LOCUS       sequence-57100-e         987 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target DNMT3a (T2 target sequence) 
            for genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 987)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE,
            Church GM
  TITLE     RNA-Guided Human Genome Engineering via Cas9.
  JOURNAL   Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033
  PUBMED    23287722
REFERENCE   2  (bases 1 to 987)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2013-02-15- 15"; volume: "339"; 
            issue: "6121"; pages: "823-6"
COMMENT     Sequence Label: gRNA_DNMT3a-T2
FEATURES             Location/Qualifiers
     source          1..987
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    56..80
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        complement(92..167)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(188..436)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(246..265)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(416..436)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        complement(585..603)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(586..603)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(621..637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(621..637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(634..656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    645..661
                     /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(669..675,676..693,694..699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    714..735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(852..869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcatgctcga gcggccgcca gtgtgatgga tatctgcaga attcgccctt taatgccaac
       61 tttgtacaag aaagctgggt ctagaaaaaa agcaccgact cggtgccact ttttcaagtt
      121 gataacggac tagccttatt ttaacttgct atttctagct ctaaaacgaa gaaggggcga
      181 tcatctcggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
      241 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
      301 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
      361 aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
      421 tgggaaatag gccctcttcc tgcccgacct tggtaccgga tccagtcgac tgaattggtt
      481 cctttaaagc ctgctttttt gtacaaaggg cgaattccag cacactggcg gccgttacta
      541 gtggatccga gctcggtacc aagcttgatg catagcttga gtattctata gtgtcaccta
      601 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
      661 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
      721 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
      781 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      841 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
      901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      961 aacatgtgag caaaaggcca gcaaaag
//
