LOCUS       sequence-496497-        6522 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  To generate inducible Flp-In cells expressing Nterm tagged miniTurbo
            deltaVHL for ProtacID..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6522)
  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 6522)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 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-VHL54:213
FEATURES             Location/Qualifiers
     source          1..6522
                     /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(2474..2491)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2480..2704
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2837..2856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2988..3035
                     /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             3043..4065
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3043..4065
                     /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    4195..4316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4232..4251)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4286..4305
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4365..4381)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4365..4381)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4378..4400)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4389..4405
                     /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(4413..4419,4420..4437,4438..4443))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4458..4479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4596..4613)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4767..5355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4847..4866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5526..6317,6318..6386))
                     /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(6387..6491)
                     /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 aagtgatgga ggccgggcgg ccgcggcccg tgctgcgctc ggtgaactcg
     1981 cgcgagccct cccaggtcat cttctgcaat cgcagtccgc gcgtcgtgct gcccgtatgg
     2041 ctcaacttcg acggcgagcc gcagccctac ccaacgctgc cgcctggcac gggccgccgc
     2101 atccacagct accgaggtca cctttggctc ttcagagatg cagggacaca cgatgggctt
     2161 ctggttaacc aaactgaatt atttgtgcca tctctcaatg ttgacggaca gcctattttt
     2221 gccaatatca cactgccagt gtatactctg aaagagcgat gcctccaggt tgtccggagc
     2281 ctagtcaagc ctgagaatta caggagactg gacatcgtca ggtcgctcta cgaagatctg
     2341 gaagaccacc caaatgtgca gaaagacctg gagcggctga cacaggagcg cattgcgcat
     2401 cagcgtatgg gagaccagat atctgcggcc gcctaactcg agtctagagg gcccgtttaa
     2461 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2521 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2581 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2641 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2701 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2761 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2821 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2881 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2941 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     3001 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     3061 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     3121 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     3181 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     3241 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     3301 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     3361 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     3421 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     3481 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     3541 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     3601 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     3661 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     3721 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     3781 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     3841 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3901 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3961 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     4021 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     4081 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4141 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4201 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4261 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4321 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4381 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4441 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4501 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4561 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4621 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4681 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4741 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4801 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4861 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4921 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4981 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5041 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5101 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5161 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5221 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5281 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5341 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5401 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5461 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5521 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5581 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5641 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5701 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5761 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5821 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5881 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5941 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6001 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6061 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6121 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6181 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6241 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6301 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6361 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6421 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6481 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
