LOCUS       sequence-498265-        6681 bp    DNA     circular SYN 26-JAN-2026
DEFINITION  To generate inducible Flp-In cells expressing Nterm tagged miniTurbo
            full length VHL for ProtacID..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6681)
  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 6681)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 26, 2026 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-3XFLAG-miniTurbo-NLS-VHL
FEATURES             Location/Qualifiers
     source          1..6681
                     /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"
     regulatory      991..1000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1000..1065
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1108..1878
                     /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             1888..1908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1915..1935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2633..2650)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2639..2863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2996..3015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3147..3194
                     /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             3202..4224
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3202..4224
                     /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    4354..4475
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4391..4410)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4445..4464
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4524..4540)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4524..4540)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4537..4559)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4548..4564
                     /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(4572..4578,4579..4596,4597..4602))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4617..4638
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4755..4772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4926..5514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5006..5025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5685..6476,6477..6545))
                     /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(6546..6650)
                     /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 cttggtaccc gccaccatgg actacaagga ccacgacggt
     1021 gactacaagg accacgacat cgactacaag gacgacgacg acaagggggg agggggagga
     1081 tctgggggag ggggaggatc tatggcgatc ccgctgctga acgctaaaca gattctggga
     1141 cagctggacg gcgggagcgt ggcagtcctg cctgtggtcg actccaccaa tcagtacctg
     1201 ctggatcgaa tcggcgagct gaagagtggg gatgcttgca ttgcagaata tcagcaggca
     1261 gggagaggaa gcagagggag gaaatggttc tctccttttg gagctaacct gtacctgagt
     1321 atgttttggc gcctgaagcg gggaccagca gcaatcggcc tgggcccggt catcggaatt
     1381 gtcatggcag aagcgctgcg aaagctggga gcagacaagg tgcgagtcaa atggcccaat
     1441 gacctgtatc tgcaggatag aaagctggca ggcatcctgg tggagctggc cggaataaca
     1501 ggcgatgctg cacagatcgt cattggcgcc gggattaacg tggctatgag gcgcgtggag
     1561 gaaagcgtgg tcaatcaggg ctggatcaca ctgcaggaag cagggattaa cctggacagg
     1621 aatactctgg ccgctatgct gatccgagag ctgcgggcag ccctggaact gttcgagcag
     1681 gaaggcctgg ctccatatct gtcacggtgg gagaagctgg ataacttcat caatagaccc
     1741 gtgaagctga tcattgggga caaagagatt ttcgggatta gccgggggat tgataaacag
     1801 ggagccctgc tgctggaaca ggacggagtt atcaaaccct ggatgggcgg agaaatcagt
     1861 ctgcggtctg ccgaaaagat gcatatgccc aagaaaaaac gcaaggtgga agatcctaag
     1921 aaaaagcgga aagtgatgcc ccggagggcg gagaactggg acgaggccga ggtaggcgcg
     1981 gaggaggcag gcgtcgaaga gtacggccct gaagaagacg gcggggagga gtcgggcgcc
     2041 gaggagtccg gcccggaaga gtccggcccg gaggaactgg gcgccgagga ggagatggag
     2101 gccgggcggc cgcggcccgt gctgcgctcg gtgaactcgc gcgagccctc ccaggtcatc
     2161 ttctgcaatc gcagtccgcg cgtcgtgctg cccgtatggc tcaacttcga cggcgagccg
     2221 cagccctacc caacgctgcc gcctggcacg ggccgccgca tccacagcta ccgaggtcac
     2281 ctttggctct tcagagatgc agggacacac gatgggcttc tggttaacca aactgaatta
     2341 tttgtgccat ctctcaatgt tgacggacag cctatttttg ccaatatcac actgccagtg
     2401 tatactctga aagagcgatg cctccaggtt gtccggagcc tagtcaagcc tgagaattac
     2461 aggagactgg acatcgtcag gtcgctctac gaagatctgg aagaccaccc aaatgtgcag
     2521 aaagacctgg agcggctgac acaggagcgc attgcgcatc agcgtatggg agaccagata
     2581 tctgcggccg cctaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     2641 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2701 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2761 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2821 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2881 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2941 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3001 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3061 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3121 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3181 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3241 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3301 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3361 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3421 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     3481 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     3541 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     3601 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     3661 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     3721 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     3781 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     3841 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     3901 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     3961 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4021 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4081 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4141 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4201 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4261 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4321 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4381 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4441 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4501 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4561 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4621 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4681 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4741 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4801 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4861 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4921 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4981 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5041 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5101 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5161 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5221 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5281 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5341 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5401 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5461 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5521 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5581 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5641 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5701 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5761 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5821 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5881 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5941 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6001 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6061 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6121 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6181 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6241 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6301 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6361 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6421 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6481 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6541 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6601 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6661 cgaaaagtgc cacctgacgt c
//
