LOCUS       sequence-109694-        7257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted FRET biosensor for monitoring mTORC1 kinase
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7257)
  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 7257)
  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-PM-TORCAR
FEATURES             Location/Qualifiers
     source          1..7257
                     /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             join(946..948,949..951,952..1662)
                     /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(991..1012)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1252..1271)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1599..1620
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2032..2742
                     /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(2809..2827)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2810..2827)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2847..2864)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2853..3077
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3123..3551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3210..3229)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3420..3441
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3560..3580)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3565..3895
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3746..3881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3808..3827
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3962..4756
                     /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(4016..4035)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4626..4645
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5021..5040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5100..5116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5100..5116)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5113..5135)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5124..5140
                     /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(5148..5154,5155..5172,5173..5178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5193..5214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5331..5348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5502..6090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5582..5601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6261..7052,7053..7121))
                     /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     6884..6903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7122..7226)
                     /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 gctgcatcaa gagcaagcgc aaggacaagg atcccatggt gagcaagggc
      961 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1021 cacaggttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1081 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1141 acctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1201 aagtccgcca tgcccgaagg ctacgtccag gagcgtacca tcttcttcaa ggacgacggc
     1261 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1321 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1381 gccatcagtg acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggcccac
     1441 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1501 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1561 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1621 accgccgccg ggatcactct cggcatggac gagctgtaca agcgcatgca tatgtccggg
     1681 ggcagcagct gcagccagac cccaagccgg gccatccccg ccactcgccg ggtggtgctc
     1741 ggcgacggcg tgcagctccc gcccggggac tacagcacga cccccggcgg cacgctcttc
     1801 agcaccaccc cgggaggtac caggatcatc tatgaccgga aattcctgat ggagtgtcgg
     1861 aactcacctg tgaccaaaac acccccaagg gatctgccca ccattccggg ggtcaccagc
     1921 ccttccagtg atgagccccc catggaagcc agccagagcc acctgcgcaa tagcccagaa
     1981 gataagcggg cgggcggtga agagtcacag tttgagatgg acattgagct ctctaaaggt
     2041 gaagaattat tcactggtgt tgtcccaatt ttggttgaat tagatggtga tgttaatggt
     2101 cacaaatttt ctgtctccgg tgaaggtgaa ggtgatgcta cgtacggtaa attgacctta
     2161 aaattactct gtactactgg taaattgcca gttccatggc caaccttagt cactacttta
     2221 ggttatggtg ttcaatgttt tgctagatac ccagatcata tgaaacaaca tgactttttc
     2281 aagtctgcca tgccagaagg ttatgttcaa gaaagaacta tttttttcaa agatgacggt
     2341 aactacaaga ccagagctga agtcaagttt gaaggtgata ccttagttaa tagaatcgaa
     2401 ttaaaaggta ttgattttaa agaagatggt aacattttag gtcacaaatt ggaatacaac
     2461 tataactctc acaatgttta catcactgct gacaaacaaa agaatggtat caaagctaac
     2521 ttcaaaatta gacacaacat tgaagatggt ggtgttcaat tagctgacca ttatcaacaa
     2581 aatactccaa ttggtgatgg tccagtcttg ttaccagaca accattactt atcctatcaa
     2641 tctgccttat tcaaagatcc aaacgaaaag agagaccaca tggtcttgtt agaatttttg
     2701 actgctgctg gtattaccga gggtatgaat gaattgtaca aatagtaaga attctgcaga
     2761 tatccatcac actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca
     2821 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2881 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2941 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3001 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3061 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3121 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3181 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3241 cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt
     3301 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3361 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3421 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3481 ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3541 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3601 ccaggcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3661 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3721 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3781 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3841 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3901 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3961 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4021 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4081 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4141 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4201 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4261 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4321 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4381 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4441 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4501 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4561 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4621 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4681 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4741 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4801 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4861 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4921 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5101 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5161 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5221 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5281 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5341 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5401 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5461 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5521 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5581 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5641 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     5701 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5761 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5821 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5881 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5941 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6001 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6061 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6121 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6181 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6361 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6601 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6661 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6721 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6781 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6841 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6901 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6961 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7021 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7081 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7141 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7201 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
