LOCUS       sequence-490356-        6532 bp    DNA     circular SYN 15-DEC-2025
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 6532)
  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 6532)
  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-3XFLAG-miniTurbo-NLS-VHL54:213
FEATURES             Location/Qualifiers
     source          1..6532
                     /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    830..848
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    851..869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     879..903
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1001..1010
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1010..1075
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1118..1888
                     /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             1898..1918
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1925..1945
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2484..2501)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2490..2714
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2847..2866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2998..3045
                     /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             3053..4075
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3053..4075
                     /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    4205..4326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4242..4261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4296..4315
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4375..4391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4375..4391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4388..4410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4399..4415
                     /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(4423..4429,4430..4447,4448..4453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4468..4489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4606..4623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4777..5365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4857..4876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5536..6327,6328..6396))
                     /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(6397..6501)
                     /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 ccctagctct ccctatcagt
      841 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
      901 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
      961 agcctccgga ctctagcgtt taaacttaag cttggtaccc gccaccatgg actacaagga
     1021 ccacgacggt gactacaagg accacgacat cgactacaag gacgacgacg acaagggggg
     1081 agggggagga tctgggggag ggggaggatc tatggcgatc ccgctgctga acgctaaaca
     1141 gattctggga cagctggacg gcgggagcgt ggcagtcctg cctgtggtcg actccaccaa
     1201 tcagtacctg ctggatcgaa tcggcgagct gaagagtggg gatgcttgca ttgcagaata
     1261 tcagcaggca gggagaggaa gcagagggag gaaatggttc tctccttttg gagctaacct
     1321 gtacctgagt atgttttggc gcctgaagcg gggaccagca gcaatcggcc tgggcccggt
     1381 catcggaatt gtcatggcag aagcgctgcg aaagctggga gcagacaagg tgcgagtcaa
     1441 atggcccaat gacctgtatc tgcaggatag aaagctggca ggcatcctgg tggagctggc
     1501 cggaataaca ggcgatgctg cacagatcgt cattggcgcc gggattaacg tggctatgag
     1561 gcgcgtggag gaaagcgtgg tcaatcaggg ctggatcaca ctgcaggaag cagggattaa
     1621 cctggacagg aatactctgg ccgctatgct gatccgagag ctgcgggcag ccctggaact
     1681 gttcgagcag gaaggcctgg ctccatatct gtcacggtgg gagaagctgg ataacttcat
     1741 caatagaccc gtgaagctga tcattgggga caaagagatt ttcgggatta gccgggggat
     1801 tgataaacag ggagccctgc tgctggaaca ggacggagtt atcaaaccct ggatgggcgg
     1861 agaaatcagt ctgcggtctg ccgaaaagat gcatatgccc aagaaaaaac gcaaggtgga
     1921 agatcctaag aaaaagcgga aagtgatgga agcgggacgc cctcgtcctg tcctgcgttc
     1981 agttaatagc cgcgaaccat cccaagtcat tttctgtaac cgcagtcctc gtgtggtact
     2041 tccggtgtgg ttgaattttg acggcgaacc ccaaccgtat ccgacacttc ctcccggtac
     2101 tggtcgccgt atccactcat atcgcgggca cttgtggctg ttccgcgatg caggtaccca
     2161 cgacggattg cttgtcaacc agaccgaatt gttcgtgcca tcgttaaatg tcgatggcca
     2221 gccgatcttt gccaacatta cattgccagt ctatacattg aaggagcgtt gtttgcaggt
     2281 ggtgcgctcc ctggtcaaac ccgagaacta tcgtcgctta gacatcgttc gctcgttgta
     2341 cgaagactta gaggaccacc ctaacgtcca aaaagacctg gagcgcttga ctcaggagcg
     2401 tattgcgcat cagcgtatgg gagaccagat atctgcggcc gcctaactcg agtctagagg
     2461 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2521 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2581 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2641 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2701 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2761 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2821 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2881 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2941 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3001 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3061 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3121 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3181 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     3241 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     3301 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     3361 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     3421 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     3481 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     3541 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     3601 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     3661 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     3721 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     3781 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     3841 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3901 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3961 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4021 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4081 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4141 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4201 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4261 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4321 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4381 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4441 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4501 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4561 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4621 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4681 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4741 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4801 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4861 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4921 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4981 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5041 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5101 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5161 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5221 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5281 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5341 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5401 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5461 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5521 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5581 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5641 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5701 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5761 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5821 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5881 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5941 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6001 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6061 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6121 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6181 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6241 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6301 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6361 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6421 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6481 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
