LOCUS       sequence-346327-        6293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human RNF216 (Triad3A) codon optimized for E. coli and
            insect cells. Catalytic helix-RBR-helix construct suitable for 
            activity assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6293)
  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 6293)
  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(457-784)
FEATURES             Location/Qualifiers
     source          1..6293
                     /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             1158..1175
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1228..1246)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1242..1289
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1326..1781
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1413..1432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1623..1644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1873..2688)
                     /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(1873..2688)
                     /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     2596..2615
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2680..2699
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2810..3398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3299..3318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3552..3569
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3584..3723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3649..3668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3809..3831
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3825..4016)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4825..5907)
                     /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     5869..5888
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5908..5985)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6191..6210
                     /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 gaaaataagc gccgtcactg ccgtagttac
      181 gatcgccgcg ccctgttgcc agcagttcag caggaacagg agttttatga acaaaaaatt
      241 aaagagatgg cagagcacga agatttcctg ctggcattac agatgaatga ggaacaatat
      301 cagaaggacg gacaacttat cgaatgtcgt tgttgctacg gagagtttcc cttcgaagaa
      361 ctgacccaat gcgccgacgc acacttgttc tgtaaggaat gccttattcg ctacgctcaa
      421 gaagccgttt ttggatctgg caagttggag ttgagctgta tggaagggtc gtgcacgtgt
      481 tcgttcccaa caagcgagct ggaaaaggtt cttccgcaaa ctattcttta caagtactat
      541 gagcgtaaag cggaagaaga ggtcgctgcg gcgtatgccg acgagctggt acgctgcccc
      601 tcttgtagct ttccagcatt gttagattcg gatgtcaaac gtttttcatg tcctaatcca
      661 cactgccgca aggaaacatg tcgtaaatgc cagggtttat ggaaggaaca caatggtctt
      721 acatgtgagg agcttgccga gaaagacgat attaaatacc gcacctcgat tgaggaaaag
      781 atgacagccg ctcgcatccg caaatgccat aagtgtggga cagggctgat caagagtgag
      841 ggttgcaacc gtatgtcttg tcgctgtggt gcgcagatgt gttatctgtg ccgtgtctcg
      901 attaacggtt acgatcattt ttgtcaacac cctcgctccc ctggtgcccc ctgtcaggaa
      961 tgcagccgtt gttccttgtg gaccgacccc acagaagatg atgagaaact gattgaagaa
     1021 attcaaaagg aggctgagga agaacagaag cgcaagaatg gtgaaaacac tttcaagcgt
     1081 attggcccgc cccttgaaaa accagtagaa aaagttcagc gtgtagaagc cttgtaaagc
     1141 tttctagacc atttaaacac caccaccacc accactgaga tccggctgct aacaaagccc
     1201 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1261 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1321 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1381 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1441 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1501 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1561 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1621 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1681 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1741 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1801 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1861 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1921 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1981 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2041 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2101 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2161 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2221 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2281 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2341 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2401 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2461 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2521 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2581 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2641 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2701 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2761 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2821 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2881 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2941 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3001 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3061 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3121 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3181 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3241 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3301 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3361 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3421 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3481 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3541 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3601 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3661 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3721 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3781 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3841 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3901 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3961 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4021 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4081 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4141 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4201 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4261 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4321 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4381 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4441 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4501 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4561 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4621 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4681 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4741 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4801 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4921 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4981 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5041 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5101 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5161 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5221 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5281 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5341 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5401 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5461 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5521 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5581 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5641 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5701 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5761 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5821 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5881 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5941 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     6001 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6061 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6121 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6181 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6241 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aat
//
