LOCUS       sequence-352552-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB-tTA-Myc tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Engineering of Extracellular Vesicles for Small Molecule-Regulated 
            Cargo Loading and Cytoplasmic Delivery of Bioactive Proteins.
  JOURNAL   Mol Pharm. 2022 May 20. doi: 10.1021/acs.molpharmaceut.2c00192.
  PUBMED    35594496
REFERENCE   2  (bases 1 to 6314)
  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: "Mol Pharm. 
            2022 May 20. doi: 10.1021/acs.molpharmaceut.2c00192."
COMMENT     Sequence Label: pCMV-FRB-tTA-Myc
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..24
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     16..33
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     17..33
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     43..62
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        43..61
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        182..561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        562..765
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     716..736
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     762..786
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          877..1449
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             1538..1816
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             join(1844..2461,2462..2845)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIE
                     GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHAD
                     ALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1914..1933)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             2846..2875
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3104..3123)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3169..3224
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3223..3242)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3582..3598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3582..3598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3595..3617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3606..3622
                     /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(3630..3636,3637..3654,3655..3660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3675..3696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3813..3830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3984..4572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4064..4083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4743..5534,5535..5603))
                     /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     5366..5385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5604..5708)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5734..6189)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5871..5892)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6083..6102
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag
       61 ggcgaattgg agctccaccg cggtggcggc cgctctagag agcttggccc attgcatacg
      121 ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt
      181 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      241 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      301 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      361 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      421 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      481 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      541 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      601 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      661 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      721 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
      781 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      841 tccagcctcc ggtcgaccga tcctgagaac ttcagggtga gtttggggac ccttgattgt
      901 tctttctttt tcgctattgt aaaattcatg ttatatggag ggggcaaagt tttcagggtg
      961 ttgtttagaa tgggaagatg tcccttgtat caccatggac cctcatgata attttgtttc
     1021 tttcactttc tactctgttg acaaccattg tctcctctta ttttcttttc attttctgta
     1081 actttttcgt taaactttag cttgcatttg taacgaattt ttaaattcac ttttgtttat
     1141 ttgtcagatt gtaagtactt tctctaatca cttttttttc aaggcaatca gggtatatta
     1201 tattgtactt cagcacagtt ttagagaaca attgttataa ttaaatgata aggtagaata
     1261 tttctgcata taaattctgg ctggcgtgga aatattctta ttggtagaaa caactacacc
     1321 ctggtcatca tcctgccttt ctctttatgg ttacaatgat atacactgtt tgagatgagg
     1381 ataaaatact ctgagtccaa accgggcccc tctgctaacc atgttcatgc cttcttctct
     1441 ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa
     1501 ttcctcgacg gatccctcga ggaattctga cactatgatc ctctggcatg agatgtggca
     1561 tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt
     1621 tgaggtgctg gagcccttgc atgctatgat ggaacggggc ccccagactc tgaaggaaac
     1681 atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt gcaggaagta
     1741 catgaaatca gggaatgtca aggacctcac ccaagcctgg gacctctatt atcatgtgtt
     1801 ccgacgaatc tcaaagggtg gtggaggcag tggcggtgga ggcagcagat tagataaaag
     1861 taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag gtttaacaac
     1921 ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa
     1981 taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc atactcactt
     2041 ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta aaagttttag
     2101 atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac ggcctacaga
     2161 aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag gtttttcact
     2221 agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt gcgtattgga
     2281 agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg atagtatgcc
     2341 gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc cagccttctt
     2401 attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc
     2461 cgcgtacagc cgcgcgcgta cgaaaaacaa ttacgggtct accatcgagg gcctgctcga
     2521 tctcccggac gacgacgccc ccgaagaggc ggggctggcg gctccgcgcc tgtcctttct
     2581 ccccgcggga cacacgcgca gactgtcgac ggcccccccg accgatgtca gcctggggga
     2641 cgagctccac ttagacggcg aggacgtggc gatggcgcat gccgacgcgc tagacgattt
     2701 cgatctggac atgttggggg acggggattc cccgggtccg ggatttaccc cccacgactc
     2761 cgccccctac ggcgctctgg atatggccga cttcgagttt gagcagatgt ttaccgatgc
     2821 ccttggaatt gacgagtacg gtggggaaca gaaactgatt agcgaagaag atctgtaact
     2881 caaatcctgc acaacagatt cttcatgttt ggaccaaatc aacttgtgat accatgctca
     2941 aagaggcctc aattatattt gagtttttaa tttttatgaa aaaaaaaaaa aaaacggaat
     3001 tcctcgaggg atccgtcgag gaattcactc ctcaggtgca ggctgcctat cagaaggtgg
     3061 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca
     3121 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     3181 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     3241 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     3301 cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac
     3361 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     3421 ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     3481 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     3541 tatggagatc cctcgacgga tcggccgcaa ttcgtaatca tgtcatagct gtttcctgtg
     3601 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3661 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3721 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3781 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3841 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3901 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3961 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4021 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4081 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4141 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4201 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4321 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4381 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4441 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4501 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4561 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4621 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4681 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4741 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4801 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4861 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4921 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4981 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5041 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5101 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5161 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5221 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5281 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5341 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5401 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5461 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5521 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5581 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5641 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5701 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5761 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5821 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5881 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5941 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6001 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6061 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6121 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6181 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6241 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     6301 taacgccagg gttt
//
