LOCUS       sequence-57096-e         949 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target human AAVS1 (T2 target 
            sequence) for genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 949)
  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 949)
  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_AAVS1-T2
FEATURES             Location/Qualifiers
     source          1..949
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        116..364
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     116..136
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     287..306
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        385..460
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(472..496)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(581..599)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(582..599)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(617..633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(617..633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(630..652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    641..657
                     /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(665..671,672..689,690..695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    710..731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(848..865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gccctttgta caaaaaagca
       61 ggctttaaag gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaagaggg
      121 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
      181 tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
      241 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
      301 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
      361 caccggggcc actagggaca ggatgtttta gagctagaaa tagcaagtta aaataaggct
      421 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt
      481 tcttgtacaa agttggcatt aaagggcgaa ttccagcaca ctggcggccg ttactagtgg
      541 atccgagctc ggtaccaagc ttgatgcata gcttgagtat tctatagtgt cacctaaata
      601 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
      661 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      721 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgc
//
