LOCUS       sequence-200417-        4391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4391)
  AUTHORS   Balleza E, Kim JM, Cluzel P
  TITLE     Systematic characterization of maturation time of fluorescent 
            proteins in living cells.
  JOURNAL   Nat Methods. 2018 Jan;15(1):47-51. doi: 10.1038/nmeth.4509. Epub 
            2017 Nov 20.
  PUBMED    29320486
REFERENCE   2  (bases 1 to 4391)
  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.4509"; journalName: "Nat Methods"; date: "2018-01"; 
            volume: "15"; issue: "1"; pages: "47-51"
COMMENT     Sequence Label: pEB1-Clover
FEATURES             Location/Qualifiers
     source          1..4391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             185..898
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(215..243)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     838..858
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      939..1025
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1563..2513)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2561..2783
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3275..3369)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3400..4194)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3511..3530)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4121..4140
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4373..4391
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctttcgtct tcaccacagc taacaccacg tcgtccctat ctgctgccct aggtctatga
       61 gtggttgctg gataacttta cgggcatgca taaggctcgt atgatatatt cagggagacc
      121 acaacggttt ccctctacaa ataattttgt ttaacttttc tagatttaag aaggagatat
      181 acatatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
      241 tggtgatgtt aatgggcaca aattttctgt cagaggagag ggtgaaggtg atgcaacaaa
      301 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac
      361 acttgtcact actttcggtt atggtgttgc atgcttttca agatacccag atcatatgaa
      421 acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatc
      481 tttcaaagat gacgggacct acaagacacg tgctgaagtc aagtttgaag gtgataccct
      541 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
      601 caaattggaa tacaacttta actcacacaa tgtatacatc acggcagaca aacaaaagaa
      661 tggaatcaaa gctaacttca aaattagaca caacgttgaa gatggaagcg ttcaactagc
      721 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
      781 ttacctgtcc caccaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt
      841 ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata
      901 aatgtccaga cctgcaggca tgcaagctct agaggcatca aataaaacga aaggctcagt
      961 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     1021 caaatccgcc gccctagacc tagggtacgg gttttgctgc ccgcaaacgg gctgttctgg
     1081 tgttgctagt ttgttatcag aatcgcagat ccggcttcag ccggtttgcc ggctgaaagc
     1141 gctatttctt ccagaattgc catgattttt tccccacggg aggcgtcact ggctcccgtg
     1201 ttgtcggcag ctttgattcg ataagcagca tcgcctgttt caggctgtct atgtgtgact
     1261 gttgagctgt aacaagttgt ctcaggtgtt caatttcatg ttctagttgc tttgttttac
     1321 tggtttcacc tgttctatta ggtgttacat gctgttcatc tgttacattg tcgatctgtt
     1381 catggtgaac agctttgaat gcaccaaaaa ctcgtaaaag ctctgatgta tctatctttt
     1441 ttacaccgtt ttcatctgtg catatggaca gttttccctt tgatatgtaa cggtgaacag
     1501 ttgttctact tttgtttgtt agtcttgatg cttcactgat agatacaaga gccataagaa
     1561 cctcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg tttttgcgtg
     1621 agccatgaga acgaaccatt gagatcatac ttactttgca tgtcactcaa aaattttgcc
     1681 tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt tcttagtccg
     1741 ttatgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt catttttatc
     1801 tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac ttggaaaatc
     1861 aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct gtaagtgttt
     1921 aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc atggtagtta
     1981 ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt tgccttgtga
     2041 gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga gtatttgttt
     2101 tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg gaaaagataa
     2161 ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt ggcatagttt
     2221 gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac agttctcgtc
     2281 atcagctctc tggttgcttt agctaataca ccataagcat tttccctact gatgttcatc
     2341 atctgagcgt attggttata agtgaacgat accgtccgtt ctttccttgt agggttttca
     2401 atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg ctccgttaag
     2461 tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga catacatctc
     2521 aattggtcta ggtgatttta atcactatac caattgagat gggctagtca atgataatta
     2581 ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt tgctggaaaa
     2641 cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt tttttttgtt
     2701 tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat aaaaagaata
     2761 gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta caaaaggatg
     2821 tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg cttaagtagc
     2881 accctcgcaa gctcgggcaa atcgctgaat attccttttg tctccgacca tcaggcacct
     2941 gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg cagtgaatgg
     3001 gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac ccataataca
     3061 agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat gtggtgctat
     3121 ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat
     3181 tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa
     3241 caggcttacc cgtcttactg tccctagtgc ttggattctc accaataaaa aacgcccggc
     3301 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca
     3361 acaggagtcc aagcgagctc tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     3421 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     3481 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     3541 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     3601 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     3661 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     3721 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     3781 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     3841 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     3901 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     3961 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     4021 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     4081 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     4141 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     4201 cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga tccttggcgg
     4261 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     4321 tggcaattcc gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     4381 tatcacgagg c
//
