LOCUS Exported 5918 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of Flag-RagC(S75N). ACCESSION . VERSION . KEYWORDS pRK5 Flag-RagC(S75N) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5918) 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 5918) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 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" protein_bind 1..22 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 37..67 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 75..91 /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 80..102 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 99..115 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 99..115 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 144..523 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 524..727 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 678..698 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 724..748 /label=LNCX /note="Human CMV promoter, forward primer" promoter 958..976 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 958..975 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 989..1008 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 1067..1090 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 2348..2357 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2361..2495 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2398..2417) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2452..2471 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2564..2921 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2587..2607) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2772..2907 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2834..2853 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2941..2958) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2941..2957) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2950..2972) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3170..3625 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3257..3276) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3467..3488 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3642..3661) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3761..3783 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3821..3839) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3907..4011 /gene="bla" /label=AmpR promoter CDS 4012..4872 /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(4230..4249) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5043..5631 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5532..5551 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5785..5802 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat 121 tacgaattaa ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat 181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag 721 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct 781 ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg 841 gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt 901 ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt 961 taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag 1021 gtccaactgc acctcggttc tatcgattga attcaccatg gaaatggact acaaggatga 1081 cgatgacaag ggtggtggtg gtggtgcgtc gacgatgtcc ctgcagtacg gggcggagga 1141 gacgcccctc gccggcagtt acggcgcggc cgattcgttt ccaaaggact tcggctacgg 1201 cgtggaggag gaggaagagg aggcggcggc ggcgggcgga ggggttgggg caggggcagg 1261 cggtggctgt ggtccggggg gcgctgacag ctccaagccg aggattctgc tcatgggact 1321 ccggcgcagc ggcaagaact ccatccagaa ggtggtgttt cataagatgt cacccaacga 1381 gaccctcttt ttggaaagta ccaacaagat ttataaggat gacatttcca atagctcctt 1441 tgtgaatttc cagatatggg attttcctgg gcaaatggac ttttttgacc caacctttga 1501 ctatgagatg atcttcaggg gaacaggagc attgatatac gtcattgacg cacaggatga 1561 ctacatggag gctttaacaa gacttcacat tactgtttct aaagcctaca aagttaaccc 1621 agacatgaat tttgaggttt ttattcacaa agttgatggt ctgtctgatg atcacaaaat 1681 agaaacacag agggacattc atcaaagggc caatgatgac cttgcagatg ctgggctaga 1741 aaaactccat cttagctttt atctgactag tatctatgac cattcaatat ttgaagcctt 1801 tagtaaggtg gtgcagaaac tcattccaca actgccgacc ttggaaaacc tattaaatat 1861 ctttatatca aattcaggta ttgaaaaagc ttttctcttt gatgttgtca gcaaaatcta 1921 cattgcaaca gacagttccc ctgtggatat gcaatcttat gaactttgct gtgacatgat 1981 cgatgttgta attgatgtgt cttgtatata tgggttaaag gaagatggaa gtggaagtgc 2041 ttatgacaaa gaatctatgg caattatcaa gctgaataat acaactgtcc tttatttaaa 2101 ggaggtgact aaatttttgg cactggtctg cattctaagg gaagaaagct ttgaaagaaa 2161 aggtttaata gactacaact tccactgttt ccgaaaagct attcatgagg tttttgaggt 2221 gggtgtgact tctcacagga gctgtggtca ccagactagt gcctccagtc tgaaagcgct 2281 gacacacaat ggcacgccac gaaacgccat ctaggcggcc gctaagtaag taaggatccc 2341 cagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca 2401 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 2461 ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat 2521 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 2581 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 2641 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 2701 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 2761 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 2821 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 2881 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc 2941 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 3001 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 3061 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct 3121 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 3181 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 3241 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 3301 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 3361 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 3421 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 3481 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 3541 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 3601 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 3661 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 3721 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 3781 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 3841 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 3901 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3961 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 4021 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 4081 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 4141 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4201 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 4261 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4321 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4381 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4441 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4501 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4561 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4621 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4681 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4741 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 4801 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4861 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4921 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4981 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 5041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 5101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 5161 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 5221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 5281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 5341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5401 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5701 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 5761 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 5821 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 5881 tttcccgact ggaaagcggg cagtgagcgc aacgcaat //