LOCUS Exported 5660 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression of Flag-RagA(R37P). ACCESSION . VERSION . KEYWORDS pRK5 Flag-RagA(R37P) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5660) 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 5660) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5660 /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 2090..2099 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2103..2237 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2140..2159) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2194..2213 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2306..2663 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2329..2349) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2514..2649 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2576..2595 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2683..2700) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2683..2699) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2692..2714) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2912..3367 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2999..3018) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3209..3230 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3384..3403) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3503..3525 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3563..3581) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3649..3753 /gene="bla" /label=AmpR promoter CDS 3754..4614 /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(3972..3991) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4785..5373 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5274..5293 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5527..5544 /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 gacgatgcca aatacagcca tgaagaaaaa 1141 ggtgctgctg atggggaaga gcgggtcggg gaagaccagc atgaggtcga taatcttcgc 1201 caattacatt gctcgcgaca cccctcgcct gggggccacc attgacgtgg aacactccca 1261 cgtccgattc ctagggaacc tggtgctgaa cctgtgggac tgtggcggtc aggacacctt 1321 catggaaaat tacttcacca gccagcgaga caatatcttc cgtaacgtgg aagttttgat 1381 ttacgtgttt gacgtggaga gccgcgaact ggaaaaggac atgcattatt accagtcgtg 1441 tctggaggcc atcctccaga actctcctga cgccaaaatc ttctgcctgg tgcacaaaat 1501 ggatctggtt caggaggatc agcgtgacct gatttttaaa gagcgagagg aagacctgag 1561 gcgtctgtct cgcccgctgg agtgtgcttg ttttcgaacg tccatctggg atgagacgct 1621 ctacaaagcc tggtccagca tcgtctacca gctgattccc aacgttcagc agctggagat 1681 gaacctcagg aattttgccc aaatcattga ggccgatgaa gttctgctgt tcgaaagagc 1741 tacattcttg gttatttccc actaccagtg caaagagcag cgcgacgtcc accggtttga 1801 gaagatcagc aacatcatca aacagttcaa gctgagctgc agtaaattgg ccgcttcctt 1861 ccagagcatg gaagttagga attccaactt cgctgctttc atcgacatct tcacctcaaa 1921 tacgtacgtg atggtggtca tgtcagatcc gtcgatccct tctgcggcca ctctgatcaa 1981 cattcgcaat gcccggaaac actttgagaa gctggagaga gtggatggcc ccaagcacag 2041 tctccttatg cgttgagcgg ccgctaagta agtaaggatc cccagcttgg ccgccatggc 2101 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 2161 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 2221 cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg 2281 aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc 2341 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 2401 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2461 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2521 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2581 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 2641 ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca 2701 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 2761 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 2821 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 2881 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 3121 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 3241 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 3361 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 3421 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 3481 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 3541 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 3601 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 3661 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 3721 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3781 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3841 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3901 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3961 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 4021 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 4081 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 4141 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 4201 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4261 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4321 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4381 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 4441 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4501 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 4561 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 4621 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 4681 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4741 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4801 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4861 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4921 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4981 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5041 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5101 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5161 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5221 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5281 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5341 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5401 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5461 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5521 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5581 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5641 ggcagtgagc gcaacgcaat //