LOCUS       sequence-86316-e        7069 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 7069)
  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 7069)
  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: 
            "10.1105/tpc.113.120972"; journalName: "Plant Cell"; date: 
            "2014-02"; volume: "26"; issue: "2"; pages: "741-53"
COMMENT     Sequence Label: pGreen_dualluc_3'UTR_sensor
FEATURES             Location/Qualifiers
     source          1..7069
                     /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(854..1789)
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        complement(1823..2002)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     2234..2256
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2248..2265
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2249..2265
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2275..2294
                     /label=T7
                     /note="T7 promoter, forward primer"
     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"
     primer_bind     2656..2676
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             2718..4370
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(2784..2802)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     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"
     primer_bind     complement(4707..4723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4720..4742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4731..4747
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(join(4755..4761,4762..4780,4781..4786))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4801..4822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4939..4956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5061..5085
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5176..5764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5256..5275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5935..6750)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6658..6677
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6742..6761
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7041..7069,1..407)
                     /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 aghcataaag tgtaaagcct ggggtgccta
     4801 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4861 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4921 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4981 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5041 aggaaagaac atgaaggcct tgacaggata tattggcggg taaactaagt cgctgtatgt
     5101 gtttgtttga gatctcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5161 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5221 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5281 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5341 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5401 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5461 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5521 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5581 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5641 gctgaagcca gttaccttcg gaagaagagt tggtagctct tgatccggca aacaaaccac
     5701 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5761 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5821 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5881 aaaatgaagt tttaaatcaa tctaaagtat atatgtgtaa cattggtcta gtgattagaa
     5941 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     6001 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     6061 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     6121 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     6181 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     6241 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     6301 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     6361 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     6421 taatacctgg aatgctgttt tccctgggat cgcagtggtg agtaaccatg catcatcagg
     6481 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6541 gaccatctca tctgtaacaa cattggcaac gctacctttg ccatgtttca gaaacaactc
     6601 tggcgcatcg ggcttcccat acaatcggta gattgtcgca cctgattgcc cgacattatc
     6661 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggccttga
     6721 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     6781 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     6841 ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat
     6901 cacgcatctt cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa
     6961 ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga
     7021 tggggcgatt caggcgatcc ccatccaaca gcccgccgtc gagcgggct
//
