LOCUS       sequence-264558-        5210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-tagged RhebL1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5210)
  AUTHORS   Angarola B, Ferguson SM
  TITLE     Weak membrane interactions allow Rheb to activate mTORC1 signaling 
            without major lysosome enrichment.
  JOURNAL   Mol Biol Cell. 2019 Oct 15;30(22):2750-2760. doi: 
            10.1091/mbc.E19-03-0146. Epub 2019 Sep 18.
  PUBMED    31532697
REFERENCE   2  (bases 1 to 5210)
  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: 
            "10.1091/mbc.E19-03-0146"; journalName: "Mol Biol Cell"; date: 
            "2019-10-15- 15"; volume: "30"; issue: "22"; pages: "2750-2760"
COMMENT     Sequence Label: pEGFP-C1-RhebL1
FEATURES             Location/Qualifiers
     source          1..5210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        449..500
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     451..471
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      539..548
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(545..547,548..550,551..1261)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(545..547,548..550,551..1261)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(590..611)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(851..870)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1198..1219
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2004..2125
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2041..2060)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2095..2114
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2132..2587)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2269..2290)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2481..2500
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2614..2718
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2720..3077
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2720..2911
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2743..2763)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2928..3063
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3112..3906
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3112..3906
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3166..3185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3776..3795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4094..4113)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4138..4185
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4514..5102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5003..5022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      481 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc
      541 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      601 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      661 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      721 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      781 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      841 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      901 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      961 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1021 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1081 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1141 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1201 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1261 gtccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc
     1321 aatgccgcta gtccgctaca ggaaggtggt catcctcgga taccgctgtg tagggaagac
     1381 atctttggca catcaatttg tggaaggcga gttctcggaa ggctacgatc ctacagtgga
     1441 gaatacttac agcaagatag tgactcttgg caaagatgag tttcacctac atctggtgga
     1501 cacagcaggg caggatgagt acagcattct gccctattca ttcatcattg gggtccatgg
     1561 ttatgtgctt gtgtattctg tcacctctct gcatagcttc caagtcattg agagtctgta
     1621 ccaaaagcta catgaaggcc atgggaaaac ccgggtgcca gtggttctag tggggaacaa
     1681 ggcagatctc tctccagaga gagaggtaca ggcagttgaa ggaaagaagc tggcagagtc
     1741 ctggggtgcg acatttatgg agtcatctgc tcgagagaat cagctgactc aaggcatctt
     1801 caccaaagtc atccaggaga ttgcccgtgt ggagaattcc tatgggcaag agcgtcgctg
     1861 ccatctcatg tgagggatcc accggatcta gataactgat cataatcagc cataccacat
     1921 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1981 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2041 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2101 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     2161 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2221 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2281 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2341 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2401 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2461 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2521 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2581 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2641 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2701 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2761 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2821 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2881 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2941 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3001 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3061 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3121 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3181 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3241 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3301 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3361 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3421 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3481 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3541 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3601 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3661 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3721 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3781 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3841 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3901 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3961 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4021 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4081 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4141 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4201 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4261 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4321 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4381 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4441 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4501 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4561 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4621 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4681 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4741 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4801 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4861 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4921 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4981 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5041 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5101 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5161 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
//
