LOCUS       sequence-233690-        6459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for cloning any gRNA to be used with the canavanine 
            co-selection system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  AUTHORS   Despres PC, Dube AK, Nielly-Thibault L, Yachie N, Landry CR
  TITLE     Double Selection Enhances the Efficiency of Target-AID and 
            Cas9-Based Genome Editing in Yeast.
  JOURNAL   G3 (Bethesda). 2018 Oct 3;8(10):3163-3171. doi: 
            10.1534/g3.118.200461.
  PUBMED    30097473
REFERENCE   2  (bases 1 to 6459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1534/g3.118"; 
            journalName: "G3 (Bethesda)"; date: "2018-10-3- 3"; volume: "8"; 
            issue: "10"; pages: "3163-3171"
COMMENT     Sequence Label: pDYSCKO
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /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)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3062..3081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3315..3332
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3449..3470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3485..3490,3491..3508,3509..3515)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3523..3539
                     /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     3528..3550
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3547..3563
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3547..3563
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3582..3602
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3584..3602
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3620..3888
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3909..3983
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3988..4007
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      4013..4260
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     terminator      complement(4266..4285)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(4290..4364)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4384..4652)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     rep_origin      4661..5116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4748..4767)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4958..4979
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5247..6050)
                     /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"
     promoter        complement(6051..6266)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6290..6309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6409..6431
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcggcatg 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 cccgcggtat 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 accaccgcta ccagcggtgg
     2641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2701 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3241 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3301 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3421 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     3481 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     3541 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     3601 gggaacaaaa gctggagctt ctttgaaaag ataatgtatg attatgcttt cactcatatt
     3661 tatacagaaa cttgatgttt tctttcgagt atatacaagg tgattacatg tacgtttgaa
     3721 gtacaactct agattttgta gtgccctctt gggctagcgg taaaggtgcg cattttttca
     3781 caccctacaa tgttctgttc aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg
     3841 cgcatgtttc ggcgttcgaa acttctccgc agtgaaagat aaatgatcat aacggaatcc
     3901 aactgggcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     3961 gaaaaagtgg caccgagtcg gtggtgcttt ttttgttttt tatgtcttcg agtcatgtaa
     4021 ttagttatgt cacgcttacg ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     4081 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     4141 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     4201 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     4261 ctcgaagaca taaaaaacaa aaaaagcacc accgactcgg tgccactttt tcaagttgat
     4321 aacggactag ccttatttta acttgctatt tctagctcta aaactgagac cagatctggt
     4381 ctcgatcatt tatctttcac tgcggagaag tttcgaacgc cgaaacatgc gcaccaactt
     4441 tcacttctac agcgtttgac caaaatcttt tgaacagaac attgtagggt gtgaaaaaat
     4501 gcgcaccttt accgctagcc caagagggca ctacaaaatc tagagttgta cttcaaacgt
     4561 acatgtaatc accttgtata tactcgaaag aaaacatcaa gtttctgtat aaatatgagt
     4621 gaaagcataa tcatacatta tcttttcaaa gaatggcgcg acgcgccctg tagcggcgca
     4681 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4741 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4801 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4861 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4921 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4981 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5041 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5101 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5161 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     5221 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
     5281 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
     5341 agtcctcttc caacaataat aatgtcagat cctgtagata ccacatcatc cacggttcta
     5401 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
     5461 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
     5521 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
     5581 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
     5641 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
     5701 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
     5761 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
     5821 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
     5881 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
     5941 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
     6001 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
     6061 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
     6121 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
     6181 tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa
     6241 gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct
     6301 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     6361 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     6421 tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
