LOCUS       sequence-154041-         767 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 767)
  AUTHORS   Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T
  TITLE     MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the 
            PITCh systems.
  JOURNAL   Nat Protoc. 2016 Jan;11(1):118-33. doi: 10.1038/nprot.2015.140. Epub
            2015 Dec 17.
  PUBMED    26678082
REFERENCE   2  (bases 1 to 767)
  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: 
            "10.1038/nprot.2015.140"; journalName: "Nat Protoc"; date: 
            "2016-01"; volume: "11"; issue: "1"; pages: "118-33"
COMMENT     Sequence Label: pCRIS-PITChv2-FBL
FEATURES             Location/Qualifiers
     source          1..767
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(46..645)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(129..149)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     626..645
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(646..699)
                     /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"
ORIGIN
        1 gagcatcgta cgcgtacgtg tttggacttc agttcttcac cttggtcagg caccgggctt
       61 gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa
      121 gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac
      181 cccggcgcgc tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc
      241 ctggtggtcg ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg
      301 cggcgccagg aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc
      361 catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca
      421 gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg
      481 cgtgaggaag agttcttgca gctcggtgac ccgctcgatg tggcggtccg ggtcgacggt
      541 gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg cccgggggac
      601 gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg gtcataggtc caggattctc
      661 ctcgacgtca ccgcatgtta gcagacttcc tctgccctct ccgcttccct tgtacagctc
      721 gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcagga
//
