LOCUS       sequence-220049-        6874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for plasmid integration using the Flp-In system. AID at C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6874)
  AUTHORS   Holland AJ, Fachinetti D, Han JS, Cleveland DW
  TITLE     Inducible, reversible system for the rapid and complete degradation 
            of proteins in mammalian cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):E3350-7. doi: 
            10.1073/pnas.1216880109. Epub 2012 Nov 12.
  PUBMED    23150568
REFERENCE   2  (bases 1 to 6874)
  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"; doi: 
            "10.1073/pnas.1216880109"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2012-12-4- 4"; volume: "109"; issue: "49"; pages: "E3350-7"
COMMENT     Sequence Label: pcDNA5-H2B-AID-EYFP
FEATURES             Location/Qualifiers
     source          1..6874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        177..556
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        557..760
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     711..731
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    762..780
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    783..801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     811..835
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             940..1317
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1324..2007
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="MGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNN
                     EPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSG
                     GPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMN
                     ERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAME
                     KCKSRA"
     CDS             join(2017..2019,2020..2733)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2059..2080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2320..2339)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2667..2688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2768..2785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2774..2998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3131..3150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3282..3329
                     /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             3337..4359
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3337..4359
                     /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    4489..4610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4526..4545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4580..4599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4659..4675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4659..4675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4672..4694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4683..4699
                     /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(4707..4713,4714..4731,4732..4737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4890..4907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5061..5649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5141..5160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5820..6611,6612..6680))
                     /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"
     primer_bind     6443..6462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6681..6785)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(6860..6874,1..5))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
       61 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      121 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      181 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      241 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      301 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      361 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      421 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      481 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      541 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      601 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      661 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      721 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
      781 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
      841 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
      901 gactctagcg tttaaactta agcttaccgt gccgccacca tgccagagcc agcgaagtct
      961 gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta aggcgcagaa gaaaggcggc
     1021 aagaagcgca agcgcagccg caaggagagc tattccatct atgtgtacaa ggttctgaag
     1081 caggtccacc ctgacaccgg catttcgtcc aaggccatgg gcatcatgaa ttcgtttgtg
     1141 aacgacattt tcgagcgcat cgcaggtgag gcttcccgcc tggcgcatta caacaagcgc
     1201 tcgaccatca cctccaggga gatccagacg gccgtgcgcc tgctgctgcc tggggagttg
     1261 gccaagcacg ccgtgtccga gggtactaag gccatcacca agtacaccag cgctaagccc
     1321 gggatgggca gtgtcgagct gaatctgagg gagactgagc tgtgtcttgg tcttcccggt
     1381 ggagatacag tggctccggt aaccggaaac aagagagggt tctcagagac ggttgatctg
     1441 aagctaaatc tgaataatga gcctgcaaac aaggaaggat ctacgactca tgacgtcgtg
     1501 acttttgatt ccaaggagaa gagtgcttgt cctaaagatc cagccaaacc tccggccaag
     1561 gcacaagttg tgggatggcc accggtgaga tcataccgga agaacgtgat ggtttcctgc
     1621 caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga aggtatcaat ggacggagca
     1681 ccgtacttga ggaaaatcga tttgaggatg tataaaagct acgatgagct ttctaatgct
     1741 ttgtccaaca tgttcagctc ttttaccatg ggcaaacatg gaggagaaga aggaatgata
     1801 gacttcatga atgagaggaa attgatggat ttggtgaata gctgggacta tgttccctct
     1861 tatgaagaca aagacggtga ttggatgctc gtcggcgacg ttccttggcc aatgttcgtc
     1921 gatacatgca agcgtttacg tctcatgaaa ggatcggatg ccattggtct cgctccgagg
     1981 gcgatggaga agtgcaagag cagagctgcg gatccagtga gcaagggcga ggagctgttc
     2041 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2101 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2161 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     2221 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2281 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2341 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2401 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2461 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2521 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2581 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc
     2641 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2701 atcactctcg gcatggacga gctgtacaag taaagcggcc gcgggcccgt ttaaacccgc
     2761 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2821 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2881 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2941 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3001 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3061 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3121 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3181 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3241 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3301 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3361 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3421 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3481 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3541 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3601 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3661 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3721 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3781 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3841 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3901 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3961 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4021 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4081 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4141 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4201 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4261 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4321 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4381 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4441 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4501 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4621 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4681 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4741 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4801 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4861 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5461 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5641 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5941 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6121 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6421 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6661 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6781 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6841 ccctatggtg cactctcagt acaatctgct ctga
//
