LOCUS       sequence-212937-        6596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of RNF144B RBR domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6596)
  AUTHORS   Michel MA, Swatek KN, Hospenthal MK, Komander D
  TITLE     Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked 
            Ubiquitin Signaling.
  JOURNAL   Mol Cell. 2017 Oct 5;68(1):233-246.e5. doi: 
            10.1016/j.molcel.2017.08.020. Epub 2017 Sep 21.
  PUBMED    28943312
REFERENCE   2  (bases 1 to 6596)
  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.2017.08.020"; journalName: "Mol Cell"; date: 
            "2017-10-5- 5"; volume: "68"; issue: "1"; pages: "233-246.e5"
COMMENT     Sequence Label: Human RNF144B (27-236)
FEATURES             Location/Qualifiers
     source          1..6596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             172..825
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             844..867
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1521..1538
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1591..1609)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1605..1652
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1689..2144
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1776..1795)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1986..2007
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2236..3051)
                     /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(2236..3051)
                     /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     2959..2978
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3043..3062
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3173..3761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3662..3681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3915..3932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3947..4086
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4012..4031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4172..4194
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4188..4379)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5188..6270)
                     /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     6232..6251
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6271..6348)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6554..6573
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atataccatg gcacatcacc atcaccatca catgtcccct
      181 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat
      241 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac
      301 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt
      361 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt
      421 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga
      481 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt
      541 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta
      601 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta
      661 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt
      721 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg
      781 cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctgagcagc
      841 ggtctggaag ttctgtttca gggcccgctc atcacttgca aactctgcct gtgtgagcag
      901 tctctggaca agatgaccac actccaggaa tgccagtgca tcttttgcac agcttgcctg
      961 aaacagtaca tgcagctggc aatccgagaa ggatgtgggt ctcccatcac ttgccctgac
     1021 atggtgtgcc taaaccacgg gaccctgcag gaagctgaga ttgcctgttt ggtacctgtg
     1081 gaccagtttc aactttatca gaggttaaaa tttgaaagag aagttcatct ggacccctac
     1141 cgaacatggt gtcctgttgc agactgtcag acagtgtgcc ctgttgcctc gagtgaccca
     1201 ggacagcctg tgctggtgga atgcccttct tgccacctga aattctgctc gtgttgcaag
     1261 gatgcttggc atgcagaggt ctcctgtaga gacagtcagc ctattgtcct gccaacagag
     1321 caccgagccc tctttgggac agatgcagaa gcccccatta agcagtgccc agtttgccgg
     1381 gtttatatcg aacgcaatga aggctgcgct cagatgatgt gcaaaaactg caagcataca
     1441 ttttgctggt actgcctcca gaacttggat aatgacattt tcctcagaca ttatgactaa
     1501 agctttctag accatttaaa caccaccacc accaccactg agatccggct gctaacaaag
     1561 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1621 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1681 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1741 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1801 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1861 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1921 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1981 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2041 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2101 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2221 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2281 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2341 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2401 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2461 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2521 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2581 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2641 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2701 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2761 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2821 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2881 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2941 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3001 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3061 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     3121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3241 tttgtttgcc ggatcaagag ctaacaactc tttttccgaa ggtaactggc ttcagcagag
     3301 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3901 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3961 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4021 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4081 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4141 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4201 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4261 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4321 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4381 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4441 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4501 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4561 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4621 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4681 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4741 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4801 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4861 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4921 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4981 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     5041 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5101 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5161 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5281 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5341 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5401 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5461 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5521 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5581 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5641 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5701 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5761 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5821 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5881 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5941 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6001 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6061 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6121 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6181 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6241 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6301 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6361 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6421 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6481 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6541 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcg
//
