LOCUS       Exported                7769 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pGrDL_SPb, complete sequence.
ACCESSION   KX758648
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7769
                     /organism="Cloning vector pGrDL_SPb"
                     /mol_type="other DNA"
                     /db_xref="taxon:1982462"
     misc_feature    533..564
                     /label=left border T-DNA
                     /note="left border T-DNA"
     misc_feature    542..564
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     regulatory      571..852
                     /regulatory_class="terminator"
                     /note="NOS terminator"
     terminator      587..839
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(854..1789)
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=Renilla luciferase
                     /protein_id="ARM20011.1"
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     regulatory      complement(1805..2811)
                     /regulatory_class="promoter"
                     /note="tomato Act7 promoter"
     regulatory      2324..2715
                     /regulatory_class="promoter"
                     /note="CaMV 35S 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"
     regulatory      3007..3398
                     /regulatory_class="promoter"
                     /note="CaMV 35S promoter"
     promoter        3055..3398
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3405..5057
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=firefly luciferase
                     /protein_id="ARM20012.1"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     regulatory      5167..5332
                     /regulatory_class="terminator"
                     /note="35S 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    5431..5447
                     /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(5455..5486)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5501..5522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    5761..5785
                     /label=right border T-DNA
                     /note="right border T-DNA"
     rep_origin      complement(5876..6464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6635..7450)
                     /codon_start=1
                     /gene="nptI"
                     /product="nptI"
                     /label=nptI
                     /note="selectable marker"
                     /protein_id="ARM20013.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(6635..7450)
                     /gene="nptI"
                     /label=nptI
     rep_origin      7741..407
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"