LOCUS Exported 7947 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of RagA-RagC(S75N). ACCESSION . VERSION . KEYWORDS pK RagA-RagC(S75N) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7947) 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 7947) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7947 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 100..129 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 160..450 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 487..510 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" promoter 1524..1542 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1543..1567 /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)." CDS 2816..2833 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2886..2904) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2900..2947 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2984..3439 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3071..3090) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3281..3302 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4254..4273 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4334..4353 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4464..5052 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4953..4972 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5206..5223 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5238..5377 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5303..5322) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5463..5485 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5479..5670) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6479..7561) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 7523..7542 /label=LacI-R /note="LacI, reverse primer" promoter complement(7562..7639) /gene="lacI" /label=lacI promoter primer_bind 7845..7864 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcaccatca ccatcaccat 121 caccatcata gccgccgccg tcgccgccgt cgcccgcgga tgtcggactc agaagtcaat 181 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag 241 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg 301 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac 361 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat 421 attattgagg ctcacagaga acagattggt ccgcggggtg gtggcgacca tcctccaaaa 481 tcggatctgg aagttctgtt ccaggggccc ggatccatgc cgaacaccgc tatgaaaaaa 541 aaagtgctgc tgatgggtaa atcgggctcg ggtaaaacct caatgcgtag tattatcttt 601 gcgaactata ttgcgcgtga tacccgtcgc ctgggtgcaa cgatcgacgt ggaacatagc 661 cacgttcgct ttctgggtaa cctggtgctg aatctgtggg attgcggcgg tcaggacacc 721 tttatggaaa attatttcac gtcgcaacgt gataacattt ttcgcaatgt cgaagtgctg 781 atctacgttt tcgatgtcga atcccgtgaa ctggaaaaag acatgcatta ttaccagtcc 841 tgcctggaag caattctgca aaactcaccg gatgctaaaa tcttttgtct ggtccacaaa 901 atggacctgg tgcaggaaga tcaacgtgac ctgattttca aagaacgcga agaagatctg 961 cgtcgcctgt cgcgtccgct ggaatgcgcg tgttttcgca ccagcatctg ggacgaaacg 1021 ctgtataaag cctggagctc tattgtttac cagctgatcc cgaatgtcca gcaactggaa 1081 atgaacctgc gtaatttcgc acagattatc gaagctgatg aagtgctgct gtttgaacgc 1141 gcaaccttcc tggttatttc ccattatcag tgcaaagaac aacgtgatgt gcaccgcttc 1201 gaaaaaatct caaacatcat caaacagttc aaactgagtt gttccaaact ggcggcctca 1261 tttcagtcga tggaagtccg caactctaat tttgcagctt tcattgatat cttcaccagt 1321 aatacgtacg ttatggtggt tatgagcgac ccgtctattc cgagtgcggc caccctgatt 1381 aacatccgta atgcccgcaa acattttgaa aaactggaac gtgttgatgg cccgaaacac 1441 agcctgctga tgcgctgagc ggccgcataa tgcttaagtc gaacagaaag taatcgtatt 1501 gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt gagcggataa 1561 caattcccca tcttagtata ttagttaagt ataagaagga gatatacata tgtcactgca 1621 atacggtgct gaagaaacgc cgctggctgg tagttatggt gctgctgatt cattcccgaa 1681 agattttggt tatggcgttg aagaagaaga agaagaagcg gcggcggcgg gcggcggcgt 1741 gggtgcaggt gccggtggcg gttgtggccc gggcggtgcc gatagctcta aaccgcgtat 1801 tctgctgatg ggcctgcgtc gcagtggtaa aaactccatc cagaaagtgg tttttcacaa 1861 aatgtcgccg aacgaaaccc tgttcctgga aagcacgaac aaaatctaca aagatgacat 1921 cagcaactca tcgtttgtga atttccagat ttgggatttt ccgggtcaaa tggatttctt 1981 tgacccgacc tttgactacg aaatgatctt ccgtggcacg ggtgcgctga tttatgttat 2041 cgatgcccaa gatgactaca tggaagcact gacccgcctg catattacgg tgtcaaaagc 2101 gtataaagtt aacccggata tgaacttcga agttttcatc cataaagtgg atggcctgtc 2161 ggatgaccac aaaattgaaa cccagcgtga tatccatcaa cgcgcaaacg atgacctggc 2221 agacgctggt ctggaaaaac tgcacctgag tttttatctg accagcattt acgatcattc 2281 aatctttgaa gcgttcagca aagtcgtgca gaaactgatt ccgcaactgc cgacgctgga 2341 aaacctgctg aacatcttca tcagtaactc cggcatcgaa aaagcctttc tgttcgatgt 2401 tgtctctaaa atctacatcg caaccgatag ctctccggtg gacatgcaga gttacgaact 2461 gtgctgtgat atgattgacg tggttatcga tgtttcctgc atttatggtc tgaaagaaga 2521 tggctcaggt tcggcttacg acaaagaatc aatggcgatt atcaaactga acaataccac 2581 ggtcctgtat ctgaaagaag tgaccaaatt tctggctctg gtgtgcattc tgcgtgaaga 2641 atctttcgaa cgcaaaggcc tgatcgatta caacttccat tgtttccgta aagccatcca 2701 cgaagttttt gaagtcggcg tgacgagcca tcgctcttgc ggtcaccaaa cgagcgcaag 2761 cagcctgaaa gccctgaccc acaatggcac cccgcgtaat gcaatctgac tcgagcacca 2821 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc 2881 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 2941 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc 3001 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 3061 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 3121 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 3181 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 3241 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 3301 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 3361 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 3421 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 3481 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac 3541 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt 3601 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca 3661 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc 3721 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt 3781 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc 3841 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg 3901 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg 3961 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat 4021 acctggaatg ctgtttttcc ggggatcgca gtggtgagta accatgcatc atcaggagta 4081 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc 4141 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc 4201 gcatcgggct tcccatacaa gcgatagatt gtcgcacctg attgcccgac attatcgcga 4261 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcganncg 4321 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt 4381 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 4441 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 4501 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 4561 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 4621 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 4681 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 4741 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 4801 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 4861 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 4921 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 4981 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 5041 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 5101 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 5161 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 5221 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 5281 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 5341 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 5401 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 5461 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct 5521 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc 5581 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat 5641 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt 5701 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga 5761 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg 5821 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt 5881 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc 5941 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc 6001 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc 6061 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac 6121 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 6181 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 6241 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 6301 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 6361 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 6421 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc 6481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 6541 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga 6601 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca 6661 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac 6721 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc 6781 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg 6841 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac 6901 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc 6961 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt 7021 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga 7081 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag 7141 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc 7201 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc 7261 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg 7321 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg 7381 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg 7441 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg 7501 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca 7561 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt 7621 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg 7681 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc 7741 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa 7801 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 7861 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 7921 aggatcgaga tctcgatccc gcgaaat //