LOCUS       sequence-166015-         659 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at 
            site X-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 659)
  AUTHORS   Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, 
            Keasling JD, Borodina I
  TITLE     EasyClone-MarkerFree: A vector toolkit for marker-less integration 
            of genes into Saccharomyces cerevisiae via CRISPR-Cas9.
  JOURNAL   Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. 
            Epub 2016 Jun 23.
  PUBMED    27166612
REFERENCE   2  (bases 1 to 659)
  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: 
            "10.1002/biot.201600147"; journalName: "Biotechnol J"; date: 
            "2016-08"; volume: "11"; issue: "8"; pages: "1110-7"
COMMENT     Sequence Label: pCfB3020(gRNA X-2)
FEATURES             Location/Qualifiers
     source          1..659
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     210..227
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(329..348)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(353..427)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
       61 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
      121 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
      181 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
      241 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggaatg
      301 cacgcgatta actaattaca tgactcgaag acataaaaaa caaaaaaagc accaccgact
      361 cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct
      421 ctaaaacgca cgtgaccact tcgagaggat catttatctt tcactgcgga gaagtttcga
      481 acgccgaaac atgcgcacca actttcactt ctacagcgtt tgaccaaaat cttttgaaca
      541 gaacattgta gggtgtgaaa aaatgcgcac ctttaccgct agcccaagag ggcactacaa
      601 aatctagagt tgtacttcaa acgtacatgt aatcaccttg tatatactcg aaagaaaac
//
