LOCUS       sequence-63426-e         978 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses a guide RNA (gRNA) to target GFP (T1 target sequence) for 
            genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 978)
  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 978)
  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-T1
FEATURES             Location/Qualifiers
     source          1..978
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        113..361
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     113..133
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     284..303
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        382..457
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(469..493)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(578..596)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(579..596)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(614..630)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(614..630)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(627..649)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    638..654
                     /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(662..668,669..686,687..692))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    707..728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(845..862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgagcggccg ccagtgtgat ggatatctgc agaattcgcc ctttgtacaa aaaagcaggc
       61 tttaaaggaa ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct
      121 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
      181 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      241 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      301 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      361 cgtgaaccgc atcgagctga agttttagag ctagaaatag caagttaaaa taaggctagt
      421 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctagac ccagctttct
      481 tgtacaaagt tggcattaaa gggcgaattc cagcacactg gcggccgtta ctagtggatc
      541 cgagctcggt accaagcttg atgcatagct tgagtattct atagtgtcac ctaaatagct
      601 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      661 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      721 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
      781 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
      841 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      901 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      961 gagcaaaagg ccagcaaa
//
