LOCUS       Exported                6263 bp DNA     circular SYN 04-AUG-2025
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 6263)
  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 6263)
  AUTHORS   Church Lab / Addgene #43803
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6263)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6263)
  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"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        1438..1542
                     /label=AmpR promoter
     CDS             1543..2400
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2574..3162
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    3450..3471
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3486..3516
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /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     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3621..3889
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA
                     gene SNR52"
     misc_feature    3890..3909
                     /label=CAN1.Y genomic target
                     /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
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3989..4008
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4
                     tRNA gene"
     terminator      4014..4261
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        complement(4280..4298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4308..4324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4465..4920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5054..5854)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(5855..6070)
                     /label=URA3 promoter
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct tctttgaaaa gataatgtat gattatgctt tcactcatat
     3661 ttatacagaa acttgatgtt ttctttcgag tatatacaag gtgattacat gtacgtttga
     3721 agtacaactc tagattttgt agtgccctct tgggctagcg gtaaaggtgc gcattttttc
     3781 acaccctaca atgttctgtt caaaagattt tggtcaaacg ctgtagaagt gaaagttggt
     3841 gcgcatgttt cggcgttcga aacttctccg cagtgaaaga taaatgatcg atacgttctc
     3901 tatggaggag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     3961 tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt ttatgtcttc gagtcatgta
     4021 attagttatg tcacgcttac gttcacgccc tccccccaca tccgctctaa ccgaaaagga
     4081 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt
     4141 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg
     4201 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg
     4261 cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4321 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4381 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4441 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4501 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4561 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4621 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4681 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4741 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4801 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4861 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     4921 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagggtaata
     4981 actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata
     5041 atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc
     5101 tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa
     5161 taataatgtc agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg
     5221 cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat
     5281 ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg
     5341 caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt
     5401 ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac
     5461 cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta
     5521 ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc
     5581 tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc
     5641 cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac
     5701 ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca
     5761 agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag
     5821 tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt
     5881 ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata
     5941 tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga
     6001 ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg
     6061 atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6121 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     6181 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     6241 accgtcatca ccgaaacgcg cga
//
