LOCUS       sequence-197074-        7469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RagA-RagC(D181N).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  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 7469)
  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: pETDuet RagA-RagC(D181N)
FEATURES             Location/Qualifiers
     source          1..7469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1045..1068
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        1137..1155
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1156..1180
                     /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             2435..2479
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2517..2535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2531..2578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2615..3070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2702..2721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(3185..3976,3977..4045))
                     /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     3808..3827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4046..4138)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4219..4807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4708..4727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4961..4978
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4993..5132
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5218..5240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5234..5425)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5997..7079)
                     /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"
     primer_bind     7041..7060
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7080..7157)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7363..7382
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gcgccgaaca ccgctatgaa aaaaaaagtg
      121 ctgctgatgg gtaaatcggg ctcgggtaaa acctcaatgc gtagtattat ctttgcgaac
      181 tatattgcgc gtgatacccg tcgcctgggt gcaacgatcg acgtggaaca tagccacgtt
      241 cgctttctgg gtaacctggt gctgaatctg tgggattgcg gcggtcagga cacctttatg
      301 gaaaattatt tcacgtcgca acgtgataac atttttcgca atgtcgaagt gctgatctac
      361 gttttcgatg tcgaatcccg tgaactggaa aaagacatgc attattacca gtcctgcctg
      421 gaagcaattc tgcaaaactc accggatgct aaaatctttt gtctggtcca caaaatggac
      481 ctggtgcagg aagatcaacg tgacctgatt ttcaaagaac gcgaagaaga tctgcgtcgc
      541 ctgtcgcgtc cgctggaatg cgcgtgtttt cgcaccagca tctgggacga aacgctgtat
      601 aaagcctgga gctctattgt ttaccagctg atcccgaatg tccagcaact ggaaatgaac
      661 ctgcgtaatt tcgcacagat tatcgaagct gatgaagtgc tgctgtttga acgcgcaacc
      721 ttcctggtta tttcccatta tcagtgcaaa gaacaacgtg atgtgcaccg cttcgaaaaa
      781 atctcaaaca tcatcaaaca gttcaaactg agttgttcca aactggcggc ctcatttcag
      841 tcgatggaag tccgcaactc taattttgca gctttcattg atatcttcac cagtaatacg
      901 tacgttatgg tggttatgag cgacccgtct attccgagtg cggccaccct gattaacatc
      961 cgtaatgccc gcaaacattt tgaaaaactg gaacgtgttg atggcccgaa acacagcctg
     1021 ctgatgcgcg gtagcggtgg cagccatcac catcatcatc accatcatta agcggccgca
     1081 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat
     1141 acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta
     1201 agtataagaa ggagatatac atatgtcact gcaatacggt gctgaagaaa cgccgctggc
     1261 tggtagttat ggtgctgctg attcattccc gaaagatttt ggttatggcg ttgaagaaga
     1321 agaagaagaa gcggcggcgg cgggcggcgg cgtgggtgca ggtgccggtg gcggttgtgg
     1381 cccgggcggt gccgatagct ctaaaccgcg tattctgctg atgggcctgc gtcgcagtgg
     1441 taaaagttcc atccagaaag tggtttttca caaaatgtcg ccgaacgaaa ccctgttcct
     1501 ggaaagcacg aacaaaatct acaaagatga catcagcaac tcatcgtttg tgaatttcca
     1561 gatttgggat tttccgggtc aaatggattt ctttgacccg acctttgact acgaaatgat
     1621 cttccgtggc acgggtgcgc tgatttatgt tatcgatgcc caagatgact acatggaagc
     1681 actgacccgc ctgcatatta cggtgtcaaa agcgtataaa gttaacccgg atatgaactt
     1741 cgaagttttc atccataaag tgaatggcct gtcggatgac cacaaaattg aaacccagcg
     1801 tgatatccat caacgcgcaa acgatgacct ggcagacgct ggtctggaaa aactgcacct
     1861 gagtttttat ctgaccagca tttacgatca ttcaatcttt gaagcgttca gcaaagtcgt
     1921 gcagaaactg attccgcaac tgccgacgct ggaaaacctg ctgaacatct tcatcagtaa
     1981 ctccggcatc gaaaaagcct ttctgttcga tgttgtctct aaaatctaca tcgcaaccga
     2041 tagctctccg gtggacatgc agagttacga actgtgctgt gatatgattg acgtggttat
     2101 cgatgtttcc tgcatttatg gtctgaaaga agatggctca ggttcggctt acgacaaaga
     2161 atcaatggcg attatcaaac tgaacaatac cacggtcctg tatctgaaag aagtgaccaa
     2221 atttctggct ctggtgtgca ttctgcgtga agaatctttc gaacgcaaag gcctgatcga
     2281 ttacaacttc cattgtttcc gtaaagccat ccacgaagtt tttgaagtcg gcgtgacgag
     2341 ccatcgctct tgcggtcacc aaacgagcgc aagcagcctg aaagccctga cccacaatgg
     2401 caccccgcgt aatgcaatct gactcgagtc tggtaaagaa accgctgctg cgaaatttga
     2461 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     2521 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2581 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2641 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2701 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2761 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2821 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2881 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2941 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3001 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3061 tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc
     3121 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3181 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3241 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3301 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3361 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3421 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3481 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3541 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3601 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3661 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3721 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3781 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3841 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3901 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3961 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4021 cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat
     4081 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4141 ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4201 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4261 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4321 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4381 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4441 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4501 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4561 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4621 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4681 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4741 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4801 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4861 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4921 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4981 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5041 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     5101 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     5161 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5221 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     5281 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     5341 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     5401 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     5461 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     5521 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     5581 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     5641 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5701 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5761 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5821 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5881 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5941 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     6001 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6061 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6121 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6181 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     6241 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     6301 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     6361 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     6421 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     6481 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     6541 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     6601 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     6661 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     6721 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6781 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6841 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6901 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6961 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     7021 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     7081 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     7141 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     7201 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7261 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7321 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7381 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7441 gatcgagatc gatctcgatc ccgcgaaat
//
