LOCUS       sequence-108215-         794 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 794)
  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 794)
  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..794
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(29..82)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(98..118)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             213..221
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 gctcttcgcc cttgctcacc attctagaag ggccgggatt ctcttccacg tcaccgcatg
       61 tcagcagact gcctctgccc tctccgccac cagacctcac ctttctcttc ttcttagggg
      121 atcccaccgg tgagtcgcca cccagctgag acaggtcaat ccttgtttcg tacaggccgg
      181 taatactctg atggatcagt gtagcgtcca gcacctcctt agtggaggtg tacctctttc
      241 tatcgattgt agtgtcgaag tacttgaaag ctgcaggtgc gcccaggttt gtcagagtga
      301 acagatgaat gatattttcg gcctgctccc tgatgggctt atctctgtgt ttgttgtaag
      361 cagacaggac cttatccaga tttgcgtcgg ccaggatcac tcttttagag aactcgctaa
      421 tctgttcaat gatctcgtcc agataatgct tgtgctgctc cacaaacagc tgtttctgtt
      481 cgttatcctc tggagatcct ttcagcttct cgtagtggct tgccagatac aggaagttca
      541 cgtacttaga aggcagggcc agttcattgc ctttctgcag ctctccagcg cttgccagca
      601 tgcgtttcct gccgttttcc agctcaaaca gactgtactt gggcagttta atgatcaggt
      661 ctttcttcac ttccttataa cctttggcct ccagaaaatc gattgggttc ttctcgaaag
      721 agcttctctc cataattgtg atacccagca gctccttgac agatttcagt ttcttgcttt
      781 tgcccttctc cacc
//
