LOCUS       sequence-326699-        7936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Flag tagged Rheb protein in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7936)
  AUTHORS   Yao Y, Hong S, Ikeda T, Mori H, MacDougald OA, Nada S, Okada M, 
            Inoki K
  TITLE     Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to 
            mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.
  JOURNAL   Mol Cell. 2020 Nov 5;80(3):437-451.e6. doi: 
            10.1016/j.molcel.2020.10.004.
  PUBMED    33157014
REFERENCE   2  (bases 1 to 7936)
  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"; doi: 
            "10.1016/j.molcel.2020.10"; journalName: "Mol Cell"; date: 
            "2020-11-5- 5"; volume: "80"; issue: "3"; pages: "437-451.e6"
COMMENT     Sequence Label: pLJM1-Flag-Rheb
FEATURES             Location/Qualifiers
     source          1..7936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     1606..1626
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1915..2118
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2069..2089
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2159..2182
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2803..2920
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2969..3479
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3398..3416
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3501..4100
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3501..3520)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3997..4017
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4228..4461
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4533..4654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4570..4589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4624..4643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4694..4829
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4849..4868)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4850..4868)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4878..4895)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4878..4894)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4887..4909)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5036..5491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5123..5142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5333..5354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5517..5621
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5622..5690,5691..6482)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5840..5859)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6653..7241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7142..7161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7395..7412
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7529..7550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7565..7570,7571..7588,7589..7595)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7603..7619
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7608..7630
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7627..7643
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7627..7643
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7662..7682
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7664..7682
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7710..7936
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1501 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga
     1561 cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc
     1621 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     1681 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1741 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgcc aagtacgccc
     1801 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     1861 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     1921 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     1981 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     2041 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     2101 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtatgga
     2161 ctacaaggat gacgatgaca agggtggtgg tggtggtgcg tcgacgatgc cgcagtccaa
     2221 gtcccggaag atcgcgatcc tgggctaccg gtctgtgggg aaatcctcat tgacgattca
     2281 atttgttgaa ggccaatttg tggactccta cgatccaacc atagaaaaca cttttacaaa
     2341 gttgatcaca gtaaatggac aagaatatca tcttcaactt gtagacacag ccgggcaaga
     2401 tgaatattct atctttcctc agacatactc catagatatt aatggctata ttcttgtgta
     2461 ttctgttaca tcaatcaaaa gttttgaagt gattaaagtt atccatggca aattgttgga
     2521 tatggtgggg aaagtacaaa tacctattat gttggttggg aataagaaag acctgcatat
     2581 ggaaagggtg atcagttatg aagaagggaa agctttggca gaatcttgga atgcagcttt
     2641 tttggaatct tctgctaaag aaaatcagac tgctgtggat gtttttcgaa ggataatttt
     2701 ggaggcagaa aaaatggacg gggcagcttc acaaggcaag tcttcatgct cggtgatgtg
     2761 agaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2821 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2881 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2941 cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc
     3001 ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg
     3061 accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta
     3121 cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc
     3181 ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag
     3241 acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg
     3301 gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt
     3361 gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc
     3421 cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg
     3481 gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3541 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3601 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3661 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     3721 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     3781 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     3841 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     3901 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     3961 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4021 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4081 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     4141 cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct gtagatctta
     4201 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     4261 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     4321 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     4381 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     4441 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
     4501 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     4561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4621 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     4681 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4741 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4801 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     4861 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4921 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4981 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     5041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5101 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5221 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     5281 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5341 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5401 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5461 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     5521 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5581 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5641 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5701 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5761 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5821 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5881 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5941 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6001 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6061 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6121 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6181 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6241 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6301 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6361 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6421 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6481 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6541 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6601 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6661 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6721 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6781 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6841 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6901 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6961 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7021 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7081 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7141 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7201 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7261 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7321 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7381 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     7441 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     7501 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     7561 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     7621 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     7681 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
     7741 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
     7801 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
     7861 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
     7921 agctcgatac ataaac
//
