LOCUS       Exported                7068 bp DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-luciferase reporter for microRNA targeting in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7068)
  AUTHORS   Liu Q, Wang F, Axtell MJ
  TITLE     Analysis of complementarity requirements for plant MicroRNA 
            targeting using a Nicotiana benthamiana quantitative transient 
            assay.
  JOURNAL   Plant Cell. 2014 Feb;26(2):741-53. doi: 10.1105/tpc.113.120972. Epub
            2014 Feb 7.
  PUBMED    24510721
REFERENCE   2  (bases 1 to 7068)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7068)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7068)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1105/tpc.113.120972"; journalName: "Plant Cell"; date: 
            "2014-02"; volume: "26"; issue: "2"; pages: "741-53"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    542..564
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA
                     (truncated)"
     terminator      complement(587..839)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(857..1789)
                     /codon_start=1
                     /label=Rluc
                     /note="luciferase from the anthozoan coelenterate Renilla 
                     reniformis (sea pansy)"
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        complement(1823..2002)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     2249..2265
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2275..2293
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2368..2711
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             2718..4367
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     promoter        complement(4668..4686)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4707..4723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4731..4747)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4755..4785)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4800..4821)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(4938..4955)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5060..5084
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5175..5763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5937..6749)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7040..7068
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
ORIGIN
        1 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc
       61 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg
      121 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg
      181 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc
      241 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat
      301 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac
      361 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc
      421 cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa
      481 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagagatc
      541 ttggcaggat atattgtggt gtaacgtatc agcttgcatg ccggtcgatc tagtaacata
      601 gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt ttctatcgcg
      661 tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa taacgtcatg
      721 cattacatgt taattattac atgcttaacg taattcaaca gaaattatat gataatcatc
      781 gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt tgaacgatct
      841 gcttgactct agattattgt tcatttttga gaactcgctc aacgaacgat ttgatatatt
      901 ttcccatttc atcaggtgca tcttcttgcg aaaaatgaag accttttact ttgacaaatt
      961 cagtattagg aaacttcttg gcaccttcaa caatagcatt ggaaaagaat cctgggtccg
     1021 attcaataaa catttttggt aaatcatcac ttgcacgtag ataagcatta taattcctaa
     1081 caatttgtac aacgtcaggt ttaccacctt ttactaacgg gatttcacga ggccatgata
     1141 atgttggacg acgaacttca cctttctctt tgaatggttc aagatatgct gcaaattctt
     1201 ctggttctaa ctttctcatg atttttgatg gcaacatggt ttccacgaag aagttattct
     1261 ccaaaaccat tttttctcct tcttcagatt tgatcaacgc aatatcttct tcaatatcag
     1321 gccattcatc ccatgattca atcacatcta ctacactttc agcgtgaact attgctttga
     1381 tcttatcttg atgctcatag ctataatgaa atgccaaaca agcaccccaa tcatggccga
     1441 caaaaatgat cttctttggt aaattaagaa gttcaaacca tgcagtaaga tatttgtaat
     1501 gatcaagtaa cctataagaa ccattaccag atttgcctga tttgcccata ccaataaggt
     1561 ctggtataat acaccgcgct actggctcaa tatgtggcac aacatgtcgc cataaataag
     1621 aagaggccgc gttaccatgt aaaaaaataa cagcattttc tgcatgtttt tctgaatcat
     1681 aataattaat aaatgaatca agaacattca tttgtttaca tctggcccac cactgcggac
     1741 cagttatcat ccgtttcctt tgttctggat cataaacttt cgaagtcatg tcgacctcga
     1801 gttgagagtg aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg
     1861 agcatttttg acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg
     1921 caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctcagt
     1981 ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat
     2041 cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcgataa cattaacgct
     2101 tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2161 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2221 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     2281 gactcactat agggcgaatt gggtaccggg cccacccccc tactccaaaa atgtcaaaga
     2341 tacagtctca gaagaccaaa gggctattga gacttttcaa caaagggtaa tttcgggaaa
     2401 cctcctcgga ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga
     2461 aggtggctcc tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatgcctc
     2521 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga
     2581 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgac atctccactg acgtaaggga
     2641 tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gtcatttcat
     2701 ttggagagga cctcgagatg gaagacgcca aaaacataaa gaaaggcccg gcgccattct
     2761 atccgctgga agatggaacc gctggagagc aactgcataa ggctatgaag agatacgccc
     2821 tggttcctgg aacaattgct tttacagatg cacatatcga ggtggacatc acttacgctg
     2881 agtacttcga aatgtccgtt cggttggcag aagctatgaa acgatatggg ctgaatacaa
     2941 atcacagaat cgtcgtatgc agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg
     3001 cgttatttat cggagttgca gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc
     3061 tcaacagtat gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa
     3121 aaattttgaa cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc atggattcta
     3181 aaacggatta ccagggattt cagtcgatgt acacgttcgt cacatctcat ctacctcccg
     3241 gttttaatga atacgatttt gtgccagagt ccttcgatag ggacaagaca attgcactga
     3301 tcatgaactc ctctggatct actggtctgc ctaaaggtgt cgctctgcct catagaactg
     3361 cctgcgtgag attctcgcat gccagagatc ctatttttgg caatcaaatc attccggata
     3421 ctgcgatttt aagtgttgtt ccattccatc acggttttgg aatgtttact acactcggat
     3481 atttgatatg tggatttcga gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga
     3541 ggagccttca ggattacaag attcaaagtg cgctgctggt gccaacccta ttctccttct
     3601 tcgccaaaag cactctgatt gacaaatacg atttatctaa tttacacgaa attgcttctg
     3661 gtggcgctcc cctctctaag gaagtcgggg aagcggttgc caagaggttc catctgccag
     3721 gtatcaggca aggatatggg ctcactgaga ctacatcagc tattctgatt acacccgagg
     3781 gggatgataa accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg
     3841 atctggatac cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc
     3901 ctatgattat gtccggttat gtaaacaatc cggaagcgac caacgccttg attgacaagg
     3961 atggatggct acattctgga gacatagctt actgggacga agacgaacac ttcttcatcg
     4021 ttgaccgcct gaagtctctg attaagtaca aaggctatca ggtggctccc gctgaattgg
     4081 aatccatctt gctccaacac cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg
     4141 atgacgccgg tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg
     4201 aaaaagagat cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag
     4261 gagttgtgtt tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa
     4321 tcagagagat cctcataaag gccaagaagg gcggaaagat cgccgtgtaa gaattcctgc
     4381 agtcgccatg cggtcattac actatcccta ggatcggtat ccagctagta caaaccggtt
     4441 ttactatctt ttactcttga actagttaat gtgtgagtag tttcccgata agggaaatta
     4501 gggttcttat agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg
     4561 tatttgtaaa atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta
     4621 aaatccagat cgcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     4681 ttaattccga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4741 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4801 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4861 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4921 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4981 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5041 ggaaagaaca tgaaggcctt gacaggatat attggcgggt aaactaagtc gctgtatgtg
     5101 tttgtttgag atctcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5161 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5221 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5281 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5341 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5401 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5461 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5521 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5581 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5641 ctgaagccag ttaccttcgg aagaagagtt ggtagctctt gatccggcaa acaaaccacc
     5701 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5761 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5821 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5881 aaatgaagtt ttaaatcaat ctaaagtata tatgtgtaac attggtctag tgattagaaa
     5941 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     6001 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     6061 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     6121 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     6181 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     6241 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     6301 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     6361 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     6421 aatacctgga atgctgtttt ccctgggatc gcagtggtga gtaaccatgc atcatcagga
     6481 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     6541 accatctcat ctgtaacaac attggcaacg ctacctttgc catgtttcag aaacaactct
     6601 ggcgcatcgg gcttcccata caatcggtag attgtcgcac ctgattgccc gacattatcg
     6661 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggccttgag
     6721 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     6781 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
     6841 tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagatc
     6901 acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa aatcaccaac
     6961 tggtccacct acaacaaagc tctcatcaac cgtggctccc tcactttctg gctggatgat
     7021 ggggcgattc aggcgatccc catccaacag cccgccgtcg agcgggct
//
