LOCUS       sequence-379616-        6143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human His-tagged HOIL-1 (RBCK1) helix-RBR (aa 233–510)
            codon optimized for E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6143)
  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 6143)
  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 helix-RBR (233–510)
FEATURES             Location/Qualifiers
     source          1..6143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      61..516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(148..167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     358..379
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(608..1423)
                     /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(608..1423)
                     /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     1331..1350
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1415..1434
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1545..2133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2034..2053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2287..2304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2319..2458
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2384..2403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2544..2566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2560..2751)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3560..4642)
                     /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     4604..4623
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4643..4720)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4926..4945
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5029..5048
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5029..5047
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5048..5072
                     /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             5087..5109
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5128..5145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5155..5178
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6036..6053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6106..6124)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(6120..6143,1..24)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
       61 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      241 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      301 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      361 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      481 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      541 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
      601 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
      661 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
      721 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
      781 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
      841 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
      901 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
      961 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     1021 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1081 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1141 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1201 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1261 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1321 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1381 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     1441 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     1501 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1561 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1621 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1681 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1741 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1801 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1861 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1921 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1981 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2041 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2101 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2161 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2221 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2281 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     2341 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     2401 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2461 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2521 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2581 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     2641 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     2701 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     2761 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     2821 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     2881 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     2941 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     3001 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3061 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     3121 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     3181 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     3241 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     3301 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     3361 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     3421 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     3481 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     3541 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3601 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     3661 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     3721 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     3781 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     3841 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     3901 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     3961 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     4021 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4081 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4141 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4201 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4261 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4321 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     4381 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     4441 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     4501 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     4561 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     4621 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     4681 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     4741 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     4801 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     4861 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4921 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4981 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     5041 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     5101 agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag cggtctggaa
     5161 gttctgtttc agggcccgga tgaggaagag cgtgctcgct tggctggaga agaagaggca
     5221 ttacgtcaat atcaacagcg caaacaacaa cagcaagagg gcaactactt acaacatgtt
     5281 cagctggacc agcgtagttt agtccttaat accgaacccg cagaatgccc ggtttgttat
     5341 tcagtactgg cacctggcga agcagtggtg ctgcgtgaat gcctgcatac gttctgccgc
     5401 gagtgtttac aaggaaccat ccgcaattca caagaggctg aggtaagttg cccgttcatc
     5461 gataatacgt acagttgttc aggaaaattg cttgagcgtg agattaaggc cctgctgacg
     5521 cctgaggact accagcgctt cctggatttg ggtattagta tcgctgagaa ccgttcggct
     5581 ttctcgtacc attgtaaaac cccggactgc aaagggtggt gcttttttga ggacgatgtt
     5641 aatgaattta cttgcccagt atgcttccat gttaattgtc tgttgtgcaa agctattcat
     5701 gaacaaatga actgcaaaga atatcaagaa gatttggcac ttcgcgccca gaacgacgta
     5761 gcggctcgcc agaccactga aatgttgaaa gttatgttac aacaggggga ggcgatgcgt
     5821 tgcccccagt gccagatcgt agtacaaaaa aaggacggct gcgattggat ccgttgcact
     5881 gtttgccata cggaaatctg ctgggtaacg aaggggcccc gttggggtcc aggaggacct
     5941 ggggatacat caggtggatg ccgttgtcgt gttaacggca tcccatgcca tccctcatgc
     6001 cagaactgcc actaaagctt tctagaccat ttaaacacca ccaccaccac cactgagatc
     6061 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     6121 tagcataacc ccttggggcc tct
//
