LOCUS       Exported                8880 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral plasmid for inducible expression of sgRNA;
            derived from tet-pLKO-puro; puromycin selection. See manual for 
            detailed protocols..
ACCESSION   .
VERSION     .
KEYWORDS    tet-pLKO-sgRNA-puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8880)
  AUTHORS   Huang HT, Seo HS, Zhang T, Wang Y, Jiang B, Li Q, Buckley DL, Nabet 
            B, Roberts JM, Paulk J, Dastjerdi S, Winter GE, McLauchlan H, Moran 
            J, Bradner JE, Eck MJ, Dhe-Paganon S, Zhao JJ, Gray NS
  TITLE     MELK is not necessary for the proliferation of basal-like breast 
            cancer cells.
  JOURNAL   Elife. 2017 Sep 19;6. pii: e26693. doi: 10.7554/eLife.26693.
  PUBMED    28926338
REFERENCE   2  (bases 1 to 8880)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2017-09-19"; volume: "6. pii: e26693. doi"; pages: " 
            10.7554/eLife.26693"
FEATURES             Location/Qualifiers
     source          1..8880
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     intron          516..1088
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     1096..1115
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1143..1161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1172..1789
                     /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
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(1245..1264)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     misc_feature    1827..2400
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1994..2011)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2221..2240
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2420..3019
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2420..2439)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2916..2936
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3147..3380
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3452..3573
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3489..3508)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3543..3562
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3613..3748
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3675..3694
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        complement(3769..3787)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3797..3814)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3797..3813)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3806..3828)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3955..4410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4042..4061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4252..4273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4436..4540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4541..5401
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4759..4778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5572..6160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6061..6080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6314..6331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6448..6469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6484..6514
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6522..6538
                     /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     6527..6549
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6546..6562
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6546..6562
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6581..6601
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6583..6601
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6629..6855
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6856..7036
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    7083..7208
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7701..7934
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8119..8163
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        8452..8546
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     8463..8482
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    8500..8518
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8528..8546
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    8711..8828
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(8877..8880,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"