LOCUS       sequence-197645-        4299 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4299)
  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 4299)
  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..4299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    851..990
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(916..935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1076..1098
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             1929..1951
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1959..2672
                     /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             1959..2672
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1989..2017)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2612..2632
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2679..2696
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2749..2767)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2763..2810
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2847..3302
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2934..2953)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3144..3165
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3394..4185,4186..4254))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4017..4036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1381 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1441 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1501 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1561 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1621 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1681 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1741 cgcgctgagg acccgtgggg ccgccatgcc ggccacagct aacaccacgt cgtccctatc
     1801 tgctgcccta ggtctatgag tggttgctgg ataactttac gggcatgcat aaggctcgta
     1861 atatatattc agggagacca caacggtttc cctctacaaa taattttgtt taactttcta
     1921 gaaataattt tgtttaactt taagaaggag atatacatat gcgtaaagga gaagaacttt
     1981 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
     2041 ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt
     2101 gcactactgg aaaactacct gttccatggc caacacttgt cactactttc ggtcatggtg
     2161 ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc aagagtgcca
     2221 tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga
     2281 cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta
     2341 ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac tataactcac
     2401 acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
     2461 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa
     2521 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt
     2581 cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg
     2641 ggattacaca tggcatggat gaactataca aactcgagca ccaccaccac caccactgag
     2701 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2761 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2821 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2881 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2941 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3001 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3061 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3121 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3181 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3241 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3301 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3361 cattcaaata tgtatccgct catgaattaa ttcttaccaa tgcttaatca gtgaggcacc
     3421 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3481 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3541 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3601 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3661 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt
     3721 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3781 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3841 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3901 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3961 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4021 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4081 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4141 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4201 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcataacacc
     4261 ccttgtatta ctgtttatgt aagcagacag ttttattgt
//
