LOCUS       sequence-13246-e        7838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral transfer plasmid for Tet-regulated shRNA expression with 
            a minimal CMV promoter. Contains DsRed2 and Venus fluorescent 
            reporters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7838)
  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. 2011 Jan . 29(1):79-83.
  PUBMED    21131983
REFERENCE   2  (bases 1 to 7838)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2011 Jan . 29(1):79-83."
COMMENT     Sequence Label: TRMPV-ns
FEATURES             Location/Qualifiers
     source          1..7838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1498..1520
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1536..1552
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    join(1618..1636,1637..1659,1660..1678,1679..1701,1702..1720,
                     1721..1743,1744..1762,1763..1785,1786..1804,1805..1827,
                     1828..1846,1847..1869,1870..1888)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1921..1959
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1956..1980
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2058..2735
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(2238..2258)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2650..2673
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           2804..2925
                     /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           2953..2967
                     /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           2995..3121
                     /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"
     promoter        3144..3643
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3179..3202)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3576..3595
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(3666..3668,3669..3671,3672..4385)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3711..3732)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3972..3991)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4319..4340
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4544..4969
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5072..5094)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5230..5250)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5235..5564
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5415..5550
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5586..5605
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5686..5703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5857..6445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5937..5956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6616..7407,7408..7476))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7239..7258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7477..7581)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7649..7667
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7767..7838,1..309)
                     /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 gatcctctag agcatcgagt ttaccactcc
     1621 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     1681 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     1741 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     1801 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
     1861 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
     1921 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
     1981 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
     2041 ctctcgacgg atccaccatg gcctcctccg agaacgtcat caccgagttc atgcgcttca
     2101 aggtgcgcat ggagggcacc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2161 gccgccccta cgagggccac aacaccgtga agctgaaggt gaccaagggc ggccccctgc
     2221 ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggtg tacgtgaagc
     2281 accccgccga catccccgac tacaagaagc tgtccttccc cgagggcttc aagtgggagc
     2341 gcgtgatgaa cttcgaggac ggcggcgtgg cgaccgtgac ccaggactcc tccctgcagg
     2401 acggctgctt catctacaag gtgaagttca tcggcgtgaa cttcccctcc gacggccccg
     2461 tgatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg
     2521 gcgtgctgaa gggcgagacc cacaaggccc tgaagctgaa ggacggcggc cactacctgg
     2581 tggagttcaa gtccatctac atggccaaga agcccgtgca gctgcccggc tactactacg
     2641 tggacgccaa gctggacatc acctcccaca acgaggacta caccatcgtg gagcagtacg
     2701 agcgcaccga gggccgccac cacctgttcc tgtagcggcc gcgactctag aatttactag
     2761 tcatatgata agatctctcg actagggata acagggtaat tgtttgaatg aggcttcagt
     2821 actttacaga atcgttgcct gcacatcttg gaaacacttg ctgggattac ttcttcaggt
     2881 taacccaaca gaaggctcga gaaggtatat tgctgttgac agtgagcgca ggaattataa
     2941 tgcttatcta tagtgaagcc acagatgtat agataagcat tataattcct atgcctactg
     3001 cctcggaatt caaggggcta ctttaggagc aattatcttg tttactaaaa ctgaatacct
     3061 tgctatctct ttgatacatt tttacaaagc tgaattaaaa tggtataaat taaatcactt
     3121 ttttcaattc tagcaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3181 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     3241 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     3301 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     3361 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     3421 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     3481 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     3541 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     3601 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcccaagctt
     3661 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3721 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     3781 cctacggcaa gctgaccctg aagctgatct gcaccaccgg caagctgccc gtgccctggc
     3841 ccaccctcgt gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca
     3901 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3961 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     4021 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     4081 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga
     4141 agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc
     4201 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     4261 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     4321 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     4381 agtaagaatt gttcaattgt aactacgtac gatgcatcga taccgtcgag atatcagtgg
     4441 tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat
     4501 agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4561 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac
     4621 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accctagaga
     4681 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     4741 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4801 agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac
     4861 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     4921 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt
     4981 ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg taagctggct
     5041 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5101 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5161 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtaga
     5221 tccggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5281 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5341 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5401 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5461 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     5521 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt actggcttaa
     5581 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     5641 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     5701 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5761 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     6301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6901 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     6961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7201 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     7261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7561 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     7621 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
     7681 agaattagct tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg
     7741 ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc
     7801 aattacgggg tcattagttc atagcccata tatggagt
//
