LOCUS       Exported               12044 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Binary vector pFMIRE6, complete sequence.
ACCESSION   KC631824
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12044)
  AUTHORS   Zhang C, Chen ZJ.
  TITLE     Differential cleavage rates of concatenated identical miRNA/siRNA 
            target sites indicate RISC approaching its target in 5 to 3 
            direction
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 12044)
  AUTHORS   Zhang C, Chen ZJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-FEB-2013) Section of Molecular Cell & Developmental 
            Biology, University of Texas at Austin, Austin, TX 78712, USA
REFERENCE   3  (bases 1 to 12044)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..12044
                     /organism="Binary vector pFMIRE6"
                     /mol_type="other DNA"
                     /note="T-DNA binary vector for expression of miRNA 
                     precursors and parallel expression of a nuclear localized 
                     RFP"
                     /db_xref="taxon:1343866"
     misc_feature    1..27
                     /note="left T-border; left border repeat from C58 T-DNA"
     misc_feature    1..25
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     terminator      111..363
                     /label=MAS terminator
                     /note="mannopine synthase terminator"
     3UTR           complement(111..362)
                     /label=contains mannopine synthase (MAS) polyA signal
                     /note="contains mannopine synthase (MAS) polyA signal"
     CDS             complement(373..924)
                     /codon_start=1
                     /product="phosphinothricin acetyltransferase"
                     /label=phosphinothricin acetyltransferase
                     /note="PAT; BAR; confers tolerance to phosphinothricin 
                     (PPT)"
                     /protein_id="AGN92880.1"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     regulatory      complement(930..1310)
                     /regulatory_class="promoter"
                     /note="mannopine synthase (MAS) promoter"
     promoter        complement(930..1310)
                     /label=MAS promoter
                     /note="mannopine synthase promoter (Velten et al., 1984)"
     misc_feature    1317..2226
                     /label=contains 35S promoter with double enhancers
                     /note="contains 35S promoter with double enhancers"
     promoter        1737..2081
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     misc_feature    2227..2256
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    2257..2468
                     /label=contains 35S polyA signal
                     /note="contains 35S polyA signal"
     misc_feature    2476..3377
                     /label=contains 35S promoter with double enhancers
                     /note="contains 35S promoter with double enhancers"
     promoter        2890..3234
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3378..4541
                     /codon_start=1
                     /product="HTB2-mCherry"
                     /label=HTB2-mCherry
                     /note="fusion protein; nuclear; marker of transformation 
                     with T-DNA"
                     /protein_id="AGN92882.1"
                     /translation="MAKADKKPAEKKPAEKTPAAEPAAAAEKKPKAGKKLPKEPAGAGD
                     KKKKRSKKNVETYKIYIFKVLKQVHPDIGISSKAMGIMNSFINDIFEKLAGESSKLARY
                     NKKPTITSREIQTAVRLVLPGELAKHAVSEGTKAVTKFTSSAAAARSMVSKGEEDNMAI
                     IKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFM
                     YGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLR
                     GTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK
                     KPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK"
     CDS             3831..4541
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     3UTR           4542..4750
                     /label=contains CaMV 35S polyA signal
                     /note="contains CaMV 35S polyA signal"
     polyA_signal    4566..4740
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    4811..1310
                     /label=sequence from pFGCGW (INSD accession DQ231581.1)
                     /note="sequence from pFGCGW (INSD accession DQ231581.1)"
     misc_feature    4811..5521
                     /label=contains octopine synthase polyA signal
                     /note="contains octopine synthase polyA signal"
     terminator      4812..5519
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(5548..5564)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    5767..5791
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     misc_feature    5767..5790
                     /note="right T-border; right border repeat from C58 T-DNA"
     rep_origin      complement(6852..7852)
                     /direction=LEFT
                     /label=pVS1
                     /note="pVS1"
     CDS             7091..7720
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             8154..9221
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      9287..9481
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     misc_feature    9825..9965
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(10151..10739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      10255..10536
                     /label=sequence from pBR325
                     /note="sequence from pBR325"
     CDS             complement(10826..11620)
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /note="AadA; sequence from pIG121Hm"
                     /protein_id="AGN92881.1"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"