LOCUS       sequence-350720-        4103 bp    DNA     circular SYN 15-APR-2025
DEFINITION  It can be used to study protein turnover in living plant cells of 
            Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4103)
  AUTHORS   Zhang H, Linster E, Gannon L, Leemhuis W, Rundle CA, Theodoulou FL, 
            Wirtz M
  TITLE     Tandem Fluorescent Protein Timers for Noninvasive Relative Protein 
            Lifetime Measurement in Plants.
  JOURNAL   Plant Physiol. 2019 Jun;180(2):718-731. doi: 10.1104/pp.19.00051. 
            Epub 2019 Mar 14.
  PUBMED    30872425
REFERENCE   2  (bases 1 to 4103)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1104/pp.19.00051"; journalName: "Plant Physiol"; date: 
            "2019-06"; volume: "180"; issue: "2"; pages: "718-731"
COMMENT     Sequence Label: pEN-R2-mCsfGFP-L3
FEATURES             Location/Qualifiers
     source          1..4103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..26)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8..26)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(31..47)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(31..47)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(149..168)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             160..969
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(227..246)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1116..1704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1605..1624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1858..1875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2034..2061)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2153..2239)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2288..2310
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2302..2319
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2303..2319
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2390..2513)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2516..3223
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2661..2679)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2929..2948
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(3978..4073)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
       61 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
      121 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
      181 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
      241 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
      301 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
      361 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
      421 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
      481 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
      541 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
      601 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
      661 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
      721 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
      781 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
      841 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
      901 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
      961 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     1021 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     1081 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1141 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1201 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1261 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1321 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1381 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1441 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1501 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1561 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1621 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1681 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1741 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1801 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1861 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     1921 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     1981 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     2041 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
     2101 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc
     2161 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact
     2221 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg
     2281 atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg gccctgcagc
     2341 tctagagctc gaattctaca ggtcactaat accatctaag tagttggttc atagtgactg
     2401 catatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     2461 attgatattt atatcatttt acgtttctcg ttcaactttc ttgtacaaag tggcaatggt
     2521 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     2581 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2641 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     2701 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     2761 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     2821 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     2881 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     2941 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3001 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3061 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3121 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3181 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagctcgacg gagcaggtgc
     3241 tggtgctggt gctggagcaa tgtccaaggg tgaagagcta tttactgggg ttgtacccat
     3301 tttggtagaa ctggacggag atgtaaacgg acataaattc tctgttagag gtgagggcga
     3361 aggcgatgcc accaatggta aattgactct gaagtttata tgcactacgg gtaaattacc
     3421 tgttccttgg ccaaccctag taacaacttt gacatatggt gttcaatgtt tctcaagata
     3481 cccagaccat atgaaaaggc atgatttctt taaaagtgct atgccagaag gctacgtgca
     3541 agagagaact atctccttta aggatgacgg tacgtataaa acacgagcag aagtgaaatt
     3601 cgaaggggat acactagtta atcgcatcga attaaagggt atagacttta aggaagatgg
     3661 taatattctc ggccataaac ttgagtataa tttcaactcg cataatgtgt acattacagc
     3721 tgacaaacaa aagaacggaa ttaaagcgaa ttttaaaatc aggcacaacg tcgaagatgg
     3781 gtctgttcaa cttgccgatc attatcagca aaacacccct attggtgatg gtccagtctt
     3841 gttacccgat aatcactact taagcacaca gtctagattg tcaaaagatc cgaatgaaaa
     3901 gcgtgatcac atggttttat tggaatttgt caccgctgca ggaataactc acggaatgga
     3961 cgagctttat aagtaagcaa ctttattata caaagttggc attataaaaa agcattgctt
     4021 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttggagctc
     4081 catggtagcg ttaacgcggc cgc
//
