LOCUS       Exported                8743 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses S. pyogenes Cas9 plus an HDV ribozyme-sgRNA for genome 
            editing in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    pCAS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8743)
  AUTHORS   Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, 
            DeLoache W, Dueber JE, Arkin AP, Cate JH
  TITLE     Selection of chromosomal DNA libraries using a multiplex CRISPR 
            system.
  JOURNAL   Elife. 2014 Aug 19;3. doi: 10.7554/eLife.03703.
  PUBMED    25139909
REFERENCE   2  (bases 1 to 8743)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8743
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    complement(749..796)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_RNA        1169..1244
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     gene            1378..2734
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1378..1721
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1722..2531
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1789..1808)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2537..2734
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             3497..7600
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7610..7630
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7646..7669
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      7673..7862
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(7919..8507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7999..8018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      8529..768
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg
       61 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt
      121 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt
      181 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc
      241 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc
      301 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata
      361 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat
      421 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt
      481 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag
      541 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat
      601 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc
      661 aatattttag tagcccgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca
      721 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact
      781 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc
      841 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata
      901 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtatat gtgttatgta
      961 gtatactctt tcttcaacaa ttaaatactc tcggtagcca agttggttta aggcgcaaga
     1021 ctgtaattta tcactacgaa atcttgagat cgggcgttcg actcgccccc gggagagatg
     1081 gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacaccttcg ggtggcgaat
     1141 gggactttca taataacgtc caataaatgt tttagagcta gaaatagcaa gttaaaataa
     1201 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tattttttgt
     1261 cactattgtt atgtaaaatg ccacctctga cagtatggaa cgcaaacttc tgtctagtgg
     1321 atataggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac
     1381 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt
     1441 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat
     1501 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag
     1561 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat
     1621 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg
     1681 ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc
     1741 acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     1801 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
     1861 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     1921 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     1981 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg
     2041 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     2101 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     2161 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     2221 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct
     2281 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     2341 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     2401 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     2461 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     2521 agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     2581 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     2641 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     2701 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca
     2761 tccagtgtcg aaaacgagct ctcgagaacc cttaatagtc gaacaagaag caggcaaagt
     2821 ttagagcact gcccctccgc actcaaaaaa gaaaaaacta ggaggaaaat aaaattctca
     2881 accacacaaa cacataaaca catacaaata caaatacaag cttatttact tgacatcgcg
     2941 cgatcttcca ctattcagcg ccgtccgccc tctctcgtgt tttttgttta cgcgacaact
     3001 atgcgaaatc cggagcaacg ggcaaccgtt tggggaaaga ccacacccac gcgcgatcgc
     3061 catggcaacg aggtcgcaca cgccccacac ccagacctcc ctgcgagcgg gcatgggtac
     3121 aatgtccccg ttgccacaga caccacttcg tagcacagcg cagagcgtag cgtgttgttg
     3181 ctgctgacaa aagaaaattt ttcttagcaa agcaaaggag gggaagcacg ggcagatagc
     3241 accgtaccat acccttggaa actcgaaatg aacgaagcag gaaatgagag aatgagagtt
     3301 ttgtaggtat atatagcggt agtgtttgcg cgttaccatc atcttctgga tctatctatt
     3361 gttcttttcc tcatcacttt cccctttttc gctcttcttc ttgtctttta tttctttctt
     3421 ttttttaatt gttccctcga ttggctatct accaaagaat ccaaacttaa tacacgtatt
     3481 tatttgtcca attaccatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag
     3541 cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct
     3601 gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag
     3661 tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg
     3721 gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga
     3781 tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg
     3841 tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat
     3901 ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta
     3961 tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa
     4021 tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt
     4081 atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg
     4141 attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa
     4201 tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa
     4261 ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt
     4321 agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa
     4381 tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc
     4441 tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt
     4501 aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc
     4561 aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt
     4621 tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg
     4681 tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca
     4741 cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga
     4801 caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt
     4861 ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc
     4921 atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat
     4981 gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta
     5041 tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg
     5101 aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac
     5161 aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt
     5221 tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca
     5281 tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat
     5341 cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag
     5401 acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg
     5461 ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc
     5521 tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca
     5581 gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg
     5641 acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa
     5701 aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa
     5761 gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa
     5821 aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat
     5881 tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta
     5941 tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga
     6001 ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa
     6061 ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt
     6121 agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg
     6181 taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg
     6241 ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt
     6301 ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt
     6361 gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt
     6421 acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac
     6481 tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt
     6541 ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa
     6601 atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg
     6661 agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga
     6721 taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt
     6781 caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa
     6841 ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga
     6901 tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa
     6961 gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga
     7021 aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat
     7081 cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc
     7141 tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt
     7201 tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa
     7261 acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga
     7321 attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa
     7381 caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt
     7441 gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg
     7501 atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct
     7561 ttatgaaaca cgcattgatt tgagtcagct aggaggtgac ggtggagggc caaaaaagaa
     7621 aagaaaagtt gaagatgctt ctgctcatca tcaccatcac catcaccatt aaatccgctc
     7681 taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag
     7741 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac
     7801 gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga
     7861 agtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     7921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     7981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     8041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     8101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     8161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     8221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     8281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     8341 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     8401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     8461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     8521 tcttttctac cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
     8581 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
     8641 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag
     8701 agcgctaatt tttcaaacaa agaatctgag ctgcattttt aca
//
