LOCUS       sequence-197070-        5707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA-RagA(T21N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5707)
  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 5707)
  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 HA-RagA(T21N)
FEATURES             Location/Qualifiers
     source          1..5707
                     /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"
     regulatory      1093..1102
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1105..1131
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2137..2146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2150..2284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2187..2206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2241..2260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2353..2710
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2353..2544
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2376..2396)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2561..2696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2730..2747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2730..2746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2739..2761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2959..3414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3046..3065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3256..3277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3431..3450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3550..3572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3610..3628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3696..3800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3801..3869,3870..4661)
                     /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(4019..4038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4832..5420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5321..5340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5574..5591
                     /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 attcccgaac cgacagtcgg tctcttcacc
     1081 aaggccattc gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat
     1141 ggtggtggtg gtgggtcgac gatgccaaat acagccatga agaaaaaggt gctgctgatg
     1201 gggaagagcg ggtcggggaa gaacagcatg aggtcgataa tcttcgccaa ttacattgct
     1261 cgcgacaccc ggcgcctggg ggccaccatt gacgtggaac actcccacgt ccgattccta
     1321 gggaacctgg tgctgaacct gtgggactgt ggcggtcagg acaccttcat ggaaaattac
     1381 ttcaccagcc agcgagacaa tatcttccgt aacgtggaag ttttgattta cgtgtttgac
     1441 gtggagagcc gcgaactgga aaaggacatg cattattacc agtcgtgtct ggaggccatc
     1501 ctccagaact ctcctgacgc caaaatcttc tgcctggtgc acaaaatgga tctggttcag
     1561 gaggatcagc gtgacctgat ttttaaagag cgagaggaag acctgaggcg tctgtctcgc
     1621 ccgctggagt gtgcttgttt tcgaacgtcc atctgggatg agacgctcta caaagcctgg
     1681 tccagcatcg tctaccagct gattcccaac gttcagcagc tggagatgaa cctcaggaat
     1741 tttgcccaaa tcattgaggc cgatgaagtt ctgctgttcg aaagagctac attcttggtt
     1801 atttcccact accagtgcaa agagcagcgc gacgtccacc ggtttgagaa gatcagcaac
     1861 atcatcaaac agttcaagct gagctgcagt aaattggccg cttccttcca gagcatggaa
     1921 gttaggaatt ccaacttcgc tgctttcatc gacatcttca cctcaaatac gtacgtgatg
     1981 gtggtcatgt cagatccgtc gatcccttct gcggccactc tgatcaacat tcgcaatgcc
     2041 cggaaacact ttgagaagct ggagagagtg gatggcccca agcacagtct ccttatgcgt
     2101 tgagcggccg ctaagtaagt aaggatcccc agcttggccg ccatggccca acttgtttat
     2161 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2221 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2281 gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg
     2341 gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2401 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2461 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2521 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2581 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2641 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2701 cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg tcgtgactgg
     2761 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2821 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2881 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2941 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3001 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3061 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3121 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3181 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3241 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3301 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3361 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3481 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3541 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3601 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3661 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3721 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3781 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3841 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3901 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3961 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4021 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4081 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4141 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4201 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4261 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4321 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4381 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4441 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4501 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4561 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4621 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4681 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4741 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4801 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4861 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4921 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4981 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5041 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5101 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5161 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5221 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5281 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5341 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5401 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5461 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5521 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5581 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5641 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5701 acgcaat
//
