LOCUS Exported 5936 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION express Flag-RagB (Gdomain) fused with RagC (C terminal domain). ACCESSION . VERSION . KEYWORDS pRK5 Flag RagB(GD)/RagC(CTD) chimera SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5936) AUTHORS Lawrence RE, Cho KF, Rappold R, Thrun A, Tofaute M, Kim DJ, Moldavski O, Hurley JH, Zoncu R TITLE A nutrient-induced affinity switch controls mTORC1 activation by its Rag GTPase-Ragulator lysosomal scaffold. JOURNAL Nat Cell Biol. 2018 Sep;20(9):1052-1063. doi: 10.1038/s41556-018-0148-6. Epub 2018 Jul 30. PUBMED 30061680 REFERENCE 2 (bases 1 to 5936) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5936 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 14..393 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 394..597 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 548..568 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 594..618 /label=LNCX /note="Human CMV promoter, forward primer" promoter 828..846 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 828..845 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 859..878 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 937..960 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 2236..2245 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2249..2383 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2286..2305) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2340..2359 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2452..2809 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2475..2495) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2660..2795 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2722..2741 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2829..2846) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2829..2845) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2838..2860) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3058..3513 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3145..3164) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3355..3376 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3530..3549) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3649..3671 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3709..3727) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3795..3899 /gene="bla" /label=AmpR promoter CDS 3900..4760 /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(4118..4137) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4931..5519 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5420..5439 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5673..5690 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5807..5828 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5843..5873 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5881..5897 /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 5886..5908 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5905..5921 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5905..5921 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 901 acctcggttc tatcgattga attcaccatg gaaatggact acaaggatga cgatgacaag 961 ggtggtggtg gtggtgtcga catggaagaa tctgactctg agaaaacgac ggagaaagaa 1021 aatctggggc cgagaatgga tccaccacta ggggaaccgg aaggatcgct tgggtgggtg 1081 ctaccaaata cagccatgaa gaaaaaggtg ctgttgatgg gtaaaagtgg gtctggtaag 1141 accagcatga ggtctattat ctttgcaaat tatattgcca gagacacacg tcgccttggc 1201 gcaacaatac tagaccgtat acatagtctt caaattaata gcagtttgag cacctactct 1261 ctcgtagact ctgttggaaa tacaaagaca tttgatgtag aacattctca tgttcgattt 1321 ctgggaaacc tggtattgaa cctgtgggat tgtggtgggc aagacacctt catggaaaat 1381 tatttcacta gccaacggga caacatcttc cgaaatgtgg aggttctgat ttatgtcttt 1441 gatgtggaga gccgcgaact ggaaaaggac atgcactatt accaatcatg cctggaggcc 1501 attctgcaga attctccaga tgccaaaata ttttgcttgg tacacaaaat ggatctggta 1561 caggaggatc aacgggacct gatttttaaa gagcgagaag aagatttgag gcgtttgtct 1621 cgcccattgg aatgttcttg tttccgaaca tctatctggg atgaaaccct ctataaggct 1681 tggtccagca tagtttatca gctgattccc cagctgccta cactggagaa cctgctgaat 1741 atctttatct ctaacagcgg catcgagaag gccttcctgt ttgacgtggt gagcaagatc 1801 tacatcgcca ccgacagctc ccccgtggat atgcagtcct atgagctgtg ctgtgacatg 1861 atcgacgtgg tcatcgacgt gagctgcatc tacggcctga aggaggacgg atccggatct 1921 gcctatgata aggagagcat ggccatcatc aagctgaaca ataccacagt gctgtacctg 1981 aaggaggtga ccaagttcct ggccctggtg tgcatcctgc gggaggagtc ctttgagaga 2041 aagggcctga tcgactataa cttccactgt tttcggaagg ccatccacga ggtgttcgaa 2101 gtgggcgtga caagccacag atcctgcggc caccagacct ccgcatcatc actgaaggca 2161 ctgacccaca acggcacacc acgaaacgct atcctcgagt aggcggccgc taagtaagta 2221 aggatcccca gcttggccgc catggcccaa cttgtttatt gcagcttata atggttacaa 2281 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 2341 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcgggaatt aattcggcgc 2401 agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg 2461 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc 2521 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 2581 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 2641 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 2701 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 2761 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctgttaac 2821 agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 2881 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 2941 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 3001 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 3061 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 3121 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 3181 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 3241 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 3301 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 3361 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag 3421 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 3481 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 3541 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 3601 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 3661 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 3721 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 3781 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3841 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 3901 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3961 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 4021 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 4081 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 4141 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4201 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4261 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4321 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4381 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4441 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4501 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4561 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 4621 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 4681 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 4741 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 4801 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4861 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 4921 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4981 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 5041 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 5101 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5161 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5221 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5281 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 5341 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5401 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5461 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5521 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 5581 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 5641 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 5701 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 5761 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 5821 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 5881 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg aattaa //