LOCUS       sequence-197057-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-RagC(S75N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  AUTHORS   Shen K, Choe A, Sabatini DM
  TITLE     Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 
            to Respond Rapidly to Amino Acid Availability.
  JOURNAL   Mol Cell. 2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026.
  PUBMED    29056322
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"; journalName: "Mol Cell. 
            2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026."
COMMENT     Sequence Label: pRK5 Flag-RagC(S75N)
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        144..523
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        524..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     678..698
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..748
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        958..976
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     958..975
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     989..1008
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1067..1090
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2348..2357
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2361..2495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2398..2417)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2452..2471
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2564..2921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2564..2755
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2587..2607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2772..2907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2941..2958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2941..2957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2950..2972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3170..3625
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3257..3276)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3467..3488
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3642..3661)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3761..3783
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3821..3839)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3907..4011
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4012..4080,4081..4872)
                     /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(4230..4249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5043..5631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5532..5551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5785..5802
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
      121 tacgaattaa ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat
      181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      721 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      781 ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg
      841 gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt
      901 ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
      961 taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag
     1021 gtccaactgc acctcggttc tatcgattga attcaccatg gaaatggact acaaggatga
     1081 cgatgacaag ggtggtggtg gtggtgcgtc gacgatgtcc ctgcagtacg gggcggagga
     1141 gacgcccctc gccggcagtt acggcgcggc cgattcgttt ccaaaggact tcggctacgg
     1201 cgtggaggag gaggaagagg aggcggcggc ggcgggcgga ggggttgggg caggggcagg
     1261 cggtggctgt ggtccggggg gcgctgacag ctccaagccg aggattctgc tcatgggact
     1321 ccggcgcagc ggcaagaact ccatccagaa ggtggtgttt cataagatgt cacccaacga
     1381 gaccctcttt ttggaaagta ccaacaagat ttataaggat gacatttcca atagctcctt
     1441 tgtgaatttc cagatatggg attttcctgg gcaaatggac ttttttgacc caacctttga
     1501 ctatgagatg atcttcaggg gaacaggagc attgatatac gtcattgacg cacaggatga
     1561 ctacatggag gctttaacaa gacttcacat tactgtttct aaagcctaca aagttaaccc
     1621 agacatgaat tttgaggttt ttattcacaa agttgatggt ctgtctgatg atcacaaaat
     1681 agaaacacag agggacattc atcaaagggc caatgatgac cttgcagatg ctgggctaga
     1741 aaaactccat cttagctttt atctgactag tatctatgac cattcaatat ttgaagcctt
     1801 tagtaaggtg gtgcagaaac tcattccaca actgccgacc ttggaaaacc tattaaatat
     1861 ctttatatca aattcaggta ttgaaaaagc ttttctcttt gatgttgtca gcaaaatcta
     1921 cattgcaaca gacagttccc ctgtggatat gcaatcttat gaactttgct gtgacatgat
     1981 cgatgttgta attgatgtgt cttgtatata tgggttaaag gaagatggaa gtggaagtgc
     2041 ttatgacaaa gaatctatgg caattatcaa gctgaataat acaactgtcc tttatttaaa
     2101 ggaggtgact aaatttttgg cactggtctg cattctaagg gaagaaagct ttgaaagaaa
     2161 aggtttaata gactacaact tccactgttt ccgaaaagct attcatgagg tttttgaggt
     2221 gggtgtgact tctcacagga gctgtggtca ccagactagt gcctccagtc tgaaagcgct
     2281 gacacacaat ggcacgccac gaaacgccat ctaggcggcc gctaagtaag taaggatccc
     2341 cagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca
     2401 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2461 ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat
     2521 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc
     2581 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2641 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2701 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2761 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2821 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2881 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc
     2941 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3001 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3061 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     3121 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3181 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3241 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3301 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3361 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3421 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3481 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg
     3541 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3601 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     3661 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3721 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3781 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3841 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3901 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3961 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4021 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4081 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4141 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4201 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4261 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4321 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4381 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4441 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4501 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4561 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4621 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4681 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4741 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4801 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4861 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4921 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4981 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5161 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5401 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5701 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5761 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5821 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5881 tttcccgact ggaaagcggg cagtgagcgc aacgcaat
//
