LOCUS       Exported               12367 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Cas9 and contains guide RNA expression cassette with 
            BclI-SwaI cloning sites for guide sequence cloning; Contains LEU2 
            marker for yeast transformation.
ACCESSION   .
VERSION     .
KEYWORDS    pML107
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12367)
  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 12367)
  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..12367
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     70..86
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     71..87
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        132..798
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     668..691
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     regulatory      827..836
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             833..2551
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4946..4966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4994..5181
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5049..5073)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      5299..5318
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(5400..5668)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(5699..5719)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5699..5717)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5738..5754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5738..5754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5751..5773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5762..5778
                     /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)."
     promoter        complement(5786..5816)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5831..5852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5969..5986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6140..6728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6220..6239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6899..7759)
                     /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     7522..7541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7760..7864)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7891..9237
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     9278..9296
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9334..9356)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     9456..9475
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(9969..11063)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(11064..11471)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(11772..12227)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11909..11930)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12121..12140
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(12353..12367,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(12367,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"