LOCUS       sequence-357727-        6820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB-Cre-HiBiT tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6820)
  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 6820)
  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-Cre-HiBiT
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             995..1273
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1307..2329
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     D"
     primer_bind     complement(2412..2429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2418..2642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2688..3116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2775..2794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2985..3006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3125..3145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3130..3458
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3309..3444
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3371..3390
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3525..4319
                     /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(3579..3598)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4189..4208
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4493..4614
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4530..4549)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4584..4603
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4663..4679)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4663..4679)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4676..4698)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4687..4703
                     /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(4711..4717,4718..4735,4736..4741))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4756..4777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4894..4911)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5065..5653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5145..5164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5824..6615,6616..6684))
                     /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     6447..6466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6685..6789)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatccag cacagtggcg gccgcgccac catgatcctc tggcatgaga tgtggcatga
     1021 aggcctggaa gaggcatctc gtttgtactt tggggaaagg aacgtgaaag gcatgtttga
     1081 ggtgctggag cccttgcatg ctatgatgga acggggcccc cagactctga aggaaacatc
     1141 ctttaatcag gcctatggtc gagatttaat ggaggcccaa gagtggtgca ggaagtacat
     1201 gaaatcaggg aatgtcaagg acctcaccca agcctgggac ctctattatc atgtgttccg
     1261 acgaatctca aagggtggtg gaggcagtgg cggtggaggc agcgccaatt tactgaccgt
     1321 acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc gcaagaacct
     1381 gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc
     1441 cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt ttcccgcaga
     1501 acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg cagtaaaaac
     1561 tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc tgccacgacc
     1621 aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa acgttgatgc
     1681 cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc aggttcgttc
     1741 actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc tggggattgc
     1801 ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag atatctcacg
     1861 tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg ttagcaccgc
     1921 aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat ggatttccgt
     1981 ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa aaaatggtgt
     2041 tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga tttttgaagc
     2101 aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc tggcctggtc
     2161 tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag tttcaatacc
     2221 ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact atatccgtaa
     2281 cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatg gaccggtggt
     2341 gagcggctgg cggctgttca agaagattag ctgactcgag tctagagggc ccgtttaaac
     2401 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2461 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2521 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2581 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2641 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2701 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2761 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2821 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2881 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2941 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3001 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3061 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3121 ctgtggaatg tgtgtcagtt agggtgtgga aagtcccagg ctccccagca ggcagaagta
     3181 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3241 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3301 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3361 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3421 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3481 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3541 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3601 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3661 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3721 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3781 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3841 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3901 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3961 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4021 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4081 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4141 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4201 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4261 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4321 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4381 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4441 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4501 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4561 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4621 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4681 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4741 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4801 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4861 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4921 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4981 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5041 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5101 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5161 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5221 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5281 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5341 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5401 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5461 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5521 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5581 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5641 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5701 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5761 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5821 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5881 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5941 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6001 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6061 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6121 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6181 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6241 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6301 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6361 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6421 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6481 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6541 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6601 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6661 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6721 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6781 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
