LOCUS       40924_22573        7769 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pGrDL_SPb, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7769)
  AUTHORS   Moyle RL, Carvalhais L, Nowak E, Pretorius L-S., Subramaniam G, 
            Dalton-Morgan J, Schenk PM.
  TITLE     Quantifying MicroRNA Directed Repression of Targeted Transcripts 
            using an Optimised Transient Dual Luciferase Assay
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7769)
  AUTHORS   Moyle RL, Carvalhais L, Nowak E, Pretorius L-S., Subramaniam G, 
            Dalton-Morgan J, Schenk PM.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-AUG-2016) School of Agriculture and Food Science, 
            University of Queensland, St Lucia, Brisbane, QLD 4072, Australia
REFERENCE   3  (bases 1 to 7769)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7769)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (20-AUG-2016) School of Agriculture and Food Science, University of 
            Queensland, St Lucia, Brisbane, QLD 4072, Australia"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7769
                     /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)
                     /label=Rluc
                     /note="luciferase from the anthozoan coelenterate Renilla 
                     reniformis (sea pansy)"
     regulatory      complement(1805..2811)
                     /label=tomato Act7 promoter
                     /note="tomato Act7 promoter"
                     /regulatory_class="promoter"
     regulatory      2324..2715
                     /label=CaMV 35S promoter
                     /note="CaMV 35S promoter"
                     /regulatory_class="promoter"
     primer_bind     2936..2952
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2962..2980
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3055..3398
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3405..5054
                     /label=luciferase
                     /note="firefly luciferase"
     regulatory      5167..5332
                     /label=35S terminator
                     /note="35S terminator"
                     /regulatory_class="terminator"
     promoter        complement(5368..5386)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5407..5423)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5431..5447)
                     /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(5455..5486)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5501..5522)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    5761..5785
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5876..6464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6638..7450)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      7741..7769
                     /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 tctgcgcaac
     2821 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     2881 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     2941 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
     3001 acccccctac tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac
     3061 ttttcaacaa agggtaattt cgggaaacct cctcggattc cattgcccag ctatctgtca
     3121 cttcatcgaa aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa
     3181 aggaaaggct atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc
     3241 cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg
     3301 atgtgacatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc
     3361 ttcctctata taaggaagtc atttcatttg gagaggacct cgagatggaa gacgccaaaa
     3421 acataaagaa aggcccggcg ccattctatc cgctggaaga tggaaccgct ggagagcaac
     3481 tgcataaggc tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac
     3541 atatcgaggt ggacatcact tacgctgagt acttcgaaat gtccgttcgg ttggcagaag
     3601 ctatgaaacg atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc
     3661 ttcaattctt tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga
     3721 acgacattta taatgaacgt gaattgctca acagtatggg catttcgcag cctaccgtgg
     3781 tgttcgtttc caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaag ctcccaatca
     3841 tccaaaaaat tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca
     3901 cgttcgtcac atctcatcta cctcccggtt ttaatgaata cgattttgtg ccagagtcct
     3961 tcgataggga caagacaatt gcactgatca tgaactcctc tggatctact ggtctgccta
     4021 aaggtgtcgc tctgcctcat agaactgcct gcgtgagatt ctcgcatgcc agagatccta
     4081 tttttggcaa tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg
     4141 gttttggaat gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt
     4201 atagatttga agaagagctg tttctgagga gccttcagga ttacaagatt caaagtgcgc
     4261 tgctggtgcc aaccctattc tccttcttcg ccaaaagcac tctgattgac aaatacgatt
     4321 tatctaattt acacgaaatt gcttctggtg gcgctcccct ctctaaggaa gtcggggaag
     4381 cggttgccaa gaggttccat ctgccaggta tcaggcaagg atatgggctc actgagacta
     4441 catcagctat tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg
     4501 ttccattttt tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc
     4561 aaagaggcga actgtgtgtg agaggtccta tgattatgtc cggttatgta aacaatccgg
     4621 aagcgaccaa cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact
     4681 gggacgaaga cgaacacttc ttcatcgttg accgcctgaa gtctctgatt aagtacaaag
     4741 gctatcaggt ggctcccgct gaattggaat ccatcttgct ccaacacccc aacatcttcg
     4801 acgcaggtgt cgcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg
     4861 ttttggagca cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag
     4921 taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc
     4981 ttaccggaaa actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg
     5041 gaaagatcgc cgtgtaagaa ttccttgtcg acttccctgc agtcgccatg cggtcattac
     5101 actatcccta ggatcggtat ccagctagta caaaccggtt ttactatctt ttactcttga
     5161 actagttaat gtgtgagtag tttcccgata agggaaatta gggttcttat agggtttcgc
     5221 tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa atacttctat
     5281 caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat cgcggccgcc
     5341 accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattccga gcttggcgta
     5401 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5461 acgagccgga aghcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5521 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5581 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5641 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5701 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgaaggcct
     5761 tgacaggata tattggcggg taaactaagt cgctgtatgt gtttgtttga gatctcatgt
     5821 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5881 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5941 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6001 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6061 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6121 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6181 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6241 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6301 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6361 gaagaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6421 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6481 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6541 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6601 tctaaagtat atatgtgtaa cattggtcta gtgattagaa aaactcatcg agcatcaaat
     6661 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     6721 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     6781 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     6841 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     6901 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     6961 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     7021 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     7081 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     7141 tccctgggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     7201 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacaa
     7261 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     7321 acaatcggta gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     7381 ataaatcagc atccatgttg gaatttaatc gcggccttga gcaagacgtt tcccgttgaa
     7441 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     7501 atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt
     7561 gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt cccgacaacg
     7621 cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc tacaacaaag
     7681 ctctcatcaa ccgtggctcc ctcactttct ggctggatga tggggcgatt caggcgatcc
     7741 ccatccaaca gcccgccgtc gagcgggct
//
