LOCUS       sequence-247045-        5576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination. May not contain 
            entire coding region of gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5576)
  AUTHORS   Munzker L, Kimani SW, Fowkes MM, Dong A, Zheng H, Li Y, Dasovich M, 
            Zak KM, Leung AKL, Elkins JM, Kessler D, Arrowsmith CH, Halabelian 
            L, Bottcher J
  TITLE     A ligand discovery toolbox for the WWE domain family of human E3 
            ligases.
  JOURNAL   Commun Biol. 2024 Jul 24;7(1):901. doi: 10.1038/s42003-024-06584-w.
  PUBMED    39048679
REFERENCE   2  (bases 1 to 5576)
  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: "Commun 
            Biol."; date: "2024-07-24"; volume: "7(1):901. doi"; pages: " 
            10.1038/s42003-024-06584-w"
COMMENT     Sequence Label: 6MIW
FEATURES             Location/Qualifiers
     source          1..5576
                     /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             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             441..458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(511..529)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      525..572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      609..1064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(696..715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     906..927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1156..1971)
                     /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(1156..1971)
                     /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     1879..1898
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1963..1982
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2093..2681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2582..2601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2835..2852
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2867..3006
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2932..2951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3092..3114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3108..3299)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4108..5190)
                     /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     5152..5171
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5191..5268)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5474..5493
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga
      121 gaaaacttgt atttccaggg ccaatccaac agcaacaact ggcgctggtt tgatgatcgc
      181 tctgggcgtt ggtgtagtta cagtgcaagc aacaatagca ctattgattc tgcctggaaa
      241 tctggagaga caagcgtgcg attcactgca ggccgaagaa gatacacggt ccaattcact
      301 acaatggtgc aggttaatga ggaaacaggg aaccgacgcc ctgtgatgct gactctcctc
      361 agggtacctc ggctgaataa aaattcaaaa aacagcaatg gacaggaact atgatgacga
      421 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
      481 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
      541 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
      601 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
      661 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
      721 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
      781 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
      841 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
      901 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
      961 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1021 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1081 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1141 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1201 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1261 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1321 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1381 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1441 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1501 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     1561 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1621 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     1681 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     1741 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     1801 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     1861 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     1921 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     1981 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2041 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2101 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2161 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2221 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2281 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2341 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2401 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2461 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2521 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2581 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2641 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2701 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     2761 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     2821 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     2881 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     2941 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3001 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3061 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3121 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3181 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3241 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3301 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3361 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3421 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3481 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3541 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3601 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3661 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3721 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     3781 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     3841 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     3901 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     3961 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4021 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4081 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4141 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4201 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4261 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4321 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4381 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4441 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4501 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4561 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     4621 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4681 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     4741 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4801 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4861 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4921 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4981 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5041 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5101 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5161 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5221 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5281 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5341 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5401 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5461 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5521 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
