LOCUS       sequence-245138-        1819 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Provides the repair template with PP2Ac microhomology on each end 
            for PP2Ac N-ter FAID tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1819)
  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 1819)
  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-FAID
FEATURES             Location/Qualifiers
     source          1..1819
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      83..92
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(89..91,92..94,95..805)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(89..91,92..94,95..805)
                     /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(134..155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(395..414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     742..763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             815..868
                     /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             869..1465
                     /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(869..888)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1365..1385
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1475..1531
                     /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             1535..1558
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1559..1762
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
ORIGIN
        1 attagttcat agcccatata tggagttccg cgttacatag catcgtacgc gtacgtgttt
       61 ggggcgggtg gcatcatgga cggccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      121 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      181 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      241 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      301 cttcagccgt taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      361 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      421 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      481 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      541 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      601 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
      661 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
      721 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
      781 tctcggcatg gacgagctgt acaagggaag cggagagggc agaggaagtc tgctaacatg
      841 cggtgacgtc gaggagaatc ctggacctat gaccgagtac aagcccacgg tgcgcctcgc
      901 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
      961 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     1021 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     1081 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     1141 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     1201 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     1261 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     1321 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     1381 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     1441 gtgcatgacc cgcaagcccg gtgccggaag cggagctact aacttcagcc tgctgaagca
     1501 ggctggcgac gtggaggaga accctggacc tatggactac aaagacgatg acgacaagaa
     1561 ggagaagagt gcttgtccta aagatccagc caaacctccg gccaaggcac aagttgtggg
     1621 atggccaccg gtgagatcat accggaagaa cgtgatggtt tcctgccaaa aatcaagcgg
     1681 tggcccggag gcggcggcgt tcgtgaaggt atcaatggac ggagcaccgt acttgaggaa
     1741 aatcgatttg aggatgtata aagacgagaa ggtgttcacc aaggagccaa acacgtacgc
     1801 gtacgatgct ctagaangc
//
