LOCUS       sequence-388717-        5543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5543)
  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 5543)
  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: TRIP12-V2_A755-A841
FEATURES             Location/Qualifiers
     source          1..5543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(56..1138)
                     /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     1100..1119
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1139..1216)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1422..1441
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1525..1544
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1525..1543
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1544..1568
                     /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             1583..1605
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1615..1632
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1645..1665
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1932..1949
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2002..2020)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2016..2063
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2100..2555
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2187..2206)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2397..2418
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2647..3462)
                     /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(2647..3462)
                     /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     3370..3389
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3454..3473
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3584..4172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4073..4092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4326..4343
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4358..4497
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4423..4442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4583..4605
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4599..4790)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
       61 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      121 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
      181 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
      241 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
      301 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
      361 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
      421 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
      481 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
      541 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
      601 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
      661 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
      721 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
      781 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
      841 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
      901 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
      961 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     1021 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     1081 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     1141 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     1201 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     1261 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     1321 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     1381 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     1441 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     1501 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     1561 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgcatcat
     1621 catcatcatc acagcagcgg cagagaaaac ttgtatttcc agggcgcaca gaacacagat
     1681 ggtgcgatat ggcagtggcg tgatgatcgg ggcctctggc atccatataa caggattgac
     1741 agccggatca ttgaggctgc ccaccaggtt ggcgaagatg aaatcagtct gtcaactctg
     1801 ggcagggttt acactattga cttcaattcc atgcagcaaa tcaatgagga cacgggaaca
     1861 gcacgtgcca ttcagagaaa acctaacccg ttagccaata gttgatgacg aagcttgcgg
     1921 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     1981 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2041 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2101 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2161 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2221 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2281 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2341 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2401 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2461 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2521 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2581 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     2641 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2701 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2761 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     2821 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2881 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     2941 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3001 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     3061 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     3121 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     3181 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     3241 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     3301 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     3361 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3421 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     3481 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     3541 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3601 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3661 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3721 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3781 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3841 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3901 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3961 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4021 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4081 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4141 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4201 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4261 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4321 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4381 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4441 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4501 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4561 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4621 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4681 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4741 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4801 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4861 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4921 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4981 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5041 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5101 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5161 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5221 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     5281 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5341 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5401 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5461 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5521 gggttgaagg ctctcaaggg cat
//
