LOCUS       sequence-305141-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AIMTOR T757 contains a cytoplasmic mTOR Activity Reporter derived 
            from hu ULK1 protein boxed between BRET-compatible entities (Ypet 
            and Nanoluciferase) to measure mTOR activity in living cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  AUTHORS   Bouquier N, Moutin E, Tintignac LA, Reverbel A, Jublanc E, Sinnreich
            M, Chastagnier Y, Averous J, Fafournoux P, Verpelli C, Boeckers T, 
            Carnac G, Perroy J, Ollendorff V
  TITLE     AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR 
            signaling and dysfunctions.
  JOURNAL   BMC Biol. 2020 Jul 3;18(1):81. doi: 10.1186/s12915-020-00790-8.
  PUBMED    32620110
REFERENCE   2  (bases 1 to 6566)
  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: "BMC Biol.";
            date: "2020-07-3"; volume: "18(1):81. doi"; pages: " 
            10.1186/s12915-020-00790-8"
COMMENT     Sequence Label: AIMTOR T757
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          665..1685
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1693..1712
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1743..1745,1746..1748,1749..2456)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     primer_bind     complement(1788..1809)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2049..2068)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2631..2978
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             3021..3533
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             3546..3572
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3588..3607)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3653..3708
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3707..3726)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4069..4085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4082..4104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4093..4109
                     /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(4117..4123,4124..4141,4142..4147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4162..4183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4242..4437
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4288..4423
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4443..4577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4480..4499)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4534..4553
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4645..4662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4816..5404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4896..4915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5575..6366,6367..6435))
                     /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     6198..6217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6436..6540)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgatcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg gggggggggg cgcgccrggg gggggggggg gggggggggg
      541 ggggggsggg gsgrggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggrgggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     1681 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcg
     1741 gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1801 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1861 acggcaagct gaccctgaag cttctatgca ccaccggcaa gctgcccgtg ccctggccca
     1921 ccctcgtgac caccctgggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
     1981 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2041 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2101 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2161 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     2221 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     2281 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2341 actacctgag ctaccagtcc gccctgttca aagaccccaa cgagaagcgc gatcacatgg
     2401 tcctgctgga gttcctgacc gccgccggga tcactgaggg catgaacgag ctgtacctcg
     2461 agatggcgga cgaggagaag ctgccgcccg gctgggagaa gcgcatgagc cgcagctcag
     2521 gccgagtgta ctacttcaac cacatcacta acgccagcca gtgggagcgg cccagcggca
     2581 acagcagcag tggtggcaaa aacgggcagg gggagcctgc caggggtacc agtgctggtg
     2641 gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt tccggcagtg
     2701 ctggtggtag tgctggtggt agtaccagtg ctggtggtag tgctggtggt agtgctggtg
     2761 gtagtgctgg tggtagtgct ggtggttccg gcagtgctgg tggtagtgct ggtggtagta
     2821 ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     2881 gttccggcag tgctggtggt agtgctggtg gtagtaccag tgctggtggt agtgctggtg
     2941 gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc cggattcacc gtgggcaccc
     3001 ccccgagcgg gggcggccgc atggtcttca cactcgaaga tttcgttggg gactggcgac
     3061 agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc
     3121 agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc
     3181 tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc
     3241 agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc
     3301 tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac
     3361 ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt
     3421 ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc
     3481 gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg gcgtctagac
     3541 tgcagctgcc tcctctggaa cgcctgactc tggattaagt cgacggatct ttttccctct
     3601 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     3661 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     3721 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     3781 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     3841 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     3901 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     3961 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     4021 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     4081 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4141 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4201 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     4261 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4321 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4381 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4441 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4501 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4561 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4621 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4681 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4741 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4801 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4861 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4921 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4981 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5041 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5101 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5161 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5221 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5281 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5341 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5401 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5461 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5521 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5581 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5641 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5701 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5761 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5821 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5881 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5941 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6001 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6061 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6121 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6181 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6241 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6301 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6361 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6421 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6481 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6541 cacatttccc cgaaaagtgc cacctg
//
