LOCUS       sequence-225023-        5360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagB (G domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5360)
  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 5360)
  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)
FEATURES             Location/Qualifiers
     source          1..5360
                     /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      1660..1669
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1673..1807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1710..1729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1764..1783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1876..2233
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1876..2067
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1899..1919)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2084..2219
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2253..2270)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2253..2269)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2262..2284)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2482..2937
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2569..2588)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2779..2800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2954..2973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3073..3095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3133..3151)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3219..3323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3324..3392,3393..4184)
                     /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(3542..3561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4355..4943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4844..4863
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5097..5114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5231..5252
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5267..5272,5273..5290,5291..5297)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5305..5321
                     /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     5310..5332
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5329..5345
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5329..5345
                     /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 cgaagaatct gactctgaga aaacgacgga gaaagaaaat
     1021 ctggggccga gaatggatcc accactaggg gaaccggaag gatcgcttgg gtgggtgcta
     1081 ccaaatacag ccatgaagaa aaaggtgctg ttgatgggta aaagtgggtc tggtaagacc
     1141 agcatgaggt ctattatctt tgcaaattat attgccagag acacacgtcg ccttggcgca
     1201 acaattgatg tagaacattc tcatgttcga tttctgggaa acctggtatt gaacctgtgg
     1261 gattgtggtg ggcaagacac cttcatggaa aattatttca ctagccaacg ggacaacatc
     1321 ttccgaaatg tggaggttct gatttatgtc tttgatgtgg agagccgcga actggaaaag
     1381 gacatgcact attaccaatc atgcctggag gccattctgc agaattctcc agatgccaaa
     1441 atattttgct tggtacacaa aatggatctg gtacaggagg atcaacggga cctgattttt
     1501 aaagagcgag aagaagattt gaggcgtttg tctcgcccat tggaatgttc ttgtttccga
     1561 acatctatct gggatgaaac cctctataag gcttggtcca gcatagttta tcagctgatt
     1621 cccttagcgg ccgctaagta agtaaggatc cccagcttgg ccgccatggc ccaacttgtt
     1681 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     1741 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     1801 ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg aaagaggaac
     1861 ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     1921 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     1981 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2041 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2101 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2161 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2221 aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca acgtcgtgac
     2281 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2341 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2401 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2461 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2521 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2581 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2641 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2701 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2761 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2821 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2881 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     2941 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3001 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3061 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3121 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3181 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3241 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3301 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3361 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3421 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3481 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3541 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3601 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3661 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3721 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3781 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3841 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3901 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3961 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4021 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4081 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4141 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4201 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4261 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4981 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5041 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5101 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5161 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5221 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5281 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5341 atgacatgat tacgaattaa
//
