LOCUS       sequence-139961-         817 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  ""All-in-one"" CRISPR/Cas9 (wt) plasmid for cloning of custom gRNA 
            with blue/white screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 817)
  AUTHORS   Findlay SD, Vincent KM, Berman JR, Postovit LM
  TITLE     A Digital PCR-Based Method for Efficient and Highly Specific 
            Screening of Genome Edited Cells.
  JOURNAL   PLoS One. 2016 Apr 18;11(4):e0153901. doi: 
            10.1371/journal.pone.0153901. eCollection 2016.
  PUBMED    27089539
REFERENCE   2  (bases 1 to 817)
  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: "PLoS One.";
            date: "2016-04-18"; volume: "11(4):e0153901. doi"; pages: " 
            10.1371/journal.pone.0153901. eCollection 2016"
COMMENT     Sequence Label: All_in_one_CRISPR/Cas9_LacZ
FEATURES             Location/Qualifiers
     source          1..817
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     115..135
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     286..305
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    349..370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(385..390,391..408,409..415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    423..439
                     /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)."
     primer_bind     428..450
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     447..463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     447..463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             459..737
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW"
     promoter        484..502
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(549..568)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(550..568)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(578..595)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(578..594)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(587..609)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccctcactaa agggaggaga agcatgaatt ccccagtgga aagacgcgca ggcaaaacgc
       61 accacgtgac ggagcgtgac cgcgcgccga gcgcgcgcca aggtcgggca ggaagagggc
      121 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      181 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      241 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      301 accgtaactc gagacgcggt ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      361 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      421 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      481 cgcaattaac cctcactaaa gggaacaaaa gctggaggac ctcgaggggg ggcccggtac
      541 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
      601 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      661 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      721 tgaatggcga atggtaggac gcgccctgta gcggcgcgtc tcacgctgct tcgcgatgta
      781 cgggccagat atacgcgttg acattgatta ttgacta
//
