LOCUS       sequence-346329-        6131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human RNF216 (Triad3A) codon optimized for E. coli and
            insect cells. Catalytic RBR-helix construct suitable for activity 
            assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6131)
  AUTHORS   Cotton TR, Cobbold SA, Bernardini JP, Richardson LW, Wang XS, 
            Lechtenberg BC
  TITLE     Structural basis of K63-ubiquitin chain formation by the 
            Gordon-Holmes syndrome RBR E3 ubiquitin ligase RNF216.
  JOURNAL   Mol Cell. 2022 Feb 3;82(3):598-615.e8. doi: 
            10.1016/j.molcel.2021.12.005. Epub 2022 Jan 7.
  PUBMED    34998453
REFERENCE   2  (bases 1 to 6131)
  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"; doi: 
            "10.1016/j.molcel.2021.12.005"; journalName: "Mol Cell"; date: 
            "2022-02-3- 3"; volume: "82"; issue: "3"; pages: "598-615.e8"
COMMENT     Sequence Label: pOPINB-hRNF216(511-784)
FEATURES             Location/Qualifiers
     source          1..6131
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..150
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             996..1013
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1066..1084)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1080..1127
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1164..1619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1251..1270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1461..1482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1711..2526)
                     /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(1711..2526)
                     /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     2434..2453
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2518..2537
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2648..3236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3137..3156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3390..3407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3422..3561
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3487..3506)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3647..3669
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3663..3854)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4663..5745)
                     /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     5707..5726
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5746..5823)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6029..6048
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggtctgg aagttctgtt tcagggcccg caacttatcg aatgtcgttg ttgctacgga
      181 gagtttccct tcgaagaact gacccaatgc gccgacgcac acttgttctg taaggaatgc
      241 cttattcgct acgctcaaga agccgttttt ggatctggca agttggagtt gagctgtatg
      301 gaagggtcgt gcacgtgttc gttcccaaca agcgagctgg aaaaggttct tccgcaaact
      361 attctttaca agtactatga gcgtaaagcg gaagaagagg tcgctgcggc gtatgccgac
      421 gagctggtac gctgcccctc ttgtagcttt ccagcattgt tagattcgga tgtcaaacgt
      481 ttttcatgtc ctaatccaca ctgccgcaag gaaacatgtc gtaaatgcca gggtttatgg
      541 aaggaacaca atggtcttac atgtgaggag cttgccgaga aagacgatat taaataccgc
      601 acctcgattg aggaaaagat gacagccgct cgcatccgca aatgccataa gtgtgggaca
      661 gggctgatca agagtgaggg ttgcaaccgt atgtcttgtc gctgtggtgc gcagatgtgt
      721 tatctgtgcc gtgtctcgat taacggttac gatcattttt gtcaacaccc tcgctcccct
      781 ggtgccccct gtcaggaatg cagccgttgt tccttgtgga ccgaccccac agaagatgat
      841 gagaaactga ttgaagaaat tcaaaaggag gctgaggaag aacagaagcg caagaatggt
      901 gaaaacactt tcaagcgtat tggcccgccc cttgaaaaac cagtagaaaa agttcagcgt
      961 gtagaagcct tgtaaagctt tctagaccat ttaaacacca ccaccaccac cactgagatc
     1021 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1081 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1141 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1201 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1261 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1321 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1381 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1441 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1501 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1561 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1621 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1681 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1741 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1801 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1861 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     1921 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     1981 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2041 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2101 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2161 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2221 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2281 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2341 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2401 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2461 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     2521 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2581 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2641 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2701 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2761 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2821 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2881 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2941 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3001 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3061 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3121 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3181 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3241 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3301 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3361 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3421 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3481 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3541 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3601 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3661 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3721 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3781 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3841 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3901 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3961 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4021 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4081 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4141 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4201 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4261 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4321 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4381 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4441 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4501 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4561 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4621 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4681 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4741 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4801 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4861 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4921 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4981 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5041 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5101 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5161 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5221 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5281 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5341 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5401 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5461 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5521 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5581 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5641 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5701 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5761 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5821 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5881 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5941 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     6001 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6061 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     6121 tcccgcgaaa t
//
