LOCUS       Exported               11574 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSG230, complete sequence.
ACCESSION   KM288852
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11574)
  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 11574)
  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 11574)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11574
                     /organism="Expression vector pSG230"
                     /mol_type="other DNA"
                     /note="G418-selectable reporter plasmid in Plasmodium 
                     falciparum with FLuc expression controlled by the PfCam 
                     promoter"
                     /db_xref="taxon:1569334"
     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="AIW62891.1"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSEL"
     regulatory      complement(2539..3168)
                     /regulatory_class="promoter"
                     /note="contains promoter and 5 UTR from P. falciparum 
                     calmodulin gene"
     regulatory      3169..4038
                     /regulatory_class="promoter"
                     /note="contains promoter and 5UTR from P. falciparum Hsp86
                     (PF07_0029)"
     misc_feature    4049..4669
                     /note="TetR protein region; tetR(B) with 4 silent 
                     mutations"
     CDS             4049..4669
                     /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    4670..4732
                     /note="Thosea asigna virus 2A-like peptide (alternate codon
                     usage)"
     CDS             4679..4732
                     /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    4733..5665
                     /label=R. reniformis (Renilla) luciferase wild-type
                     /note="R. reniformis (Renilla) luciferase wild-type"
     CDS             4733..5665
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_feature    5666..5728
                     /note="Thosea asigna virus 2A-like peptide, P. falciparum 
                     optimized codon usage"
     CDS             5675..5728
                     /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             5729..6520
                     /codon_start=1
                     /product="neomycin phosphotransferase II"
                     /label=neomycin phosphotransferase II
                     /note="neomycin phosphotransferase II selectable marker"
                     /protein_id="AIW62893.1"
                     /translation="MEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      6556..7119
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum HRPII 3 UTR"
     gene            complement(7167..8523)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      7167..7364
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(7370..8179)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=aminoglycoside phosphotransferase
                     /note="KanMX selectable marker for E. coli and S. 
                     cerevisiae"
                     /protein_id="AIW62892.1"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      complement(8180..8530)
                     /regulatory_class="promoter"
                     /note="yeast TEF promoter"
     promoter        complement(8180..8523)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     repeat_region   8531..9069
     repeat_region   9070..9624
     misc_feature    9715..10337
                     /label=S. cerevisiae CEN6/ARS4
                     /note="S. cerevisiae CEN6/ARS4"
     misc_feature    9778..10281
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     rep_origin      10338..11539
     rep_origin      10532..11120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    11408..11429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        11444..11474
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11482..11498
                     /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     11506..11522
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        11540..11558
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"