LOCUS       sequence-164852-        7774 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 7774)
  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 7774)
  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_SPb
FEATURES             Location/Qualifiers
     source          1..7774
                     /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"
     primer_bind     2925..2947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2939..2956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2940..2956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2966..2985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2966..2984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3059..3403
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3347..3367
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             3410..5062
                     /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(3476..3494)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(5373..5393)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5373..5391)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5412..5428)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5412..5428)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5425..5447)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5436..5452
                     /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(5460..5466,5467..5485,5486..5491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5506..5527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5644..5661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5766..5790
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5881..6469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5961..5980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6640..7455)
                     /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     7363..7382
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7447..7466
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7746..7774,1..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 tcgactttta atcaactgaa agtaaaagca agttaaatca gcacacttag tctaacctaa
     1861 acatatacat gaaggtatca acacataaca caacatcata agatcgattc agaactcaac
     1921 taaatgtctc tagtcatata aggttttact tgttagtaag taaactgata tatctaaatc
     1981 ccagacaatt tggagctcag tcaacaggcg gacacttgta ataataaaaa catgcttcgt
     2041 ttttccgtat agcagccaaa aaaaaaagat ccatcaaagt ttagctaaat ttgctggtaa
     2101 ccaaaatgag tatgcagaga tcaagtacta ataacacagt agatctagga aaattaagag
     2161 cagatcaagt tcatgacaga ttgaccataa aaacaacaga tctggaaaaa tcaaagagaa
     2221 tatcacacct aaatctaagt tttactaagt aattagagca gtagagtaat cagaagaaca
     2281 aaaagcagcg attcaacatg agtaagtaga gcagatctca aagcagaaat tgaaagaaaa
     2341 gaagcaagta aataaagcaa gatctgatga agaagatagg ttagattacc gaaaaggaaa
     2401 tgaaaatgga tctgaaaagc aaaggcaaac ggatgatgac ttgtggccga cttgctttct
     2461 ctctctacag tctggaagag gatgaatgag ttaagacaat acacaagtat atatatagtc
     2521 gtaaaaactg gctgagacgg cctttgaatt tataattttc ttttttattc tattcaatgt
     2581 atttttgaat ttgaatgata agaggtggtg agcacaaaga caaccaaatt gacagattcg
     2641 ccctccacta ttgaatggag taaacaagga aaatctttgt tcaaattatt ttcaattttt
     2701 ttaaaatcta attcctatca atttgagttg attttttcga tcttctttca caaaatttaa
     2761 tttttttttt caactatgtg tgcgctcgag caaaaattta cgaactatta tctgcgcaag
     2821 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     2881 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     2941 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
     3001 gcccaccccc ctactccaaa aatgtcaaag atacagtctc agaagaccaa agggctattg
     3061 agacttttca acaaagggta atttcgggaa acctcctcgg attccattgc ccagctatct
     3121 gtcacttcat cgaaaggaca gtagaaaagg aaggtggctc ctacaaatgc catcattgcg
     3181 ataaaggaaa ggctatcatt caagatgcct ctgccgacag tggtcccaaa gatggacccc
     3241 cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg
     3301 attgatgtga catctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag
     3361 acccttcctc tatataagga agttcatttc atttggagag gacctcgaga tggaagacgc
     3421 caaaaacata aagaaaggcc cggcgccatt ctatccgctg gaagatggaa ccgctggaga
     3481 gcaactgcat aaggctatga agagatacgc cctggttcct ggaacaattg cttttacaga
     3541 tgcacatatc gaggtggaca tcacttacgc tgagtacttc gaaatgtccg ttcggttggc
     3601 agaagctatg aaacgatatg ggctgaatac aaatcacaga atcgtcgtat gcagtgaaaa
     3661 ctctcttcaa ttctttatgc cggtgttggg cgcgttattt atcggagttg cagttgcgcc
     3721 cgcgaacgac atttataatg aacgtgaatt gctcaacagt atgggcattt cgcagcctac
     3781 cgtggtgttc gtttccaaaa aggggttgca aaaaattttg aacgtgcaaa aaaagctccc
     3841 aatcatccaa aaaattatta tcatggattc taaaacggat taccagggat ttcagtcgat
     3901 gtacacgttc gtcacatctc atctacctcc cggttttaat gaatacgatt ttgtgccaga
     3961 gtccttcgat agggacaaga caattgcact gatcatgaac tcctctggat ctactggtct
     4021 gcctaaaggt gtcgctctgc ctcatagaac tgcctgcgtg agattctcgc atgccagaga
     4081 tcctattttt ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca
     4141 tcacggtttt ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt
     4201 aatgtataga tttgaagaag agctgtttct gaggagcctt caggattaca agattcaaag
     4261 tgcgctgctg gtgccaaccc tattctcctt cttcgccaaa agcactctga ttgacaaata
     4321 cgatttatct aatttacacg aaattgcttc tggtggcgct cccctctcta aggaagtcgg
     4381 ggaagcggtt gccaagaggt tccatctgcc aggtatcagg caaggatatg ggctcactga
     4441 gactacatca gctattctga ttacacccga gggggatgat aaaccgggcg cggtcggtaa
     4501 agttgttcca ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt
     4561 taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt atgtccggtt atgtaaacaa
     4621 tccggaagcg accaacgcct tgattgacaa ggatggatgg ctacattctg gagacatagc
     4681 ttactgggac gaagacgaac acttcttcat cgttgaccgc ctgaagtctc tgattaagta
     4741 caaaggctat caggtggctc ccgctgaatt ggaatccatc ttgctccaac accccaacat
     4801 cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc ggtgaacttc ccgccgccgt
     4861 tgttgttttg gagcacggaa agacgatgac ggaaaaagag atcgtggatt acgtcgccag
     4921 tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg tttgtggacg aagtaccgaa
     4981 aggtcttacc ggaaaactcg acgcaagaaa aatcagagag atcctcataa aggccaagaa
     5041 gggcggaaag atcgccgtgt aagaattcct tgtcgacttc cctgcagtcg ccatgcggtc
     5101 attacactat ccctaggatc ggtatccagc tagtacaaac cggttttact atcttttact
     5161 cttgaactag ttaatgtgtg agtagtttcc cgataaggga aattagggtt cttatagggt
     5221 ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact
     5281 tctatcaata aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatcgcgg
     5341 ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     5401 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5461 aacatacgag ccggaaghca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5521 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5581 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5641 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5701 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgaa
     5761 ggccttgaca ggatatattg gcgggtaaac taagtcgctg tatgtgtttg tttgagatct
     5821 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5881 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5941 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6001 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6061 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6121 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6181 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6241 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6301 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6361 cttcggaaga agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6421 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6481 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6541 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6601 atcaatctaa agtatatatg tgtaacattg gtctagtgat tagaaaaact catcgagcat
     6661 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     6721 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     6781 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     6841 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     6901 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     6961 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     7021 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     7081 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     7141 tgttttccct gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     7201 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     7261 aacaacattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     7321 cccatacaat cggtagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     7381 cccatataaa tcagcatcca tgttggaatt taatcgcggc cttgagcaag acgtttcccg
     7441 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     7501 tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg
     7561 gctttgttga ataaatcgaa cttttgctga gttgaaggat cagatcacgc atcttcccga
     7621 caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc accaactggt ccacctacaa
     7681 caaagctctc atcaaccgtg gctccctcac tttctggctg gatgatgggg cgattcaggc
     7741 gatccccatc caacagcccg ccgtcgagcg ggct
//
