LOCUS       p414-TEF1p-Cas9-        9524 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Yeast centromeric plasmid for expressing human codon-optimized Cas9 
            with the constitutive TEF1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    p414-TEF1p-Cas9-CYC1t
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9524)
  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 9524)
  AUTHORS   Church Lab / Addgene #43802
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9524)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2013"; volume: "41"; pages: "4336-43"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(69..572)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        609..713
                     /label=AmpR promoter
     CDS             714..1571
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1745..2333
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2657..2687
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /label=lac operator
                     /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     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2791..3190
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3194..3210
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3222..7325
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             7338..7358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     terminator      7374..7621
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        complement(7640..7658)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7668..7684)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      7829..8284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(8386..9057)
                     /label=TRP1
                     /note="phosphoribosylanthranilate isomerase, required for 
                     tryptophan biosynthesis"
     promoter        complement(9058..9338)
                     /label=TRP1 promoter
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcc tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tcatagcttc aaaatgtttc tactcctttt
     2821 ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc
     2881 acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa
     2941 ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa
     3001 aaatttttat cacgtttctt tttcttgaaa attttttttt tgattttttt ctctttcgat
     3061 gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat
     3121 ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa gcatagcaat
     3181 ctaatctaag ttttctagaa ctagtggatc ccccgggaaa aatggacaag aagtactcca
     3241 ttgggctcga tatcggcaca aacagcgtcg gttgggccgt cattacggac gagtacaagg
     3301 tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc
     3361 tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg ctcaaaagaa
     3421 cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta
     3481 gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg
     3541 tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg
     3601 cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg
     3661 ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac
     3721 acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc
     3781 aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg
     3841 acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg
     3901 cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg
     3961 ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga
     4021 gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg
     4081 cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc
     4141 gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg
     4201 agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt
     4261 acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag
     4321 caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg
     4381 aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca
     4441 atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag
     4501 aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc
     4561 ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc
     4621 gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct
     4681 ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg
     4741 tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg
     4801 tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag
     4861 ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag
     4921 actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct
     4981 tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc
     5041 tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg
     5101 aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag
     5161 tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga
     5221 tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg
     5281 gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg
     5341 acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc
     5401 ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac
     5461 tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga
     5521 accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg
     5581 gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc
     5641 agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg
     5701 aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc cagtcttttc
     5761 tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga
     5821 gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc
     5881 tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg
     5941 gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga
     6001 tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg
     6061 acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca
     6121 gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg
     6181 cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg
     6241 aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc
     6301 aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg aattttttca
     6361 agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg
     6421 gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc
     6481 tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca
     6541 aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg
     6601 accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg
     6661 ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca
     6721 caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat
     6781 ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg
     6841 aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg
     6901 cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag
     6961 ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg
     7021 atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc
     7081 tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag
     7141 aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact
     7201 tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac
     7261 tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg
     7321 gagacagcag ggctgacccc aagaagaaga ggaaggtgtg atctcttctc gagtcatgta
     7381 attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga
     7441 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt
     7501 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg
     7561 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg
     7621 cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     7681 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     7741 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     7801 ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta gcggcgcatt aagcgcggcg
     7861 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     7921 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     7981 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8041 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8101 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8161 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     8221 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     8281 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataggca
     8341 agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct tagcattttt
     8401 gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac ctccgcttac
     8461 atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg gcgtcagtcc
     8521 accagctaac ataaaatgta agctttcggg gctctcttgc cttccaaccc agtcagaaat
     8581 cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca agggaataaa
     8641 cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg gaaatacgag
     8701 tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact catctccatg
     8761 cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct ccttaggttg
     8821 attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat atgcttttac
     8881 aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat tgggcacaca
     8941 tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct ctgtgctctg
     9001 caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa cagacatact
     9061 ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc accaatgccc
     9121 tccctcttgg ccctctcctt ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa
     9181 gaaaagctcc ggatcaagat tgtacgtaag gtgacaagct atttttcaat aaagaatatc
     9241 ttccactact gccatctggc gtcataactg caaagtacac atatattacg atgctgtcta
     9301 ttaaatgctt cctatattat atatatagta atgtcgttta tggtgcactc tcagtacaat
     9361 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     9421 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     9481 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcga
//
