pCFD2-dU6:2gRNA vector (V012277)

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LOCUS       pCFD2-dU6-2gRNA.        6227 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Drosophila expression vector in which a guide RNA (gRNA) is 
            expressed from the U6-2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD2-dU6-2gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6227)
  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 6227)
  AUTHORS   Bullock Lab / Addgene #49409
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6227)
  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..6227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..406
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2
                     snRNA (Das et al., 1987)"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      501..506
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     promoter        complement(811..829)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(850..866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(874..890)
                     /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(898..928)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(943..964)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1252..1840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2014..2871)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2872..2976)
                     /label=AmpR promoter
     rep_origin      complement(3002..3457)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     3599..3615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3622..3640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(3832..3949,4026..4119,4211..4817,4872..5005,
                     5066..5226,5300..5325))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=tryptophan oxygenase
                     /note="vermilion"
                     /note="selectable marker, required to synthesize the brown
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    5640..5709
                     /label=attB
                     /note="attB site for the phi-C31 integrase (Groth et al.,
                     2000)"