LOCUS       sequence-456989-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB protein in fusion with a bipartite SSD degron, consisting of 
            fragments of human Skp1 and SOCS2 proteins, through a flexible 
            glycine-serine linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  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 6273)
  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-FRB:GSlinker:SSD
FEATURES             Location/Qualifiers
     source          1..6273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(213..242)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      242..251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(313..332)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(314..332)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(377..580)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(406..426)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        581..960
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1132..1151
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1226..1330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1331..1399,1400..2191)
                     /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(1549..1568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2362..2950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2851..2870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3104..3121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3238..3259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3274..3279,3280..3297,3298..3304)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3312..3328
                     /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     3317..3339
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3336..3352
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3336..3352
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3401..3522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3412..3431)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3466..3485
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3696..4490)
                     /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(3807..3826)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4417..4436
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4557..4886)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4571..4706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4625..4644)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4871..4891
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4900..5328)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5010..5031)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5222..5241
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5374..5598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5587..5604
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        5624..5642
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5624..5641
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(6198..6273,1..203))
                     /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"
ORIGIN
        1 tacttcctgc accactcttg ggcctccatt aaatctcgac cataggcctg attaaaggat
       61 gtttccttca gagtctgggg gccccgttcc atcatagcat gcaagggctc cagcacctca
      121 aacatgcctt tcacgttcct ttccccaaag tacaaacgag atgcctcttc caggccttca
      181 tgccacatct catgccagag gattctagaa gccaggtcct cctcgctgat cagcttctgc
      241 tccatggtgg cgaattccag cacactggcg gccgttacta gtggatccga gctcggtacc
      301 aagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa gcagtgggtt
      361 ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca
      421 tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
      481 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
      541 gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta
      601 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
      661 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
      721 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
      781 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
      841 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
      901 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
      961 aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat
     1021 tcttcatgca attgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact
     1081 actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag
     1141 agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc
     1201 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1261 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1321 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1381 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1441 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1501 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1561 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1621 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1681 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1741 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1801 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1861 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1921 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1981 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2041 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2101 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2161 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2221 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2281 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2341 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2401 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2461 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     2521 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2581 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2641 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2701 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2761 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2821 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2881 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2941 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3001 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3061 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3121 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     3181 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3241 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     3301 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     3361 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
     3421 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     3481 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
     3541 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
     3601 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt
     3661 cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     3721 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     3781 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     3841 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     3901 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     3961 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     4021 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     4081 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     4141 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     4201 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     4261 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     4321 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     4381 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     4441 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     4501 cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     4561 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     4621 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     4681 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     4741 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     4801 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     4861 gactttccac accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt
     4921 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     4981 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5041 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5101 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5161 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5221 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5281 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc
     5341 tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat
     5401 gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa
     5461 tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag
     5521 tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct
     5581 ggcaactaga aggcacagtc gaggctgatc agcgagctct agcatttagg tgacactata
     5641 gaatagggcc ctctagatta gatgtccagg tagttggcgg ccaggatcag ctcgaacagg
     5701 gtgccctggt ccaccttcag gaactcctgg tcccacacgg ggatgtcgtc ggtcctcttc
     5761 tccttgttct cgtcgtcctc aggaggagga gggtcgtcct tgtggtgggt gcaccactgg
     5821 atcaccttct tcaggatggc ggcgttcacg ttgggcaggg gcacggggtc gtcgtcgccc
     5881 tcgtcgtcca tgcccaggtc ctccagcatg gtcttgatgg tcacgctctg cttggcgatc
     5941 tccacgtcca cctcgaagat ctcgccgtcg ctgctctgca gcttgatgct ggggctcccg
     6001 ccagatccgc ctccggatcc ccccacctgg aacttgtact cctccaggta gtccttcagc
     6061 ctggtgggca ggggcaggcc ccagatggcg ccggtgcact tgttgatggt cagcctgcac
     6121 aggtgctgca ggctgggggc gctggtagag cctccggagc cgccgccaga gcctccggag
     6181 ccgccgccag agccgccctt tgagattcgt cggaacacat gataatagag gtcccaggct
     6241 tggaggaggt ccttgacatt ccctgatttc atg
//
