LOCUS       sequence-393646-        6423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of N-terminal HiBIT-tagged human WDR5 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6423)
  AUTHORS   Schwalm MP, Kramer A, Dolle A, Weckesser J, Yu X, Jin J, Saxena K, 
            Knapp S
  TITLE     Tracking the PROTAC degradation pathway in living cells highlights 
            the importance of ternary complex measurement for PROTAC 
            optimization.
  JOURNAL   Cell Chem Biol. 2023 Jun 20:S2451-9456(23)00157-5. doi: 
            10.1016/j.chembiol.2023.06.002.
  PUBMED    37354907
REFERENCE   2  (bases 1 to 6423)
  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. 2023 Jun 20:S2451-9456(23)00157-5. doi: 
            10.1016/j.chembiol.2023.06.002."
COMMENT     Sequence Label: pNIC28-HiBIT-WDR5
FEATURES             Location/Qualifiers
     source          1..6423
                     /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             1288..1305
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1358..1376)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1372..1419
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1456..1911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1543..1562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1753..1774
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2003..2818)
                     /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(2003..2818)
                     /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     2726..2745
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2810..2829
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2940..3528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3429..3448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3682..3699
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3714..3853
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3779..3798)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3939..3961
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3955..4146)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4955..6037)
                     /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     5999..6018
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6038..6115)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6321..6340
                     /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 atccatggtg agcggctggc ggctgttcaa
      181 gaagattagc aacaggatca ggggcagcag cggcggcagc agcgcgacgg aggagaagaa
      241 gcccgagacc gaggccgcca gagcacagcc aaccccttcg tcatccgcca ctcagagcaa
      301 gcctacacct gtgaagccaa actatgctct aaagttcacc cttgctggcc acaccaaagc
      361 agtgtcctcc gtgaaattca gcccgaatgg agagtggctg gcaagttcat ctgctgataa
      421 acttattaaa atttggggcg cgtatgatgg gaaatttgag aaaaccatat ctggtcacaa
      481 gctgggaata tccgatgtag cctggtcgtc agattctaac cttcttgttt ctgcctcaga
      541 tgacaaaacc ttgaagatat gggacgtgag ctcgggcaag tgtctgaaaa ccctgaaggg
      601 acacagtaat tatgtctttt gctgcaactt caatccccag tccaacctta ttgtctcagg
      661 atcctttgac gaaagcgtga ggatatggga tgtgaaaaca gggaagtgcc tcaagacttt
      721 gccagctcac tcggatccag tctcggccgt tcattttaat cgtgatggat ccttgatagt
      781 ttcaagtagc tatgatggtc tctgtcgcat ctgggacacc gcctcgggcc agtgcctgaa
      841 gacgctcatc gatgacgaca acccccccgt gtcttttgtg aagttctccc cgaacggcaa
      901 atacatcctg gccgccacgc tggacaacac tctgaagctc tgggactaca gcaaggggaa
      961 gtgcctgaag acgtacactg gccacaagaa tgagaaatac tgcatatttg ccaatttctc
     1021 tgttactggt gggaagtgga ttgtgtctgg ctcagaggat aaccttgttt acatctggaa
     1081 ccttcagacg aaagagattg tacagaaact acaaggccac acagatgtcg tgatctcaac
     1141 agcttgtcac ccaacagaaa acatcatcgc ctctgctgcg ctagaaaatg acaaaacaat
     1201 taaactgtgg aagagtgact gctaacagta aaggtggata cggatccgaa ttcgagctcc
     1261 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     1321 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1381 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1441 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1501 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1561 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1621 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1681 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1741 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1801 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1861 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1921 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1981 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2041 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2101 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2161 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2221 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2281 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2341 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2401 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2461 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2521 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2581 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2641 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2701 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2761 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2821 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2881 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2941 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3001 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3061 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3121 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3181 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3241 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3301 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3361 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3421 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3481 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3541 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3601 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3661 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3721 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3781 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3841 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3901 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3961 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4021 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4081 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4141 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4201 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4261 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4321 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4381 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4441 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4501 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4561 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4621 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4681 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4741 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4801 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4861 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4921 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4981 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5041 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5101 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5161 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5221 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5281 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5341 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5401 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5461 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5521 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5581 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5641 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5701 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5761 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5821 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5881 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5941 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6001 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6061 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6121 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6181 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6241 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6301 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6361 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     6421 aat
//
