LOCUS       sequence-203039-        6489 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 6489)
  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 6489)
  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..6489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..38
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1533..2032
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1568..1591)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1965..1984
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2050..3075
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2050..3075
                     /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    3205..3326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3242..3261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3296..3315
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3375..3391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3375..3391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3388..3410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3399..3415
                     /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(3423..3429,3430..3447,3448..3453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3468..3489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3606..3623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3777..4365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3857..3876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4536..5327,5328..5396))
                     /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     5159..5178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5397..5501)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5576..5595)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5767..6146
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6147..6350
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6301..6321
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6395..6414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6395..6413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6449..6458
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6458..6481
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 tcgaggttat caaacaagtt tgtacaaaaa agttggcatg gccggcgaag gagatcagca
       61 ggacgctgcg cacaacatgg gcaaccacct gccgctcctg cctgagagtg aggaagaaga
      121 tgaaatggaa gttgaagacc aggatagtaa agaagccaaa aaaccaaaca tcataaattt
      181 tgacaccagt ctgccgacat cacatacata cctaggtgct gatatggaag aatttcatgg
      241 caggactttg cacgatgacg acagctgtca ggtgattcca gttcttccac aagtgatgat
      301 gatcctgatt cccggacaga cattacctct tcagcttttt caccctcaag aagtcagtat
      361 ggtgcggaat ttaattcaga aagatagaac ctttgctgtt cttgcataca gcaatgtaca
      421 ggaaagggaa gcacagtttg gaacaacagc agagatatat gcctatcgag aagaacagga
      481 ttttggaatt gagatagtga aagtgaaagc aattggaaga caaaggttca aagtccttga
      541 gctaagaaca cagtcagatg gaatccagca agctaaagtg caaattcttc ccgaatgtgt
      601 gttgccttca accatgtctg cagttcaatt agaatccctc aataagtgcc agatatttcc
      661 ttcaaaacct gtctcaagag aagaccaatg ttcatataaa tggtggcaga aataccagaa
      721 gagaaagttt cattgtgcaa atctaacttc atggcctcgc tggctgtatt ccttatatga
      781 tgctgagacc ttaatggaca gaatcaagaa acagctacgt gaatgggatg aaaatctaaa
      841 agatgattct cttccttcaa atccaataga tttttcttac agagtagctg cttgtcttcc
      901 tattgatgat gtattgagaa ttcagctcct taaaattggc agtgctatcc agcgacttcg
      961 ctgtgaatta gacattatga ataaatgtac ttccctttgc tgtaaacaat gtcaagaaac
     1021 agaaataaca accaaaaatg aaatattcag tttatcctta tgtgggccga tggcagctta
     1081 tgtgaatcct catggatatg tgcatgagac acttactgtg tataaggctt gcaacttgaa
     1141 tctgataggc cggccttcta cagaacacag ctggtttcct gggtatgcct ggactgttgc
     1201 ccagtgtaag atctgtgcaa gccatattgg atggaagttt acggccacca aaaaagacat
     1261 gtcacctcaa aaattttggg gcttaacgcg atctgctctg ttgcccacga tcccagacac
     1321 tgaagatgaa ataagtccag acaaagtaat actttgcttg taagacccag atttattgta
     1381 caaagtggtt cgataacgaa ttccgccccc cccccctaac gttactggcc gaagccgctt
     1441 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     1501 caatgtgagg gccgcgttta acgaattcta ccgggtaggg gaggcgcttt tcccaaggca
     1561 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     1621 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     1681 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     1741 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     1801 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     1861 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     1921 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     1981 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat
     2041 cccgccacca tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2101 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2161 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2221 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2281 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2341 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2401 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2461 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2521 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     2581 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     2641 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     2701 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     2761 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     2821 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     2881 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     2941 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3001 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3061 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3121 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3181 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3241 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3301 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3361 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3421 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3481 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3541 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3601 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3661 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3721 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3781 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3841 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3901 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3961 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4021 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4081 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4141 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4201 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4261 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4321 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4381 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4441 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4501 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4561 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4621 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4681 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4741 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4801 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4861 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4921 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4981 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5041 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5101 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5161 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5221 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5281 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5341 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5401 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5461 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5521 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     5581 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     5641 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     5701 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     5761 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     5821 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     5881 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     5941 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     6001 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     6061 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     6121 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     6181 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     6241 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     6301 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     6361 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     6421 aagctggcta gcgtttaaac ttaagcttgc caccatggac tacaaggacg acgatgacaa
     6481 gggcggagc
//
