LOCUS       sequence-426717-        6339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRBN midi construct for use in crystallography and biophysical 
            studies enabling ligand and degrader design.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6339)
  AUTHORS   Kroupova A, Spiteri VA, Rutter ZJ, Furihata H, Darren D, 
            Ramachandran S, Chakraborti S, Haubrich K, Pethe J, Gonzales D, 
            Wijaya AJ, Rodriguez-Rios M, Sturbaut M, Lynch DM, Farnaby W, 
            Nakasone MA, Zollman D, Ciulli A
  TITLE     Design of a Cereblon construct for crystallographic and biophysical 
            studies of protein degraders.
  JOURNAL   Nat Commun. 2024 Oct 15;15(1):8885. doi: 10.1038/s41467-024-52871-9.
  PUBMED    39406745
REFERENCE   2  (bases 1 to 6339)
  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: "2024-10-15"; volume: "15(1):8885. doi"; pages: " 
            10.1038/s41467-024-52871-9"
COMMENT     Sequence Label: His6-TEV-CRBN-midi
FEATURES             Location/Qualifiers
     source          1..6339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1204..1221
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1274..1292)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1288..1335
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1372..1827
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1459..1478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1669..1690
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1919..2734)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1919..2734)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2642..2661
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2726..2745
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2856..3444
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3345..3364
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3598..3615
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3630..3769
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3695..3714)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3855..3877
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3871..4062)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4871..5953)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5915..5934
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5954..6031)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6237..6256
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccgccaaa aagccaaaca tcatcaactt
      181 cgacaccagt ctgccgacat cacacaccta cctgggtgcc gatatggaag aatttcatgg
      241 ccgcacgttg cacgatgacg acagcattca ggtgattccg gttctgccgc aagtgatgat
      301 gatcctggtt cccggacaga ccttaccgct gcagctgttt cacccgcagg aagtcagtat
      361 ggtccggaat ctgattcaga acgatcgcac ctttgctgtt ttagcataca gcaatgtaca
      421 ggaaagggaa gcagaatttg gcactacagc cgagatttac gcctatcgtg aagaacagga
      481 tttcggtatt gagatcgtga aagttaaagc aattggccgt caacgtttca aagtccttga
      541 gctacgcacc cagtcagatg gtatccagca agctaaagtg cagattcttc cggaaggctc
      601 gggcgatgcg gagaccttaa tggaccgtat caagaaacag ctccgtgaat gggatgagaa
      661 tctcaaagat gattcgctgc cttcaaatcc aattgatttt agctactggg tagcggcgaa
      721 ccttcctatt gatgatagct tgcgtattca gctcctgaaa attgattcgg cgatccagcg
      781 acttcgctgt gaattagaca ttatgaataa atgtacttcc ctgtgctgca aacaatgtca
      841 agaaaccgaa atcacaacca aaaacgaaat tttctcgctg tcccgcgagg gtccgatggc
      901 agcgtatgtg aatccgcatg ggtatgtgca tgagattctg actgtgtata aggcgtgcaa
      961 cctgaacctg ataggccgcc cgtctaccga acacagctgg tttccagggt atgcctggac
     1021 ggttgcccag tgcaagatct gtgcgagcca tattggatgg aagtttacgg ccaccaaaaa
     1081 agacatgagc cctcaaaaat tctggggctt aacgcgctct gcgctgattc ccacgatcta
     1141 acagtaaagg tggatacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc
     1201 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1261 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1321 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1381 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1441 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1501 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1561 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1621 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1681 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1741 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1801 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1861 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1921 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1981 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     2041 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     2101 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     2161 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2221 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2281 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2341 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2401 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2461 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2521 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2581 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2641 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2701 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2761 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2821 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2881 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2941 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3001 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3061 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3121 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3181 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3241 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3301 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3361 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3421 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3481 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3541 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3601 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3661 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3721 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3781 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3841 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3901 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3961 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4021 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4081 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4141 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4201 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4261 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4321 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4381 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4441 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4501 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4561 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4621 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4681 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4741 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4801 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4861 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4921 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4981 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5041 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5101 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5161 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5221 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5281 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5341 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5401 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5461 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5521 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5581 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5641 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5701 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5761 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5821 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5881 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5941 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6001 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     6061 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     6121 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6181 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6241 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6301 cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
