LOCUS       sequence-326718-        7936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Flag-Rheb-12Rs mutant protein in mammalian cells (all 
            the lysine residues on Rheb except the K19 and K120 are mutated to 
            arginines)..
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-12Rs
FEATURES             Location/Qualifiers
     source          1..7936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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     1833..1853
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2142..2345
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2296..2316
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2386..2409
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    3030..3147
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3196..3706
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3625..3643
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3728..4327
                     /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(3728..3747)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4224..4244
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4455..4688
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4760..4881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4797..4816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4851..4870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4921..5056
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5076..5095)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5077..5095)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5105..5122)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5105..5121)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5114..5136)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5263..5718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5350..5369)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5560..5581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5744..5848
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5849..5917,5918..6709)
                     /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(6067..6086)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6880..7468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7369..7388
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7622..7639
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7756..7777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7792..7797,7798..7815,7816..7822)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7830..7846
                     /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     7835..7857
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7854..7870
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7854..7870
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7889..7909
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7891..7909
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt rgyrsyygaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     1801 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     1861 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1921 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1981 ttttaaaatt atgttttaaa atggactatc atatgccaag tacgccccct attgacgtca
     2041 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     2101 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     2161 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     2221 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     2281 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     2341 gagctggttt agtgaaccgt cagatccgct agcgctaccg gtatggacta caaggatgac
     2401 gatgacaagg gtggtggtgg tggtgcgtcg acgatgccgc agtccaggtc ccggaggatc
     2461 gcgatcctgg gctaccggtc tgtggggaaa tcctcattga cgattcaatt tgttgaaggc
     2521 caatttgtgg actcctacga tccaaccata gaaaacactt ttacaaggtt gatcacagta
     2581 aatggacaag aatatcatct tcaacttgta gacacagccg ggcaagatga atattctatc
     2641 tttcctcaga catactccat agatattaat ggctatattc ttgtgtattc tgttacatca
     2701 atcaggagtt ttgaagtgat tagggttatc catggcaggt tgttggatat ggtggggagg
     2761 gtacaaatac ctattatgtt ggttgggaat aagagggacc tgcatatgga aagggtgatc
     2821 agttatgaag aagggagggc tttggcagaa tcttggaatg cagctttttt ggaatcttct
     2881 gctagggaaa atcagactgc tgtggatgtt tttcgaagga taattttgga ggcagaaagg
     2941 atggacgggg cagcttcaca aggcaggtct tcatgctcgg tgatgtgaga attctcgacc
     3001 tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     3061 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     3121 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     3181 tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca
     3241 gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg
     3301 cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc
     3361 cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg
     3421 ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca
     3481 gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg
     3541 gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt
     3601 gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc
     3661 ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga
     3721 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     3781 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     3841 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     3901 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     3961 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     4021 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     4081 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     4141 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     4201 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     4261 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     4321 tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     4381 atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     4441 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     4501 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4561 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4621 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4681 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4741 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4801 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4861 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     4921 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4981 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5041 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     5101 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     5161 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5221 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     5281 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5341 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5401 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5461 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5521 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5581 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5641 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5701 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5761 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5821 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5881 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5941 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6001 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6061 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6121 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6181 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6241 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6301 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6361 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6421 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6481 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6541 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6601 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6661 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6721 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6781 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6841 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6901 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6961 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7021 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7081 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7141 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7201 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7261 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7321 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7381 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7441 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7501 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7561 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7621 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7681 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7741 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7801 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7861 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     7921 ggagctgcaa gcttaa
//
