LOCUS       sequence-225000-        5429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagC (G domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5429)
  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 5429)
  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)
FEATURES             Location/Qualifiers
     source          1..5429
                     /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      1729..1738
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1742..1876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1779..1798)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1833..1852
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1945..2302
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1945..2136
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1968..1988)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2153..2288
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2322..2339)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2322..2338)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2331..2353)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2551..3006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2638..2657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2848..2869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3023..3042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3142..3164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3202..3220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3288..3392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3393..3461,3462..4253)
                     /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(3611..3630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4424..5012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4913..4932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5166..5183
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5300..5321
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5336..5341,5342..5359,5360..5366)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5374..5390
                     /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     5379..5401
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5398..5414
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5398..5414
                     /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 ctccctgcag tacggggcgg aggagacgcc cctcgccggc
     1021 agttacggcg cggccgattc gtttccaaag gacttcggct acggcgtgga ggaggaggaa
     1081 gaggaggcgg cggcggcggg cggaggggtt ggggcagggg caggcggtgg ctgtggtccg
     1141 gggggcgctg acagctccaa gccgaggatt ctgctcatgg gactccggcg cagcggcaag
     1201 tcctccatcc agaaggtggt gtttcataag atgtcaccca acgagaccct ctttttggaa
     1261 agtaccaaca agatttataa ggatgacatt tccaatagct cctttgtgaa tttccagata
     1321 tgggattttc ctgggcaaat ggactttttt gacccaacct ttgactatga gatgatcttc
     1381 aggggaacag gagcattgat atacgtcatt gacgcacagg atgactacat ggaggcttta
     1441 acaagacttc acattactgt ttctaaagcc tacaaagtta acccagacat gaattttgag
     1501 gtttttattc acaaagttga tggtctgtct gatgatcaca aaatagaaac acagagggac
     1561 attcatcaaa gggccaatga tgaccttgca gatgctgggc tagaaaaact ccatcttagc
     1621 ttttatctga ctagtatcta tgaccattca atatttgaag cctttagtaa ggtggtgcag
     1681 aaactcattc cattagcggc cgctaagtaa gtaaggatcc ccagcttggc cgccatggcc
     1741 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1801 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1861 ttatcatgtc tggatcggga attaattcgg cgcagcacca tggcctgaaa taacctctga
     1921 aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca
     1981 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2041 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2101 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2161 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2221 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     2281 tggaggccta ggcttttgca aaaagctgtt aacagcttgg cactggccgt cgttttacaa
     2341 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     2401 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     2461 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2521 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     2581 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2641 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2701 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2761 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2821 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2881 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2941 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3001 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3061 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3121 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3181 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3241 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3301 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3361 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3421 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3481 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3541 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3601 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3661 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3721 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3781 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3841 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3901 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3961 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4021 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     4081 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4141 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4201 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4261 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4321 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4381 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4441 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4501 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4561 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4621 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4681 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4741 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4801 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4861 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4921 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4981 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5041 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5101 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5161 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     5221 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5281 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     5341 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     5401 gaaacagcta tgacatgatt acgaattaa
//
