LOCUS       sequence-456691-        6093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human Rbx1 protein in fusion with FRB domain through a flexible 
            glycine-serine linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6093)
  AUTHORS   Verbic A, Lebar T, Praznik A, Jerala R
  TITLE     Subunits of an E3 Ligase Complex as Degrons for Efficient 
            Degradation of Cytosolic, Nuclear, and Membrane Proteins.
  JOURNAL   ACS Synth Biol. 2024 Mar 15;13(3):792-803. doi: 
            10.1021/acssynbio.3c00588. Epub 2024 Feb 26.
  PUBMED    38404221
REFERENCE   2  (bases 1 to 6093)
  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/acssynbio.3c00588"; journalName: "ACS Synth Biol"; date: 
            "2024-03-15- 15"; volume: "13"; issue: "3"; pages: "792-803"
COMMENT     Sequence Label: pcDNA3.1-myc:Rbx1:gs10:FRB:HA
FEATURES             Location/Qualifiers
     source          1..6093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..90
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(309..413)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(488..507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        679..1058
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1059..1262
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1213..1233
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1307..1326
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1307..1325
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1388..1397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1397..1426
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      1430..1439
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1787..2065
                     /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             2066..2092
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2111..2129)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2112..2129)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2149..2166)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2155..2379
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2425..2853
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2512..2531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2722..2743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2862..2882)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3027..3161
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3088..3107
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3242..4036
                     /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(3296..3315)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3906..3925
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4210..4331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4247..4266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4301..4320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4380..4396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4380..4396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4393..4415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4404..4420
                     /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(4428..4434,4435..4452,4453..4458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4473..4494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4611..4628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4782..5370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4862..4881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5541..6093,1..239,240..308))
                     /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"
ORIGIN
        1 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
       61 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      301 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      361 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      421 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
      481 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
      541 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      601 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      661 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
      721 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      781 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      841 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      901 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      961 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1021 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1081 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1141 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1201 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1261 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     1321 atagggagac ccaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct
     1381 ggaattcgcc accatggagc agaagctgat cagcgaggag gacctggccg ccgccatgga
     1441 cgtggacacc cccagcggca ccaacagcgg cgccggcaag aagaggttcg aggtgaagaa
     1501 gtggaacgcc gtggccctgt gggcctggga catcgtggtg gacaactgcg ccatctgcag
     1561 gaaccacatc atggacctgt gcatcgagtg ccaggccaac caggccagcg ccaccagcga
     1621 ggagtgcacc gtggcctggg gcgtgtgcaa ccacgccttc cacttccact gcatcagcag
     1681 gtggctgaag accaggcagg tgtgccccct ggacaacagg gagtgggagt tccagaagta
     1741 cggccacggc ggctctggcg gcggctccgg aggctctgct tctagaatcc tctggcatga
     1801 gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa
     1861 aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct
     1921 gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg
     1981 caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta
     2041 tcatgtgttc cgacgaatct caaagtaccc atacgatgtt ccagattacg cttaatctag
     2101 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     2161 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2221 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2281 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2341 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     2401 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2461 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2521 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2581 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2641 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2701 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2761 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2821 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     2881 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2941 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3001 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccagtt ccgcccattc
     3061 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3121 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3181 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3241 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3301 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3361 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3421 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3481 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3541 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3601 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3661 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3721 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3781 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3841 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3901 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3961 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4021 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4081 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4141 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4201 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4261 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4321 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4381 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4441 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4501 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4561 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4621 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4681 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4741 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4801 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4861 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4921 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4981 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5041 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5101 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5161 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5221 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5281 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5341 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5401 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5461 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5521 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5581 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5641 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5701 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5761 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5821 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5881 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5941 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6001 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6061 ccatccgtaa gatgcttttc tgtgactggt gag
//
