LOCUS       40924_25306        8505 bp DNA     circular SYN 18-DEC-2018
DEFINITION  RNA interference vector pIBB103, complete sequence.
ACCESSION   .
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  JOURNAL   Unpublished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  JOURNAL   Unpublished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)
  TITLE     Direct Submission
REFERENCE   7  (bases 1 to 8505)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. 
            Microbiol. Methods"; date: "2011"; volume: "86"; issue: "2"; pages: 
            "156-159"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 4; type: "Journal Article"; journalName: "Submitted 
            (01-NOV-2010) Molecular Microbiology, Washington University School 
            of Medicine, 660 S. Euclid Ave. Campus Box 8230, St. Louis, MO 
            63110, USA"
COMMENT     SGRef: number: 5; type: "Journal Article"; journalName: "Submitted 
            (24-MAY-2011) Molecular Microbiology, Washington University School 
            of Medicine, 660 S. Euclid Ave. Campus Box 8230, St. Louis, MO 
            63110, USA"
COMMENT     SGRef: number: 6; type: "Journal Article"
COMMENT     On May 24, 2011 this sequence version replaced HQ455038.1.
FEATURES             Location/Qualifiers
     source          1..8505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /label=AmpR promoter
     CDS             126..983
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1157..1745
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /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
                     /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"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     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"
     regulatory      complement(3194..4129)
                     /note="Cryptococcal terminator; derived from region
                     downstream from the NMT1 (N-myristoyltransferase I) coding 
                     region in Cryptococcus neoformans var. grubii H99"
                     /regulatory_class="terminator"
     CDS             complement(4139..4930)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     regulatory      complement(4937..5769)
                     /note="ACT1 promoter; derived from a region upstream of the
                     ACT1 (actin) coding sequence in Cryptococcus neoformans 
                     var. grubii H99"
                     /regulatory_class="promoter"
     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)
                     /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_class="terminator"
     regulatory      6140..6693
                     /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_class="promoter"
     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)
                     /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_class="promoter"
     regulatory      7541..7847
                     /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"
                     /regulatory_class="terminator"
     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"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc
     2221 gcggtgggtt agggggttag ggggttaggg ggggctgcaa ttaccctgtt atccctatgc
     2281 aggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc
     2341 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
     2401 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc
     2461 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg
     2521 ccgtcccgtc aagtcagcgt aatgctctgc cartgttaca accaattaac caattctgat
     2581 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2641 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2701 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2761 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2821 gaatccggtg agaatggcaa aagcttacga gcccatttat acccatataa atcagcatcc
     2881 atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg gctcataaca
     2941 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     3001 tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttcccc cccccccctg
     3061 cattaccctg ttatccctat gcagcccccc ctaaccccct aaccccctaa ccccctaacc
     3121 ccctaacccc ctaaccccct aaccccctaa ccctaacccc ctaacccacc gcggccgctc
     3181 tagggaatta gcttggggga cagatgatat ccgacaagag ggtgaagagg gaggaaagca
     3241 ggactctggt gattctgggg ttttgagagt cccgaaccgg ttgggaaaac cccaaaggga
     3301 tagcgatgga aaaagtttaa aaaaagtgga agaagaggta gatggaaatg aggatgaaaa
     3361 aggaggagaa gaagaagaag aagatggaaa agttgtggag aaggaagaga ctagaagcaa
     3421 ggggaaacgg aggcataaac aaactcacaa gcatggtaca gagagcgacc aaactcgtcc
     3481 cgcggacgaa aaatccaaaa gaaaagaaag cacgcacaca aggcccccaa ataagacacc
     3541 gccgctcctc tgcctcctcc tcgtccaaga agaagaaaaa gagacttcat aaagaagacg
     3601 agctagaatc gccgtccacg ccatggtttg aaattgcctt tgaagggctt ccttttggac
     3661 cagatgtacc cgcggtaagt ctgattccca tctgctgtca ttttccattc gaatgctttg
     3721 atcgtcgttt ccgggataaa gggcgctgac gcgatttgaa ttttgggcgt gatcaatccc
     3781 agttcaagat gctcggctgg atgaggaaac agtggagact cttgttggca gaggcggtgt
     3841 tcattcttgg gttagtgtac atcattcaag ctacaggact gtaaaatcag actccttcta
     3901 cctttttttc cttggaatgt tttacattgt gtatgtccat gtttgcaggt tcttgtctcc
     3961 cgttgtaaat gggaaattac aattagtgtc gagtgacact tttgtacttg ggcaaaatat
     4021 gcatgtcata gcacagcgtt agcaatgtat caagaatgtc aagaagactg tctatccgct
     4081 tgttgcggtc gagagaaatt gagagaatga agagacgggg tttgaggggg aattctcaga
     4141 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     4201 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     4261 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     4321 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     4381 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     4441 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     4501 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     4561 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     4621 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     4681 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     4741 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     4801 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     4861 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     4921 gttcaatcat ggatccgggc gagtttacta atggaaaaag acaattagca aagaggctgc
     4981 atattaggag acgcggtgaa gattgggagg acaatagggt ccgtgcacca aaaagtcctg
     5041 acggcagctc tgttcaacgc acatccatct cttccagcta tgcgtgtggc accagcacgg
     5101 atggattgtt ccaccaaaag gacaaggcag atggatgcag gtcgaggcga tgcggtgcta
     5161 tcggccctcg ttgtcgaacg ccacgtcgtc ctctcggcga ccgtcaatcc tggaaggaaa
     5221 tgaccaagcg tgcacccagc atccagcatc caacatccag cacccagcac ctagcaccca
     5281 tgatccaatg gccggcatgc agatcagcgt ggggagagca ccatctagca gcccctggca
     5341 ggcccagcat tgtgctcgga tgtgcgatag gcggtgggtg cgataaggtg gattagtgga
     5401 ggatggagga ctgagtggcg ggcaagggaa gggggcggga agatgactca ccttgggcag
     5461 tgggtggggg atgcggtata ggggatgtat gtggatgctg gcggaggata gaagctgtaa
     5521 gctgagctgg tgccaacggt gacgctgtga gagtggtctc ggcgaccctg acggcgactc
     5581 ggcgttaccc cgtgtggcac accggacgcg cgtcgagtga cgttgctgct attgttgtta
     5641 ccatcatcct ctcctccgac aaccatacac tcagcgcaca ccatgccact cgaatcctgc
     5701 atgcttatgt gagtcctccc cttcccgtgc cccgctctcc agctcacatc ctcgcagcct
     5761 ggacaataga agctaattcg attcgacctc gagggggggc ccggtacccg caaccatgtg
     5821 atgtgcaaac ctcgtactgc agacaaagat ggacctgttt cgtctttgca gatgcacagg
     5881 attaaaacgc tgtttctttt ttaaccaaga tgcaatctgt tctacgtagg agaggcaccg
     5941 ttagctatca acttgttatg aaactctgtc aaacaatgtc gttacagcgg ccagccgcca
     6001 ttacaaatat ttagaagaaa gccaatacca gcaggaacca gctgcacaga gtacggatct
     6061 ttgaacttga atgaagactg gaaactacag tacatgcttc gagacaacaa ctgagtggcg
     6121 cgccagatca accatcgata gaagcaggtc ttgtgaaccc cggggatgcg acagcagagg
     6181 ttcgaacacg caaaatcgta atttgatatt tatatgcgcg cccaccttcg ctgacaattc
     6241 ccgttcatga atcgtttgtc agtaattgac gcgccggggg tgccttgggg gagtgttgtc
     6301 aattaacata caaatattcc cgttcgtatc catttgcata tccatctcat cacatagtag
     6361 tattcccgtt cgtatccatt tgcatatctc gtcacatagt cttcccgttc gtatccattt
     6421 catatcgtaa catcatacag tcttcacaat cttcccgttc gtcctctcat cgtcaagcca
     6481 ttgattgtca atacacgcct acatattccc gttctcttgt ctgccacatc gctcgtgcgt
     6541 tcttcccgtt tttatgggaa gactggggtc atgtcgtcaa tacagcagca gacataatgg
     6601 gtgttatata tgtcgagttt cacatctggc tgtactaaga tatccgtgac ccactttcac
     6661 aacgatcact tatcagcgct caatcccctc ttgcatatga ctagtgcaaa ccggcaaaga
     6721 tatcggctac tgctacaaca ggaaggagaa gaaggacgtg agtctgtcct aaccagtgcg
     6781 acagcgatga gctcataagc cagtagcacg gtgagggcgg tactatggtc ggtgcgcctc
     6841 tcaagggacg aatcgtcatc atcgacgatg ttctcacctc tggcaaggcc atccgtgaag
     6901 ctattgacat tctcaaggcc tcccctgaag cgaagcttgt cggaattgtc cagcttgatt
     6961 atgcaagagg ggattgagcg ctgataagtg atcgttgtga aagtgggtca cggatatctt
     7021 agtacagcca gatgtgaaac tcgacatata taacacccat tatgtctgct gctgtattga
     7081 cgacatgacc ccagtcttcc cataaaaacg ggaagaacgc acgagcgatg tggcagacaa
     7141 gagaacggga atatgtaggc gtgtattgac aatcaatggc ttgacgatga gaggacgaac
     7201 gggaagattg tgaagactgt atgatgttac gatatgaaat ggatacgaac gggaagacta
     7261 tgtgacgaga tatgcaaatg gatacgaacg ggaatactac tatgtgatga gatggatatg
     7321 caaattgata cgaacgggaa tatttgtatg ttaattgaca acactccccc aaggcacccc
     7381 cggcgcgtca attactgaca aacgattcat gaacgggaat tgtcagcgaa ggtgggcgcg
     7441 catataaata tcaaattacg attttgcgtg ttcgaacctc tgctgtcgca tccccggggt
     7501 tcgacaagac ctgcttctat cgatgaagat ctggcgcgcc actcagttgt tgtctcgaag
     7561 catgtactgt agtttccagt cttcattcaa gttcaaagat ccgtactctg tgcagctggt
     7621 tcctgctggt attggctttc ttctaaatat ttgtaatggc ggctggccgc tgtaacgaca
     7681 ttgtttgaca gagtttcata acaagttgat agctaacggt gcctctccta cgtagaacag
     7741 attgcatctt ggttaaaaaa gaaacagcgt tttaatcctg tgcatctgca aagacgaaac
     7801 aggtccatct ttgtctgcag taccaggttt gcacatcaca tggttgcggt acccaattcg
     7861 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     7921 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     7981 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     8041 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     8101 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     8161 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     8221 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     8281 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     8341 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     8401 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     8461 tttaacgcga attttaacaa aatattaacg cttacaattt aggtg
//
