LOCUS       sequence-225033-        6533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express GFP-RagB (Q99L mutant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6533)
  AUTHORS   Lawrence RE, Cho KF, Rappold R, Thrun A, Tofaute M, Kim DJ, 
            Moldavski O, Hurley JH, Zoncu R
  TITLE     A nutrient-induced affinity switch controls mTORC1 activation by its
            Rag GTPase-Ragulator lysosomal scaffold.
  JOURNAL   Nat Cell Biol. 2018 Sep;20(9):1052-1063. doi: 
            10.1038/s41556-018-0148-6. Epub 2018 Jul 30.
  PUBMED    30061680
REFERENCE   2  (bases 1 to 6533)
  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: 
            "10.1038/s41556-018-0148-6"; journalName: "Nat Cell Biol"; date: 
            "2018-09"; volume: "20"; issue: "9"; pages: "1052-1063"
COMMENT     Sequence Label: pRK5 GFP RagB Q99L
FEATURES             Location/Qualifiers
     source          1..6533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      925..934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1009..1011,1012..1722)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1009..1011,1012..1722)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2833..2842
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2846..2980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2883..2902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2937..2956
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3049..3406
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3049..3240
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3072..3092)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3257..3392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3426..3443)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3426..3442)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3435..3457)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3655..4110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3742..3761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3952..3973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4127..4146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4246..4268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4306..4324)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4392..4496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4497..4565,4566..5357)
                     /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(4715..4734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5528..6116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6017..6036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6270..6287
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6404..6425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6440..6445,6446..6463,6464..6470)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6478..6494
                     /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     6483..6505
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6502..6518
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6502..6518
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgccacc atggactaca aggatgccgc tgccaagggc
      961 ggaggtgggg actacaagga tgccgctgcc aagggcggag gtggatctgt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1201 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccg ggatcactct cggcatggac gagctgtaca agggaggcgg tggatcgggc
     1741 gggggtggat cagtcgacat ggaagaatct gactctgaga aaacgacgga gaaagaaaat
     1801 ctggggccga gaatggatcc accactaggg gaaccggaag gatcgcttgg gtgggtgcta
     1861 ccaaatacag ccatgaagaa aaaggtgctg ttgatgggta aaagtgggtc tggtaagacc
     1921 agcatgaggt ctattatctt tgcaaattat attgccagag acacacgtcg ccttggcgca
     1981 acaattgatg tagaacattc tcatgttcga tttctgggaa acctggtatt gaacctgtgg
     2041 gattgtggtg ggctagacac cttcatggaa aattatttca ctagccaacg ggacaacatc
     2101 ttccgaaatg tggaggttct gatttatgtc tttgatgtgg agagccgcga actggaaaag
     2161 gacatgcact attaccaatc atgcctggag gccattctgc agaattctcc agatgccaaa
     2221 atattttgct tggtacacaa aatggatctg gtacaggagg atcaacggga cctgattttt
     2281 aaagagcgag aagaagattt gaggcgtttg tctcgcccat tggaatgttc ttgtttccga
     2341 acatctatct gggatgaaac cctctataag gcttggtcca gcatagttta tcagctgatt
     2401 cccaatgttc agcagctgga aatgaaccta aggaattttg ctgaaattat cgaagctgat
     2461 gaagtacttc tttttgagag agctactttt ctggtaattt ctcactatca gtgtaaagag
     2521 cagcgtgatg cccatagatt tgagaaaata agcaacatta ttaagcagtt caagctgagc
     2581 tgcagcaagc tggctgcctc tttccagagt atggaagtca ggaactctaa cttcgctgct
     2641 ttcattgaca tctttacatc caacacttat gtgatggttg tgatgtctga tccgtccatt
     2701 ccttctgcag ctactctgat caacatccgc aatgccagga aacactttga aaagctggaa
     2761 agagtggatg gaccaaagca gtgtcttctc atgcgctaag cggccgctaa gtaagtaagg
     2821 atccccagct tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata
     2881 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2941 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc
     3001 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     3061 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3181 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3241 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3301 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3361 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc
     3421 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     3481 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     3541 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     3601 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     3661 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3721 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3781 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3841 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     3901 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3961 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     4021 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4081 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4141 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4201 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4261 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4321 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4381 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4441 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4501 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4561 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4621 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4681 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4741 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4801 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4861 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4921 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4981 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5041 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5101 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5161 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5221 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5281 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5341 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5401 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5461 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5521 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5581 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5641 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5701 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5761 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5821 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5881 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5941 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6001 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6061 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6121 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6181 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6241 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6301 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6361 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     6421 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     6481 tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat taa
//
