LOCUS       sequence-357726-        6255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB-LgBiT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6255)
  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 6255)
  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: pcDNA3.1-FRB-LgBiT
FEATURES             Location/Qualifiers
     source          1..6255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(101..120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        292..671
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        672..875
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     826..846
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     920..939
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        920..938
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1043..1052
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1047..1056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1058..1333
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     primer_bind     complement(1902..1919)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1908..2133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2179..2607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2266..2285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2476..2497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2616..2636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2621..2950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2801..2936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2863..2882
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3017..3811
                     /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(3071..3090)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3681..3700
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3985..4106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4022..4041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4076..4095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4155..4171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4155..4171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4168..4190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4179..4195
                     /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(4203..4209,4210..4227,4228..4233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4248..4269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4386..4403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4557..5145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4637..4656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5316..6107,6108..6176))
                     /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     5939..5958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6177..6255,1..26))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
       61 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
      121 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      181 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      241 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      301 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      361 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      421 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      481 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      541 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      601 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      661 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      721 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      781 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      841 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
      901 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt
      961 taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga attctgcaga
     1021 tatccagcac agtggcggcc gcgccaccat ggtggccatc ctctggcatg agatgtggca
     1081 tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt
     1141 tgaggtgctg gagcccttgc atgctatgat ggaacggggc ccccagactc tgaaggaaac
     1201 atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt gcaggaagta
     1261 catgaaatca gggaatgtca aggacctcac ccaagcctgg gacctctatt atcatgtgtt
     1321 ccgacgaatc tcagtttctc aaggcagttc aggtggtggc gggagcggag gtggaggctc
     1381 gagcggtgtc ttcacactcg aagatttcgt tggggactgg gaacagacag ccgcctacaa
     1441 cctggaccaa gtccttgaac agggaggtgt gtccagtttg ctgcagaatc tcgccgtgtc
     1501 cgtaactccg atccaaagga ttgtccggag cggtgaaaat gccctgaaga tcgacatcca
     1561 tgtcatcatc ccgtatgaag gtctgagcgc cgaccaaatg gcccagatcg aagaggtgtt
     1621 taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgccct atggcacact
     1681 ggtaatcgac ggggttacgc cgaacatgct gaactatttc ggacggccgt atgaaggcat
     1741 cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat
     1801 tatcgacgag cgcctgatca cccccgacgg ctccatgctg ttccgagtaa ccatcaacag
     1861 ctaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     1921 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1981 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2041 ttctattctg ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2101 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2161 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2221 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2281 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctcgaaat cgggggctcc
     2341 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2401 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2461 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2521 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2581 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2641 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2701 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2761 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2821 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2881 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2941 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3001 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3061 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3121 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3181 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3241 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3301 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3361 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3421 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3481 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3541 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3601 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3661 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3721 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3781 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3841 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3901 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3961 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4021 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4081 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4141 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4201 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4261 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4321 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4381 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4441 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4501 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4561 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4621 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4681 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4741 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4801 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4861 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4921 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4981 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5041 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5101 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5161 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5221 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5281 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5341 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5401 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5461 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5521 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5581 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5641 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5701 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5761 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5821 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5881 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5941 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6001 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6061 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6121 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6181 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6241 tttgaatgta tttag
//
