LOCUS       sequence-472953-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human HOIL-1L NZF (195-252).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  AUTHORS   Michel MA, Scutts S, Komander D
  TITLE     Secondary interactions in ubiquitin-binding domains achieve linkage 
            or substrate specificity.
  JOURNAL   Cell Rep. 2024 Aug 27;43(8):114545. doi: 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23.
  PUBMED    39052481
REFERENCE   2  (bases 1 to 6140)
  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 Rep.";
            date: "2024-08-27"; volume: "43(8):114545. doi"; pages: " 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23"
COMMENT     Sequence Label: pOPINK-HOIL-1L NZF (195-252)
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(398..415)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             428..450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    465..489
                     /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(489..508)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(490..508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(592..611)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        817..894
                     /gene="lacI"
                     /label=lacI promoter
     CDS             895..1977
                     /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(914..933)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2786..2977
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2971..2993)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3079..3218
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3134..3153
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3233..3250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3404..3992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3484..3503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4103..4122)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4114..4929
                     /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             4114..4929
                     /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(4187..4206)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5021..5476)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5158..5179)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5370..5389
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5513..5560)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5556..5574
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5627..5644)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5842..5865)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(5884..6140,1..397))
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
ORIGIN
        1 acattttcag catttcaggt agcttgctaa gaaaatcaac tttgagagtt tcaaagtctt
       61 tactatatgc aattctcgaa acaccgtatc taatatccaa aaccgctcct tcaagcattg
      121 aaatctctgc acgctctttt ggacaaccac ccaacatgtt gtgcttgtca gctatataac
      181 gtatgatggc catagactgt gttaatttaa catcaccatc aatataataa ggaagattgg
      241 gaaactccaa acccaattca aactttttgt ttcgccattt atcaccttca tcgcgctcat
      301 acaaatgctc ttcatatttt tcttcaagat attccaaaag aagtcgagtg ggttgcacaa
      361 ggcccttaat tttccaataa cctagtatag gggacatgtg atggtgatgg tgatgtgcca
      421 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
      481 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
      541 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
      601 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
      661 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
      721 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
      781 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
      841 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
      901 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
      961 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     1021 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     1081 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     1141 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     1201 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     1261 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     1321 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     1381 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     1441 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     1501 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     1561 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     1621 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     1681 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     1741 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     1801 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     1861 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     1921 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     1981 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     2041 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     2101 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     2161 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     2221 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     2281 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     2341 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     2401 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     2461 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     2521 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     2581 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     2641 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     2701 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     2761 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     2821 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     2881 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     2941 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3001 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3061 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     3121 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     3181 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     3241 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3301 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3361 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3421 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3481 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3541 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3601 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3661 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3721 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3781 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3841 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3901 ttagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3961 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4021 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     4081 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     4141 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     4201 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     4261 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     4321 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     4381 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     4441 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     4501 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     4561 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     4621 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     4681 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     4741 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     4801 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     4861 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     4921 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5041 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5101 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5161 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5221 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5281 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5341 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5401 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5461 ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac
     5521 ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag
     5581 cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt
     5641 ggtgtttaaa tggtctagaa agctttactg ctggtactga cgcagcgcct cctcctcgcc
     5701 cgccaggcgc gctcgctcct cctcgtcggg ctggtatgag gcggggacct ggtaggcctc
     5761 ggggcgcgcc cggcagcaca tctcacagcc aggccgcgtg ggcttgttga tgaaggtgca
     5821 cccggggcac tgccagccca ccgggccctg aaacagaact tccagaccgc tgctcagatc
     5881 cgattttgga ggatggtcgc caccaccaaa cgtggcttgc cagccctgca aaggccatgc
     5941 tatatacttg ctggatttca agtacttatc aatttgtggg atagcttcaa tacgtttttt
     6001 aaaacaaact aattttggga acgcatccag gcacattggg tccatgtata aaacaacatc
     6061 aagagcgtca tacaacatga agtcaggatg ggttacatga tcaccattta aatatgtttt
     6121 atgacataaa cgatcttcga
//
