LOCUS       Exported                8505 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  RNA interference vector pIBB103, complete sequence.
ACCESSION   HQ455038
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8505)
  AUTHORS   Bose I, Doering TL.
  TITLE     Efficient implementation of RNA interference in the pathogenic yeast
            Cryptococcus neoformans
  JOURNAL   J. Microbiol. Methods 86 (2), 156-159 (2011)
  PUBMED    21554906
REFERENCE   2  (bases 1 to 8505)
  AUTHORS   Bose I, Reilly MC, Skowyra ML, Doering TL.
  TITLE     A convergent promoter vector for RNAi in Cryptococcus neoformans 
            that allows 5-FOA selection for colonies with efficient RNAi
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 8505)
  AUTHORS   Skowyra ML, Doering TL.
  TITLE     RNA interference in Cryptococcus neoformans
  JOURNAL   Unpublished
REFERENCE   4  (bases 1 to 8505)
  AUTHORS   Bose I, Reilly MC, Skowyra ML, Doering TL.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-NOV-2010) Molecular Microbiology, Washington 
            University School of Medicine, 660 S. Euclid Ave. Campus Box 8230, 
            St. Louis, MO 63110, USA
REFERENCE   5  (bases 1 to 8505)
  AUTHORS   Bose I, Reilly MC, Skowyra ML, Doering TL.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-MAY-2011) Molecular Microbiology, Washington 
            University School of Medicine, 660 S. Euclid Ave. Campus Box 8230, 
            St. Louis, MO 63110, USA
REFERENCE   6  (bases 1 to 8505)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     On May 24, 2011 this sequence version replaced HQ455038.1.
FEATURES             Location/Qualifiers
     source          1..8505
                     /organism="RNA interference vector pIBB103"
                     /mol_type="other DNA"
                     /note="designed to mediate RNA interference in Cryptococcus
                     neoformans; includes a cloning site for a target sequence 
                     adjacent to a fragment from the cryptococcal URA5 gene, 
                     both flanked by convergent GAL7 promoters; in appropriate 
                     media, the vector generates dsRNA that can mediate 
                     silencing of both the target gene of interest and 
                     endogenous URA5; the latter allows 5-FOA selection for 
                     colonies in which RNAi is most effective"
                     /db_xref="taxon:944336"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             126..986
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /EC_number="3.5.2.6"
                     /label=bla
                     /note="ampicillin-resistance determinant derived from 
                     bacterial transposon Tn3; used as a selective marker in 
                     Escherichia coli"
                     /protein_id="ADV36643.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            126..986
                     /gene="bla"
                     /label=bla
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1169..1768
                     /note="Escherichia coli origin of replication; derived from
                     pMB1"
     regulatory      2021..2142
                     /regulatory_class="promoter"
                     /note="Lac promoter"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2069..2099
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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)."
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            2143..2199
                     /gene="lacZ alpha"
                     /label=lacZ alpha
                     /note="disrupted by insertion of cryptococcal telomeric 
                     repeats, geneticin resistance determinant, and the RNA 
                     interference cassette"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2200..7852
                     /note="cryptococcal-specific insert region containing 
                     cryptococcal telomeric repeats, cryptococcal 
                     geneticin-resistance marker, and RNA interference cassette;
                     artificially assembled"
     repeat_region   2223..2259
                     /note="telomeric repeat; derived from Cryptococcus 
                     neoformans var. neoformans JEC21"
     gene            complement(2294..3047)
                     /gene="npt1"
                     /label=npt1
     CDS             complement(2580..2936)
                     /codon_start=1
                     /gene="npt1"
                     /product="nonfunctional neomycin phosphotransferase I"
                     /label=npt1
                     /note="truncated kanamycin-resistance determinant; derived 
                     from bacterial transposon Tn903; expressed in Escherichia 
                     coli does not confer resistance to the aminoglycoside 
                     antibiotics neomycin and kanamycin"
                     /protein_id="ADV36645.2"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARKLLPFSPDSVVTHGD
                     FSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPD
                     MNKLQFHLMLDEFF"
     repeat_region   3080..3170
                     /note="telomeric repeat; derived from Cryptococcus 
                     neoformans var. neoformans JEC21"
     misc_feature    complement(3190..5774)
                     /note="synthetic geneticin-resistance determinant for use 
                     as a selectable marker in Cryptococcus neoformans; confers 
                     resistance to geneticin (G418)"
     regulatory      complement(3194..4129)
                     /regulatory_class="terminator"
                     /note="Cryptococcal terminator; derived from region 
                     downstream from the NMT1 (N-myristoyltransferase I) coding 
                     region in Cryptococcus neoformans var. grubii H99"
     CDS             complement(4136..4930)
                     /codon_start=1
                     /gene="nptII"
                     /product="aminoglycoside-3-O-phosphotransferase II"
                     /EC_number="2.7.1.95"
                     /label=nptII
                     /note="confers resistance to the aminoglycoside antifungal 
                     geneticin (G418) in Cryptococcus neoformans; 
                     aminoglycoside-resistance determinant derived from the 
                     bacterial transposon Tn5; confers resistance to kanamycin 
                     when placed under control of a bacterial promoter; here 
                     driven by the ACT1 promoter specific to Cryptococcus 
                     neoformans"
                     /protein_id="ADV36644.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            complement(4136..4930)
                     /gene="nptII"
                     /label=nptII
     regulatory      complement(4937..5769)
                     /regulatory_class="promoter"
                     /note="ACT1 promoter; derived from a region upstream of the
                     ACT1 (actin) coding sequence in Cryptococcus neoformans 
                     var. grubii H99"
     misc_feature    5810..7852
                     /note="RNA interference cassette; uses convergent promoters
                     to drive the transcription of an interfering construct in 
                     Cryptococcus neoformans"
     regulatory      complement(5810..6116)
                     /regulatory_class="terminator"
                     /note="Gal7 (FNU16994) terminator; derived from a region 
                     downstream of the GAL7 (UDP glucose-D-galactose-1-phosphate
                     uridylyltransferase) coding sequence in C. neoformans var. 
                     neoformans JEC21"
     regulatory      6140..6693
                     /regulatory_class="promoter"
                     /note="Gal7 (FNU16994) promoter; derived from a region 
                     upstream of the GAL7 (UDP glucose-D-galactose-1-phosphate 
                     uridylyltransferase) coding sequence in Cryptococcus 
                     neoformans var. neoformans JEC21."
     misc_feature    6706..6957
                     /note="ura5 gene fragment; derived from the unspliced URA5 
                     (orotidine monophosphate pyrophosphorylase) coding sequence
                     in Cryptococcus neoformans var. neoformans JEC21; this 
                     fragment is not translated and functions only as a sentinel
                     for RNA interference; CPCURA5A"
     regulatory      complement(6964..7518)
                     /regulatory_class="promoter"
                     /note="Gal7 (FNU16994) promoter; derived from a region 
                     upstream of the GAL7 (UDP glucose-D-galactose-1-phosphate 
                     uridylyltransferase) coding sequence in Cryptococcus 
                     neoformans var. neoformans JEC21"
     regulatory      7541..7847
                     /regulatory_class="terminator"
                     /note="Gal7 (FNU16994) terminator; derived from a region 
                     downstream of the GAL7 (UDP glucose-D-galactose-1-phosphate
                     uridylyltransferase) coding sequence in Cryptococcus 
                     neoformans var. neoformans JEC21"
     misc_feature    7853..2199
                     /note="vector backbone; derived from pBlueScript-SK(+)"
     gene            7853..8043
                     /gene="lacZ alpha"
                     /label=lacZ alpha
                     /note="disrupted by insertion of cryptococcal telomeric 
                     repeats, geneticin resistance determinant, and the RNA 
                     interference cassette"
     promoter        complement(7862..7880)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7887..7903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      8045..8500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      8062..8368
                     /note="F1+; origin of replication for Bacteriophage F1"