LOCUS       Exported                7951 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression of RagA-RagC(K84T).
ACCESSION   .
VERSION     .
KEYWORDS    pK RagA-RagC(K84T)
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7951
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             100..129
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             160..450
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     CDS             487..510
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1524..1542
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1543..1567
                     /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             2816..2833
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2886..2904)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2900..2947
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2984..3439
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3071..3090)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3281..3302
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3531..4346)
                     /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     4254..4273
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4338..4357
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4468..5056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4957..4976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5210..5227
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5242..5381
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5307..5326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5467..5489
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5483..5674)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6483..7565)
                     /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     7527..7546
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7566..7643)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7849..7868
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcaccatca ccatcaccat
      121 caccatcata gccgccgccg tcgccgccgt cgcccgcgga tgtcggactc agaagtcaat
      181 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
      241 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
      301 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
      361 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
      421 attattgagg ctcacagaga acagattggt ccgcggggtg gtggcgacca tcctccaaaa
      481 tcggatctgg aagttctgtt ccaggggccc ggatccatgc cgaacaccgc tatgaaaaaa
      541 aaagtgctgc tgatgggtaa atcgggctcg ggtaaaacct caatgcgtag tattatcttt
      601 gcgaactata ttgcgcgtga tacccgtcgc ctgggtgcaa cgatcgacgt ggaacatagc
      661 cacgttcgct ttctgggtaa cctggtgctg aatctgtggg attgcggcgg tcaggacacc
      721 tttatggaaa attatttcac gtcgcaacgt gataacattt ttcgcaatgt cgaagtgctg
      781 atctacgttt tcgatgtcga atcccgtgaa ctggaaaaag acatgcatta ttaccagtcc
      841 tgcctggaag caattctgca aaactcaccg gatgctaaaa tcttttgtct ggtccacaaa
      901 atggacctgg tgcaggaaga tcaacgtgac ctgattttca aagaacgcga agaagatctg
      961 cgtcgcctgt cgcgtccgct ggaatgcgcg tgttttcgca ccagcatctg ggacgaaacg
     1021 ctgtataaag cctggagctc tattgtttac cagctgatcc cgaatgtcca gcaactggaa
     1081 atgaacctgc gtaatttcgc acagattatc gaagctgatg aagtgctgct gtttgaacgc
     1141 gcaaccttcc tggttatttc ccattatcag tgcaaagaac aacgtgatgt gcaccgcttc
     1201 gaaaaaatct caaacatcat caaacagttc aaactgagtt gttccaaact ggcggcctca
     1261 tttcagtcga tggaagtccg caactctaat tttgcagctt tcattgatat cttcaccagt
     1321 aatacgtacg ttatggtggt tatgagcgac ccgtctattc cgagtgcggc caccctgatt
     1381 aacatccgta atgcccgcaa acattttgaa aaactggaac gtgttgatgg cccgaaacac
     1441 agcctgctga tgcgctgagc ggccgcataa tgcttaagtc gaacagaaag taatcgtatt
     1501 gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt gagcggataa
     1561 caattcccca tcttagtata ttagttaagt ataagaagga gatatacata tgtcactgca
     1621 atacggtgct gaagaaacgc cgctggctgg tagttatggt gctgctgatt cattcccgaa
     1681 agattttggt tatggcgttg aagaagaaga agaagaagcg gcggcggcgg gcggcggcgt
     1741 gggtgcaggt gccggtggcg gttgtggccc gggcggtgcc gatagctcta aaccgcgtat
     1801 tctgctgatg ggcctgcgtc gcagtggtaa aagttccatc cagaaagtgg tttttcacac
     1861 catgtcgccg aacgaaaccc tgttcctgga aagcacgaac aaaatctaca aagatgacat
     1921 cagcaactca tcgtttgtga atttccagat ttgggatttt ccgggtcaaa tggatttctt
     1981 tgacccgacc tttgactacg aaatgatctt ccgtggcacg ggtgcgctga tttatgttat
     2041 cgatgcccaa gatgactaca tggaagcact gacccgcctg catattacgg tgtcaaaagc
     2101 gtataaagtt aacccggata tgaacttcga agttttcatc cataaagtgg atggcctgtc
     2161 ggatgaccac aaaattgaaa cccagcgtga tatccatcaa cgcgcaaacg atgacctggc
     2221 agacgctggt ctggaaaaac tgcacctgag tttttatctg accagcattt acgatcattc
     2281 aatctttgaa gcgttcagca aagtcgtgca gaaactgatt ccgcaactgc cgacgctgga
     2341 aaacctgctg aacatcttca tcagtaactc cggcatcgaa aaagcctttc tgttcgatgt
     2401 tgtctctaaa atctacatcg caaccgatag ctctccggtg gacatgcaga gttacgaact
     2461 gtgctgtgat atgattgacg tggttatcga tgtttcctgc atttatggtc tgaaagaaga
     2521 tggctcaggt tcggcttacg acaaagaatc aatggcgatt atcaaactga acaataccac
     2581 ggtcctgtat ctgaaagaag tgaccaaatt tctggctctg gtgtgcattc tgcgtgaaga
     2641 atctttcgaa cgcaaaggcc tgatcgatta caacttccat tgtttccgta aagccatcca
     2701 cgaagttttt gaagtcggcg tgacgagcca tcgctcttgc ggtcaccaaa cgagcgcaag
     2761 cagcctgaaa gccctgaccc acaatggcac cccgcgtaat gcaatctgac tcgagcacca
     2821 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2881 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2941 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     3001 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3061 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3121 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3181 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3241 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3301 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3361 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3421 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3481 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     3541 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     3601 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     3661 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     3721 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     3781 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     3841 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     3901 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     3961 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4021 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     4081 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     4141 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4201 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4261 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     4321 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     4381 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4441 ccccgtagaa aagatcaaag aatcttcttg agatcctttt tttctgcgcg taatctgctg
     4501 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4561 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4621 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4681 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4741 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4801 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4861 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4921 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4981 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5041 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5101 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5161 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5221 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5281 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5341 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     5401 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5461 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5521 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     5581 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     5641 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     5701 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     5761 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     5821 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     5881 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     5941 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     6001 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     6061 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     6121 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     6181 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     6241 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     6301 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     6361 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     6421 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     6481 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6541 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     6601 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     6661 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     6721 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     6781 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     6841 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     6901 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     6961 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     7021 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     7081 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     7141 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     7201 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     7261 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     7321 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     7381 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     7441 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     7501 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     7561 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     7621 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     7681 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     7741 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     7801 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     7861 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     7921 gtagaggatc gagatctcga tcccgcgaaa t
//
