LOCUS       sequence-194521-        7951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RagA(Q66L)-RagC(Q120L).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7951)
  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 7951)
  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: pK RagA(Q66L)-RagC(Q120L)
FEATURES             Location/Qualifiers
     source          1..7951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     907..926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(946..1023)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1229..1248
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1332..1350
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1351..1375
                     /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)."
     RBS             1390..1412
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1431..1460
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1491..1781
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     CDS             1818..1841
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        2855..2873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2874..2898
                     /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)."
     CDS             4147..4164
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4217..4235)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4231..4278
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4315..4770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4402..4421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(4862..5677)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4862..5677)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5585..5604
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5669..5688
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5799..6387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6288..6307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6541..6558
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6573..6712
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6638..6657)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6798..6820
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6814..7005)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7814..7951,1..945))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
       61 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
      121 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
      181 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
      241 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
      301 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
      361 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
      421 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
      481 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
      541 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
      601 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
      661 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
      721 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
      781 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
      841 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
      901 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
      961 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     1021 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     1081 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     1141 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     1201 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     1261 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     1321 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1381 agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc catcaccatc
     1441 accatcacca tcaccatcat agccgccgcc gtcgccgccg tcgcccgcgg atgtcggact
     1501 cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct gagactcaca
     1561 tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa aagaccactc
     1621 ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg gactccttaa
     1681 gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat ttggacatgg
     1741 aggataacga tattattgag gctcacagag aacagattgg tccgcggggt ggtggcgacc
     1801 atcctccaaa atcggatctg gaagttctgt tccaggggcc cggatccatg ccgaacaccg
     1861 ctatgaaaaa aaaagtgctg ctgatgggta aatcgggctc gggtaaaacc tcaatgcgta
     1921 gtattatctt tgcgaactat attgcgcgtg atacccgtcg cctgggtgca acgatcgacg
     1981 tggaacatag ccacgttcgc tttctgggta acctggtgct gaatctgtgg gattgcggcg
     2041 gtctggacac ctttatggaa aattatttca cgtcgcaacg tgataacatt tttcgcaatg
     2101 tcgaagtgct gatctacgtt ttcgatgtcg aatcccgtga actggaaaaa gacatgcatt
     2161 attaccagtc ctgcctggaa gcaattctgc aaaactcacc ggatgctaaa atcttttgtc
     2221 tggtccacaa aatggacctg gtgcaggaag atcaacgtga cctgattttc aaagaacgcg
     2281 aagaagatct gcgtcgcctg tcgcgtccgc tggaatgcgc gtgttttcgc accagcatct
     2341 gggacgaaac gctgtataaa gcctggagct ctattgttta ccagctgatc ccgaatgtcc
     2401 agcaactgga aatgaacctg cgtaatttcg cacagattat cgaagctgat gaagtgctgc
     2461 tgtttgaacg cgcaaccttc ctggttattt cccattatca gtgcaaagaa caacgtgatg
     2521 tgcaccgctt cgaaaaaatc tcaaacatca tcaaacagtt caaactgagt tgttccaaac
     2581 tggcggcctc atttcagtcg atggaagtcc gcaactctaa ttttgcagct ttcattgata
     2641 tcttcaccag taatacgtac gttatggtgg ttatgagcga cccgtctatt ccgagtgcgg
     2701 ccaccctgat taacatccgt aatgcccgca aacattttga aaaactggaa cgtgttgatg
     2761 gcccgaaaca cagcctgctg atgcgctgag cggccgcata atgcttaagt cgaacagaaa
     2821 gtaatcgtat tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg
     2881 tgagcggata acaattcccc atcttagtat attagttaag tataagaagg agatatacat
     2941 atgtcactgc aatacggtgc tgaagaaacg ccgctggctg gtagttatgg tgctgctgat
     3001 tcattcccga aagattttgg ttatggcgtt gaagaagaag aagaagaagc ggcggcggcg
     3061 ggcggcggcg tgggtgcagg tgccggtggc ggttgtggcc cgggcggtgc cgatagctct
     3121 aaaccgcgta ttctgctgat gggcctgcgt cgcagtggta aaagttccat ccagaaagtg
     3181 gtttttcaca aaatgtcgcc gaacgaaacc ctgttcctgg aaagcacgaa caaaatctac
     3241 aaagatgaca tcagcaactc atcgtttgtg aatttccaga tttgggattt tccgggtctg
     3301 atggatttct ttgacccgac ctttgactac gaaatgatct tccgtggcac gggtgcgctg
     3361 atttatgtta tcgatgccca agatgactac atggaagcac tgacccgcct gcatattacg
     3421 gtgtcaaaag cgtataaagt taacccggat atgaacttcg aagttttcat ccataaagtg
     3481 gatggcctgt cggatgacca caaaattgaa acccagcgtg atatccatca acgcgcaaac
     3541 gatgacctgg cagacgctgg tctggaaaaa ctgcacctga gtttttatct gaccagcatt
     3601 tacgatcatt caatctttga agcgttcagc aaagtcgtgc agaaactgat tccgcaactg
     3661 ccgacgctgg aaaacctgct gaacatcttc atcagtaact ccggcatcga aaaagccttt
     3721 ctgttcgatg ttgtctctaa aatctacatc gcaaccgata gctctccggt ggacatgcag
     3781 agttacgaac tgtgctgtga tatgattgac gtggttatcg atgtttcctg catttatggt
     3841 ctgaaagaag atggctcagg ttcggcttac gacaaagaat caatggcgat tatcaaactg
     3901 aacaatacca cggtcctgta tctgaaagaa gtgaccaaat ttctggctct ggtgtgcatt
     3961 ctgcgtgaag aatctttcga acgcaaaggc ctgatcgatt acaacttcca ttgtttccgt
     4021 aaagccatcc acgaagtttt tgaagtcggc gtgacgagcc atcgctcttg cggtcaccaa
     4081 acgagcgcaa gcagcctgaa agccctgacc cacaatggca ccccgcgtaa tgcaatctga
     4141 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     4201 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4261 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     4321 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4381 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4441 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4501 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4561 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4621 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4681 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4741 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     4801 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     4861 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     4921 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4981 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     5041 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     5101 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     5161 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     5221 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     5281 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     5341 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     5401 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     5461 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     5521 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     5581 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     5641 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     5701 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5761 tgagcgtcag accccgtaga aaagatcaaa gaatcttctt gagatccttt ttttctgcgc
     5821 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5881 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5941 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6001 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6061 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6121 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6181 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6241 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6301 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6361 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6421 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6481 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6541 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     6601 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     6661 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     6721 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6781 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6841 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6901 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6961 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     7021 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     7081 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     7141 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     7201 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     7261 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     7321 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     7381 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     7441 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     7501 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     7561 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     7621 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     7681 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     7741 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     7801 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7861 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     7921 ttttcaccag tgagacgggc aacagctgat t
//
