LOCUS       sequence-218310-        6025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA to target URA3 knockout locus and has KanMX marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6025)
  TITLE     Plasmids for Cas9-mediated genome editing in S. cerevisiae with 
            dominant markers
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6025)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCB30
FEATURES             Location/Qualifiers
     source          1..6025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(76..867,868..936))
                     /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     699..718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(937..1041)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1068..2410
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(2031..2078)
                     /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     2451..2469
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     gene            3480..4836
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        3480..3823
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3824..4633
                     /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(3891..3910)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4639..4836
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        4908..5176
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5197..5271
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5276..5295
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      complement(5342..5930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5422..5441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
       61 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
      121 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
      181 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
      241 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
      301 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
      361 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
      421 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
      481 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
      541 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
      601 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
      661 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
      721 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
      781 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
      841 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
      901 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
      961 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1021 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg
     1081 tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
     1141 tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa
     1201 tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa
     1261 gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca
     1321 aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa
     1381 caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata
     1441 actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc
     1501 ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg
     1561 tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat
     1621 actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg
     1681 gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt
     1741 ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag taatactaga
     1801 gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga
     1861 tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa
     1921 tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg
     1981 gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta
     2041 tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg
     2101 cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat
     2161 ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt
     2221 aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg
     2281 atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt
     2341 ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc atttcctttg
     2401 atattggatc atactaagaa accattatta tcatgacatt aacctataaa aataggcgta
     2461 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2521 agcggcgcgc caacaaatat attgcaagta gtgacctatg gtgtgtagat atacgtacag
     2581 ttagttacga gcctaaagac acaacgtgtt tgttaattat actgtcgctg taatatcttc
     2641 tcttccatta tcaccggtca ttccttgcag gggcggtagt acccggagac cctgaacttt
     2701 tctttttttt tttgcgaaat taaaaagttc attttcaatt cgacaatgag gtctacaagc
     2761 cattgtttta tgttgatgag agccagctta aagagttaaa aatttcatag ctaatggtga
     2821 cagggggaat ggcaagcaag tgggatcaga agggtatgga cattgcctat gaggaggcgg
     2881 ccttaggtta caaagagggt ggtgttccta ttggcggatg tcttatcaat aacaaagacg
     2941 gaagtgttct cggtcgtggt cacaacatga gatttcaaaa gggatccgcc acactacatg
     3001 gtgagatctc cactttggaa aactgtggga gattagaggg caaagtgtac aaagatacca
     3061 ctttgtatac gacgctgtct ccatgcgaca tgtgtacagg tgccatcatc atgtatggta
     3121 ttccacgctg tgttgtcggt gagaacgtta atttcaaaag taagggcgag aaatatttac
     3181 aaactagagg tcacgaggtt gttgttgttg acgatgagag gtgtaaaaag atcatgaaac
     3241 aatttatcga tgaaagacct caggattggt ttgaagatat tggtgagtag agcacgcagc
     3301 acgctgtatt tacgtattta attttatata tttgtgcata cactactagg gaagacttga
     3361 aaaaaaccta ggaaatgaaa aaacgacaca ggaagtcccg tatttactat tttttccttc
     3421 cttttgatgg ggcagggcgg aaatagagga taggataagc cattaccctg ttatccctag
     3481 acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat
     3541 gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac
     3601 attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc
     3661 agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa
     3721 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     3781 tgctaggata cagttctcac atcacatccg aacataaaca accatgggta aggaaaagac
     3841 tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     3901 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     3961 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     4021 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     4081 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa cagcattcca
     4141 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     4201 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     4261 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     4321 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt
     4381 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     4441 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     4501 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     4561 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     4621 tgagtttttc taatcagtac tgacaataaa aagattcttg ttttcaagaa cttgtcattt
     4681 gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt gatttatatt
     4741 ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca
     4801 tgcgtcaatc gtatgtgaat gctggtcgct atactgttaa ttaagacggt cacagcttgt
     4861 ctgtaagcgg atctccacgg gacccacagt cgtagatgcg tcacgtgtct ttgaaaagat
     4921 aatgtatgat tatgctttca ctcatattta tacagaaact tgatgttttc tttcgagtat
     4981 atacaaggtg attacatgta cgtttgaagt acaactctag attttgtagt gccctcttgg
     5041 gctagcggta aaggtgcgca ttttttcaca ccctacaatg ttctgttcaa aagattttgg
     5101 tcaaacgctg tagaagtgaa agttggtgcg catgtttcgg cgttcgaaac ttctccgcag
     5161 tgaaagataa atgatcacca tcaaagaagg ttaatggttt tagagctaga aatagcaagt
     5221 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt ggtgcttttt
     5281 ttgtttttta tgtctgttta aacaatgtat gaaaccctgt atggagagtg attctggcgt
     5341 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5401 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5461 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5521 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5581 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5641 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5701 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5761 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5821 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5881 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5941 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6001 tcatgagatt atcaaaaagg atctt
//
