LOCUS       sequence-173676-        6384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  proximity-directed biotin labeling on the FRB domain of mTOR in 
            mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  AUTHORS   Lee SY, Lee H, Lee HK, Lee SW, Ha SC, Kwon T, Seo JK, Lee C, Rhee HW
  TITLE     Proximity-Directed Labeling Reveals a New Rapamycin-Induced 
            Heterodimer of FKBP25 and FRB in Live Cells.
  JOURNAL   ACS Cent Sci. 2016 Aug 24;2(8):506-16. doi: 
            10.1021/acscentsci.6b00137. Epub 2016 Aug 12.
  PUBMED    27610411
REFERENCE   2  (bases 1 to 6384)
  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"; doi: 
            "10.1021/acscentsci.6b00137"; journalName: "ACS Cent Sci"; date: 
            "2016-08-24- 24"; volume: "2"; issue: "8"; pages: "506-16"
COMMENT     Sequence Label: pCDNA5_FRB-pBirA-HA
FEATURES             Location/Qualifiers
     source          1..6384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..711
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     662..682
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    713..731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    734..752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     762..786
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             895..1173
                     /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             join(1192..1539,1540..1542,1543..2151)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2155..2181
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2229..2246)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2235..2459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2592..2611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2743..2790
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2798..3820
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2798..3820
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3950..4071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3987..4006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4041..4060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4120..4136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4120..4136)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4133..4155)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4144..4160
                     /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(4168..4174,4175..4192,4193..4198))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4213..4234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4351..4368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4522..5110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4602..4621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5281..6072,6073..6141))
                     /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     5904..5923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6142..6246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6321..6340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc
      721 agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt
      781 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      841 tccagcctcc ggactctagc gtttaaactt aagcttggta ccatggcttc tagaatcctc
      901 tggcatgaga tgtggcatga aggcctggaa gaggcatctc gtttgtactt tggggaaagg
      961 aacgtgaaag gcatgtttga ggtgctggag cccttgcatg ctatgatgga acggggcccc
     1021 cagactctga aggaaacatc ctttaatcag gcctatggtc gagatttaat ggaggcccaa
     1081 gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg acctcctcca agcctgggac
     1141 ctctattatc atgtgttccg acgaatctca aagactagtg aattcggatc caaggacaac
     1201 accgtgcccc tgaagctgat cgccctgctg gccaacggcg agttccactc tggcgagcag
     1261 ctgggagaga ccctgggaat gagcagagcc gccatcaaca agcacatcca gacactgaga
     1321 gactggggag tggacgtgtt caccgtgcct ggcaagggct acagcctgcc tgagcctatc
     1381 cagctgctga acgccaagca gatcctggga cagctggatg gcggaagcgt ggccgtgctg
     1441 cctgtgatcg actccaccaa tcagtacctg ctggacagaa tcggagagct gaagtccggc
     1501 gacgcctgca tcgccgagta ccagcaggct ggcagaggag gcagaggacg gaagtggttc
     1561 agcccattcg gagccaacct gtacctgtcc atgttctgga gactggagca gggacctgct
     1621 gctgccatcg gactgagtct ggtgatcgga atcgtgatgg ccgaggtgct gagaaagctg
     1681 ggagccgaca aggtgagagt gaagtggcct aatgacctgt acctccagga ccgcaagctg
     1741 gctggcatcc tggtggagct gacaggcaag acaggcgatg ccgctcagat cgtgatcgga
     1801 gccggaatca acatggccat gagaagagtg gaggagagcg tggtgaacca gggctggatc
     1861 accctgcagg aggctggcat caacctggac cggaacaccc tggccgccat gctgatcaga
     1921 gagctgagag ccgctctgga gctgttcgag caggagggac tggctcctta cctgagcaga
     1981 tgggagaagc tggacaactt catcaacaga cctgtgaagc tgatcatcgg cgacaaggaa
     2041 atcttcggca tctccagagg aatcgacaag cagggagctc tgctgctgga gcaggacgga
     2101 atcatcaagc cctggatggg cggagaaatc tccctgagaa gcgcagagaa ggcttaccct
     2161 tacgatgtac cggattacgc ataggcggcc gctcgagtct agagggcccg tttaaacccg
     2221 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2281 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2341 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2401 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2461 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2521 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2581 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2641 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2701 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     2761 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     2821 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     2881 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     2941 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3001 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3061 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3121 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3181 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3241 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3301 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3361 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3421 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     3481 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     3541 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     3601 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     3661 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     3721 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     3781 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     3841 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3901 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     3961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4021 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4081 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4141 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4201 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4261 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4321 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4381 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4441 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4501 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4561 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4621 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4681 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4741 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4801 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4861 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4921 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4981 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5041 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5101 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5161 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5221 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5281 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5341 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5401 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5461 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5521 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5581 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5641 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5701 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5761 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5821 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5881 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5941 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6001 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6061 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6121 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6181 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6241 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     6301 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     6361 ctccctgctt gtgtgttgga ggtc
//
