LOCUS       sequence-379617-        6839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human His-tagged HOIL-1 (RBCK1) full-length (1–510) 
            codon optimized for E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6839)
  AUTHORS   Wang XS, Cotton TR, Trevelyan SJ, Richardson LW, Lee WT, Silke J, 
            Lechtenberg BC
  TITLE     The unifying catalytic mechanism of the RING-between-RING E3 
            ubiquitin ligase family.
  JOURNAL   Nat Commun. 2023 Jan 11;14(1):168. doi: 10.1038/s41467-023-35871-z.
  PUBMED    36631489
REFERENCE   2  (bases 1 to 6839)
  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: "Nat 
            Commun."; date: "2023-01-11"; volume: "14(1):168. doi"; pages: " 
            10.1038/s41467-023-35871-z"
COMMENT     Sequence Label: pOPINB-HOIL-1 full-length (1–510)
FEATURES             Location/Qualifiers
     source          1..6839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..59)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      55..102
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      139..594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(226..245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     436..457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(686..1501)
                     /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(686..1501)
                     /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     1409..1428
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1493..1512
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1623..2211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2112..2131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2365..2382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2397..2536
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2462..2481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2622..2644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2638..2829)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3638..4720)
                     /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     4682..4701
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4721..4798)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5004..5023
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5107..5126
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5107..5125
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5126..5150
                     /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             5165..5187
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5206..5223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5233..5256
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6810..6827
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
       61 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      121 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      181 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      241 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      301 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      361 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      421 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
      481 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
      541 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
      601 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      661 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
      721 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
      781 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
      841 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
      901 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
      961 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1021 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     1081 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1141 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1201 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1261 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1321 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1381 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1441 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     1501 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     1561 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1621 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1681 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1741 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     1801 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1861 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1921 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1981 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2041 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2101 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2161 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2221 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2281 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2341 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2401 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     2461 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     2521 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2581 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2641 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     2701 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     2761 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     2821 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     2881 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     2941 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     3001 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3061 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3121 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3181 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3241 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3301 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3361 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3421 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3481 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3541 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3601 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3661 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3721 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     3781 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     3841 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     3901 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     3961 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4021 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4081 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4141 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4201 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4261 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4321 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4381 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4441 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4501 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4561 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4621 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     4681 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     4741 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     4801 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     4861 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     4921 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     4981 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5041 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5101 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5161 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     5221 cacagcagcg gtctggaagt tctgtttcag ggcccgatgg atgagaaaac caagaaagcg
     5281 gaagagatgg cgctgtcgct gacccgcgcg gtggcgggcg gcgatgaaca ggtggcgatg
     5341 aaatgcgcca tctggctggc ggaacagcgc gtgccgctgt cggtgcagct gaaaccggaa
     5401 gtcagcccga cgcaggatat tcgcctgtgg gtcagcgtgg aagatgcgca gatgcatacc
     5461 gtcaccatct ggctgaccgt gcgcccggat atgaccgttg ccagcctgaa agatatggtg
     5521 ttcctggatt acggcttccc gccggtgctg caacagtggg tgattggtca gcgtctggcg
     5581 cgcgatcagg aaacgctgca cagccacggc gtgcgccaga acggcgacag cgcctatctg
     5641 tatctgctgt cggcgcgtaa caccagcctg aatccgcagg aactgcaacg tgaacgccag
     5701 ctgcgtatgc tggaagatct gggctttaaa gacctgacgc tgcaaccgcg cggcccgctg
     5761 gaaccgggtc cgccgaaacc gggcgtgccg caggaaccgg gtcgtggtca gccggatgcg
     5821 gtgccggaac cgccgccggt tggctggcag tgccctggct gcacctttat caacaaaccg
     5881 acgcgtccgg gctgcgaaat gtgttgccgt gcgcgtccgg aagcgtatca ggttcctgcc
     5941 agctatcagc cggatgaaga agaacgtgcg cgtctggcgg gcgaagaaga agcgctgcgc
     6001 cagtatcagc agcgtaaaca gcagcagcag gaaggtaact atctgcaaca tgtgcagctg
     6061 gatcagcgca gcctggtgct gaataccgaa ccggcagaat gcccggtttg ctacagcgtg
     6121 ctggcaccgg gcgaagcggt ggtgctgcgt gaatgcctgc ataccttctg ccgtgaatgc
     6181 ctgcagggca ccattcgcaa cagccaggaa gcggaagtga gctgcccgtt tattgataac
     6241 acctacagct gcagcggcaa actgctggaa cgtgaaatca aagcgctgct gacgccggaa
     6301 gattatcagc gttttctgga tctgggcatc agcattgccg aaaaccgcag cgcctttagc
     6361 tatcactgta aaacgccgga ttgcaaaggc tggtgcttct ttgaagatga tgttaacgaa
     6421 tttacctgcc cggtttgctt ccacgttaac tgcctgctgt gcaaagcgat tcatgaacag
     6481 atgaactgca aagagtatca ggaagatctg gcgctgcgtg cgcagaacga tgtggcggcg
     6541 cgtcagacca ccgaaatgct gaaagtgatg ctgcaacagg gcgaagcgat gcgctgcccg
     6601 cagtgccaga ttgtggtgca gaagaaagat ggctgcgact ggatccgctg caccgtttgc
     6661 cataccgaaa tctgctgggt gaccaaaggt ccgcgctggg ggccgggcgg cccgggtgat
     6721 accagcggcg gctgccgctg ccgcgtgaac ggtattccgt gccatccttc ctgccagaac
     6781 tgtcactaaa gctttctaga ccatttaaac accaccacca ccaccactga gatccggct
//
