LOCUS       sequence-160228-         989 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses gRNA of interest under U6 promoter (cloning by BpiI) and 
            GFP under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 989)
  AUTHORS   Daneshvar K, Pondick JV, Kim BM, Zhou C, York SR, Macklin JA, 
            Abualteen A, Tan B, Sigova AA, Marcho C, Tremblay KD, Mager J, Choi 
            MY, Mullen AC
  TITLE     DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC 
            Expression to Control Definitive Endoderm Differentiation of 
            Embryonic Stem Cells.
  JOURNAL   Cell Rep. 2016 Oct 4;17(2):353-365. doi: 
            10.1016/j.celrep.2016.09.017.
  PUBMED    27705785
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: 
            "10.1016/j.celrep.2016.09"; journalName: "Cell Rep"; date: 
            "2016-10-4- 4"; volume: "17"; issue: "2"; pages: "353-365"
COMMENT     Sequence Label: pgRGFP
FEATURES             Location/Qualifiers
     source          1..989
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(join(14..727,728..730,731..733))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(17..727,728..730,731..733))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(59..80)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     408..427
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     667..688
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      730..739
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(815..834)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 ggttatgcgg ccgctacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
       61 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
      121 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
      181 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
      241 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
      301 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
      361 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
      421 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
      481 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
      541 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
      601 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
      661 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
      721 ccttgctcac catggtggcg accgggagat ctagatggat gcaggtcgaa aggcccggag
      781 atgaggaaga ggagaacagc gcggcagacg tgcgcttttg aagcgtgcag aatgccgggc
      841 ctccggagga ccttcgggcg cccgccccgc ccctgagccc gcccctgagc ccagaaagcg
      901 aaggagcaaa gctgctattg gccgctgccc caaaggccta cccgcttcca ttgctcagcg
      961 gtgctgtcca tctgcacgag actagtgag
//
