LOCUS Exported 8740 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION express FRB-myc-RagA. ACCESSION . VERSION . KEYWORDS pLJM1 FRB-myc-RagA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8740) 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 8740) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8740 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 1..589 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 490..509 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 743..760 /label=L4440 /note="L4440 vector, forward primer" protein_bind 877..898 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 913..943 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 951..967 /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 956..978 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 975..991 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 975..991 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 1010..1030 /label=T3 /note="T3 promoter, forward primer" promoter 1012..1030 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 1058..1284 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 1285..1465 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1512..1637 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 2130..2363 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2548..2592 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" primer_bind 2890..2910 /label=hU6-F /note="Human U6 promoter, forward primer" promoter 3199..3402 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 3353..3373 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 3449..3727 /codon_start=1 /product="T2098L mutant of FKBP-rapamycin binding domain of human FRAP" /label=FRB* /note="binds synthetic ligands such as AP21967" /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK" CDS 3872..3901 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" misc_feature 4891..5008 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 5057..5567 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 5486..5504 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 5589..6188 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(5589..5608) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6085..6105 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 6316..6549 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 6621..6742 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6658..6677) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6712..6731 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 6782..6917 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6844..6863 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(6937..6956) /label=T7 /note="T7 promoter, forward primer" promoter complement(6938..6956) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6966..6983) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6966..6982) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6975..6997) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7124..7579 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7211..7230) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7421..7442 /label=F1ori-F /note="F1 origin, forward primer" promoter 7605..7709 /gene="bla" /label=AmpR promoter CDS 7710..8570 /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(7928..7947) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc 1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg 1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac 1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag 1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt 1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag 1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt 1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt 1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga 1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg 1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag 1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg 1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa 2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg 2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca 2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg 2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg 2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga 2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc 2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac 2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta 2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa 2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc 2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg 2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg 2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc 2821 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc 2881 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2941 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg 3001 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 3061 gactatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 3121 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 3181 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 3241 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 3301 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 3361 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag 3421 atccgctagc gctaccggtg ccaccatgat cctctggcat gagatgtggc atgaaggcct 3481 ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct 3541 ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa 3601 tcaggcctat ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc 3661 agggaatgtc aaggacctcc tccaagcctg ggacctctat tatcatgtgt tccgacgaat 3721 ctcaaagggt ggttctgccg gtggctccgg ttctggctcc agcggtggca gctctggtgc 3781 gtccggcacg ggtactgcgg gtggcactgg cagcggttcc ggtggaggct ctggaggcgg 3841 ttctgaaggt ggcggatccg aaggcggcgg cgaacaaaag ctcattagcg aggaagacct 3901 tgtcgacatg cccaacaccg ccatgaagaa gaaggtgctg ctgatgggca agtccggctc 3961 tggcaagaca tctatgcgga gcatcatctt cgccaattat atcgccagag ataccaggcg 4021 cctgggagca acaatcgacg tggagcacag ccacgtgcgg tttctgggca acctggtgct 4081 gaatctgtgg gactgcggcg gccaggatac cttcatggag aactacttta catctcagcg 4141 ggacaacatc ttcagaaatg tggaggtgct gatctacgtg ttcgatgtgg agagcagaga 4201 gctggagaag gacatgcact actatcagag ctgcctggag gccatcctgc agaactcccc 4261 cgatgccaag atcttctgtc tggtgcacaa gatggacctg gtgcaggagg accagcggga 4321 tctgatcttt aaggagagag aggaggatct gcggagactg tctaggcctc tggagtgcgc 4381 ctgtttccgc accagcatct gggacgagac actgtacaag gcctggtcta gcatcgtgta 4441 tcagctgatc ccaaacgtgc agcagctgga gatgaacctg aggaattttg cccagatcat 4501 cgaggccgat gaggtgctgc tgttcgagcg cgccaccttc ctggtcatca gccactacca 4561 gtgcaaggag cagagggacg tgcaccgctt cgagaagatc tccaacatca tcaagcagtt 4621 taagctgagc tgttccaagc tggccgcctc cttccagtct atggaggtgc ggaactccaa 4681 tttcgccgcc ttcatcgaca tcttcacctc taatacatat gtgatggtgg tcatgagcga 4741 cccctccatc ccttctgccg ccacactgat caacatcaga aatgcccgga aacactttga 4801 aaaactggag cgggtggacg gacctaaaca tagcctgctg atgcgataag aattctcgac 4861 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta 4921 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 4981 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact 5041 ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc 5101 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc 5161 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc 5221 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt 5281 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc 5341 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag 5401 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag 5461 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt 5521 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg 5581 agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca 5641 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 5701 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 5761 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 5821 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 5881 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 5941 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg 6001 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 6061 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 6121 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 6181 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg 6241 catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 6301 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg 6361 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag 6421 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc 6481 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa 6541 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat 6601 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca 6661 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 6721 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc 6781 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 6841 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 6901 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg 6961 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 7021 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 7081 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc 7141 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 7201 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 7261 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 7321 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 7381 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 7441 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 7501 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 7561 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 7621 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 7681 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 7741 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 7801 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 7861 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 7921 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 7981 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 8041 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 8101 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 8161 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 8221 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 8281 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 8341 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 8401 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 8461 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 8521 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 8581 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 8641 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 8701 actgagcgtc agaccccgta gaaaagatca aaggatcttc //