LOCUS       sequence-495091-        6668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate inducible Flp-In cells expressing Cterm tagged miniTurbo
            full length VHL for ProtacID..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6668)
  AUTHORS   Shrestha S, Maitland MER, Jing L, Duan S, Nie DY, St-Germain J, 
            Kanaris M, Barsyte-Lovejoy D, Arrowsmith CH, Raught B
  TITLE     Characterization of PROTAC specificity and endogenous protein 
            interactomes using ProtacID.
  JOURNAL   Nat Commun. 2025 Aug 29;16(1):8089. doi: 10.1038/s41467-025-63357-7.
  PUBMED    40883309
REFERENCE   2  (bases 1 to 6668)
  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"; journalName: "Nat 
            Commun."; date: "2025-08-29"; volume: "16(1):8089. doi"; pages: " 
            10.1038/s41467-025-63357-7"
COMMENT     Sequence Label: pCDNA5-FRT/TO-NLS-VHL-miniTurbo-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6668
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             987..1007
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1014..1034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1689..2459
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             2508..2573
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2620..2637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2626..2850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2983..3002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3134..3181
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3189..4211
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3189..4211
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4341..4462
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4378..4397)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4432..4451
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4511..4527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4511..4527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4524..4546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4535..4551
                     /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(4559..4565,4566..4583,4584..4589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4604..4625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4742..4759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4913..5501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4993..5012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5672..6463,6464..6532))
                     /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"
     promoter        complement(6533..6637)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttatgccca agaaaaaacg caaggtggaa gatcctaaga
     1021 aaaagcggaa agtgatgccc cggagggcgg agaactggga cgaggccgag gtaggcgcgg
     1081 aggaggcagg cgtcgaagag tacggccctg aagaagacgg cggggaggag tcgggcgccg
     1141 aggagtccgg cccggaagag tccggcccgg aggaactggg cgccgaggag gagatggagg
     1201 ccgggcggcc gcggcccgtg ctgcgctcgg tgaactcgcg cgagccctcc caggtcatct
     1261 tctgcaatcg cagtccgcgc gtcgtgctgc ccgtatggct caacttcgac ggcgagccgc
     1321 agccctaccc aacgctgccg cctggcacgg gccgccgcat ccacagctac cgaggtcacc
     1381 tttggctctt cagagatgca gggacacacg atgggcttct ggttaaccaa actgaattat
     1441 ttgtgccatc tctcaatgtt gacggacagc ctatttttgc caatatcaca ctgccagtgt
     1501 atactctgaa agagcgatgc ctccaggttg tccggagcct agtcaagcct gagaattaca
     1561 ggagactgga catcgtcagg tcgctctacg aagatctgga agaccaccca aatgtgcaga
     1621 aagacctgga gcggctgaca caggagcgca ttgcgcatca gcgtatggga gacgcggccg
     1681 caatggcgat cccgctgctg aacgctaaac agattctggg acagctggac ggcgggagcg
     1741 tggcagtcct gcctgtggtc gactccacca atcagtacct gctggatcga atcggcgagc
     1801 tgaagagtgg ggatgcttgc attgcagaat atcagcaggc agggagagga agcagaggga
     1861 ggaaatggtt ctctcctttt ggagctaacc tgtacctgag tatgttttgg cgcctgaagc
     1921 ggggaccagc agcaatcggc ctgggcccgg tcatcggaat tgtcatggca gaagcgctgc
     1981 gaaagctggg agcagacaag gtgcgagtca aatggcccaa tgacctgtat ctgcaggata
     2041 gaaagctggc aggcatcctg gtggagctgg ccggaataac aggcgatgct gcacagatcg
     2101 tcattggcgc cgggattaac gtggctatga ggcgcgtgga ggaaagcgtg gtcaatcagg
     2161 gctggatcac actgcaggaa gcagggatta acctggacag gaatactctg gccgctatgc
     2221 tgatccgaga gctgcgggca gccctggaac tgttcgagca ggaaggcctg gctccatatc
     2281 tgtcacggtg ggagaagctg gataacttca tcaatagacc cgtgaagctg atcattgggg
     2341 acaaagagat tttcgggatt agccggggga ttgataaaca gggagccctg ctgctggaac
     2401 aggacggagt tatcaaaccc tggatgggcg gagaaatcag tctgcggtct gccgaaaaga
     2461 tgcatggggg agggggagga tctgggggag ggggaggatc tgcagctgac tacaaggacc
     2521 acgacggtga ctacaaggac cacgacatcg actacaagga cgacgacgac aaggcagcgt
     2581 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2641 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2701 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2761 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2821 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2881 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2941 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3001 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3061 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3121 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     3181 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     3241 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     3301 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     3361 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     3421 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     3481 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     3541 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     3601 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     3661 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     3721 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     3781 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     3841 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     3901 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3961 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     4021 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     4081 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     4141 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     4201 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4261 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4321 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4441 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4501 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4561 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4621 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4681 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4741 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4801 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4861 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4921 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4981 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5041 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5101 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5161 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5221 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5281 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5341 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5401 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5461 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5521 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5581 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5641 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5701 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5761 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5821 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5881 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5941 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6001 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6061 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6121 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6181 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6241 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6301 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6361 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6421 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6481 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6541 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6601 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6661 ctgacgtc
//
