LOCUS       Exported                8452 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSG586, complete sequence.
ACCESSION   KM288858
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8452)
  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 8452)
  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 8452)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8452
                     /organism="Expression vector pSG586"
                     /mol_type="other DNA"
                     /note="blasticidin S-selectable reporter plasmid in 
                     Plasmodium falciparum for assessing control of GFP 
                     expression from a mutated TetR aptamer-regulated PfCam 
                     promoter after Bxb1 chromosomal integration"
                     /db_xref="taxon:1569340"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_recomb     join(88..138,144..194)
                     /label=tandem Bxb1 attP sites
                     /note="tandem Bxb1 attP sites"
     promoter        complement(230..248)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     rep_origin      complement(249..1450)
                     /direction=LEFT
     primer_bind     complement(266..282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    290..306
                     /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(314..344)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    359..380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(668..1256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             1845..2654
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=aminoglycoside phosphotransferase
                     /note="KanMX4 selectable marker for E. coli"
                     /protein_id="AIW62905.1"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      2660..2857
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     regulatory      complement(2905..3468)
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum HRPII 3 UTR"
     misc_feature    complement(3499..3894)
                     /label=blasticidin deaminase selectable marker
                     /note="blasticidin deaminase selectable marker"
     CDS             complement(3499..3894)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    complement(3901..3963)
                     /note="Thosea asigna virus 2A-like peptide, P. falciparum 
                     optimized codon usage"
     CDS             complement(3901..3954)
                     /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    complement(3964..4896)
                     /label=R. reniformis (Renilla) luciferase wild-type
                     /note="R. reniformis (Renilla) luciferase wild-type"
     CDS             complement(3964..4896)
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     misc_feature    complement(4897..4959)
                     /note="Thosea asigna virus 2A-like peptide (alternate codon
                     usage)"
     CDS             complement(4897..4950)
                     /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    complement(4960..5580)
                     /note="TetR protein region; tetR(B) with 4 silent 
                     mutations"
     CDS             complement(4960..5580)
                     /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"
     regulatory      complement(5591..6195)
                     /regulatory_class="promoter"
                     /note="promoter and 5UTR from P. chabaudi dihydrofolate 
                     reductase-thymidylate synthase gene"
     regulatory      6196..6825
                     /regulatory_class="promoter"
                     /note="contains promoter and 5 UTR from P. falciparum 
                     calmodulin gene"
     misc_feature    6828..6874
                     /label=mutated TetR aptamer
                     /note="mutated TetR aptamer"
     misc_feature    6897..7610
                     /label=green fluorescent protein GFPmut2
                     /note="green fluorescent protein GFPmut2"
     CDS             6897..7610
                     /codon_start=1
                     /product="Aequoria victoria green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     regulatory      7617..8452
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum hsp86 3 UTR"