LOCUS Exported 7224 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION FRET-based biosensor for monitoring mTORC1 kinase activity.. ACCESSION . VERSION . KEYWORDS pcDNA3-TORCAR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7224) AUTHORS Zhou X, Clister TL, Lowry PR, Seldin MM, Wong GW, Zhang J TITLE Dynamic Visualization of mTORC1 Activity in Living Cells. JOURNAL Cell Rep. 2015 Mar 11. pii: S2211-1247(15)00177-1. doi: 10.1016/j.celrep.2015.02.031. PUBMED 25772363 REFERENCE 2 (bases 1 to 7224) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7224 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(54..73) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 245..624 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 625..828 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 779..799 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 873..892 /label=T7 /note="T7 promoter, forward primer" promoter 873..891 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 924..1640 /codon_start=1 /product="enhanced monomeric variant of CFP (Rizzo et al., 2004)" /label=mCerulean /note=" mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(969..990) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1230..1249) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1577..1598 /label=EGFP-C /note="EGFP, forward primer" CDS 2010..2720 /codon_start=1 /product="yellow fluorescent protein optimized for FRET with CyPet (Nguyen and Daugherty, 2005)" /label=YPet /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF LTAAGITEGMNELYK" promoter complement(2787..2805) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2788..2805) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2825..2842) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2831..3055 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3101..3529 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3188..3207) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3398..3419 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3538..3558) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3543..3872 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3723..3858 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3785..3804 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3939..4733 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3993..4012) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4603..4622 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4907..5028 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4944..4963) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4998..5017 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5077..5093) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5077..5093) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5090..5112) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5101..5117 /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)." promoter complement(5125..5155) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5170..5191 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5308..5325) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5479..6067) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5559..5578) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6238..7098) /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 6861..6880 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7099..7203) /gene="bla" /label=AmpR promoter ORIGIN 1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc 61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct 121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg 181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg 241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat 301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact 841 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa 901 gcttggtacc gagctcggat cccatggtga gcaagggcga ggagctgttc accggggtgg 961 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgtccggcg 1021 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 1081 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttcg 1141 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 1201 acgtccagga gcgtaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 1261 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1321 aggacggcaa catcctgggg cacaagctgg agtacaacgc catcagtgac aacgtctata 1381 tcaccgccga caagcagaag aacggcatca aggcccactt caagatccgc cacaacatcg 1441 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 1501 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 1561 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 1621 gcatggacga gctgtacaag cgcatgcata tgtccggggg cagcagctgc agccagaccc 1681 caagccgggc catccccgcc actcgccggg tggtgctcgg cgacggcgtg cagctcccgc 1741 ccggggacta cagcacgacc cccggcggca cgctcttcag caccaccccg ggaggtacca 1801 ggatcatcta tgaccggaaa ttcctgatgg agtgtcggaa ctcacctgtg accaaaacac 1861 ccccaaggga tctgcccacc attccggggg tcaccagccc ttccagtgat gagcccccca 1921 tggaagccag ccagagccac ctgcgcaata gcccagaaga taagcgggcg ggcggtgaag 1981 agtcacagtt tgagatggac attgagctct ctaaaggtga agaattattc actggtgttg 2041 tcccaatttt ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg 2101 aaggtgaagg tgatgctacg tacggtaaat tgaccttaaa attactctgt actactggta 2161 aattgccagt tccatggcca accttagtca ctactttagg ttatggtgtt caatgttttg 2221 ctagataccc agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt 2281 atgttcaaga aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag 2341 tcaagtttga aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag 2401 aagatggtaa cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca 2461 tcactgctga caaacaaaag aatggtatca aagctaactt caaaattaga cacaacattg 2521 aagatggtgg tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc 2581 cagtcttgtt accagacaac cattacttat cctatcaatc tgccttattc aaagatccaa 2641 acgaaaagag agaccacatg gtcttgttag aatttttgac tgctgctggt attaccgagg 2701 gtatgaatga attgtacaaa tagtaagaat tctgcagata tccatcacac tggcggccgc 2761 tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga 2821 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 2881 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 2941 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3001 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 3061 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca 3121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 3181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 3241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 3301 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 3361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 3421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 3481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga 3541 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 3601 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 3661 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 3721 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 3781 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag 3841 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt 3901 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc 3961 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga 4021 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 4081 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 4141 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 4201 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 4261 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 4321 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 4381 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 4441 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 4501 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg 4561 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata 4621 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg 4681 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac 4741 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc 4801 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 4861 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc 4921 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 4981 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat 5041 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 5101 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 5161 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 5221 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 5281 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 5341 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 5401 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 5461 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 5521 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 5581 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 5641 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 5701 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 5761 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 5821 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5881 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 5941 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 6001 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 6061 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 6121 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 6181 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 6241 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 6301 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 6361 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 6421 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 6481 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 6541 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 6601 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 6661 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 6721 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 6781 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 6841 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6901 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6961 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 7021 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 7081 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 7141 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 7201 gcgcacattt ccccgaaaag tgcc //