LOCUS       sequence-109697-        7314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear-targeted FRET biosensor for monitoring mTORC1 kinase 
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7314)
  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 7314)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep. 
            2015 Mar 11. pii: S2211-1247(15)00177-1. doi: 
            10.1016/j.celrep.2015.02.031."
COMMENT     Sequence Label: pcDNA3-TORCAR-NLS
FEATURES             Location/Qualifiers
     source          1..7314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1009..1011,1012..1014,1015..1725)
                     /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     1662..1683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2818..2838
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2866..2884)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2867..2884)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2904..2921)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2910..3134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3180..3608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3267..3286)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3477..3498
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3617..3637)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3622..3952
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3803..3938
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3865..3884
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4019..4813
                     /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(4073..4092)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4683..4702
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4987..5108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5024..5043)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5078..5097
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5157..5173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5157..5173)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5170..5192)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5181..5197
                     /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(join(5205..5211,5212..5229,5230..5235))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5250..5271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5388..5405)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5559..6147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5639..5658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6318..7109,7110..7178))
                     /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     6941..6960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7179..7283)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgacctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aacgccatca gtgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1621 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcatatgtcc
     1741 gggggcagca gctgcagcca gaccccaagc cgggccatcc ccgccactcg ccgggtggtg
     1801 ctcggcgacg gcgtgcagct cccgcccggg gactacagca cgacccccgg cggcacgctc
     1861 ttcagcacca ccccgggagg taccaggatc atctatgacc ggaaattcct gatggagtgt
     1921 cggaactcac ctgtgaccaa aacaccccca agggatctgc ccaccattcc gggggtcacc
     1981 agcccttcca gtgatgagcc ccccatggaa gccagccaga gccacctgcg caatagccca
     2041 gaagataagc gggcgggcgg tgaagagtca cagtttgaga tggacattga gctcatggtg
     2101 agcaaaggcg aagagctgtt caccggcgtg gtgcccatcc tggtggagct ggacggcgac
     2161 gtgaacggcc acaagttcag cgtgagcggc gagggcgagg gcgacgccac ctacggcaag
     2221 ctgaccctga agctgctgtg caccaccggc aagctgcccg tgccctggcc caccctggtg
     2281 accaccctgg gctacggcgt gcagtgcttc gcccggtacc ccgaccacat gaagcagcac
     2341 gacttcttca agagcgccat gcccgagggc tacgtgcagg agcggaccat cttcttcaag
     2401 gacgacggca actacaagac ccgggccgag gtgaagttcg agggcgacac cctggtgaac
     2461 cggatcgagc tgaagggcat cgacttcaag gaggacagca acatcctggg ccacaagctg
     2521 gagtacaact acaacagcca caacgtgtac atcaccgccg acaagcagaa gaacggcatc
     2581 aaggccaact tcaagatccg gcacaacatc gaggacggcg gcgtgcagct ggccgaccac
     2641 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgataa ccactacctg
     2701 agctaccaga gcgccctgtt caaggacccc aacgagaagc gggaccacat ggtgctgctg
     2761 gagttcctga ccgccgccgg catcaccgag ggtatgaatg aattgtacaa agaattccca
     2821 aaaaagaaga gaaaggtgga agacgcataa tctagagggc cctattctat agtgtcacct
     2881 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2941 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3001 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3061 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3121 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3181 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3241 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3301 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     3361 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3421 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3481 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3541 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3601 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3661 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3721 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3781 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3841 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3901 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3961 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4021 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4081 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4141 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4201 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4261 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4321 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4381 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4441 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4501 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4561 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4621 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4681 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4741 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4801 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4861 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4921 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4981 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5041 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5101 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5161 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5221 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5281 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5341 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5401 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5461 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5521 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5581 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5641 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5701 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     5761 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5821 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5881 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5941 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6001 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6061 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6121 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6181 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6241 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6301 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6361 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6421 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6481 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6541 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6601 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6661 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6721 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6781 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6841 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6901 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6961 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7021 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7081 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7141 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7201 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7261 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
