LOCUS Exported 5918 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION express Flag-RagC (D181N mutant). ACCESSION . VERSION . KEYWORDS pRK5 Flag RagC (D181N) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5918) 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 5918) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5918 /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 2218..2227 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2231..2365 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2268..2287) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2322..2341 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2434..2791 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2457..2477) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2642..2777 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2704..2723 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2811..2828) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2811..2827) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2820..2842) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3040..3495 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3127..3146) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3337..3358 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3512..3531) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3631..3653 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3691..3709) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3777..3881 /gene="bla" /label=AmpR promoter CDS 3882..4742 /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(4100..4119) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4913..5501 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5402..5421 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5655..5672 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5789..5810 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5825..5855 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5863..5879 /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 5868..5890 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5887..5903 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5887..5903 /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 gtggtgcgtc gacgatgtcc ctgcagtacg gggcggagga gacgcccctc 1021 gccggcagtt acggcgcggc cgattcgttt ccaaaggact tcggctacgg cgtggaggag 1081 gaggaagagg aggcggcggc ggcgggcgga ggggttgggg caggggcagg cggtggctgt 1141 ggtccggggg gcgctgacag ctccaagccg aggattctgc tcatgggact ccggcgcagc 1201 ggcaagtcct ccatccagaa ggtggtgttt cataagatgt cacccaacga gaccctcttt 1261 ttggaaagta ccaacaagat ttataaggat gacatttcca atagctcctt tgtgaatttc 1321 cagatatggg attttcctgg gcaaatggac ttttttgacc caacctttga ctatgagatg 1381 atcttcaggg gaacaggagc attgatatac gtcattgacg cacaggatga ctacatggag 1441 gctttaacaa gacttcacat tactgtttct aaagcctaca aagttaaccc agacatgaat 1501 tttgaggttt ttattcacaa agttaatggt ctgtctgatg atcacaaaat agaaacacag 1561 agggacattc atcaaagggc caatgatgac cttgcagatg ctgggctaga aaaactccat 1621 cttagctttt atctgactag tatctatgac cattcaatat ttgaagcctt tagtaaggtg 1681 gtgcagaaac tcattccaca actgccgacc ttggaaaacc tattaaatat ctttatatca 1741 aattcaggta ttgaaaaagc ttttctcttt gatgttgtca gcaaaatcta cattgcaaca 1801 gacagttccc ctgtggatat gcaatcttat gaactttgct gtgacatgat cgatgttgta 1861 attgatgtgt cttgtatata tgggttaaag gaagatggaa gtggaagtgc ttatgacaaa 1921 gaatctatgg caattatcaa gctgaataat acaactgtcc tttatttaaa ggaggtgact 1981 aaatttttgg cactggtctg cattctaagg gaagaaagct ttgaaagaaa aggtttaata 2041 gactacaact tccactgttt ccgaaaagct attcatgagg tttttgaggt gggtgtgact 2101 tctcacagga gctgtggtca ccagactagt gcctccagtc tgaaagcgct gacacacaat 2161 ggcacgccac gaaacgccat ctaggcggcc gctaagtaag taaggatccc cagcttggcc 2221 gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 2281 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 2341 caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat 2401 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa 2461 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 2521 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 2581 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 2641 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 2701 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 2761 aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc 2821 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 2881 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 2941 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 3001 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 3061 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 3121 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3181 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3241 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3301 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3361 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 3421 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 3481 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 3541 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 3601 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 3661 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 3721 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 3781 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 3841 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 3901 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 3961 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 4021 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 4081 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 4141 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 4201 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 4261 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 4321 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 4381 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 4441 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 4501 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 4561 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 4621 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 4681 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 4741 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 4801 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 4861 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 4921 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 4981 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 5041 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 5101 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 5161 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5221 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5281 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5341 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5401 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5461 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 5521 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 5581 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 5641 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 5701 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 5761 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 5821 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 5881 ttcacacagg aaacagctat gacatgatta cgaattaa //