LOCUS       sequence-334690-        6263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS426-SNR52p-gRNA.MAT-alpha_option1-SUP4t; 20-bp gRNA sequence 
            targeting MAT-alpha cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6263)
  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 6263)
  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: pXZX351
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..72)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      113..1455
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    445..492
                     /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        1482..1586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1587..1655,1656..2447)
                     /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(1805..1824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2618..3206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3107..3126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3360..3377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3494..3515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3530..3535,3536..3553,3554..3560)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3568..3584
                     /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     3573..3595
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3592..3608
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3592..3608
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3627..3647
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3629..3647
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3665..3933
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3954..4028
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4033..4052
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      4058..4305
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4323..4342)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4324..4342)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4352..4369)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4352..4368)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4361..4383)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4509..4964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4596..4615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4806..4827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5095..5898)
                     /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(5899..6114)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6138..6157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(6257..6263,1..16)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
       61 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttagt atgatccaat
      121 atcaaaggaa atgatagcat tgaaggatga gactaatcca attgaggagt ggcagcatat
      181 agaacagcta aagggtagtg ctgaaggaag catacgatac cccgcatgga atgggataat
      241 atcacaggag gtactagact acctttcatc ctacataaat agacgcatat aagtacgcat
      301 ttaagcataa acacgcacta tgccgttctt ctcatgtata tatatataca ggcaacacgc
      361 agatataggt gcgacgtgaa cagtgagctg tatgtgcgca gctcgcgttg cattttcgga
      421 agcgctcgtt ttcggaaacg ctttgaagtt cctattccga agttcctatt ctctagaaag
      481 tataggaact tcagagcgct tttgaaaacc aaaagcgctc tgaagacgca ctttcaaaaa
      541 accaaaaacg caccggactg taacgagcta ctaaaatatt gcgaataccg cttccacaaa
      601 cattgctcaa aagtatctct ttgctatata tctctgtgct atatccctat ataacctacc
      661 catccacctt tcgctccttg aacttgcatc taaactcgac ctctacattt tttatgttta
      721 tctctagtat tactctttag acaaaaaaat tgtagtaaga actattcata gagtgaatcg
      781 aaaacaatac gaaaatgtaa acatttccta tacgtagtat atagagacaa aatagaagaa
      841 accgttcata attttctgac caatgaagaa tcatcaacgc tatcactttc tgttcacaaa
      901 gtatgcgcaa tccacatcgg tatagaatat aatcggggat gcctttatct tgaaaaaatg
      961 cacccgcagc ttcgctagta atcagtaaac gcgggaagtg gagtcaggct ttttttatgg
     1021 aagagaaaat agacaccaaa gtagccttct tctaacctta acggacctac agtgcaaaaa
     1081 gttatcaaga gactgcatta tagagcgcac aaaggagaaa aaaagtaatc taagatgctt
     1141 tgttagaaaa atagcgctct cgggatgcat ttttgtagaa caaaaaagaa gtatagattc
     1201 tttgttggta aaatagcgct ctcgcgttgc atttctgttc tgtaaaaatg cagctcagat
     1261 tctttgtttg aaaaattagc gctctcgcgt tgcatttttg ttttacaaaa atgaagcaca
     1321 gattcttcgt tggtaaaata gcgctttcgc gttgcatttc tgttctgtaa aaatgcagct
     1381 cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgttcta caaaatgaag
     1441 cacagatgct tcgttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1501 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1561 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1621 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1681 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1741 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1801 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1861 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1921 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1981 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2041 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2101 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     2161 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2221 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2281 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2341 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2401 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2461 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2521 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2581 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2641 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2701 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2761 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2821 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2881 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2941 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3001 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3061 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3121 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3181 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3241 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3301 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3361 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3421 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3481 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     3541 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     3601 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     3661 agcttctttg aaaagataat gtatgattat gctttcactc atatttatac agaaacttga
     3721 tgttttcttt cgagtatata caaggtgatt acatgtacgt ttgaagtaca actctagatt
     3781 ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt tttcacaccc tacaatgttc
     3841 tgttcaaaag attttggtca aacgctgtag aagtgaaagt tggtgcgcat gtttcggcgt
     3901 tcgaaacttc tccgcagtga aagataaatg atccaaatca tacagaaaca caggttttag
     3961 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     4021 agtcggtggt gctttttttg ttttttatgt cttcgagtca tgtaattagt tatgtcacgc
     4081 ttacgttcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     4141 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     4201 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     4261 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt
     4321 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     4381 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4441 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4501 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4561 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4621 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4681 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4741 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4801 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4861 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4921 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     4981 ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat
     5041 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     5101 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     5161 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     5221 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     5281 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     5341 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     5401 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     5461 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     5521 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     5581 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     5641 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     5701 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     5761 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     5821 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     5881 atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg
     5941 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     6001 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     6061 atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg
     6121 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     6181 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6241 caagctgtga ccgtctccgg gag
//
