LOCUS       sequence-174915-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rapamycin binding motif of mTOR (FRB) anchored to the plasma 
            membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  AUTHORS   Liu N, Yang Y, Ge L, Liu M, Colecraft HM, Liu X
  TITLE     Cooperative and acute inhibitionby multiple C-terminal motifs of 
            L-type Ca2+ channels.
  JOURNAL   Elife. 2017 Jan 6;6. pii: e21989. doi: 10.7554/eLife.21989.
  PUBMED    28059704
REFERENCE   2  (bases 1 to 6455)
  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: "Elife."; 
            date: "2017-01-6"; volume: "6. pii: e21989. doi"; pages: " 
            10.7554/eLife.21989"
COMMENT     Sequence Label: FRB-CFP-ras
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             688..966
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             973..1683
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(1012..1033)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1273..1292)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1620..1641
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1774..1792)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1775..1792)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1812..1829)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1818..2042
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2088..2516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2175..2194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2385..2406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2525..2545)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2530..2859
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2710..2845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2772..2791
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2926..3720
                     /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(2980..2999)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3590..3609
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3894..4015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3931..3950)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3985..4004
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4064..4080)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4064..4080)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4077..4099)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4088..4104
                     /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(4112..4118,4119..4136,4137..4142))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4157..4178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4295..4312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4466..5054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4546..4565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5225..6016,6017..6085))
                     /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     5848..5867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6086..6190)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6265..6284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccgc caccatgatc ctctggcatg agatgtggca tgaaggcctg
      721 gaagaggcat ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg
      781 gagcccttgc atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat
      841 caggcctatg gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca
      901 gggaatgtca aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc
      961 tcaaagaccg gtagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1021 ctggacggcg acgtaaacgg ccacaggttc agcgtgtccg gcgagggcga gggcgatgcc
     1081 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1141 cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac
     1201 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgtacc
     1261 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1321 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1381 gggcacaagc tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag
     1441 aagaacggca tcaaggccca cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1501 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1561 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1621 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1681 aagaagctga accctcctga tgagagtggc cccggctgca tgagctgcaa gtgtgtgctc
     1741 tcctaactcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc
     1801 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1861 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1921 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1981 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2041 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2281 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2401 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2521 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2581 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2701 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2761 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     2821 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     2881 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     2941 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3001 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3061 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3121 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3181 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3241 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3301 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3361 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3421 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3481 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3541 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3601 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3661 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3721 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3781 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3841 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3901 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3961 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4021 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4081 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4141 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4201 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4261 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4321 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4381 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4441 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4501 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4561 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4621 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4681 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4741 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4801 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4861 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4921 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4981 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5041 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5101 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5161 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5221 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5281 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5341 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5401 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5461 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5521 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5581 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5641 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5701 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5761 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5821 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5881 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5941 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6001 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6061 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6121 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6181 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     6241 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     6301 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     6361 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     6421 gctgcttcgc gatgtacggg ccagatatac gcgtt
//
