LOCUS       sequence-224853-        5936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagB (Gdomain) fused with RagC (C terminal domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5936)
  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 5936)
  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 RagB(GD)/RagC(CTD) chimera
FEATURES             Location/Qualifiers
     source          1..5936
                     /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      2236..2245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2249..2383
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2286..2305)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2340..2359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2452..2809
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2452..2643
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2475..2495)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2660..2795
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2829..2846)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2829..2845)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2838..2860)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3058..3513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3145..3164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3355..3376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3530..3549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3649..3671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3709..3727)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3795..3899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3900..3968,3969..4760)
                     /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(4118..4137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4931..5519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5420..5439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5673..5690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5807..5828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5843..5848,5849..5866,5867..5873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5881..5897
                     /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     5886..5908
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5905..5921
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5905..5921
                     /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 catggaagaa tctgactctg agaaaacgac ggagaaagaa
     1021 aatctggggc cgagaatgga tccaccacta ggggaaccgg aaggatcgct tgggtgggtg
     1081 ctaccaaata cagccatgaa gaaaaaggtg ctgttgatgg gtaaaagtgg gtctggtaag
     1141 accagcatga ggtctattat ctttgcaaat tatattgcca gagacacacg tcgccttggc
     1201 gcaacaatac tagaccgtat acatagtctt caaattaata gcagtttgag cacctactct
     1261 ctcgtagact ctgttggaaa tacaaagaca tttgatgtag aacattctca tgttcgattt
     1321 ctgggaaacc tggtattgaa cctgtgggat tgtggtgggc aagacacctt catggaaaat
     1381 tatttcacta gccaacggga caacatcttc cgaaatgtgg aggttctgat ttatgtcttt
     1441 gatgtggaga gccgcgaact ggaaaaggac atgcactatt accaatcatg cctggaggcc
     1501 attctgcaga attctccaga tgccaaaata ttttgcttgg tacacaaaat ggatctggta
     1561 caggaggatc aacgggacct gatttttaaa gagcgagaag aagatttgag gcgtttgtct
     1621 cgcccattgg aatgttcttg tttccgaaca tctatctggg atgaaaccct ctataaggct
     1681 tggtccagca tagtttatca gctgattccc cagctgccta cactggagaa cctgctgaat
     1741 atctttatct ctaacagcgg catcgagaag gccttcctgt ttgacgtggt gagcaagatc
     1801 tacatcgcca ccgacagctc ccccgtggat atgcagtcct atgagctgtg ctgtgacatg
     1861 atcgacgtgg tcatcgacgt gagctgcatc tacggcctga aggaggacgg atccggatct
     1921 gcctatgata aggagagcat ggccatcatc aagctgaaca ataccacagt gctgtacctg
     1981 aaggaggtga ccaagttcct ggccctggtg tgcatcctgc gggaggagtc ctttgagaga
     2041 aagggcctga tcgactataa cttccactgt tttcggaagg ccatccacga ggtgttcgaa
     2101 gtgggcgtga caagccacag atcctgcggc caccagacct ccgcatcatc actgaaggca
     2161 ctgacccaca acggcacacc acgaaacgct atcctcgagt aggcggccgc taagtaagta
     2221 aggatcccca gcttggccgc catggcccaa cttgtttatt gcagcttata atggttacaa
     2281 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2341 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcgggaatt aattcggcgc
     2401 agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg
     2461 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2521 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2581 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     2641 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     2701 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     2761 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctgttaac
     2821 agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     2881 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2941 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     3001 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     3061 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3121 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3181 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3241 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3301 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3361 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag
     3421 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3481 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     3541 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     3601 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3661 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     3721 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     3781 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3841 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3901 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3961 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4021 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4081 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4141 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4201 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4261 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4321 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4381 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4441 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4501 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4561 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4621 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4681 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4741 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4801 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4861 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4921 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4981 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5041 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5101 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5161 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5221 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5281 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5341 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5401 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5461 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5521 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5581 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5641 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5701 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5761 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5821 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5881 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg aattaa
//
