LOCUS       sequence-163429-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  receives miRNA target sequences for testing using the dual 
            luciferase assay system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  AUTHORS   Moyle RL, Carvalhais LC, Pretorius LS, Nowak E, Subramaniam G, 
            Dalton-Morgan J, Schenk PM
  TITLE     An Optimized Transient Dual Luciferase Assay for Quantifying 
            MicroRNA Directed Repression of Targeted Sequences.
  JOURNAL   Front Plant Sci. 2017 Sep 20;8:1631. doi: 10.3389/fpls.2017.01631. 
            eCollection 2017.
  PUBMED    28979287
REFERENCE   2  (bases 1 to 7087)
  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"; journalName: "Front Plant
            Sci."; date: "2017-09-20"; volume: "8:1631. doi"; pages: " 
            10.3389/fpls.2017.01631. eCollection 2017"
COMMENT     Sequence Label: pGrDL_SP
FEATURES             Location/Qualifiers
     source          1..7087
                     /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(1827..2006)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     2238..2260
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2252..2269
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2253..2269
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2279..2298
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2279..2297
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2372..2716
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     2660..2680
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             2723..4375
                     /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(2789..2807)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(4686..4706)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4686..4704)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4725..4741)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4725..4741)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4738..4760)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4749..4765
                     /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(4773..4779,4780..4798,4799..4804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4819..4840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4957..4974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5079..5103
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5953..6768)
                     /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     6676..6695
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6760..6779
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7059..7087,1..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 tcgatcgacc
     1801 tcgagttgag agtgaatatg agactctaat tggataccga ggggaattta tggaacgtca
     1861 gtggagcatt tttgacaaga aatatttgct agctgatagt gaccttaggc gacttttgaa
     1921 cgcgcaataa tggtttctga cgtatgtgct tagctcatta aactccagaa acccgcggct
     1981 cagtggctcc ttcaacgttg cggttctgtc agttccaaac gtaaaacggc ttgtcccgcg
     2041 tcatcggcgg gggtcataac gtgactccct taattctccg ctcatgatcg ataacattaa
     2101 cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2161 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2221 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     2281 atacgactca ctatagggcg aattgggtac cgggcccacc cccctactcc aaaaatgtca
     2341 aagatacagt ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatttcgg
     2401 gaaacctcct cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa
     2461 aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg
     2521 cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag
     2581 aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa
     2641 gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat
     2701 ttcatttgga gaggacctcg agatggaaga cgccaaaaac ataaagaaag gcccggcgcc
     2761 attctatccg ctggaagatg gaaccgctgg agagcaactg cataaggcta tgaagagata
     2821 cgccctggtt cctggaacaa ttgcttttac agatgcacat atcgaggtgg acatcactta
     2881 cgctgagtac ttcgaaatgt ccgttcggtt ggcagaagct atgaaacgat atgggctgaa
     2941 tacaaatcac agaatcgtcg tatgcagtga aaactctctt caattcttta tgccggtgtt
     3001 gggcgcgtta tttatcggag ttgcagttgc gcccgcgaac gacatttata atgaacgtga
     3061 attgctcaac agtatgggca tttcgcagcc taccgtggtg ttcgtttcca aaaaggggtt
     3121 gcaaaaaatt ttgaacgtgc aaaaaaagct cccaatcatc caaaaaatta ttatcatgga
     3181 ttctaaaacg gattaccagg gatttcagtc gatgtacacg ttcgtcacat ctcatctacc
     3241 tcccggtttt aatgaatacg attttgtgcc agagtccttc gatagggaca agacaattgc
     3301 actgatcatg aactcctctg gatctactgg tctgcctaaa ggtgtcgctc tgcctcatag
     3361 aactgcctgc gtgagattct cgcatgccag agatcctatt tttggcaatc aaatcattcc
     3421 ggatactgcg attttaagtg ttgttccatt ccatcacggt tttggaatgt ttactacact
     3481 cggatatttg atatgtggat ttcgagtcgt cttaatgtat agatttgaag aagagctgtt
     3541 tctgaggagc cttcaggatt acaagattca aagtgcgctg ctggtgccaa ccctattctc
     3601 cttcttcgcc aaaagcactc tgattgacaa atacgattta tctaatttac acgaaattgc
     3661 ttctggtggc gctcccctct ctaaggaagt cggggaagcg gttgccaaga ggttccatct
     3721 gccaggtatc aggcaaggat atgggctcac tgagactaca tcagctattc tgattacacc
     3781 cgagggggat gataaaccgg gcgcggtcgg taaagttgtt ccattttttg aagcgaaggt
     3841 tgtggatctg gataccggga aaacgctggg cgttaatcaa agaggcgaac tgtgtgtgag
     3901 aggtcctatg attatgtccg gttatgtaaa caatccggaa gcgaccaacg ccttgattga
     3961 caaggatgga tggctacatt ctggagacat agcttactgg gacgaagacg aacacttctt
     4021 catcgttgac cgcctgaagt ctctgattaa gtacaaaggc tatcaggtgg ctcccgctga
     4081 attggaatcc atcttgctcc aacaccccaa catcttcgac gcaggtgtcg caggtcttcc
     4141 cgacgatgac gccggtgaac ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat
     4201 gacggaaaaa gagatcgtgg attacgtcgc cagtcaagta acaaccgcga aaaagttgcg
     4261 cggaggagtt gtgtttgtgg acgaagtacc gaaaggtctt accggaaaac tcgacgcaag
     4321 aaaaatcaga gagatcctca taaaggccaa gaagggcgga aagatcgccg tgtaagaatt
     4381 ccttgtcgac ttccctgcag tcgccatgcg gtcattacac tatccctagg atcggtatcc
     4441 agctagtaca aaccggtttt actatctttt actcttgaac tagttaatgt gtgagtagtt
     4501 tcccgataag ggaaattagg gttcttatag ggtttcgctc atgtgttgag catataagaa
     4561 acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc taattcctaa
     4621 aaccaaaatc cagtactaaa atccagatcg cggccgccac cgcggtggag ctccagcttt
     4681 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     4741 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag hcataaagtg
     4801 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4861 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4921 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5041 agaatcaggg gataacgcag gaaagaacat gaaggccttg acaggatata ttggcgggta
     5101 aactaagtcg ctgtatgtgt ttgtttgaga tctcatgtga gcaaaaggcc agcaaaaggc
     5161 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga agaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5821 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5881 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgtgtaaca
     5941 ttggtctagt gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     6001 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     6061 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     6121 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     6181 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     6241 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     6301 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     6361 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     6421 tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag
     6481 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     6541 cgtcagccag tttagtctga ccatctcatc tgtaacaaca ttggcaacgc tacctttgcc
     6601 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc
     6661 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     6721 atttaatcgc ggccttgagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     6781 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     6841 aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc
     6901 tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca
     6961 aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct
     7021 cactttctgg ctggatgatg gggcgattca ggcgatcccc atccaacagc ccgccgtcga
     7081 gcgggct
//
