LOCUS       Exported                9830 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSG75, complete sequence.
ACCESSION   KM288849
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9830)
  AUTHORS   Goldfless SJ, Wagner JC, Niles JC.
  TITLE     Versatile control of Plasmodium falciparum gene expression with an 
            inducible protein-RNA interaction
  JOURNAL   Nat Commun 5, 5329 (2014)
  PUBMED    25370483
REFERENCE   2  (bases 1 to 9830)
  AUTHORS   Goldfless SJ, Niles JC.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-AUG-2014) Biological Engineering, MIT, 77 
            Massachusetts Avenue, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 9830)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9830
                     /organism="Expression vector pSG75"
                     /mol_type="other DNA"
                     /note="TetR-aptamer regulated Fluc reporter in Plasmodium 
                     falciparum"
                     /db_xref="taxon:1569331"
     promoter        complement(17..35)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     rep_origin      complement(36..1237)
                     /direction=LEFT
     primer_bind     complement(53..69)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    77..93
                     /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(101..131)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    146..167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(455..1043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(1238..1860)
                     /label=S. cerevisiae CEN6/ARS4
                     /note="S. cerevisiae CEN6/ARS4"
     misc_feature    1294..1797
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     repeat_region   1951..2505
     repeat_region   2506..3044
     regulatory      3045..3395
                     /regulatory_class="promoter"
                     /note="yeast TEF promoter"
     gene            3052..4408
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        3052..3395
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3396..4205
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=aminoglycoside phosphotransferase
                     /note="KanMX selectable marker for E. coli and S. 
                     cerevisiae"
                     /protein_id="AIW62884.1"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      4211..4408
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     regulatory      complement(4456..5019)
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum HRPII 3 UTR"
     CDS             complement(5055..5990)
                     /codon_start=1
                     /product="luciferase"
                     /label=luciferase
                     /note="R. reniformis (Renilla) luciferase, wild-type"
                     /protein_id="AIW62883.1"
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     regulatory      complement(6007..6611)
                     /regulatory_class="promoter"
                     /note="5UTR from PcDT gene"
     regulatory      6612..7241
                     /regulatory_class="promoter"
                     /note="contains promoter and 5 UTR from P. falciparum 
                     calmodulin gene"
     misc_feature    7244..7290
                     /label=TetR-binding aptamer
                     /note="TetR-binding aptamer"
     CDS             7313..8965
                     /codon_start=1
                     /product="luciferase"
                     /label=luciferase
                     /note="P. pyralis (firefly) luciferase, wild-type with 
                     K549E mutation to abolish peroxisomal targeting in yeast"
                     /protein_id="AIW62882.1"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSEL"
     regulatory      complement(8972..9807)
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum hsp86 3 UTR"