LOCUS       sequence-145742-         988 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 988)
  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 988)
  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..988
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     137..157
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     308..327
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    371..392
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(407..412,413..430,431..437)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    445..461
                     /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     450..472
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     469..485
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     469..485
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             481..759
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW"
     promoter        506..524
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(571..590)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(572..590)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(600..617)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(600..616)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(609..631)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tatagggaga gagagagaat taccctcact aaagggagga gaagcatgaa ttccccagtg
       61 gaaagacgcg caggcaaaac gcaccacgtg acggagcgtg accgcgcgcc gagcgcgcgc
      121 caaggtcggg caggaagagg gcctatttcc catgattcct tcatatttgc atatacgata
      181 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
      241 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
      301 taaaatggac tatcatatgc ttaccgtaac tcgagacgcg gtggaaagcg ggcagtgagc
      361 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
      421 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
      481 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagg
      541 acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
      601 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      661 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      721 ccttcccaac agttgcgcag cctgaatggc gaatggtagg acgcgccctg tagcggcgcg
      781 tctcacgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      841 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      901 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      961 gtcaataatg acgtatgttc ccatagta
//
