LOCUS       sequence-496997-        4451 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Expresses sgRNA targeting the fcy gene in S. cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4451)
  AUTHORS   Gupta D, Patinios C, Bassett HV, Kibe A, Collins SP, Kamm C, Wang Y,
            Zhao C, Vollen K, Toussaint C, Calvin I, Cullot G, Aird EJ, Polkoff 
            KM, Nguyen-Vo TP, Migur A, Schut F, Al'Abri IS, Achmedov T, Del Re 
            A, Corn JE, Saliba AE, Crook N, Stepanova AN, Alonso JM, Beisel CL
  TITLE     Targeted DNA ADP-ribosylation triggers templated repair in bacteria 
            and base mutagenesis in eukaryotes.
  JOURNAL   Nat Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w.
  PUBMED    40908325
REFERENCE   2  (bases 1 to 4451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w."
COMMENT     Sequence Label: pCBS-5740
FEATURES             Location/Qualifiers
     source          1..4451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        672..940
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        961..1036
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1389..1490
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1491..2165
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      2235..3217
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(join(3342..4133,4134..4202))
                     /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     3965..3984
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4203..4307)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4410..4451,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtgcg
      661 ggctgccaat gtctttgaaa agataatgta tgattatgct ttcactcata tttatacaga
      721 aacttgatgt tttctttcga gtatatacaa ggttattaca tgtacgtttg aagtacaact
      781 ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt cacaccctac
      841 aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt
      901 tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc taatctccca cagttttcca
      961 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     1021 ggcaccgagt cggtgctttt gttttttatg tctggactga cagctgtggt agagccacaa
     1081 acagccggta caagcaacga tctccaggac catctgaatc atgcgcggat gacacgaact
     1141 cacgacggcg atcacagaca ttaacccaca gtacagacac tgcgacaacg tggcaattcg
     1201 tcgcaatact ggccaacggg gtcatcacgg ctcatcatgc gccaaacaaa tgtgtgcaat
     1261 acacgctcgg atgactgcat gatgaccgca ctgactgggg acagcagatc cacctaagcc
     1321 tgtgagagaa gcagacaccc gacagatcaa ggcagttaac tagtgcactg cagtacagcc
     1381 gattaagaat tccggtcgaa aaaagaaaag gagagggcca agagggaggg cattggtgac
     1441 tattgagcac gtgagtatac gtgattaagc acacaaaggc agcttggagt atgtctgtta
     1501 ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag agcacagagg
     1561 ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt gtgcccaata
     1621 gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta aaagcatata
     1681 aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa cctaaggagg
     1741 atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat ggagatgagt
     1801 cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc gtatttccaa
     1861 aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt attcccttgt
     1921 ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt tctgactggg
     1981 ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga ctgacgccag
     2041 aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta agcggaggtg
     2101 tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa aatgctaaga
     2161 aataggttat tactgagtag tatttattta agtattgttt gtgcacttgc ctgcaggcct
     2221 tttgaaaagc gagtaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga
     2281 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg
     2341 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa
     2401 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg
     2461 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa
     2521 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg
     2581 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa
     2641 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg
     2701 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta
     2761 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat
     2821 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat
     2881 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa
     2941 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag
     3001 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat
     3061 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt
     3121 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcatcag agcgcttttg
     3181 gttttcaaaa gcgctctgaa gttcctatac tttctagcta gagaatagga acttcccgag
     3241 cggccgcgat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3301 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3361 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3421 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3481 cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3541 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3601 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3661 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3721 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3781 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3841 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3901 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3961 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4021 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4081 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4141 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4201 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4261 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4321 aaagtgccac ctgtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4441 tgcttgcaaa c
//
