LOCUS       sequence-232047-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminus of plum pox virus protease (PPVp) fused to 
            FKBP-rapamycin binding (FRB) domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  AUTHORS   Fink T, Lonzaric J, Praznik A, Plaper T, Merljak E, Leben K, Jerala 
            N, Lebar T, Strmsek Z, Lapenta F, Bencina M, Jerala R
  TITLE     Design of fast proteolysis-based signaling and logic circuits in 
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2019 Feb;15(2):115-122. doi: 
            10.1038/s41589-018-0181-6. Epub 2018 Dec 10.
  PUBMED    30531965
REFERENCE   2  (bases 1 to 6139)
  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: 
            "10.1038/s41589-018-0181-6"; journalName: "Nat Chem Biol"; date: 
            "2019-02"; volume: "15"; issue: "2"; pages: "115-122"
COMMENT     Sequence Label: FRB_nPPVp
FEATURES             Location/Qualifiers
     source          1..6139
                     /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"
     regulatory      958..967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             967..996
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             997..1275
                     /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"
     promoter        complement(1692..1710)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1693..1710)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1730..1747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1736..1960
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2006..2434
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2093..2112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2303..2324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2443..2463)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2448..2777
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2628..2763
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2690..2709
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2844..3638
                     /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(2898..2917)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3508..3527
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3812..3933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3849..3868)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3903..3922
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3982..3998)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3982..3998)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3995..4017)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4006..4022
                     /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(4030..4036,4037..4054,4055..4060))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4075..4096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4213..4230)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4384..4972)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4464..4483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5143..5934,5935..6003))
                     /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     5766..5785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6004..6108)
                     /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 gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgcggccg ctctagagcc
      961 accatggagc agaagctgat cagcgaggag gatctgatcc tctggcatga gatgtggcat
     1021 gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt
     1081 gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct gaaggaaaca
     1141 tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac
     1201 atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta tcatgtgttc
     1261 cgacgaatct caaagggatc cggaagtagc aagagcctgt tccgcggcct gcgcgactac
     1321 aaccccatcg ccagcagcat ctgccagctg aacaacagca gcggcgcccg ccagagcgag
     1381 atgttcggcc tgggcttcgg cggcctgatc gtgaccaacc agcacctgtt caagcgcaac
     1441 gacggcgagc tgaccatccg cagccaccac ggcgagttcg tggtgaagga caccaagacc
     1501 ctgaagctgc tgccctgcaa gggccgcgac atcgtgatca tccgcctgcc caaggacttc
     1561 ccccccttcc ccaagcgcct gcaattccgc acccccacca ccgaggaccg cgtgtgcctg
     1621 atcggcagca acttccagac ctaaactagt gcggccgcct gcagctcgag catgcatcta
     1681 gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     1741 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1801 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1861 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1921 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1981 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2041 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2101 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2161 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2221 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2281 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2341 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2401 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2461 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2521 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2581 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2641 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2701 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2761 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2821 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2881 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2941 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3001 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3061 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3121 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3181 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3241 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3301 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3361 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3421 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3481 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3541 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3601 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3661 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3721 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3781 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3841 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3901 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3961 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4021 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4081 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4141 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4201 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4261 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4321 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4381 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4441 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4501 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4561 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4621 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4681 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4741 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4801 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4861 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4921 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4981 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5041 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5101 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5161 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5221 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5281 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5341 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5401 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5461 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5521 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5581 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5641 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5701 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5761 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5821 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5881 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5941 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6001 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6061 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6121 aaaagtgcca cctgacgtc
//
