LOCUS       sequence-245134-        1682 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Provides the repair template with PP2Ac microhomology on each end 
            for PP2Ac N-ter HA tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1682)
  AUTHORS   Lin DW, Chung BP, Huang JW, Wang X, Huang L, Kaiser P
  TITLE     Microhomology based CRISPR tagging tools for protein tracking, 
            purification, and depletion.
  JOURNAL   J Biol Chem. 2019 May 28. pii: RA119.008422. doi: 
            10.1074/jbc.RA119.008422.
  PUBMED    31138654
REFERENCE   2  (bases 1 to 1682)
  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: "J Biol 
            Chem. 2019 May 28. pii: RA119.008422. doi: 
            10.1074/jbc.RA119.008422."
COMMENT     Sequence Label: pN-PITCh-3XHA
FEATURES             Location/Qualifiers
     source          1..1682
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(91..93,94..96,97..807)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(91..93,94..96,97..807)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(136..157)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(397..416)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     744..765
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             817..870
                     /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             871..1467
                     /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(871..890)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1367..1387
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1477..1533
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1537..1617
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
ORIGIN
        1 attagttcat agcccatata tggagttccg cgttacatag catcgtacgc gtacgtgttt
       61 ggggcgggtg gcatcatgga cgccggatcc atggtgagca agggcgagga gctgttcacc
      121 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      181 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      241 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      301 tgcttcagcc gttaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      361 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      421 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      481 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      541 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      601 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      661 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      721 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
      781 actctcggca tggacgagct gtacaaggga agcggagagg gcagaggaag tctgctaaca
      841 tgcggtgacg tcgaggagaa tcctggacct atgaccgagt acaagcccac ggtgcgcctc
      901 gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
      961 cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa
     1021 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     1081 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     1141 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     1201 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     1261 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     1321 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     1381 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     1441 tggtgcatga cccgcaagcc cggtgccgga agcggagcta ctaacttcag cctgctgaag
     1501 caggctggcg acgtggagga gaaccctgga cctatgtatc cttacgacgt gcctgactac
     1561 gcctatcctt acgacgtgcc tgactacgcc tatccttacg acgtgcctga ctacgccgac
     1621 gagaaggtgt tcaccaagga gccaaacacg tacgcgtacg atgctctaga atgctgatgg
     1681 gc
//
