LOCUS       sequence-210646-        6502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of FLAG-YWAA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6502)
  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 6502)
  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 -YWAA-pGK-HYG
FEATURES             Location/Qualifiers
     source          1..6502
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        95..474
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..678
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     629..649
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     723..742
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        723..741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      777..786
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             786..809
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    843..867
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2194..2218)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        2362..2862
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2397..2420)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2795..2814
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2880..3905
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2880..3905
                     /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    4035..4156
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4072..4091)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4126..4145
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4205..4221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4205..4221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4229..4245
                     /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(4253..4259,4260..4277,4278..4283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4298..4319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4436..4453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4607..5195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4687..4706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5366..6157,6158..6226))
                     /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     5989..6008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6227..6331)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6406..6425)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      121 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      241 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      301 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      361 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      421 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      481 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      541 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      601 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      661 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
      721 attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttgcca
      781 ccatggacta caaggacgac gatgacaagc tcgatggagg tgcggccgct cgaggttatc
      841 aaacaagttt gtacaaaaaa gttggcatgg ccggcgaagg agatcagcag gacgctgcgc
      901 acaacatggg caaccacctg ccgctcctgc ctgagagtga ggaagaagat gaaatggaag
      961 ttgaagacca ggatagtaaa gaagccaaaa aaccaaacat cataaatttt gacaccagtc
     1021 tgccgacatc acatacatac ctaggtgctg atatggaaga atttcatggc aggactttgc
     1081 acgatgacga cagctgtcag gtgattccag ttcttccaca agtgatgatg atcctgattc
     1141 ccggacagac attacctctt cagctttttc accctcaaga agtcagtatg gtgcggaatt
     1201 taattcagaa agatagaacc tttgctgttc ttgcatacag caatgtacag gaaagggaag
     1261 cacagtttgg aacaacagca gagatatatg cctatcgaga agaacaggat tttggaattg
     1321 agatagtgaa agtgaaagca attggaagac aaaggttcaa agtccttgag ctaagaacac
     1381 agtcagatgg aatccagcaa gctaaagtgc aaattcttcc cgaatgtgtg ttgccttcaa
     1441 ccatgtctgc agttcaatta gaatccctca ataagtgcca gatatttcct tcaaaacctg
     1501 tctcaagaga agaccaatgt tcatataaat ggtggcagaa ataccagaag agaaagtttc
     1561 attgtgcaaa tctaacttca tggcctcgct ggctgtattc cttatatgat gctgagacct
     1621 taatggacag aatcaagaaa cagctacgtg aatgggatga aaatctaaaa gatgattctc
     1681 ttccttcaaa tccaatagat ttttcttaca gagtagctgc ttgtcttcct attgatgatg
     1741 tattgagaat tcagctcctt aaaattggca gtgctatcca gcgacttcgc tgtgaattag
     1801 acattatgaa taaatgtact tccctttgct gtaaacaatg tcaagaaaca gaaataacaa
     1861 ccaaaaatga aatattcagt ttatccttat gtgggccgat ggcagcttat gtgaatcctc
     1921 atggatatgt gcatgagaca cttactgtgt ataaggcttg caacttgaat ctgataggcc
     1981 ggccttctac agaacacagc tggtttcctg gggctgccgc gactgttgcc cagtgtaaga
     2041 tctgtgcaag ccatattgga tggaagttta cggccaccaa aaaagacatg tcacctcaaa
     2101 aattttgggg cttaacgcga tctgctctgt tgcccacgat cccagacact gaagatgaaa
     2161 taagtccaga caaagtaata ctttgcttgt aagacccagc tttcttgtac aaagtggttc
     2221 gataacgaat tccgcccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2281 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2341 ccgcgtttaa cgaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     2401 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     2461 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     2521 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     2581 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     2641 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     2701 gggctcagag gctgggaagg ggtgggtccg gggggcgggc tcaggggcgg gctcaggggc
     2761 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     2821 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat cccgccacca
     2881 tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     2941 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     3001 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     3061 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     3121 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     3181 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     3241 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     3301 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     3361 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     3421 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     3481 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     3541 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     3601 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     3661 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     3721 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     3781 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     3841 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     3901 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3961 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4021 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4081 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4141 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4201 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4261 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4321 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4381 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5041 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5221 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5341 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5401 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5461 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5521 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5581 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5641 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5701 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5761 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5821 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5881 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5941 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6001 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6061 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6121 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6181 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6241 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6301 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6361 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     6421 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     6481 cgcgagcaaa atttaagcta ca
//
