LOCUS       pDYSCKO-ADE1               6460 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pDYSCKO canavanine co-selection vector with a guide targeting the 
            ADE1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    pDYSCKO-ADE1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  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 6460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..6460
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             1543..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        3485..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      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(4385..4653)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     rep_origin      4662..5117
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4749..4768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4959..4980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5248..6051)
                     /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(6052..6267)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6291..6310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6410..6432
                     /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 aaacgttcag tcttcgtaat
     4381 tgacgatcat ttatctttca ctgcggagaa gtttcgaacg ccgaaacatg cgcaccaact
     4441 ttcacttcta cagcgtttga ccaaaatctt ttgaacagaa cattgtaggg tgtgaaaaaa
     4501 tgcgcacctt taccgctagc ccaagagggc actacaaaat ctagagttgt acttcaaacg
     4561 tacatgtaat caccttgtat atactcgaaa gaaaacatca agtttctgta taaatatgag
     4621 tgaaagcata atcatacatt atcttttcaa agaatggcgc gacgcgccct gtagcggcgc
     4681 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4741 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4801 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4861 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4921 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4981 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5041 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5101 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5161 caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat
     5221 acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc
     5281 ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa
     5341 tagtcctctt ccaacaataa taatgtcaga tcctgtagat accacatcat ccacggttct
     5401 atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa
     5461 ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa
     5521 atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga
     5581 gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc
     5641 tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat
     5701 gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc
     5761 aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt
     5821 tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag
     5881 taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac
     5941 atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc
     6001 aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct
     6061 tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt
     6121 ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc
     6181 ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa
     6241 agaataaaaa aaaaatgatg aattgaaaag gtggtatggt gcactctcag tacaatctgc
     6301 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     6361 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     6421 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//