LOCUS       sequence-225029-        5942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express GFP-RagC (C terminal domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5942)
  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 5942)
  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 (CTD)
FEATURES             Location/Qualifiers
     source          1..5942
                     /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      2242..2251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2255..2389
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2292..2311)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2346..2365
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2458..2815
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2458..2649
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2481..2501)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2666..2801
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2835..2852)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2835..2851)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2844..2866)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3064..3519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3151..3170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3361..3382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3536..3555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3655..3677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3715..3733)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3801..3905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3906..3974,3975..4766)
                     /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(4124..4143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4937..5525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5426..5445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5679..5696
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5813..5834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5849..5854,5855..5872,5873..5879)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5887..5903
                     /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     5892..5914
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5911..5927
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5911..5927
                     /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 accaactgcc gaccttggaa
     1741 aacctattaa atatctttat atcaaattca ggtattgaaa aagcttttct ctttgatgtt
     1801 gtcagcaaaa tctacattgc aacagacagt tcccctgtgg atatgcaatc ttatgaactt
     1861 tgctgtgaca tgatcgatgt tgtaattgat gtgtcttgta tatatgggtt aaaggaagat
     1921 ggaagtggaa gtgcttatga caaagaatct atggcaatta tcaagctgaa taatacaact
     1981 gtcctttatt taaaggaggt gactaaattt ttggcactgg tctgcattct aagggaagaa
     2041 agctttgaaa gaaaaggttt aatagactac aacttccact gtttccgaaa agctattcat
     2101 gaggtttttg aggtgggtgt gacttctcac aggagctgtg gtcaccagac tagtgcctcc
     2161 agtctgaaag cgctgacaca caatggcacg ccacgaaacg ccatcttagc ggccgctaag
     2221 taagtaagga tccccagctt ggccgccatg gcccaacttg tttattgcag cttataatgg
     2281 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2341 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggaattaatt
     2401 cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg
     2461 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2521 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2581 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2641 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2701 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2761 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2821 gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2881 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2941 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     3001 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3061 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3121 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3181 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3241 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3301 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3361 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     3421 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3481 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3541 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3601 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3661 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3721 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3781 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3841 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3901 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3961 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4021 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4081 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4141 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4201 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4261 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4321 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4381 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4441 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4501 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4561 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4621 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4681 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4741 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4801 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4861 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4921 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4981 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5041 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5101 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5161 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5221 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5281 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5341 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5401 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5461 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5521 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5581 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5641 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5701 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     5761 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     5821 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     5881 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt
     5941 aa
//
