LOCUS       sequence-456990-        5970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  F-box domain of human Skp2 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 5970)
  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 5970)
  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-F-box:gs10:FRB:HA
FEATURES             Location/Qualifiers
     source          1..5970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     354..373
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    658..779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(695..714)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     749..768
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(828..844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(828..844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(841..863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    852..868
                     /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(876..882,883..900,901..906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    921..942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1059..1076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1230..1818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1310..1329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1989..2780,2781..2849))
                     /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     2612..2631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2850..2954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3029..3048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3220..3599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3600..3803
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3754..3774
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3848..3867
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3848..3866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4184..4462
                     /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             4463..4489
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(4508..4526)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4509..4526)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4546..4563)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4552..4776
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4822..5250
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4909..4928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5119..5140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5259..5279)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5264..5593
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5444..5579
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5506..5525
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(5660..5970,1..484)
                     /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(5714..5733)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
       61 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
      121 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
      181 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
      241 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
      301 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
      361 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
      421 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
      481 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
      541 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
      601 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
      661 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
      721 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
      781 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
      841 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
      901 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
      961 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1021 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     1981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2281 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2341 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2401 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2461 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2521 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2581 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2641 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2701 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2761 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2821 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2941 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     3001 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3061 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     3121 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     3181 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     3241 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     3301 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3361 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3421 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     3481 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     3541 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     3601 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     3661 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     3721 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     3781 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
     3841 tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct cggatccact
     3901 agtaacggcc gccagtgtgc tggaattcgc caccatgagg gagaacttcc ccggcgtgag
     3961 ctgggacagc ctgcccgacg agctgctgct gggcatcttc agctgcctgt gcctgcccga
     4021 gctgctgaag gtgagcggcg tgtgcaagag gtggtacagg ctggccagcg acgagagcct
     4081 gtggcagacc ctggacctga ccggcaagaa cctgcacccc gacgtgaccg gcaggctgct
     4141 gagcggcggc tctggcggcg gctccggagg ctctgcttct agaatcctct ggcatgagat
     4201 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     4261 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     4321 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag
     4381 gaagtacatg aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca
     4441 tgtgttccga cgaatctcaa agtacccata cgatgttcca gattacgctt aatctagagg
     4501 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     4561 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4621 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4681 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4741 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     4801 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4861 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4921 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4981 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5041 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5101 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5161 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5221 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     5281 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5341 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5401 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     5461 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5521 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5581 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     5641 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     5701 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     5761 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     5821 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     5881 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     5941 gggcgaagtg ccggggcagg atctcctgtc
//
