LOCUS       sequence-57098-e         989 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target GFP (T2 target sequence) for 
            genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 989)
  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 989)
  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_GFP-T2
FEATURES             Location/Qualifiers
     source          1..989
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    58..82
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        complement(94..169)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(190..438)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(248..267)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(418..438)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        complement(587..605)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(588..605)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(623..639)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(623..639)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(636..658)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    647..663
                     /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(671..677,678..695,696..701))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    716..737
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(854..871)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atgcatgctc gagcggccgc cagtgtgatg gatatctgca gaattcgccc tttaatgcca
       61 actttgtaca agaaagctgg gtctagaaaa aaagcaccga ctcggtgcca ctttttcaag
      121 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaact gaagaagatg
      181 gtgcgctccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt
      241 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta
      301 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt
      361 caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat
      421 catgggaaat aggccctctt cctgcccgac cttggtaccg gatccagtcg actgaattgg
      481 ttcctttaaa gcctgctttt ttgtacaaag ggcgaattcc agcacactgg cggccgttac
      541 tagtggatcc gagctcggta ccaagcttga tgcatagctt gagtattcta tagtgtcacc
      601 taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      661 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      721 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      781 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      841 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      901 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      961 agaacatgtg agcaaaaggc cagcaaaag
//
