LOCUS       Exported               13320 bp ds-DNA    circular SYN 29-1-2014
DEFINITION  Empty lentiviral vector for doxycycline-inducible expression of an 
            shRNAmir together with TurboRFP.
ACCESSION   .
VERSION     .
KEYWORDS    pTRIPZ
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13320)
  AUTHORS   Open Biosystems
  TITLE     Direct Submission
COMMENT     Excision of rtTA3 using Cre recombinase or BamHI yields a vector 
            that can be used for Tet-repressible expression.
FEATURES             Location/Qualifiers
     source          1..13320
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     LTR             1..634
                     /note="5 LTR"
                     /note="5 long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /note="HIV-1 psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2063..2180
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(2245..2619)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(2639..2686)
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter "
     protein_bind    2722..2740
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2757..2775
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2793..2811
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2829..2847
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2864..2882
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2900..2918
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2930..2968
                     /note="minimal CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3005..3700
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /note="TurboRFP"
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     misc_feature    3806..4064
                     /note="shRNAmir insertion site"
                     /note="A fragment encoding a microRNA-adapted shRNA can be 
                     inserted between the XhoI and MluI sites."
     promoter        4070..5281
                     /note="UbC promoter"
                     /note="human ubiquitin C promoter"
     protein_bind    5294..5327
                     /bound_moiety="Cre recombinase"
                     /note="loxP511"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACAT). loxP511 sites are compatible with each
                     other, but incompatible with wild-type loxP sites."
     CDS             5334..6041
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /note="rtTA3"
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     protein_bind    6042..6075
                     /bound_moiety="Cre recombinase"
                     /note="loxP511"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACAT). loxP511 sites are compatible with each
                     other, but incompatible with wild-type loxP sites."
     misc_feature    6098..6666
                     /note="IRES"
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             6673..7275
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGSRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    7291..7879
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             8086..8319
                     /note="3 LTR (Delta-U3)"
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8426..8650
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8696..9124
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        9138..9467
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      9318..9453
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             9516..10545
                     /note="HygR (inactivated)"
                     /note="The hygromycin resistance gene was inactivated by 
                     destroying an EcoRI site within the coding sequence."
     polyA_signal    10675..10796
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(11247..11835)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(12006..12866)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(12867..12971)
                     /gene="bla"
                     /note="AmpR promoter"
     polyA_signal    13019..13153
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"