LOCUS       sequence-346323-        7904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human RNF216 (Triad3A) codon optimized for E. coli and
            insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7904)
  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 7904)
  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
FEATURES             Location/Qualifiers
     source          1..7904
                     /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             2769..2786
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2839..2857)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2853..2900
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2937..3392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3024..3043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3234..3255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3484..4299)
                     /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(3484..4299)
                     /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     4207..4226
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4291..4310
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4421..5009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4910..4929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5163..5180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5195..5334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5260..5279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5420..5442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5436..5627)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6436..7518)
                     /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     7480..7499
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7519..7596)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7802..7821
                     /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 gaggaaggaa ataacaacga ggaggtgatc
      181 catctgaaca attttcactg ccaccgcggc caagagtgga tcaacttacg cgacgggcct
      241 attacaattt ccgattcttc cgatgaagaa cgcattccta tgcttgttac acccgcacca
      301 caacagcacg aagaggaaga ccttgacgac gatgtgattc ttacggaaga cgacagcgaa
      361 gacgactacg gcgagttcct ggatcttggg ccaccgggca ttagtgagtt taccaaacct
      421 agcggccaaa cggaacgcga accaaagcca gggccgtctc ataatcaggc agctaacgac
      481 atcgttaatc cccgctccga acaaaaagtc attatcttag aggaaggaag cctgttgtac
      541 actgaatcgg atccgttaga aacacaaaat cagagcagtg aagatagtga aacagaattg
      601 ctgtcgaatt taggcgaatc tgcggcgctt gctgatgatc aagccattga agaggactgt
      661 tggctggatc acccttattt ccagtcattg aatcagcaac cgcgcgaaat cacaaaccaa
      721 gtcgttccac aagagcgtca acccgaggct gaattgggcc gcctgctttt ccaacatgag
      781 tttccaggac cagccttccc acgccctgaa ccacagcagg ggggcattag tggaccgtca
      841 tcgccgcagc ctgcgcaccc gttaggcgaa tttgaagacc agcaactggc aagcgatgac
      901 gaagagcctg ggcctgcgtt tccgatgcaa gaatcgcaag agccaaacct ggaaaatatc
      961 tggggccagg aggctgccga agtagatcaa gaattagttg aactgttagt aaaggaaacc
     1021 gaagcgcgct ttccggacgt tgccaatggt ttcatcgagg agattattca ctttaagaat
     1081 tactacgact taaatgtact ttgtaatttt ttattggaaa acccggatta tcccaagcgt
     1141 gaagatcgca ttattatcaa tccatcttct tctttattgg cgtcacaaga tgagacgaaa
     1201 ctgcccaaga tcgacttttt tgattacagc aaacttacgc cgcttgatca acgttgcttc
     1261 attcaggcag cagatcttct gatggctgat tttaaagtcc tttcttcgca ggatattaag
     1321 tgggctctgc acgagcttaa aggtcattat gccattaccc gtaaagctct gtccgatgct
     1381 atcaagaaat ggcaagagtt atcacctgaa acatcgggaa agcgcaaaaa acgtaaacaa
     1441 atgaatcagt actcctacat tgacttcaag tttgaacagg gtgacatcaa gatcgagaag
     1501 cgtatgtttt tcctggaaaa taagcgccgt cactgccgta gttacgatcg ccgcgccctg
     1561 ttgccagcag ttcagcagga acaggagttt tatgaacaaa aaattaaaga gatggcagag
     1621 cacgaagatt tcctgctggc attacagatg aatgaggaac aatatcagaa ggacggacaa
     1681 cttatcgaat gtcgttgttg ctacggagag tttcccttcg aagaactgac ccaatgcgcc
     1741 gacgcacact tgttctgtaa ggaatgcctt attcgctacg ctcaagaagc cgtttttgga
     1801 tctggcaagt tggagttgag ctgtatggaa gggtcgtgca cgtgttcgtt cccaacaagc
     1861 gagctggaaa aggttcttcc gcaaactatt ctttacaagt actatgagcg taaagcggaa
     1921 gaagaggtcg ctgcggcgta tgccgacgag ctggtacgct gcccctcttg tagctttcca
     1981 gcattgttag attcggatgt caaacgtttt tcatgtccta atccacactg ccgcaaggaa
     2041 acatgtcgta aatgccaggg tttatggaag gaacacaatg gtcttacatg tgaggagctt
     2101 gccgagaaag acgatattaa ataccgcacc tcgattgagg aaaagatgac agccgctcgc
     2161 atccgcaaat gccataagtg tgggacaggg ctgatcaaga gtgagggttg caaccgtatg
     2221 tcttgtcgct gtggtgcgca gatgtgttat ctgtgccgtg tctcgattaa cggttacgat
     2281 catttttgtc aacaccctcg ctcccctggt gccccctgtc aggaatgcag ccgttgttcc
     2341 ttgtggaccg accccacaga agatgatgag aaactgattg aagaaattca aaaggaggct
     2401 gaggaagaac agaagcgcaa gaatggtgaa aacactttca agcgtattgg cccgcccctt
     2461 gaaaaaccag tagaaaaagt tcagcgtgta gaagccttgc cccgccctgt gccacaaaat
     2521 ctgccacaac ctcagatgcc accatatgcc tttgcacacc cccccttccc attacctcct
     2581 gtacgtcccg ttttcaataa tttcccattg aatatgggcc ctatccctgc accttatgtt
     2641 ccacccttgc ctaatgtacg tgtcaattat gacttcggtc ctatccacat gcctttagag
     2701 cacaatcttc ccatgcactt tggaccgcaa ccgcgccatc gtttctaaag ctttctagac
     2761 catttaaaca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     2821 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2881 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2941 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3001 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3061 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3121 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3181 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3241 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3301 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3361 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     3421 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     3481 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3541 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3601 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3661 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3721 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3781 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3841 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     3901 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3961 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     4021 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     4081 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     4141 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     4201 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     4261 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     4321 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4381 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4441 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4501 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4561 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5041 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5101 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5161 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5221 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5281 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5341 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5401 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5461 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5521 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5581 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5641 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5701 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5761 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5821 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5881 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5941 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6001 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6061 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     6121 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     6181 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     6241 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     6301 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     6361 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     6421 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6481 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     6541 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     6601 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     6661 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     6721 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     6781 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     6841 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     6901 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     6961 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     7021 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     7081 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     7141 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     7201 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     7261 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     7321 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     7381 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     7441 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     7501 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     7561 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     7621 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     7681 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     7741 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     7801 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     7861 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
