LOCUS       sequence-326717-        7936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Flag-Rheb-4Rs mutant protein in mammalian cells (the 
            lysine residues on the position 109, 135, 151 and 178 on Rheb 
            protein 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-4Rs
FEATURES             Location/Qualifiers
     source          1..7936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..133
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    250..271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(286..291,292..309,310..316)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    324..340
                     /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     329..351
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     348..364
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     348..364
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     383..403
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        385..403
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        431..657
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             658..838
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    885..1010
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1503..1736
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1921..1965
                     /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     2263..2283
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        2572..2775
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2726..2746
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2816..2839
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    3460..3577
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3626..4136
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4055..4073
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4158..4757
                     /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(4158..4177)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4654..4674
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4885..5118
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5190..5311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5227..5246)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5281..5300
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5351..5486
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5506..5525)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5507..5525)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5535..5552)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5535..5551)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5544..5566)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5693..6148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5780..5799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5990..6011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6174..6278
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6279..6347,6348..7139)
                     /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(6497..6516)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7310..7898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7799..7818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
       61 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      121 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      181 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      241 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      301 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      361 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
      421 gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt
      481 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg
      541 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
      601 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
      661 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      721 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      781 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      841 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      901 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      961 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     1021 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1081 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1141 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1201 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1261 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1321 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1381 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1441 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1501 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1561 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1621 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1681 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1741 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1801 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1861 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1921 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1981 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2041 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2101 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2161 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg
     2221 tatcgatcac gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg
     2281 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     2341 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     2401 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgccaag tacgccccct
     2461 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     2521 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     2581 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     2641 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     2701 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     2761 tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg gtatggacta
     2821 caaggatgac gatgacaagg gtggtggtgg tggtgcgtcg acgatgccgc agtccaagtc
     2881 ccggaagatc gcgatcctgg gctaccggtc tgtggggaaa tcctcattga cgattcaatt
     2941 tgttgaaggc caatttgtgg actcctacga tccaaccata gaaaacactt ttacaaagtt
     3001 gatcacagta aatggacaag aatatcatct tcaacttgta gacacagccg ggcaagatga
     3061 atattctatc tttcctcaga catactccat agatattaat ggctatattc ttgtgtattc
     3121 tgttacatca atcaaaagtt ttgaagtgat taaagttatc catggcaaat tgttggatat
     3181 ggtggggagg gtacaaatac ctattatgtt ggttgggaat aagaaagacc tgcatatgga
     3241 aagggtgatc agttatgaag aagggagggc tttggcagaa tcttggaatg cagctttttt
     3301 ggaatcttct gctagggaaa atcagactgc tgtggatgtt tttcgaagga taattttgga
     3361 ggcagaaaaa atggacgggg cagcttcaca aggcaggtct tcatgctcgg tgatgtgaga
     3421 attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     3481 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     3541 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     3601 agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg
     3661 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
     3721 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
     3781 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
     3841 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
     3901 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
     3961 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
     4021 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
     4081 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg
     4141 atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     4201 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     4261 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     4321 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     4381 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     4441 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     4501 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     4561 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     4621 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     4681 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4741 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     4801 gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     4861 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     4921 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     4981 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5041 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5101 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
     5161 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5281 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     5341 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     5401 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     5461 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     5521 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     5581 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     5641 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     5701 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5761 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5821 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5881 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5941 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6001 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     6061 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6121 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     6181 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6241 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6301 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6361 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6421 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6481 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6541 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6601 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6661 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6721 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6781 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6841 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6901 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6961 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7021 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7081 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7141 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7201 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7261 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7321 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7381 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7441 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7501 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7561 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7621 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7681 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7741 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7801 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7861 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7921 tacggttcct ggcctt
//
