LOCUS       sequence-496494-        7049 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  To generate inducible Flp-In cells expressing Cterm tagged miniTurbo
            CRBNmidi for ProtacID..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7049)
  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 7049)
  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-NLS-CRBNmidi-miniTurbo-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7049
                     /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             2070..2840
                     /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             2889..2954
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3001..3018)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3007..3231
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3364..3383)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3515..3562
                     /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             3570..4592
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3570..4592
                     /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    4722..4843
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4759..4778)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4813..4832
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4892..4908)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4892..4908)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4905..4927)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4916..4932
                     /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(4940..4946,4947..4964,4965..4970))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4985..5006
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5123..5140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5294..5882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5374..5393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6053..6844,6845..6913))
                     /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(6914..7018)
                     /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 agtggccaaa aagccaaaca tcatcaactt cgacaccagt ctgccgacat
     1081 cacacaccta cctgggtgcc gatatggaag aatttcatgg ccgcacgttg cacgatgacg
     1141 acagcattca ggtgattccg gttctgccgc aagtgatgat gatcctggtt cccggacaga
     1201 ccttaccgct gcagctgttt cacccgcagg aagtcagtat ggtccggaat ctgattcaga
     1261 acgatcgcac ctttgctgtt ttagcataca gcaatgtaca ggaaagggaa gcagaatttg
     1321 gcactacagc cgagatttac gcctatcgtg aagaacagga tttcggtatt gagatcgtga
     1381 aagttaaagc aattggccgt caacgtttca aagtccttga gctacgcacc cagtcagatg
     1441 gtatccagca agctaaagtg cagattcttc cggaaggctc gggcgatgcg gagaccttaa
     1501 tggaccgtat caagaaacag ctccgtgaat gggatgagaa tctcaaagat gattcgctgc
     1561 cttcaaatcc aattgatttt agctactggg tagcggcgaa ccttcctatt gatgatagct
     1621 tgcgtattca gctcctgaaa attgattcgg cgatccagcg acttcgctgt gaattagaca
     1681 ttatgaataa atgtacttcc ctgtgctgca aacaatgtca agaaaccgaa atcacaacca
     1741 aaaacgaaat tttctcgctg tcccgcgagg gtccgatggc agcgtatgtg aatccgcatg
     1801 ggtatgtgca tgagattctg actgtgtata aggcgtgcaa cctgaacctg ataggccgcc
     1861 cgtctaccga acacagctgg tttccagggt atgcctggac ggttgcccag tgcaagatct
     1921 gtgcgagcca tattggatgg aagtttacgg ccaccaaaaa agacatgagc cctcaaaaat
     1981 tctggggctt aacgcgctct gcgctgattc ccacgatcgg tggcggaggg ggttctgggg
     2041 gcggaggtgg atcagcggcc gcaatggcga tcccgctgct gaacgctaaa cagattctgg
     2101 gacagctgga cggcgggagc gtggcagtcc tgcctgtggt cgactccacc aatcagtacc
     2161 tgctggatcg aatcggcgag ctgaagagtg gggatgcttg cattgcagaa tatcagcagg
     2221 cagggagagg aagcagaggg aggaaatggt tctctccttt tggagctaac ctgtacctga
     2281 gtatgttttg gcgcctgaag cggggaccag cagcaatcgg cctgggcccg gtcatcggaa
     2341 ttgtcatggc agaagcgctg cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca
     2401 atgacctgta tctgcaggat agaaagctgg caggcatcct ggtggagctg gccggaataa
     2461 caggcgatgc tgcacagatc gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg
     2521 aggaaagcgt ggtcaatcag ggctggatca cactgcagga agcagggatt aacctggaca
     2581 ggaatactct ggccgctatg ctgatccgag agctgcgggc agccctggaa ctgttcgagc
     2641 aggaaggcct ggctccatat ctgtcacggt gggagaagct ggataacttc atcaatagac
     2701 ccgtgaagct gatcattggg gacaaagaga ttttcgggat tagccggggg attgataaac
     2761 agggagccct gctgctggaa caggacggag ttatcaaacc ctggatgggc ggagaaatca
     2821 gtctgcggtc tgccgaaaag atgcatgggg gagggggagg atctggggga gggggaggat
     2881 ctgcagctga ctacaaggac cacgacggtg actacaagga ccacgacatc gactacaagg
     2941 acgacgacga caaggcagcg taactcgagt ctagagggcc cgtttaaacc cgctgatcag
     3001 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3061 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3121 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3181 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3241 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3301 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3361 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3421 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3481 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     3541 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     3601 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     3661 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3721 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3781 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     3841 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3901 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     3961 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4021 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4081 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4141 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4201 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4261 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4321 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4381 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4441 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4501 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4561 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     4621 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4681 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4741 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4801 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4861 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4921 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4981 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5041 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5101 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5161 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5221 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5281 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5341 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5401 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5461 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5521 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5581 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5641 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5701 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5761 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5821 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5881 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5941 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6001 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6061 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6121 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6181 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6241 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6301 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6361 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6421 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6481 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6541 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6601 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6661 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6721 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6781 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6841 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6901 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6961 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7021 catttccccg aaaagtgcca cctgacgtc
//
