LOCUS       sequence-210775-        6501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of FLAG-CRBN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6501)
  AUTHORS   Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, Wong KK,
            Bradner JE, Kaelin WG Jr
  TITLE     The myeloma drug lenalidomide promotes the cereblon-dependent 
            destruction of Ikaros proteins.
  JOURNAL   Science. 2014 Jan 17;343(6168):305-9. doi: 10.1126/science.1244917. 
            Epub 2013 Nov 29.
  PUBMED    24292623
REFERENCE   2  (bases 1 to 6501)
  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: 
            "10.1126/science.1244917"; journalName: "Science"; date: 
            "2014-01-17- 17"; volume: "343"; issue: "6168"; pages: "305-9"
COMMENT     Sequence Label: pcDNA3-FLAG-CRBN-pGK-HYG
FEATURES             Location/Qualifiers
     source          1..6501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     761..780
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        761..779
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      815..824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             824..847
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    881..905
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2232..2256)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        2400..2899
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2435..2458)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2832..2851
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2917..3942
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2917..3942
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4072..4193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4109..4128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4163..4182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4242..4258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4242..4258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4255..4277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4266..4282
                     /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(4290..4296,4297..4314,4315..4320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4335..4356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4473..4490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4644..5232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4724..4743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5403..6194,6195..6263))
                     /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     6026..6045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6264..6368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6443..6462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
       61 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      121 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      721 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      781 agacccaagc tggctagcgt ttaaacttaa gcttgccacc atggactaca aggacgacga
      841 tgacaagctc gatggaggtg cggccgctcg aggttatcaa acaagtttgt acaaaaaagt
      901 tggcatggcc ggcgaaggag atcagcagga cgctgcgcac aacatgggca accacctgcc
      961 gctcctgcct gagagtgagg aagaagatga aatggaagtt gaagaccagg atagtaaaga
     1021 agccaaaaaa ccaaacatca taaattttga caccagtctg ccgacatcac atacatacct
     1081 aggtgctgat atggaagaat ttcatggcag gactttgcac gatgacgaca gctgtcaggt
     1141 gattccagtt cttccacaag tgatgatgat cctgattccc ggacagacat tacctcttca
     1201 gctttttcac cctcaagaag tcagtatggt gcggaattta attcagaaag atagaacctt
     1261 tgctgttctt gcatacagca atgtacagga aagggaagca cagtttggaa caacagcaga
     1321 gatatatgcc tatcgagaag aacaggattt tggaattgag atagtgaaag tgaaagcaat
     1381 tggaagacaa aggttcaaag tccttgagct aagaacacag tcagatggaa tccagcaagc
     1441 taaagtgcaa attcttcccg aatgtgtgtt gccttcaacc atgtctgcag ttcaattaga
     1501 atccctcaat aagtgccaga tatttccttc aaaacctgtc tcaagagaag accaatgttc
     1561 atataaatgg tggcagaaat accagaagag aaagtttcat tgtgcaaatc taacttcatg
     1621 gcctcgctgg ctgtattcct tatatgatgc tgagacctta atggacagaa tcaagaaaca
     1681 gctacgtgaa tgggatgaaa atctaaaaga tgattctctt ccttcaaatc caatagattt
     1741 ttcttacaga gtagctgctt gtcttcctat tgatgatgta ttgagaattc agctccttaa
     1801 aattggcagt gctatccagc gacttcgctg tgaattagac attatgaata aatgtacttc
     1861 cctttgctgt aaacaatgtc aagaaacaga aataacaacc aaaaatgaaa tattcagttt
     1921 atccttatgt gggccgatgg cagcttatgt gaatcctcat ggatatgtgc atgagacact
     1981 tactgtgtat aaggcttgca acttgaatct gataggccgg ccttctacag aacacagctg
     2041 gtttcctggg tatgcctgga ctgttgccca gtgtaagatc tgtgcaagcc atattggatg
     2101 gaagtttacg gccaccaaaa aagacatgtc acctcaaaaa ttttggggct taacgcgatc
     2161 tgctctgttg cccacgatcc cagacactga agatgaaata agtccagaca aagtaatact
     2221 ttgcttgtaa gacccagctt tcttgtacaa agtggttcga taacgaattc cgcccccccc
     2281 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     2341 attttccacc atattgccgt cttttggcaa tgtgagggcc gcgtttaacg aattctaccg
     2401 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     2461 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     2521 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     2581 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     2641 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     2701 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     2761 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     2821 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     2881 ctcatctccg ggcctttcga cctgcatccc gccaccatga aaaagcctga actcaccgcg
     2941 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3001 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     3061 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     3121 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     3181 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3241 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     3301 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3361 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3421 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     3481 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     3541 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     3601 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     3661 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     3721 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     3781 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     3841 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     3901 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     3961 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4021 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4201 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4261 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4321 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4381 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4441 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4501 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4561 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4621 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4681 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4741 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4801 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4861 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4921 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4981 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5041 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5101 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5161 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5221 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5281 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5341 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5401 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5461 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5521 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5581 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5641 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5701 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5761 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5821 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5881 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5941 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6001 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6061 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6121 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6181 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6241 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6301 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6361 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     6421 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     6481 tgctccctgc ttgtgtgttg g
//
