LOCUS       sequence-197067-        5660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flag-RagA(T21N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5660)
  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 5660)
  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-RagA(T21N)
FEATURES             Location/Qualifiers
     source          1..5660
                     /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      2090..2099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2103..2237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2140..2159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2194..2213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2306..2663
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2306..2497
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2329..2349)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2514..2649
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2683..2700)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2683..2699)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2692..2714)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2912..3367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2999..3018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3209..3230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3384..3403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3503..3525
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3563..3581)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3649..3753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3754..3822,3823..4614)
                     /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(3972..3991)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4785..5373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5274..5293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5527..5544
                     /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 gacgatgcca aatacagcca tgaagaaaaa
     1141 ggtgctgctg atggggaaga gcgggtcggg gaagaacagc atgaggtcga taatcttcgc
     1201 caattacatt gctcgcgaca cccggcgcct gggggccacc attgacgtgg aacactccca
     1261 cgtccgattc ctagggaacc tggtgctgaa cctgtgggac tgtggcggtc aggacacctt
     1321 catggaaaat tacttcacca gccagcgaga caatatcttc cgtaacgtgg aagttttgat
     1381 ttacgtgttt gacgtggaga gccgcgaact ggaaaaggac atgcattatt accagtcgtg
     1441 tctggaggcc atcctccaga actctcctga cgccaaaatc ttctgcctgg tgcacaaaat
     1501 ggatctggtt caggaggatc agcgtgacct gatttttaaa gagcgagagg aagacctgag
     1561 gcgtctgtct cgcccgctgg agtgtgcttg ttttcgaacg tccatctggg atgagacgct
     1621 ctacaaagcc tggtccagca tcgtctacca gctgattccc aacgttcagc agctggagat
     1681 gaacctcagg aattttgccc aaatcattga ggccgatgaa gttctgctgt tcgaaagagc
     1741 tacattcttg gttatttccc actaccagtg caaagagcag cgcgacgtcc accggtttga
     1801 gaagatcagc aacatcatca aacagttcaa gctgagctgc agtaaattgg ccgcttcctt
     1861 ccagagcatg gaagttagga attccaactt cgctgctttc atcgacatct tcacctcaaa
     1921 tacgtacgtg atggtggtca tgtcagatcc gtcgatccct tctgcggcca ctctgatcaa
     1981 cattcgcaat gcccggaaac actttgagaa gctggagaga gtggatggcc ccaagcacag
     2041 tctccttatg cgttgagcgg ccgctaagta agtaaggatc cccagcttgg ccgccatggc
     2101 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2161 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2221 cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg
     2281 aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     2341 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2401 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2461 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2521 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2581 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     2641 ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca
     2701 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2761 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2821 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2881 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3121 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3241 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     3361 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3421 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3481 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3541 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3601 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3661 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3721 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3781 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3841 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3901 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3961 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4021 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4081 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4141 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4201 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4261 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4321 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4381 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4441 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4501 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4561 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4621 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4681 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4741 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4801 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4861 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4921 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4981 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5041 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5101 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5161 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5221 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5281 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5341 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5401 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5461 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5521 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     5581 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5641 ggcagtgagc gcaacgcaat
//
