LOCUS       sequence-385991-        6336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression of the GST-tagged Cereblon Thalidomide binding 
            domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6336)
  TITLE     Knapp lab plasmids
REFERENCE   2  (bases 1 to 6336)
  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"
COMMENT     Sequence Label: pGTVL2-CRBN-TBD
FEATURES             Location/Qualifiers
     source          1..6336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     80..102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(96..287)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1096..2178)
                     /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     2140..2159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2179..2256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2462..2481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2565..2584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2565..2583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2584..2608
                     /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             2623..2645
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2656..2673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2680..3333
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK"
     CDS             3361..3381
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3765..3782
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3835..3853)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3849..3896
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3933..4388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4020..4039)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4230..4251
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4480..5295)
                     /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(4480..5295)
                     /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     5203..5222
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5287..5306
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5417..6005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5906..5925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6159..6176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6191..6330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6256..6275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
       61 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      121 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
      181 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
      241 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
      301 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
      361 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
      421 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
      481 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
      541 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
      601 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
      661 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
      721 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
      781 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
      841 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
      901 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
      961 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     1021 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     1081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     1201 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     1261 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     1321 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     1381 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     1441 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     1501 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     1561 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     1621 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     1681 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     1741 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     1801 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     1861 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     1921 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     1981 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     2041 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     2101 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     2161 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     2221 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     2281 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     2341 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     2401 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     2461 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     2521 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     2581 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     2641 ggagatatac atatgcacca tcatcatcat cattcttcta tgtcccctat actaggttat
     2701 tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa
     2761 tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa
     2821 ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag
     2881 tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa
     2941 gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg
     3001 agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct
     3061 gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat
     3121 gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca
     3181 atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca
     3241 caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa
     3301 gccacgtttg gtggtgccga ccatcctcca aaatcgagct caggtgtaga tctgggtacc
     3361 gagaacctgt acttccaatc catgacttcc ctttgctgta aacaatgtca agaaacagaa
     3421 ataacaacca aaaatgaaat attcagttta tccttatgtg ggccgatggc agcttatgtg
     3481 aatcctcatg gatatgtgca tgagacactt actgtgtata aggcttgcaa cttgaatctg
     3541 ataggccggc cttctacaga acacagctgg tttcctgggt atgcctggac tgttgcccag
     3601 tgtaagatct gtgcaagcca tattggatgg aagtttacgg ccaccaaaaa agacatgtca
     3661 cctcaaaaat tttggggctt aacgcgatct gctctgttgc ccacgtaaag gtggatacgg
     3721 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     3781 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     3841 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     3901 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     3961 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4021 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4081 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4141 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4201 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4261 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4321 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4381 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4441 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     4501 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     4561 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     4621 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     4681 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     4741 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     4801 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     4861 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     4921 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     4981 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     5041 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     5101 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     5161 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     5221 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     5281 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     5341 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5401 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5461 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5521 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     5581 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5641 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5701 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5761 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5821 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5881 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5941 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6001 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6061 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6121 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6181 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6241 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     6301 ctatcgctac gtgactgggt catggctgcg ccccga
//
