LOCUS       sequence-86322-e        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GUS-based microRNA target competitor used for testing function as 
            target mimicry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  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 6849)
  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_GUS_competitor
FEATURES             Location/Qualifiers
     source          1..6849
                     /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(588..840)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(860..1411)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(1453..1632)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     1864..1886
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1878..1895
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1879..1895
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1905..1924
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1905..1923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1998..2342
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     2286..2306
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             2349..4160
                     /codon_start=1
                     /gene="uidA (or gusA) from E. coli"
                     /product="beta-glucuronidase"
                     /label=GUS
                     /translation="MVRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAI
                     AVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEV
                     MEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHD
                     FFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQ
                     VVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQF
                     LINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDW
                     ADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARD
                     KNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISD
                     LFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMY
                     TDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRK
                     PKSAAFLLQKRWTGMNFGEKPQQGGKQ"
     primer_bind     complement(4449..4469)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4449..4467)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4488..4504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4488..4504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4501..4523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4512..4528
                     /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(4536..4542,4543..4560,4561..4566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4581..4602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4719..4736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4841..4865
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(4956..5544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5036..5055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5715..6530)
                     /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     6438..6457
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6522..6541
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6821..6849,1..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 gtaacgttat cagcttgcat gccggtcgat ctagtaacat
      601 agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc
      661 gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat
      721 gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat
      781 cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc
      841 tgcttgactc taggggtcat cagatttcgg tgacgggcag gaccggacgg ggcggcaccg
      901 gcaggctgaa gtccagctgc cagaaaccca cgtcatgcca gttcccgtgc ttgaagccgg
      961 ccgcccgcag catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc acgctcgggt
     1021 cgttgggcag cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc agggacttca
     1081 gcaggtgggt gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg gagacgtaca
     1141 cggtcgactc ggccgtccag tcgtaggcgt tgcgtgcctt ccagggaccc gcgtaggcga
     1201 tgccggcgac ctcgccgtcc acctcggcga cgagccaggg atagcgctcc cgcagacgga
     1261 cgaggtcgtc cgtccactcc tgcggttcct gcggctcggt acggaagttg accgtgcttg
     1321 tctcgatgta gtggttgacg atggtgcaga ccgccggcat gtccgcctcg gtggcacggc
     1381 ggatgtcggc cgggcgtcgt tctgggctca tggtagatcc cctcgatcga gttgagagtg
     1441 aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg agcatttttg
     1501 acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg caataatggt
     1561 ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctcagt ggctccttca
     1621 acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat cggcgggggt
     1681 cataacgtga ctcccttaat tctccgctca tgatcgataa cattaacgct tacaatttcc
     1741 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1801 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1861 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     1921 agggcgaatt gggtaccggg cccacccccc tactccaaaa atgtcaaaga tacagtctca
     1981 gaagaccaaa gggctattga gacttttcaa caaagggtaa tttcgggaaa cctcctcgga
     2041 ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga aggtggctcc
     2101 tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatgcctc tgccgacagt
     2161 ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc
     2221 acgtcttcaa agcaagtgga ttgatgtgac atctccactg acgtaaggga tgacgcacaa
     2281 tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg
     2341 acctcgagat ggtacgtcct gtagaaaccc caacccgtga aatcaaaaaa ctcgacggcc
     2401 tgtgggcatt cagtctggat cgcgaaaact gtggaattga tcagcgttgg tgggaaagcg
     2461 cgttacaaga aagccgggca attgctgtgc caggcagttt taacgatcag ttcgccgatg
     2521 cagatattcg taattatgcg ggcaacgtct ggtatcagcg cgaagtcttt ataccgaaag
     2581 gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt cactcattac ggcaaagtgt
     2641 gggtcaataa tcaggaagtg atggagcatc agggcggcta tacgccattt gaagccgatg
     2701 tcacgccgta tgttattgcc gggaaaagtg tacgtatcac cgtttgtgtg aacaacgaac
     2761 tgaactggca gactatcccg ccgggaatgg tgattaccga cgaaaacggc aagaaaaagc
     2821 agtcttactt ccatgatttc tttaactatg ccggaatcca tcgcagcgta atgctctaca
     2881 ccacgccgaa cacctgggtg gacgacatca ccgtggtgac gcatgtcgcg caagactgta
     2941 accacgcgtc tgttgactgg caggtggtgg ccaatggtga tgtcagcgtt gaactgcgtg
     3001 atgcggatca acaggtggtt gcaactggac aaggcactag cgggactttg caagtggtga
     3061 atccgcacct ctggcaaccg ggtgaaggtt atctctatga actgtgcgtc acagccaaaa
     3121 gccagacaga gtgtgacatc tacccgcttc gcgtcggcat ccggtcagtg gcagtgaagg
     3181 gcgaacagtt cctgattaac cacaaaccgt tctactttac tggctttggt cgtcatgaag
     3241 atgcggactt gcgtggcaaa ggattcgata acgtgctgat ggtgcacgac cacgcattaa
     3301 tggactggat tggggccaac tcctaccgta cctcgcatta cccttacgct gaagagatgc
     3361 tcgactgggc agatgaacat ggcatcgtgg tgattgatga aactgctgct gtcggcttta
     3421 acctctcttt aggcattggt ttcgaagcgg gcaacaagcc gaaagaactg tacagcgaag
     3481 aggcagtcaa cggggaaact cagcaagcgc acttacaggc gattaaagag ctgatagcgc
     3541 gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc caacgaaccg gatacccgtc
     3601 cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc aacgcgtaaa ctcgacccga
     3661 cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc tcacaccgat accatcagcg
     3721 atctctttga tgtgctgtgc ctgaaccgtt attacggatg gtatgtccaa agcggcgatt
     3781 tggaaacggc agagaaggta ctggaaaaag aacttctggc ctggcaggag aaactgcatc
     3841 agccgattat catcaccgaa tacggcgtgg atacgttagc cgggctgcac tcaatgtaca
     3901 ccgacatgtg gagtgaagag tatcagtgtg catggctgga tatgtatcac cgcgtctttg
     3961 atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt cgccgatttt gcgacctcgc
     4021 aaggcatatt gcgcgttggc ggtaacaaga aagggatctt cactcgcgac cgcaaaccga
     4081 agtcggcggc ttttctgctg caaaaacgct ggactggcat gaacttcggt gaaaaaccgc
     4141 agcagggagg caaacaatga gaattcctgc agtcgccatg cggtcattac actatcccta
     4201 ggatcggtat gcagaccggt tttactatct tttactcttg aactagttaa tgtgtgagta
     4261 gtttcccgat aagggaaatt agggttctta tagggtttcg ctcatgtgtt gagcatataa
     4321 gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc
     4381 taaaaccaaa atccagtact aaaatccaga tcgcggccgc caccgcggtg gagctccagc
     4441 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     4501 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4561 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4621 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4681 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4801 acagaatcag gggataacgc aggaaagaac atgaaggcct tgacaggata tattggcggg
     4861 taaactaagt cgctgtatgt gtttgtttga gatctcatgt gagcaaaagg ccagcaaaag
     4921 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4981 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5041 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5101 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5161 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5221 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5281 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5341 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5401 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaagaagagt tggtagctct
     5461 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5521 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5581 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5641 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgtgtaa
     5701 cattggtcta gtgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     5761 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     5821 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     5881 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     5941 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     6001 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     6061 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     6121 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     6181 cctgaatcag gatattcttc taatacctgg aatgctgttt tccctgggat cgcagtggtg
     6241 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     6301 tccgtcagcc agtttagtct gaccatctca tctgtaacaa cattggcaac gctacctttg
     6361 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcggta gattgtcgca
     6421 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     6481 gaatttaatc gcggccttga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     6541 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     6601 gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa tcgaactttt
     6661 gctgagttga aggatcagat cacgcatctt cccgacaacg cagaccgttc cgtggcaaag
     6721 caaaagttca aaatcaccaa ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc
     6781 ctcactttct ggctggatga tggggcgatt caggcgatcc ccatccaaca gcccgccgtc
     6841 gagcgggct
//
