LOCUS       sequence-337031-        7197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS426-SNR52p-gRNA.MATa_option3-SUP4t; 20-bp gRNA sequence targeting
            MATa cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7197)
  AUTHORS   Xie ZX, Mitchell LA, Liu HM, Li BZ, Liu D, Agmon N, Wu Y, Li X, Zhou
            X, Li B, Xiao WH, Ding MZ, Wang Y, Yuan YJ, Boeke JD
  TITLE     Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of 
            Saccharomyces cerevisiae.
  JOURNAL   G3 (Bethesda). 2018 Jan 4;8(1):173-183. doi: 10.1534/g3.117.300347.
  PUBMED    29150593
REFERENCE   2  (bases 1 to 7197)
  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/g3.117"; 
            journalName: "G3 (Bethesda)"; date: "2018-01-4- 4"; volume: "8"; 
            issue: "1"; pages: "173-183"
COMMENT     Sequence Label: pXZX501
FEATURES             Location/Qualifiers
     source          1..7197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1935..3277
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(2898..2945)
                     /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)."
     primer_bind     3318..3336
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3766..3828)
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     misc_RNA        complement(3841..3916)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3924..4330)
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     primer_bind     4430..4449
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4473..4688
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             4689..5492
                     /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"
     rep_origin      complement(5623..6078)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5760..5781)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5972..5991
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6204..6226
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6218..6235
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6219..6235
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6245..6264
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6245..6263
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(6282..6529)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     terminator      complement(6535..6554)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(6559..6633)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(6654..6922)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(6940..6960)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6940..6958)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6979..6995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6979..6995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6992..7014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7003..7019
                     /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)."
     promoter        complement(join(7027..7033,7034..7051,7052..7057))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7072..7093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga
     1981 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     2041 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa
     2101 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     2161 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa
     2221 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg
     2281 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa
     2341 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg
     2401 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta
     2461 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat
     2521 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat
     2581 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa
     2641 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag
     2701 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat
     2761 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt
     2821 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg
     2881 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga
     2941 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac
     3001 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga
     3061 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg
     3121 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc
     3181 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct
     3241 catccttcaa tgctatcatt tcctttgata ttggatcata ctaagaaacc attattatca
     3301 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     3361 atgacggtga aaacctctga cacatgcagc aaagaacaac aagagttcca aaaagtagtg
     3421 gaacaaaagc aaatgaagga tttcatgcgt ttgtactcta atctggtaga aagatgtttc
     3481 acagactgtg tcaatgactt cacaacatca aagctaacca ataaggaaca aacatgcatc
     3541 atgaagtgct cagaaaagtt cttgaagcat agcgaacgtg tagggcagcg tttccaagaa
     3601 caaaacgctg ccttgggaca aggcttgggc cgataaggtg tactggcgta tatatatcta
     3661 attatgtatc tctggtgtag cccattttta gcatgtaaat ataaagagaa accatatcta
     3721 atctaaccaa atccaaacaa aattcaatag ttactatcgc ttttttcttt ctgtatcgca
     3781 aataagtgaa aattaaaaaa gaaagattaa attggaagtt ggatatggct cgagaaaaaa
     3841 gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
     3901 tttctagctc taaaacaagc ttctgccaat cgcagctccc agagtttcgt tcccagccgc
     3961 gagcaccaca gcgtaccatg tgcgccacga ggcctcaaac gtaaaacaat cgaaacgaaa
     4021 agaaacagac tataggggag tatagagaca gccggccaat aagaagagga aaaagaaata
     4081 ctagcgttta tcaatgtggg tcgttaacat atcctgttga caatgattac aggttaaaag
     4141 gtagcgtaag tgaatattaa ctatggatat tcttatactt agataagaga catataaaac
     4201 acacgatgat tgattgattt ttatgaccaa tatatgtaat tcgtaattca gatagttttt
     4261 atacttttaa tgtgtgccga accatattac tagtatatgt aactaccatg ttatgttcaa
     4321 ttggcagatc cgacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4381 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     4441 cagattgtac tgagagtgca ccataccacc ttttcaattc atcatttttt ttttattctt
     4501 ttttttgatt tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag
     4561 aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa
     4621 catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa
     4681 gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg
     4741 ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg
     4801 ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac
     4861 taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa
     4921 aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg
     4981 gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca
     5041 ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag
     5101 aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct
     5161 ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt
     5221 ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt
     5281 tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata
     5341 gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat
     5401 ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag
     5461 catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta
     5521 tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt
     5581 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
     5641 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5701 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5761 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5821 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5881 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5941 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     6001 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6061 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     6121 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     6181 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
     6241 cgcgtaatac gactcactat agggcgaatt gggtaccggc cgcaaattaa agccttcgag
     6301 cgtcccaaaa ccttctcaag caaggttttc agtataatgt tacatgcgta cacgcgtctg
     6361 tacagaaaaa aaagaaaaat ttgaaatata aataacgttc ttaatactaa cataactata
     6421 aaaaaataaa tagggaccta gacttcaggt tgtctaactc cttccttttc ggttagagcg
     6481 gatgtggggg gagggcgtga acgtaagcgt gacataacta attacatgac tcgaagacat
     6541 aaaaaacaaa aaaagcacca ccgactcggt gccacttttt caagttgata acggactagc
     6601 cttattttaa cttgctattt ctagctctaa aacagcacgg gcatttttag aacgatcatt
     6661 tatctttcac tgcggagaag tttcgaacgc cgaaacatgc gcaccaactt tcacttctac
     6721 agcgtttgac caaaatcttt tgaacagaac attgtagggt gtgaaaaaat gcgcaccttt
     6781 accgctagcc caagagggca ctacaaaatc tagagttgta cttcaaacgt acatgtaatc
     6841 accttgtata tactcgaaag aaaacatcaa gtttctgtat aaatatgagt gaaagcataa
     6901 tcatacatta tcttttcaaa gaagctccag cttttgttcc ctttagtgag ggttaattgc
     6961 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     7021 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     7081 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7141 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcg
//
