LOCUS Exported 7469 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of RagA-RagC(L91P). ACCESSION . VERSION . KEYWORDS pETDuet RagA-RagC(L91P) 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7469 /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 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" primer_bind 2912..2933 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3185..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 ccccgttcct 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 tggatggcct 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 //