LOCUS       sequence-225026-        5111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagB (C terminal domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5111)
  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 5111)
  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 (CTD)
FEATURES             Location/Qualifiers
     source          1..5111
                     /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      1411..1420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1424..1558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1461..1480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1515..1534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1627..1984
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1627..1818
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1650..1670)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1835..1970
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2004..2021)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2004..2020)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2013..2035)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2233..2688
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2320..2339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2530..2551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2705..2724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2824..2846
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2884..2902)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2970..3074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3075..3143,3144..3935)
                     /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(3293..3312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4106..4694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4595..4614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4848..4865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4982..5003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5018..5023,5024..5041,5042..5048)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5056..5072
                     /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     5061..5083
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5080..5096
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5080..5096
                     /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 caatgttcag cagctggaaa tgaacctaag gaattttgct
     1021 gaaattatcg aagctgatga agtacttctt tttgagagag ctacttttct ggtaatttct
     1081 cactatcagt gtaaagagca gcgtgatgcc catagatttg agaaaataag caacattatt
     1141 aagcagttca agctgagctg cagcaagctg gctgcctctt tccagagtat ggaagtcagg
     1201 aactctaact tcgctgcttt cattgacatc tttacatcca acacttatgt gatggttgtg
     1261 atgtctgatc cgtccattcc ttctgcagct actctgatca acatccgcaa tgccaggaaa
     1321 cactttgaaa agctggaaag agtggatgga ccaaagcagt gtcttctcat gcgcttagcg
     1381 gccgctaagt aagtaaggat ccccagcttg gccgccatgg cccaacttgt ttattgcagc
     1441 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1501 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgg
     1561 gaattaattc ggcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg
     1621 taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     1681 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     1741 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     1801 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     1861 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     1921 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     1981 caaaaagctg ttaacagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2041 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2101 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2161 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     2221 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2281 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2341 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2401 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     2461 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2521 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     2581 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2641 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     2701 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     2761 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2821 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     2881 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2941 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3001 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3061 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3121 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3181 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3241 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3301 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3361 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3421 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3481 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3541 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3601 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3661 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3721 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3781 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3841 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3901 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3961 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4021 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4081 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4141 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4201 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4261 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4321 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4381 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4441 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4501 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4561 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4621 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4681 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4741 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4801 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4861 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4921 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4981 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     5041 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga
     5101 ttacgaatta a
//
