LOCUS       40924_48977        9040 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Retroviral Tet-shRNA expression vector TtRMPVIR, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9040)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol. (2010) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (01-NOV-2010) Scott Lowe Lab, Cold Spring Harbor Laboratory, 1 
            Bungtown Road, Cold Spring Harbor, NY 11724, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..309
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     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)"
     CDS             1152..1568
                     /codon_start=1
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
                     /translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
                     NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
                     PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
     promoter        1600..1914
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     CDS             1953..2627
                     /codon_start=1
                     /label=DsRed2
                     /note="improved tetrameric variant of DsRed fluorescent
                     protein"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     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           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)"
     promoter        3011..3510
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             3533..4249
                     /codon_start=1
                     /label=Venus
                     /note="yellow fluorescent protein (YFP) with fast and
                     efficient maturation (Nagai et al., 2002)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    4269..4844
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             4845..5588
                     /codon_start=1
                     /label=rtTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     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      complement(7059..7647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7821..8678)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8679..8783)
                     /label=AmpR promoter
     enhancer        8969..9040
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgctctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctcaataaa agagcccaca acccctcact
      541 cggcgcgcca gtcttccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct
      601 cttgctgttt gcatccgaat cgtggtctcg ctgttccttg ggagggtctc ctctgagtga
      661 ttgactaccc acgacggggg tctttcattt gggggctcgt ccgggatttg gagacccctg
      721 cccagggacc accgacccac caccgggagg taagctggcc agcaacttat ctgtgtctgt
      781 ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg tctgtactag ttagctaact
      841 agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc
      901 ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga ggaagggagt
      961 cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag aacctaaaac
     1021 agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc
     1081 tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt
     1141 ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt cactggaaag
     1201 atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct
     1261 tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc
     1321 gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag
     1381 accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca
     1441 agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc
     1501 ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc
     1561 taggcgccgg aattgaagat ctgggggatc gataccgtcg agtttactcc ctatcagtga
     1621 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgatgtc gagtttactc
     1681 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
     1741 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttatccct atcagtgata
     1801 gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg aggtaggcgt
     1861 gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
     1921 attaattcga gctcggtacc cggggatcca ccatggcctc ctccgagaac gtcatcaccg
     1981 agttcatgcg cttcaaggtg cgcatggagg gcaccgtgaa cggccacgag ttcgagatcg
     2041 agggcgaggg cgagggccgc ccctacgagg gccacaacac cgtgaagctg aaggtgacca
     2101 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca
     2161 aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg
     2221 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggcgacc gtgacccagg
     2281 actcctccct gcaggacggc tgcttcatct acaaggtgaa gttcatcggc gtgaacttcc
     2341 cctccgacgg ccccgtgatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc
     2401 tgtacccccg cgacggcgtg ctgaagggcg agacccacaa ggccctgaag ctgaaggacg
     2461 gcggccacta cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc
     2521 ccggctacta ctacgtggac gccaagctgg acatcacctc ccacaacgag gactacacca
     2581 tcgtggagca gtacgagcgc accgagggcc gccaccacct gttcctgtag cggccagcta
     2641 agcctcgact agggataaca gggtaattgt ttgaatgagg cttcagtact ttacagaatc
     2701 gttgcctgca catcttggaa acacttgctg ggattacttc ttcaggttaa cccaacagaa
     2761 ggctcgagaa ggtatattgc tgttgacagt gagcgcagga attataatgc ttatctatag
     2821 tgaagccaca gatgtataga taagcattat aattcctatg cctactgcct cggaattcaa
     2881 ggggctactt taggagcaat tatcttgttt actaaaactg aataccttgc tatctctttg
     2941 atacattttt acaaagctga attaaaatgg tataaattaa atcacttttt tcaattctag
     3001 caattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     3061 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     3121 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     3181 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     3241 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     3301 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     3361 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     3421 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     3481 ttctcctctt cctcatctcc gggcctttcg acctgcagcc caagcttcca ccatggtgag
     3541 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     3601 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     3661 gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     3721 caccctgggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
     3781 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     3841 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     3901 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3961 gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     4021 ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta
     4081 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4141 ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4201 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattgtt
     4261 caattccgcc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     4321 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     4381 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     4441 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     4501 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     4561 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     4621 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     4681 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     4741 catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga
     4801 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatgagt agactggaca
     4861 agagcaaagt cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga
     4921 cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga
     4981 agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc
     5041 acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat
     5101 accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa
     5161 cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct
     5221 ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat
     5281 tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta
     5341 tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct
     5401 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg
     5461 gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     5521 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     5581 tccccgggta atccggactc gacctgcagc caagcttatc gataccgtcg agatatcagt
     5641 ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc
     5701 atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     5761 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata
     5821 actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagggt cgaccctaga
     5881 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     5941 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     6001 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt
     6061 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     6121 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat ttgggggctc
     6181 gtccgggatc gggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg
     6241 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     6301 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     6361 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     6421 gatccggctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     6481 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     6541 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     6601 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6661 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     6721 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttactggctt
     6781 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     6841 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     6901 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6961 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     7021 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     7081 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     7141 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7201 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7261 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7321 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7381 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7441 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     7501 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7561 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7621 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7681 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7741 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7801 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7861 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7921 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7981 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     8041 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     8101 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     8161 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     8221 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8281 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8341 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8401 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     8461 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8521 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8581 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8641 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8701 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8761 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     8821 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     8881 caagaattag cttggccatt gcatacgttg tatccatatc ataatatgta catttatatt
     8941 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
     9001 tcaattacgg ggtcattagt tcatagccca tatatggagt
//
