LOCUS       sequence-153408-         805 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Positive control plasmid for Nicking Mutagenesis. Constitutively 
            expresses a non-fluorescent variant of GFPmut3 (Y66H) that is 
            mutated to fluorescent (H66Y) in the protocol..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 805)
  AUTHORS   Wrenbeck EE, Klesmith JR, Stapleton JA, Adeniran A, Tyo KE, 
            Whitehead TA
  TITLE     Plasmid-based one-pot saturation mutagenesis.
  JOURNAL   Nat Methods. 2016 Nov;13(11):928-930. doi: 10.1038/nmeth.4029. Epub 
            2016 Oct 10.
  PUBMED    27723752
REFERENCE   2  (bases 1 to 805)
  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/nmeth.4029"; journalName: "Nat Methods"; date: "2016-11"; 
            volume: "13"; issue: "11"; pages: "928-930"
COMMENT     Sequence Label: pEDA5_GFPmut3_Y66H
FEATURES             Location/Qualifiers
     source          1..805
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(20..37)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(44..757)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(44..757)
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(84..104)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     699..727
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     RBS             765..787
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtttgtat agttcatcca
       61 tgccatgtgt aatcccagca gctgttacaa actcaagaag gaccatgtgg tctctctttt
      121 cgttgggatc tttcgaaagg gcagattgtg tggacaggta atggttgtct ggtaaaagga
      181 cagggccatc gccaattgga gtattttgtt gataatggtc tgctagttga acgcttccat
      241 cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc attcttttgt ttgtctgcca
      301 tgatgtatac attgtgtgag ttatagttgt attccaattt gtgtccaaga atgtttccat
      361 cttctttaaa atcaatacct tttaactcga ttctattaac aagggtatca ccttcaaact
      421 tgacttcagc acgtgtcttg tagttcccgt catctttgaa aaatatagtt ctttcctgta
      481 cataaccttc gggcatggca ctcttgaaaa agtcatgctg tttcatatga tctgggtatc
      541 tcgcaaagca ttgaacacca tgaccgaaag tagtgacaag tgttggccat ggaacaggta
      601 gttttccagt agtgcaaata aatttaaggg taagttttcc gtatgttgca tcaccttcac
      661 cctctccact gacagaaaat ttgtgcccat taacatcacc atctaattca acaagaattg
      721 ggacaactcc agtgaaaagt tcttctcctt tacgcatatg tatatctcct tcttaaagtt
      781 aaacaaaatt atttctagaa agtta
//
