LOCUS       sequence-225015-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagC (D181N mutant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  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 (D181N)
FEATURES             Location/Qualifiers
     source          1..5918
                     /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      2218..2227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2231..2365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2268..2287)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2322..2341
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2434..2791
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2434..2625
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2457..2477)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2642..2777
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2811..2828)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2811..2827)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2820..2842)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3040..3495
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3127..3146)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3337..3358
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3512..3531)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3631..3653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3691..3709)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3777..3881
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3882..3950,3951..4742)
                     /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(4100..4119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4913..5501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5402..5421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5655..5672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5789..5810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5825..5830,5831..5848,5849..5855)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5863..5879
                     /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     5868..5890
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5887..5903
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5887..5903
                     /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 gtggtgcgtc gacgatgtcc ctgcagtacg gggcggagga gacgcccctc
     1021 gccggcagtt acggcgcggc cgattcgttt ccaaaggact tcggctacgg cgtggaggag
     1081 gaggaagagg aggcggcggc ggcgggcgga ggggttgggg caggggcagg cggtggctgt
     1141 ggtccggggg gcgctgacag ctccaagccg aggattctgc tcatgggact ccggcgcagc
     1201 ggcaagtcct ccatccagaa ggtggtgttt cataagatgt cacccaacga gaccctcttt
     1261 ttggaaagta ccaacaagat ttataaggat gacatttcca atagctcctt tgtgaatttc
     1321 cagatatggg attttcctgg gcaaatggac ttttttgacc caacctttga ctatgagatg
     1381 atcttcaggg gaacaggagc attgatatac gtcattgacg cacaggatga ctacatggag
     1441 gctttaacaa gacttcacat tactgtttct aaagcctaca aagttaaccc agacatgaat
     1501 tttgaggttt ttattcacaa agttaatggt ctgtctgatg atcacaaaat agaaacacag
     1561 agggacattc atcaaagggc caatgatgac cttgcagatg ctgggctaga aaaactccat
     1621 cttagctttt atctgactag tatctatgac cattcaatat ttgaagcctt tagtaaggtg
     1681 gtgcagaaac tcattccaca actgccgacc ttggaaaacc tattaaatat ctttatatca
     1741 aattcaggta ttgaaaaagc ttttctcttt gatgttgtca gcaaaatcta cattgcaaca
     1801 gacagttccc ctgtggatat gcaatcttat gaactttgct gtgacatgat cgatgttgta
     1861 attgatgtgt cttgtatata tgggttaaag gaagatggaa gtggaagtgc ttatgacaaa
     1921 gaatctatgg caattatcaa gctgaataat acaactgtcc tttatttaaa ggaggtgact
     1981 aaatttttgg cactggtctg cattctaagg gaagaaagct ttgaaagaaa aggtttaata
     2041 gactacaact tccactgttt ccgaaaagct attcatgagg tttttgaggt gggtgtgact
     2101 tctcacagga gctgtggtca ccagactagt gcctccagtc tgaaagcgct gacacacaat
     2161 ggcacgccac gaaacgccat ctaggcggcc gctaagtaag taaggatccc cagcttggcc
     2221 gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2281 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2341 caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat
     2401 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     2461 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2521 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2581 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2641 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2701 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2761 aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc
     2821 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2881 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2941 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     3001 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     3061 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3121 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3181 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3241 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3301 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3361 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     3421 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3481 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3541 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3601 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3661 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3721 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3781 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3841 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3901 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3961 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4021 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4081 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4141 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4201 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4261 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4321 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4381 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4441 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4501 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4561 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4621 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4681 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4741 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4801 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4861 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4921 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4981 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5041 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5101 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5161 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5221 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5281 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5341 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5401 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5461 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5521 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5581 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5641 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     5701 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5761 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     5821 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     5881 ttcacacagg aaacagctat gacatgatta cgaattaa
//
