LOCUS       sequence-387445-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoding guide RNAs for the knock out of TPS2 and GSY1 genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  AUTHORS   Takhaveev V, Ozsezen S, Smith EN, Zylstra A, Chaillet ML, Chen H, 
            Papagiannakis A, Milias-Argeitis A, Heinemann M
  TITLE     Temporal segregation of biosynthetic processes is responsible for 
            metabolic oscillations during the budding yeast cell cycle.
  JOURNAL   Nat Metab. 2023 Feb;5(2):294-313. doi: 10.1038/s42255-023-00741-x. 
            Epub 2023 Feb 27.
  PUBMED    36849832
REFERENCE   2  (bases 1 to 5809)
  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: 
            "10.1038/s42255-023-00741-x"; journalName: "Nat Metab"; date: 
            "2023-02"; volume: "5"; issue: "2"; pages: "294-313"
COMMENT     Sequence Label: pROS13-Tps2/Gsy1
FEATURES             Location/Qualifiers
     source          1..5809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..269)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(402..419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(573..1161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(653..672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1332..2123,2124..2192))
                     /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     1955..1974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2193..2297)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2389..2422
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            2477..3833
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2477..2820
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2821..3630
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2888..2907)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3636..3833
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3896..3929
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4052..4320
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        4341..4415
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4420..4439
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4664..5645
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      complement(5691..5710)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5715..5789)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gatcatttat ctttcactgc ggagaagttt cgaacgccga aacatgcgca ccaactttca
       61 cttctacagc gtttgaccaa aatcttttga acagaacatt gtagggtgtg aaaaaatgcg
      121 cacctttacc gctagcccaa gagggcacta caaaatctag agttgtactt caaacgtaca
      181 tgtaatcacc ttgtatatac tcgaaagaaa acatcaagtt tctgtataaa tatgagtgaa
      241 agcataatca tacattatct tttcaaagaa gctccagctt ttgttccctt tagtgagggt
      301 attcacgtag acggataggt atagccagac atcagcagca tacttcggga accgtaggct
      361 ctagagagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
      421 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
      481 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
      541 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
      601 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
      661 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      721 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      781 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
      841 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      901 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      961 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     1021 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     1081 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1141 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1201 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1261 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1321 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1381 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     1441 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     1501 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     1561 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1621 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1681 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1741 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1801 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     1861 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     1921 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     1981 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2041 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2101 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2161 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2221 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2281 caaatagggg ttccgcgcac tcgagactat atgtgaaggc atggctatgg cacggcagac
     2341 attccgccag atcatcaata ggcacgcggc cggtcgacaa cccttaatat aacttcgtat
     2401 aatgtatgct atacgaagtt attaggtcta gagatctgtt tagcttgcct cgtccccgcc
     2461 gggtcacccg gccagcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg
     2521 cagctcaggg gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata
     2581 atcatttgca tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc
     2641 gctgcagacc tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc
     2701 cgcgcccctg tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat
     2761 acttcctttt aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc
     2821 atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat
     2881 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     2941 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     3001 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     3061 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     3121 ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     3181 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     3241 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     3301 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     3361 cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     3421 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     3481 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     3541 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     3601 tttcatttga tgctcgatga gtttttctaa tcagtactga caataaaaag attcttgttt
     3661 tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg
     3721 ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg
     3781 cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga
     3841 ttcgatacta acgccgccat ccagtgtcga aaacgagctc tcgagaaccc ttaatataac
     3901 ttcgtataat gtatgctata cgaagttatt aggtgatatc agatccacta gtggcctatg
     3961 ccacctttcg agaggacgat gcccgtgtct aaatgattcg accagcctaa gaatgttcaa
     4021 cccctcacta aagggaacaa aagctggagc ttctttgaaa agataatgta tgattatgct
     4081 ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca
     4141 tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg
     4201 cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag
     4261 tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc
     4321 caatctacag tattttgatg gttttagagc tagaaatagc aagttaaaat aaggctagtc
     4381 cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctt
     4441 cgagtcatgt aattagttat gtcacgctta cgttcacgcc ctccacgcat ttaagcataa
     4501 acacgcacta tgccgttctt ctcatgtata tatatataca ggcaacacgc agatataggt
     4561 gcgacgtgaa cagtgagctg tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt
     4621 ttcggaaacg ctttgaagtt cctattccga agttcctatt ctgtagaaag tataggaacc
     4681 tcagagcgct tttgaaaacc aaaagcgctc tgaagacgca ctttcaaaaa accaaaaacg
     4741 caccggactg taacgagcta ctaaaatatt gcgaataccg cttccacaaa cattgctcaa
     4801 aagtatctct ttgctatata tctctgtgct atatccctat ataacctacc catccacctt
     4861 tcgctccttg aacttgcatc taaactcgac ctctacattt tttatgttta tctctagtat
     4921 tactctttag acaaaaaaat tgtagtaaga actattcata gagtgaatcg aaaacaatac
     4981 gaaaatgtaa acatttccta tacgtagtat atagagacaa aatagaagaa accgttcata
     5041 attttctgac caatgaagaa tcatcaacgc tatcactttc tgttcacaaa gtatgcgcaa
     5101 tccacatcgg tatagaatat aatcggggat gcctttatct tgaaaaaatg cacccgcagc
     5161 ttcgctagta atcagtaaac gcgggaagtg gagtcaggct ttttttatgg aagagaaaat
     5221 agacaccaaa gtagccttct tctaacctta acggacctac agtgcaaaaa gttatcaaga
     5281 gactgcatta tagagcgcac aaaggagaaa aaaagtaatc taagatgctt tgttagaaaa
     5341 atagcgctct cgggatgcat ttttgtagaa caaaaaagaa gtatagattc tttgttggta
     5401 aaatagcgct ctcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg
     5461 aaaaattagc gctctcgcgt tgcatttttg ttttacaaaa atgaagcaca gattcttcgt
     5521 tggtaaaata gcgctttcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt
     5581 gtttgaaaaa ttagcgctct cgcgttgcat ttttgttcta caaaatgaag cacagatgct
     5641 tcgttggagg gcgtgaacgt aagcgtgaca taactaatta catgactcga agacataaaa
     5701 aacaaaaaaa gcaccaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
     5761 ttttaacttg ctatttctag ctctaaaaca tagaatttgt ttccaaaat
//
