LOCUS       sequence-136394-         846 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Middle Entry clone for Gateway containing a zebrafish 
            codon-optimized Cas9 flanked by 2 NLS and followed by inframe GFP. 
            Cas9 and GFP are separated by the sequence of a T2A self-cleaving 
            peptide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 846)
  AUTHORS   Ablain J, Durand EM, Yang S, Zhou Y, Zon LI
  TITLE     A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in 
            Zebrafish.
  JOURNAL   Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032.
  PUBMED    25752963
REFERENCE   2  (bases 1 to 846)
  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: "Dev Cell. 
            2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032."
COMMENT     Sequence Label: pME-Cas9-T2A-GFP
FEATURES             Location/Qualifiers
     source          1..846
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    69..93
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     94..111
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     95..111
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     121..140
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        121..139
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      175..184
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             193..213
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgatgag caatgctttt
       61 ttataatgcc aactttgtac aaaaaagcag gcttgtaaaa cgacggccag tgagcgcgcg
      121 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cgacgccacc
      181 atggcttctc cacctaagaa gaagagaaag gtgggaagca tggataagaa gtatagcatc
      241 ggcctggata ttggaactaa ctccgtgggt tgggcagtga ttacagacga ctacaaggtc
      301 cctagcaaga aatttaaggt gctgggtaac accgacaggc acagcatcaa gaaaaatctg
      361 attggagccc tgctgttcgg ttctggagag actgccgaag caacacgcct gaaaagaaca
      421 gcaagaaggc gctataccag aaggaagaat agaatctgtt acctgcagga gattttctct
      481 aacgaaatgg ctaaggtgga cgattcattc tttcataggc tggaggaaag tttcctggtc
      541 gaggaagata agaaacacga gcgccatcct atctttggaa acattgtgga cgaggtcgcc
      601 tatcacgaaa aatacccaac catctatcat ctgcgcaaga aactggctga ctctactgat
      661 aaagccgacc tgagactgat ctatctggct ctggcccaca tgattaagtt caggggtcat
      721 tttctgatcg agggcgatct gaaccccgac aattccgatg tggacaagct gttcatccag
      781 ctggtccaga tttacaatca gctgtttgag gaaaacccta ttaatgcttc cagagtggac
      841 gcaaaa
//
