LOCUS       sequence-197063-        5965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA-RagC(S75N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5965)
  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 5965)
  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-RagC(S75N)
FEATURES             Location/Qualifiers
     source          1..5965
                     /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      2395..2404
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2408..2542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2445..2464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2499..2518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2611..2968
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2611..2802
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2634..2654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2819..2954
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2988..3005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2988..3004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2997..3019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3217..3672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3304..3323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3514..3535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3689..3708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3808..3830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3868..3886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3954..4058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4059..4127,4128..4919)
                     /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(4277..4296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5090..5678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5579..5598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5832..5849
                     /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 gatgtccctg cagtacgggg cggaggagac gcccctcgcc
     1201 ggcagttacg gcgcggccga ttcgtttcca aaggacttcg gctacggcgt ggaggaggag
     1261 gaagaggagg cggcggcggc gggcggaggg gttggggcag gggcaggcgg tggctgtggt
     1321 ccggggggcg ctgacagctc caagccgagg attctgctca tgggactccg gcgcagcggc
     1381 aagaactcca tccagaaggt ggtgtttcat aagatgtcac ccaacgagac cctctttttg
     1441 gaaagtacca acaagattta taaggatgac atttccaata gctcctttgt gaatttccag
     1501 atatgggatt ttcctgggca aatggacttt tttgacccaa cctttgacta tgagatgatc
     1561 ttcaggggaa caggagcatt gatatacgtc attgacgcac aggatgacta catggaggct
     1621 ttaacaagac ttcacattac tgtttctaaa gcctacaaag ttaacccaga catgaatttt
     1681 gaggttttta ttcacaaagt tgatggtctg tctgatgatc acaaaataga aacacagagg
     1741 gacattcatc aaagggccaa tgatgacctt gcagatgctg ggctagaaaa actccatctt
     1801 agcttttatc tgactagtat ctatgaccat tcaatatttg aagcctttag taaggtggtg
     1861 cagaaactca ttccacaact gccgaccttg gaaaacctat taaatatctt tatatcaaat
     1921 tcaggtattg aaaaagcttt tctctttgat gttgtcagca aaatctacat tgcaacagac
     1981 agttcccctg tggatatgca atcttatgaa ctttgctgtg acatgatcga tgttgtaatt
     2041 gatgtgtctt gtatatatgg gttaaaggaa gatggaagtg gaagtgctta tgacaaagaa
     2101 tctatggcaa ttatcaagct gaataataca actgtccttt atttaaagga ggtgactaaa
     2161 tttttggcac tggtctgcat tctaagggaa gaaagctttg aaagaaaagg tttaatagac
     2221 tacaacttcc actgtttccg aaaagctatt catgaggttt ttgaggtggg tgtgacttct
     2281 cacaggagct gtggtcacca gactagtgcc tccagtctga aagcgctgac acacaatggc
     2341 acgccacgaa acgccatcta ggcggccgct aagtaagtaa ggatccccag cttggccgcc
     2401 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2461 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2521 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac
     2581 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     2641 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2701 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2761 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2821 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2881 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2941 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt
     3001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     3061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3121 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3541 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4741 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4921 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4981 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5161 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5221 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5701 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5761 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5821 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5881 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5941 aagcgggcag tgagcgcaac gcaat
//
