LOCUS       sequence-471112-        5713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein overexpression of Thalidomide Binding domain of hCRBN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5713)
  AUTHORS   Lloyd HC, Li Y, Payne NC, Zhao Z, Xu W, Kroupova A, Zollman D, Long 
            T, Kabir F, Chen M, Freeman R, Feng EY, Xi SY, Hsu YC, Ciulli A, 
            Mazitschek R, Woo CM
  TITLE     A method for the detection and enrichment of endogenous cereblon 
            substrates.
  JOURNAL   Cell Chem Biol. 2025 Aug 5:S2451-9456(25)00225-9. doi: 
            10.1016/j.chembiol.2025.07.002.
  PUBMED    40782806
REFERENCE   2  (bases 1 to 5713)
  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: "Cell Chem 
            Biol. 2025 Aug 5:S2451-9456(25)00225-9. doi: 
            10.1016/j.chembiol.2025.07.002."
COMMENT     Sequence Label: pET28a-TBD
FEATURES             Location/Qualifiers
     source          1..5713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(766..1221)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(903..924)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1115..1134
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1258..1305)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1301..1319
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1372..1389)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1823..1840)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1850..1867)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1886..1908
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1923..1947
                     /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)."
     primer_bind     complement(1947..1966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1948..1966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2050..2069)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2275..2352
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2353..3435
                     /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     complement(2372..2391)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4244..4435
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4429..4451)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4537..4676
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4592..4611
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4691..4708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4862..5450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4942..4961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5561..5580)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(5572..5713,1..674)
                     /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             join(5572..5713,1..674)
                     /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     complement(5645..5664)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
       61 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
      121 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
      181 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
      241 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
      301 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
      361 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
      421 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
      481 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
      541 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
      601 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
      661 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
      721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
      781 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
      841 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
      901 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
      961 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     1021 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     1081 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     1141 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     1201 atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa
     1261 aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt
     1321 ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg
     1381 gtggtggtgc tcgagtgcgg ccgcaagctt gtcgacggag ctcgaattct tacaggcaca
     1441 gaataacttt gtccgggcta atttcatctt cggtgtccgg aatggtcggc agcagcgcgc
     1501 ttctcgtcag accccagaat ttctgcgggg acatatcttt tttggtggcc gtaaatttcc
     1561 aaccaatgtg gctcgcgcaa atcttacact gcgccacggt ccacgcgtag cccgggaacc
     1621 aagagtgctc ggtgctcgga cgaccgatca gattcagatt gcaggccttg taaacggtga
     1681 gcgtttcgtg cacgtaaccg tgcgggttca cgtaagctgc catcgggcca cacaggctca
     1741 gggaaaagat ttcgtttttg gtagtaattt ccgtttcttg gcattgtttg cagcacaggc
     1801 tcgtacactt gttcatcata tggctgccgc gcggcaccag gccgctgctg tgatgatgat
     1861 gatgatggct gctgcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     1921 ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg
     1981 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     2041 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     2101 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     2161 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     2221 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     2281 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     2341 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     2401 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     2461 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     2521 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     2581 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     2641 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     2701 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     2761 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     2821 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2881 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2941 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     3001 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     3061 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     3121 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     3181 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     3241 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     3301 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     3361 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3421 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     3481 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     3541 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     3601 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     3661 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     3721 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     3781 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3841 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3901 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3961 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     4021 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     4081 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     4141 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     4201 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     4261 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     4321 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     4381 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     4441 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     4501 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     4561 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4621 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4681 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4741 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4801 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4861 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4921 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4981 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5041 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5101 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5161 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5221 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5281 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     5341 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5401 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5461 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5521 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     5581 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     5641 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     5701 tatgggaagc ccg
//
