LOCUS       Exported                7255 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Drosophila expression vector for simultaneously expressing two guide
            RNA (gRNA) sequences from the U6-1 and U6-3 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    pCFD4-U6-1_U6-3tandemgRNAs
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
FEATURES             Location/Qualifiers
     source          1..7255
                     /organism="synthetic DNA construct"
                     /lab_host="Drosophila melanogaster"
                     /mol_type="other DNA"
     CDS             complement(join(445..562,639..732,824..1430,1485..1618,
                     1679..1839,1913..1938))
                     /codon_start=1
                     /product="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    2253..2322
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    2564..2949
                     /bound_moiety="Su(Hw), CP190, and Mod(mdg4)67.2"
                     /note="gypsy insulator"
                     /note="chromatin insulator from Drosophila"
     promoter        2963..3759
                     /note="dU6-1 promoter"
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        3778..3853
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3854..4274
                     /note="dU6-3 promoter"
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        4293..4368
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4369..4374
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5879..6739)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6740..6844)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(6870..70)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"