LOCUS Exported 5965 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of HA-RagC(S75N). ACCESSION . VERSION . KEYWORDS pRK5 HA-RagC(S75N) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5965) 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 5965) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5965 /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" regulatory 1093..1102 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1105..1131 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" regulatory 2395..2404 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 2408..2542 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2445..2464) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2499..2518 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 2611..2968 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2634..2654) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2819..2954 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2881..2900 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2988..3005) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2988..3004) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2997..3019) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3217..3672 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3304..3323) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3514..3535 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3689..3708) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3808..3830 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3868..3886) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3954..4058 /gene="bla" /label=AmpR promoter CDS 4059..4919 /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(4277..4296) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5090..5678 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5579..5598 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5832..5849 /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 attcccgaac cgacagtcgg tctcttcacc 1081 aaggccattc gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat 1141 ggtggtggtg gtgggtcgac gatgtccctg cagtacgggg cggaggagac gcccctcgcc 1201 ggcagttacg gcgcggccga ttcgtttcca aaggacttcg gctacggcgt ggaggaggag 1261 gaagaggagg cggcggcggc gggcggaggg gttggggcag gggcaggcgg tggctgtggt 1321 ccggggggcg ctgacagctc caagccgagg attctgctca tgggactccg gcgcagcggc 1381 aagaactcca tccagaaggt ggtgtttcat aagatgtcac ccaacgagac cctctttttg 1441 gaaagtacca acaagattta taaggatgac atttccaata gctcctttgt gaatttccag 1501 atatgggatt ttcctgggca aatggacttt tttgacccaa cctttgacta tgagatgatc 1561 ttcaggggaa caggagcatt gatatacgtc attgacgcac aggatgacta catggaggct 1621 ttaacaagac ttcacattac tgtttctaaa gcctacaaag ttaacccaga catgaatttt 1681 gaggttttta ttcacaaagt tgatggtctg tctgatgatc acaaaataga aacacagagg 1741 gacattcatc aaagggccaa tgatgacctt gcagatgctg ggctagaaaa actccatctt 1801 agcttttatc tgactagtat ctatgaccat tcaatatttg aagcctttag taaggtggtg 1861 cagaaactca ttccacaact gccgaccttg gaaaacctat taaatatctt tatatcaaat 1921 tcaggtattg aaaaagcttt tctctttgat gttgtcagca aaatctacat tgcaacagac 1981 agttcccctg tggatatgca atcttatgaa ctttgctgtg acatgatcga tgttgtaatt 2041 gatgtgtctt gtatatatgg gttaaaggaa gatggaagtg gaagtgctta tgacaaagaa 2101 tctatggcaa ttatcaagct gaataataca actgtccttt atttaaagga ggtgactaaa 2161 tttttggcac tggtctgcat tctaagggaa gaaagctttg aaagaaaagg tttaatagac 2221 tacaacttcc actgtttccg aaaagctatt catgaggttt ttgaggtggg tgtgacttct 2281 cacaggagct gtggtcacca gactagtgcc tccagtctga aagcgctgac acacaatggc 2341 acgccacgaa acgccatcta ggcggccgct aagtaagtaa ggatccccag cttggccgcc 2401 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 2461 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 2521 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac 2581 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt 2641 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 2701 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 2761 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 2821 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 2881 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg 2941 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt 3001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 3061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 3121 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 3181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa 3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 3541 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct 3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 3721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 3841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 3901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 4321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 4381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 4441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 4501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4741 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 4801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 4861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 4921 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 4981 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5161 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5221 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 5401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 5521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5701 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5761 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 5821 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 5881 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 5941 aagcgggcag tgagcgcaac gcaat //