LOCUS       Exported                9040 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Retroviral Tet-shRNA expression vector TtRMPVIR, complete sequence.
ACCESSION   HQ456318
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9040)
  AUTHORS   Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ, Lowe 
            SW.
  TITLE     Toolkit for evaluating genes required for proliferation and survival
            using tetracycline-regulated RNAi
  JOURNAL   Nat. Biotechnol. (2010) In press
  PUBMED    21131983
REFERENCE   2  (bases 1 to 9040)
  AUTHORS   Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ, Lowe 
            SW.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-NOV-2010) Scott Lowe Lab, Cold Spring Harbor 
            Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
REFERENCE   3  (bases 1 to 9040)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9040
                     /organism="Retroviral Tet-shRNA expression vector
                     TtRMPVIR"
                     /mol_type="other DNA"
                     /db_xref="taxon:931128"
     repeat_region   1..728
                     /label=5-LTR
                     /note="5-LTR"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             514..689
                     /label=5 LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    752..951
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     regulatory      758..1567
                     /regulatory_class="other"
                     /note="packaging signal"
     CDS             1152..1568
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     regulatory      1599..1914
                     /regulatory_class="promoter"
                     /note="TREtight"
     promoter        1600..1914
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1608..1626
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1644..1662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1679..1697
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1715..1733
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1751..1769
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1786..1804
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1822..1840
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1953..2630
                     /codon_start=1
                     /gene="dsRed"
                     /product="dsRed"
                     /label=dsRed
                     /protein_id="ADR71687.1"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     gene            1953..2630
                     /gene="dsRed"
                     /label=dsRed
     misc_RNA        2644..2991
                     /product="miR30-shRNA"
                     /label=miR30-shRNA shRen.713 targeting Renilla luciferase
                     /note="miR30-shRNA shRen.713 targeting Renilla luciferase"
     ncRNA           2671..2792
                     /label=5 miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2820..2834
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2862..2988
                     /label=3 miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     regulatory      3002..3510
                     /regulatory_class="promoter"
                     /note="PGK"
     promoter        3011..3510
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             3533..4252
                     /codon_start=1
                     /gene="Venus"
                     /product="Venus"
                     /label=Venus
                     /protein_id="ADR71688.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     gene            3533..4252
                     /gene="Venus"
                     /label=Venus
     misc_feature    4282..4833
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             4845..5591
                     /codon_start=1
                     /gene="rtTA3"
                     /product="rtTA3"
                     /label=rtTA3
                     /protein_id="ADR71689.1"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     gene            4845..5591
                     /gene="rtTA3"
                     /label=rtTA3
     LTR             5746..6171
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     promoter        6437..6766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6617..6752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(7059..7647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7818..8678)
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /protein_id="ADR71690.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(7818..8678)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(8679..8783)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        8969..309
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"