LOCUS       Exported                6274 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Yeast high-copy plasmid for gRNA targeting of the negatively 
            selectable CAN1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    p426-SNR52p-gRNA.CAN1.Y-SUP4t
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6274)
  AUTHORS   DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
  TITLE     Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas 
            systems.
  JOURNAL   Nucleic Acids Res. 2013;41:4336-43.
  PUBMED    23460208
REFERENCE   2  (bases 1 to 6274)
  AUTHORS   Church Lab / Addgene #43803
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6274
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     rep_origin      69..1411
                     /note="2u ori"
                     /note="yeast 2u plasmid origin of replication"
     promoter        1438..1542
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             1543..2403
                     /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"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3621..3889
                     /note="SNR52 promoter"
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_feature    3890..3909
                     /note="CAN1.Y genomic target"
                     /note="genomic target sequence located 207 bp downstream of
                     the start codon in the yeast CAN1 gene"
     misc_RNA        3910..3984
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3989..4008
                     /note="SUP4 terminator"
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      4014..4261
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     rep_origin      4469..4924
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(5055..5858)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /note="URA3"
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5859..6074)
                     /gene="S. cerevisiae URA3"
                     /note="URA3 promoter"