LOCUS       sequence-225006-        6170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express GFP-RagC (G domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6170)
  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 6170)
  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 RagC (GD)
FEATURES             Location/Qualifiers
     source          1..6170
                     /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      2470..2479
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2483..2617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2520..2539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2574..2593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2686..3043
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2686..2877
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2709..2729)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2894..3029
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3063..3080)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3063..3079)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3072..3094)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3292..3747
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3379..3398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3589..3610
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3764..3783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3883..3905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3943..3961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4029..4133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4134..4202,4203..4994)
                     /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(4352..4371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5165..5753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5654..5673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5907..5924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6041..6062
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6077..6082,6083..6100,6101..6107)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6115..6131
                     /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     6120..6142
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6139..6155
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6139..6155
                     /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 actccctgca gtacggggcg
     1741 gaggagacgc ccctcgccgg cagttacggc gcggccgatt cgtttccaaa ggacttcggc
     1801 tacggcgtgg aggaggagga agaggaggcg gcggcggcgg gcggaggggt tggggcaggg
     1861 gcaggcggtg gctgtggtcc ggggggcgct gacagctcca agccgaggat tctgctcatg
     1921 ggactccggc gcagcggcaa gtcctccatc cagaaggtgg tgtttcataa gatgtcaccc
     1981 aacgagaccc tctttttgga aagtaccaac aagatttata aggatgacat ttccaatagc
     2041 tcctttgtga atttccagat atgggatttt cctgggcaaa tggacttttt tgacccaacc
     2101 tttgactatg agatgatctt caggggaaca ggagcattga tatacgtcat tgacgcacag
     2161 gatgactaca tggaggcttt aacaagactt cacattactg tttctaaagc ctacaaagtt
     2221 aacccagaca tgaattttga ggtttttatt cacaaagttg atggtctgtc tgatgatcac
     2281 aaaatagaaa cacagaggga cattcatcaa agggccaatg atgaccttgc agatgctggg
     2341 ctagaaaaac tccatcttag cttttatctg actagtatct atgaccattc aatatttgaa
     2401 gcctttagta aggtggtgca gaaactcatt ccattagcgg ccgctaagta agtaaggatc
     2461 cccagcttgg ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag
     2521 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2581 gtccaaactc atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc
     2641 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
     2701 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2761 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2821 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2881 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2941 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3001 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg
     3061 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3121 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3181 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     3241 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3301 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3361 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3421 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3481 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3541 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3601 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     3661 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3721 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3781 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3841 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3901 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3961 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4021 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4081 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4141 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4201 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4261 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4321 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4381 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4441 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4501 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4561 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4621 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4681 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4741 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4801 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4861 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4921 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4981 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5041 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5101 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5161 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5221 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5281 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5341 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5401 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5461 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5521 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5581 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5641 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5701 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5761 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5821 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5881 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5941 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6001 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     6061 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6121 gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattaa
//
