LOCUS       sequence-224985-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express GFP-RagB (G domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  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 6101)
  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 (GD)
FEATURES             Location/Qualifiers
     source          1..6101
                     /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(973..975,976..1686)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(973..975,976..1686)
                     /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(1015..1036)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1276..1295)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1623..1644
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2401..2410
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2414..2548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2451..2470)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2505..2524
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2617..2974
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2617..2808
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2640..2660)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2825..2960
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2994..3011)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2994..3010)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3003..3025)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3223..3678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3310..3329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3520..3541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3695..3714)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3814..3836
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3874..3892)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3960..4064
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4065..4133,4134..4925)
                     /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(4283..4302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5096..5684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5585..5604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5838..5855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5972..5993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6008..6013,6014..6031,6032..6038)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6046..6062
                     /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     6051..6073
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6070..6086
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6070..6086
                     /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 ggaggtggat ctgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1021 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1081 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1141 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1201 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1261 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1321 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1381 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1441 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1501 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1561 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1621 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1681 tacaagggag gcggtggatc gggcgggggt ggatcagtcg acgaagaatc tgactctgag
     1741 aaaacgacgg agaaagaaaa tctggggccg agaatggatc caccactagg ggaaccggaa
     1801 ggatcgcttg ggtgggtgct accaaataca gccatgaaga aaaaggtgct gttgatgggt
     1861 aaaagtgggt ctggtaagac cagcatgagg tctattatct ttgcaaatta tattgccaga
     1921 gacacacgtc gccttggcgc aacaattgat gtagaacatt ctcatgttcg atttctggga
     1981 aacctggtat tgaacctgtg ggattgtggt gggcaagaca ccttcatgga aaattatttc
     2041 actagccaac gggacaacat cttccgaaat gtggaggttc tgatttatgt ctttgatgtg
     2101 gagagccgcg aactggaaaa ggacatgcac tattaccaat catgcctgga ggccattctg
     2161 cagaattctc cagatgccaa aatattttgc ttggtacaca aaatggatct ggtacaggag
     2221 gatcaacggg acctgatttt taaagagcga gaagaagatt tgaggcgttt gtctcgccca
     2281 ttggaatgtt cttgtttccg aacatctatc tgggatgaaa ccctctataa ggcttggtcc
     2341 agcatagttt atcagctgat tcccttagcg gccgctaagt aagtaaggat ccccagcttg
     2401 gccgccatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2461 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2521 catcaatgta tcttatcatg tctggatcgg gaattaattc ggcgcagcac catggcctga
     2581 aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg
     2641 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2701 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2761 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2821 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2881 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2941 aggaggcttt tttggaggcc taggcttttg caaaaagctg ttaacagctt ggcactggcc
     3001 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     3061 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     3121 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     3181 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     3241 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3301 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3361 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3421 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3481 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3541 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     3601 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3661 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3721 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3781 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3841 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3901 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3961 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4021 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4081 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4141 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4201 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4261 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4321 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4381 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4441 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4501 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4561 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4621 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4681 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4741 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4801 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4861 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4921 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4981 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5041 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5101 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5161 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5221 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5281 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5341 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5401 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5461 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5521 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5581 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5641 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5701 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5761 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5821 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5881 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5941 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6001 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6061 aatttcacac aggaaacagc tatgacatga ttacgaatta a
//
