LOCUS       Exported                8676 bp ds-DNA    circular SYN 11-9-2014
DEFINITION  Lentiviral vector for inducible barcoded shRNA libraries, with an 
            HTS6 cassette for high-throughput sequencing plus TagGFP2 and 
            puromycin resistance markers.
ACCESSION   .
VERSION     .
KEYWORDS    pRSIT17-U6Tet-sh-HTS6-CMV-TetRep-2A-TagGFP2-2A-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8676)
  AUTHORS   Cellecta
  TITLE     Direct Submission
COMMENT     
FEATURES             Location/Qualifiers
     source          1..8676
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     promoter        6..232
                     /note="RSV promoter"
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        1819..2064
                     /note="U6-Tet promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA, 
                     modified to contain two tetracycline operator (tetO) sites"
     protein_bind    2020..2037
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2046..2064
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    2066..2095
                     /note="cloning site"
                     /note="shRNA cloning site"
     misc_feature    2123..2238
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2387..2590
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             2664..3284
                     /codon_start=1
                     /gene="synthetic gene"
                     /product="tetracycline repressor TetR"
                     /note="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"
     CDS             3291..3344
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /protein_id="
                     "
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3351..4064
                     /codon_start=1
                     /product="monomeric green fluorescent protein, also known 
                     as mTagGFP"
                     /note="TagGFP2"
                     /note="mammalian codon-optimized"
                     /translation="MSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIK
                     FICTTGKLPVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDG
                     KYKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEA
                     NFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLE
                     SFSACCHTHGMDELYR"
     CDS             4083..4136
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /protein_id="
                     "
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4143..4742
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    4755..5343
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             5417..5650
                     /note="3 LTR (Delta-U3)"
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5722..5843
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5883..6018
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     rep_origin      complement(6453..7041)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7212..8072)
                     /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(8073..8177)
                     /gene="bla"
                     /note="AmpR promoter"