: "E2967-76"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     212..228
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        235..253
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2253..2322
                     /label=attB
                     /note="attB site for the phi-C31 integrase (Groth et al.,
                     2000)"
     protein_bind    2564..2949
                     /label=gypsy insulator
                     /note="chromatin insulator from Drosophila"
     promoter        2963..3759
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1
                     snRNA (Port et al., 2014)"
     misc_RNA        3778..3853
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3854..4274
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3
                     snRNA (Port et al., 2014)"
     promoter        complement(4679..4697)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4718..4734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4742..4758)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4766..4796)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4811..4832)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5882..6739)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6740..6844)
                     /label=AmpR promoter
     rep_origin      complement(join(6870..7255,1..70))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"