LOCUS       Exported                9593 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSG594, complete sequence.
ACCESSION   KM288860
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9593)
  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 9593)
  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 9593)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9593
                     /organism="Expression vector pSG594"
                     /mol_type="other DNA"
                     /note="Plasmid allows modification of native PfCRT locus in
                     Plasmodium falciparum to achieve TetR aptamer-regulated 
                     expression of a PfCRT Dd2 cDNA allele"
                     /db_xref="taxon:1569342"
     regulatory      9..428
                     /regulatory_class="promoter"
                     /note="native upstream sequence from P. falciparum CRT 
                     gene"
     misc_feature    435..503
                     /label=TetR aptamer
                     /note="TetR aptamer"
     exon            504..594
                     /number=1
                     /note="P. falciparum CRT recoded"
     exon            595..863
                     /number=2
                     /note="P. falciparum CRT (Dd2 type)"
     exon            864..1034
                     /number=3
                     /note="P. falciparum CRT (Dd2 type)"
     exon            1035..1169
                     /number=4
                     /note="P. falciparum CRT (Dd2 type)"
     exon            1170..1241
                     /number=5
                     /note="P. falciparum CRT (Dd2 type)"
     exon            1242..1316
                     /number=6
                     /note="P. falciparum CRT (Dd2 type)"
     exon            1317..1400
                     /number=7
                     /note="P. falciparum CRT (Dd2 type)"
     exon            1401..1451
                     /number=8
     exon            1452..1508
                     /number=9
     exon            1509..1601
                     /number=10
     exon            1602..1646
                     /number=11
     exon            1647..1700
                     /number=12
     exon            1701..1778
                     /number=13
     regulatory      1787..2490
                     /regulatory_class="terminator"
                     /note="derived from P. berghei CRT gene"
     regulatory      2510..3499
                     /regulatory_class="promoter"
                     /note="promoter and 5UTR from P. falciparum calmodulin 
                     gene"
     CDS             3512..5392
                     /codon_start=1
                     /product="hDHFR, tetR, GLucSDEL"
                     /label=hDHFR, tetR, GLucSDEL
                     /note="Human dihydrofolate reductase/thymidylate synthase 
                     selectable marker; tetR(B); Gaussia luciferase reporter"
                     /protein_id="AIW62910.1"
                     /translation="MHGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVE
                     GKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQP
                     ELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEY
                     PGVLSDVQEEKGIKYKFEVYEKNDAGGSGEGRGSLLTCGDVEENPGPSRLDKSKVINSA
                     LELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEG
                     ESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENAL
                     YALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGL
                     ELIICGLEKQLKCESGSPGSGEGRGSLLTCGDVEENPGPGVKVLFALICIAVAEAKPTE
                     NNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHI
                     KCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVD
                     CTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDSDEL"
     CDS             4088..4141
                     /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             4142..4759
                     /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="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     CDS             4772..4825
                     /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             4826..5377
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="GVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGK
                     LPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQ
                     GGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQR
                     CATFASKIQGQVDKIKGAGGD"
     regulatory      5435..6006
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum HRPII gene"
     regulatory      6013..7062
                     /regulatory_class="terminator"
                     /note="derived from P. falciparum CRT gene"
     promoter        7425..7529
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7530..8390
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="blasticidin deaminase selectable marker for E. coli"
                     /protein_id="AIW62911.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8561..9149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    9437..9458
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9473..9503
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9511..9527
                     /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     9535..9551
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        9569..9587
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"