LOCUS       Exported                5186 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  URA3 vector for cloning guide sequences using BclI-SwaI sites into 
            guide RNA expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    pT040
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5186)
  AUTHORS   Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T, 
            Wyrick JJ
  TITLE     New vectors for simple and streamlined CRISPR-Cas9 genome editing in
            Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep 
            21.
  PUBMED    26305040
REFERENCE   2  (bases 1 to 5186)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1002/yea.3098"; 
            journalName: "Yeast"; date: "2015-12"; volume: "32"; issue: "12"; 
            pages: "711-20"
FEATURES             Location/Qualifiers
     source          1..5186
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        27..131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             132..992
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(350..369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1163..1751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1652..1671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1905..1922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2039..2060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2075..2105
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2113..2129
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2118..2140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2137..2153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2137..2153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2174..2192
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2224..2240
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2225..2241
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2287..2303)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2287..2303)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2378..2397
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(2479..2747)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(2773..2792)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2774..2792)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2799..2816)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2799..2815)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2808..2830)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    3331..3623
                     /label=CEN4
                     /note="S. cerevisiae CEN4 centromere"
     rep_origin      3624..4077
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1"
     promoter        4089..4304
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             4305..5108
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"