LOCUS       sequence-99322-e        1019 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses Cas9 nickase and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1019)
  AUTHORS   Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T
  TITLE     Multiplex genome engineering in human cells using all-in-one 
            CRISPR/Cas9 vector system.
  JOURNAL   Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400.
  PUBMED    24954249
REFERENCE   2  (bases 1 to 1019)
  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: "Sci Rep."; 
            date: "2014-06-23"; volume: "4:5400. doi"; pages: " 
            10.1038/srep05400"
COMMENT     Sequence Label: pX330A_D10A-1x2
FEATURES             Location/Qualifiers
     source          1..1019
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     126..145
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        222..297
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(383..688)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     538..560
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     552..569
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     553..569
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     579..598
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        579..597
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(645..665)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(645..663)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(684..700)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(684..700)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(697..719)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    708..724
                     /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)."
     promoter        complement(join(732..738,739..756,757..762))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    777..798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
       61 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      121 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      181 tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag
      241 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      301 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg
      361 ccaattctgc agacaaatgg ctctagagac cggcgccgct acagggcgcg tcccattcgc
      421 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      481 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      541 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
      601 aattgggtac cgggcccccc ctcgaggtcc tccagctttt gttcccttta gtgagggtta
      661 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      721 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
      781 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccaccg gtggtctctt
      841 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      901 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      961 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtac
//
