LOCUS       Exported               11625 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSG231, complete sequence.
ACCESSION   KM288853
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11625)
  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 11625)
  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 11625)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11625
                     /organism="Expression vector pSG231"
                     /mol_type="other DNA"
                     /note="G418-selectable reporter plasmid in Plasmodium 
                     falciparum for assessing regulation of FLuc expression from
                     a TetR aptamer-regulated PfCam promoter"
                     /db_xref="taxon:1569335"
     regulatory      complement(24..859)
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum hsp86 3 UTR"
     CDS             complement(866..2518)
                     /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="AIW62894.1"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSEL"
     misc_feature    complement(2541..2587)
                     /label=TetR aptamer
                     /note="TetR aptamer"
     regulatory      complement(2590..3219)
                     /regulatory_class="promoter"
                     /note="contains promoter and 5 UTR from P. falciparum 
                     calmodulin gene"
     regulatory      3220..4089
                     /regulatory_class="promoter"
                     /note="contains promoter and 5UTR from P. falciparum Hsp86
                     (PF07_0029)"
     misc_feature    4100..4720
                     /note="TetR protein region; tetR(B) with 4 silent 
                     mutations"
     CDS             4100..4720
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     misc_feature    4721..4783
                     /note="Thosea asigna virus 2A-like peptide (alternate codon
                     usage)"
     CDS             4730..4783
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     misc_feature    4784..5716
                     /label=R. reniformis (Renilla) luciferase wild-type
                     /note="R. reniformis (Renilla) luciferase wild-type"
     CDS             4784..5716
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_feature    5717..5779
                     /note="Thosea asigna virus 2A-like peptide, P. falciparum 
                     optimized codon usage"
     CDS             5726..5779
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5780..6571
                     /codon_start=1
                     /product="neomycin phosphotransferase"
                     /label=neomycin phosphotransferase
                     /note="neomycin phosphotransferase II selectable marker"
                     /protein_id="AIW62896.1"
                     /translation="MEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      6607..7170
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum HRPII 3 UTR"
     gene            complement(7218..8574)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      7218..7415
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(7421..8230)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=aminoglycoside phosphotransferase
                     /note="KanMX selectable marker for E. coli and S. 
                     cerevisiae"
                     /protein_id="AIW62895.1"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      complement(8231..8581)
                     /regulatory_class="promoter"
                     /note="yeast TEF promoter"
     promoter        complement(8231..8574)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     repeat_region   8582..9120
     repeat_region   9121..9675
     misc_feature    9766..10388
                     /label=S. cerevisiae CEN6/ARS4
                     /note="S. cerevisiae CEN6/ARS4"
     misc_feature    9829..10332
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     rep_origin      10389..11590
     rep_origin      10583..11171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    11459..11480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        11495..11525
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11533..11549
                     /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     11557..11573
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        11591..11609
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"