LOCUS       sequence-225008-        5825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagC (G domain) fused with with RagB (C terminal 
            domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5825)
  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 5825)
  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 Flag RagC(GD)/RagB(CTD) chimera
FEATURES             Location/Qualifiers
     source          1..5825
                     /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"
     CDS             937..960
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2125..2134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2138..2272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2175..2194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2229..2248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2341..2698
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2341..2532
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2364..2384)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2549..2684
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2718..2735)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2718..2734)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2727..2749)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2947..3402
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3034..3053)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3244..3265
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3419..3438)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3538..3560
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3598..3616)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3684..3788
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3789..3857,3858..4649)
                     /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(4007..4026)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4820..5408
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5309..5328
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5562..5579
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5696..5717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5732..5737,5738..5755,5756..5762)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5770..5786
                     /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     5775..5797
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5794..5810
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5794..5810
                     /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 attcaccatg gaaatggact acaaggatga cgatgacaag
      961 ggtggtggtg gtggtgtcga catgagcctg cagtatggcg ccgaggagac ccctctggcc
     1021 ggatcctacg gagcagcaga ctctttccca aaggattttg gctatggcgt ggaagaggag
     1081 gaggaggagg cagcagcagc aggaggagga gtgggagccg gcgccggcgg cggctgtggc
     1141 ccaggcggcg ccgacagctc caagccaagg atcctgctga tgggactgcg gagatctggc
     1201 aagtctagca tccagaaggt ggtgttccac aagatgtctc ccaatgagac cctgtttctg
     1261 gagagcacaa acaagatcta caaggacgac atcagcaact cctctttcgt gaattttcag
     1321 atctgggact tccctggcca gatggacttc tttgatccaa ccttcgatta tgagatgatc
     1381 tttaggggca caggcgccct gatctacgtg atcgacgccc aggacgatta tatggaggcc
     1441 ctgacccgcc tgcacatcac agtgagcaag gcctacaagg tgaaccctga catgaatttc
     1501 gaggtgttta tccacaaggt ggatggcctg tccgacgatc acaagatcga gacccagagg
     1561 gacatccacc agcgcgccaa tgacgatctg gccgatgccg gcctggagaa gctgcacctg
     1621 tccttctacc tgacctctat ctatgatcac agcatcttcg aggccttttc caaggtggtg
     1681 cagaagctga tccccaatgt tcagcagctg gaaatgaacc taaggaattt tgctgaaatt
     1741 atcgaagctg atgaagtact tctttttgag agagctactt ttctggtaat ttctcactat
     1801 cagtgtaaag agcagcgtga tgcccataga tttgagaaaa taagcaacat tattaagcag
     1861 ttcaagctga gctgcagcaa gctggctgcc tctttccaga gtatggaagt caggaactct
     1921 aacttcgctg ctttcattga catctttaca tccaacactt atgtgatggt tgtgatgtct
     1981 gatccgtcca ttccttctgc agctactctg atcaacatcc gcaatgccag gaaacacttt
     2041 gaaaagctgg aaagagtgga tggaccaaag cagtgtcttc tcatgcgcta agcggccgct
     2101 aagtaagtaa ggatccccag cttggccgcc atggcccaac ttgtttattg cagcttataa
     2161 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2221 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgggaatta
     2281 attcggcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
     2341 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2401 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2461 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2521 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2581 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2641 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2701 gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2761 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2821 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     2881 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     2941 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3001 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3061 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3121 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3181 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3241 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg
     3301 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3361 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3421 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3481 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3541 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3601 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3661 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3721 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3781 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3841 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3901 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3961 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4021 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4081 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4141 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4201 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4261 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4321 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4381 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4441 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4501 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4561 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4621 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4681 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4741 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4801 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4861 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4921 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4981 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5041 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5101 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5161 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5221 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5281 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5341 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5401 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5461 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5521 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5581 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5641 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5701 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     5761 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     5821 attaa
//
