LOCUS       sequence-200474-        4394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding 2ndVal mGFPmut2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4394)
  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 4394)
  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: pEB1-2ndVal mGFPmut2
FEATURES             Location/Qualifiers
     source          1..4394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             171..887
                     /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="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(204..232)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     827..847
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      928..1014
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1552..2502)
                     /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      2550..2772
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3264..3358)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3389..4183)
                     /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(3500..3519)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4110..4129
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4362..4380
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cacagctaac accacgtcgt ccctatctgc tgccctaggt ctatgagtgg ttgctggata
       61 actttacggg catgcataag gctcgtatga tatattcagg gagaccacaa cggtttccct
      121 ctacaaataa ttttgtttaa cttttctaga tttaagaagg agatatacat atggtgagta
      181 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
      241 atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta
      301 cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta
      361 ctttcgcgta tggtcttcaa tgctttgcga gatacccaga tcatatgaaa cagcatgact
      421 ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg
      481 acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa
      541 tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat
      601 acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag
      661 ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc
      721 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca
      781 cacaatctaa gctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt
      841 ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaataa atgtccagac
      901 ctgcaggcat gcaagctcta gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg
      961 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     1021 ccctagacct agggtacggg ttttgctgcc cgcaaacggg ctgttctggt gttgctagtt
     1081 tgttatcaga atcgcagatc cggcttcagc cggtttgccg gctgaaagcg ctatttcttc
     1141 cagaattgcc atgatttttt ccccacggga ggcgtcactg gctcccgtgt tgtcggcagc
     1201 tttgattcga taagcagcat cgcctgtttc aggctgtcta tgtgtgactg ttgagctgta
     1261 acaagttgtc tcaggtgttc aatttcatgt tctagttgct ttgttttact ggtttcacct
     1321 gttctattag gtgttacatg ctgttcatct gttacattgt cgatctgttc atggtgaaca
     1381 gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt tacaccgttt
     1441 tcatctgtgc atatggacag ttttcccttt gatatgtaac ggtgaacagt tgttctactt
     1501 ttgtttgtta gtcttgatgc ttcactgata gatacaagag ccataagaac ctcagatcct
     1561 tccgtattta gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa
     1621 cgaaccattg agatcatact tactttgcat gtcactcaaa aattttgcct caaaactggt
     1681 gagctgaatt tttgcagtta aagcatcgtg tagtgttttt cttagtccgt tatgtaggta
     1741 ggaatctgat gtaatggttg ttggtatttt gtcaccattc atttttatct ggttgttctc
     1801 aagttcggtt acgagatcca tttgtctatc tagttcaact tggaaaatca acgtatcagt
     1861 cgggcggcct cgcttatcaa ccaccaattt catattgctg taagtgttta aatctttact
     1921 tattggtttc aaaacccatt ggttaagcct tttaaactca tggtagttat tttcaagcat
     1981 taacatgaac ttaaattcat caaggctaat ctctatattt gccttgtgag ttttcttttg
     2041 tgttagttct tttaataacc actcataaat cctcatagag tatttgtttt caaaagactt
     2101 aacatgttcc agattatatt ttatgaattt ttttaactgg aaaagataag gcaatatctc
     2161 ttcactaaaa actaattcta atttttcgct tgagaacttg gcatagtttg tccactggaa
     2221 aatctcaaag cctttaacca aaggattcct gatttccaca gttctcgtca tcagctctct
     2281 ggttgcttta gctaatacac cataagcatt ttccctactg atgttcatca tctgagcgta
     2341 ttggttataa gtgaacgata ccgtccgttc tttccttgta gggttttcaa tcgtggggtt
     2401 gagtagtgcc acacagcata aaattagctt ggtttcatgc tccgttaagt catagcgact
     2461 aatcgctagt tcatttgctt tgaaaacaac taattcagac atacatctca attggtctag
     2521 gtgattttaa tcactatacc aattgagatg ggctagtcaa tgataattac tagtcctttt
     2581 cctttgagtt gtgggtatct gtaaattctg ctagaccttt gctggaaaac ttgtaaattc
     2641 tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt atattcaagt
     2701 ggttataatt tatagaataa agaaagaata aaaaaagata aaaagaatag atcccagccc
     2761 tgtgtataac tcactacttt agtcagttcc gcagtattac aaaaggatgt cgcaaacgct
     2821 gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag
     2881 ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat caggcacctg agtcgctgtc
     2941 tttttcgtga cattcagttc gctgcgctca cggctctggc agtgaatggg ggtaaatggc
     3001 actacaggcg ccttttatgg attcatgcaa ggaaactacc cataatacaa gaaaagcccg
     3061 tcacgggctt ctcagggcgt tttatggcgg gtctgctatg tggtgctatc tgactttttg
     3121 ctgttcagca gttcctgccc tctgattttc cagtctgacc acttcggatt atcccgtgac
     3181 aggtcattca gactggctaa tgcacccagt aaggcagcgg tatcatcaac aggcttaccc
     3241 gtcttactgt ccctagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc
     3301 gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca
     3361 agcgagctct cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
     3421 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     3481 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     3541 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     3601 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct
     3661 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     3721 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     3781 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     3841 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     3901 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     3961 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
     4021 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     4081 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     4141 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     4201 ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
     4261 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
     4321 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4381 cctttcgtct tcac
//
