LOCUS       sequence-456985-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A fragment of human Skp1 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 6126)
  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 6126)
  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:Skp1(dC111):gs10:FRB:HA
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      132..720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     621..640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     874..891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1008..1029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1044..1049,1050..1067,1068..1074)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1082..1098
                     /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)."
     primer_bind     1087..1109
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1106..1122
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1106..1122
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1171..1292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1182..1201)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1236..1255
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1466..2260)
                     /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(1577..1596)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2187..2206
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2327..2656)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2341..2476
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2395..2414)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2641..2661
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2670..3098)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2780..2801)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2992..3011
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3144..3368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3357..3374
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        3394..3412
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3394..3411
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(3431..3457)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3458..3736)
                     /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             complement(4109..4138)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      4138..4147
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4209..4228)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4210..4228)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4273..4476)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4302..4322)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4477..4856
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5028..5047
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5122..5226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5227..5295,5296..6087)
                     /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     complement(5445..5464)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
       61 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
      121 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      181 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      241 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
      301 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      361 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      421 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      481 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      541 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      601 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      661 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      721 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      781 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      841 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      901 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      961 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     1021 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     1081 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     1141 tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca
     1201 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     1261 ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc cagcatgaga
     1321 tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt
     1381 tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg
     1441 gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga
     1501 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
     1561 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     1621 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     1681 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga
     1741 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
     1801 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     1861 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     1921 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
     1981 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     2041 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
     2101 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     2161 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     2221 cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg
     2281 tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg caaaagccta
     2341 ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag gcggcctcgg
     2401 cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg
     2461 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
     2521 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
     2581 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     2641 accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt aaatcagctc
     2701 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2761 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2821 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2881 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2941 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3001 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3061 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc tagagcccca
     3121 gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat gcctgctatt
     3181 gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca
     3241 cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc
     3301 accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga
     3361 aggcacagtc gaggctgatc agcgagctct agcatttagg tgacactata gaatagggcc
     3421 ctctagatta agcgtaatct ggaacatcgt atgggtactt tgagattcgt cggaacacat
     3481 gataatagag gtcccaggct tggaggaggt ccttgacatt ccctgatttc atgtacttcc
     3541 tgcaccactc ttgggcctcc attaaatctc gaccataggc ctgattaaag gatgtttcct
     3601 tcagagtctg ggggccccgt tccatcatag catgcaaggg ctccagcacc tcaaacatgc
     3661 ctttcacgtt cctttcccca aagtacaaac gagatgcctc ttccaggcct tcatgccaca
     3721 tctcatgcca gaggattcta gaagcagagc ctccggagcc gccgccagag ccgccgatgt
     3781 ccaggtagtt ggcggccagg atcagctcga acagggtgcc ctggtccacc ttcaggaact
     3841 cctggtccca cacggggatg tcgtcggtcc tcttctcctt gttctcgtcg tcctcaggag
     3901 gaggagggtc gtccttgtgg tgggtgcacc actggatcac cttcttcagg atggcggcgt
     3961 tcacgttggg caggggcacg gggtcgtcgt cgccctcgtc gtccatgccc aggtcctcca
     4021 gcatggtctt gatggtcacg ctctgcttgg cgatctccac gtccacctcg aagatctcgc
     4081 cgtcgctgct ctgcagcttg atgctgggca ggtcctcctc gctgatcagc ttctgctcca
     4141 tggtggcgaa ttccagcaca ctggcggccg ttactagtgg atccgagctc ggtaccaagc
     4201 ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct
     4261 agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     4321 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     4381 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     4441 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     4501 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     4561 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     4621 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     4681 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     4741 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     4801 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg
     4861 cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt
     4921 catgcaattg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc
     4981 agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg catcagagca
     5041 gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt
     5101 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5161 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5221 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5281 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5341 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5401 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5461 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5521 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5581 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5641 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5701 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5761 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5821 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5881 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5941 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6001 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6061 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6121 attgat
//
