LOCUS       sequence-473106-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human RYBP NZF (20-51).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  AUTHORS   Michel MA, Scutts S, Komander D
  TITLE     Secondary interactions in ubiquitin-binding domains achieve linkage 
            or substrate specificity.
  JOURNAL   Cell Rep. 2024 Aug 27;43(8):114545. doi: 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23.
  PUBMED    39052481
REFERENCE   2  (bases 1 to 6062)
  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: "Cell Rep.";
            date: "2024-08-27"; volume: "43(8):114545. doi"; pages: " 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23"
COMMENT     Sequence Label: pOPINK-RYBP NZF (20-51)
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             186..209
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             329..346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(399..417)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      413..460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      497..952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(584..603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     794..815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1044..1859)
                     /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(1044..1859)
                     /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     1767..1786
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1851..1870
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1981..2569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2470..2489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2723..2740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2755..2894
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2820..2839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2980..3002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2996..3187)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3996..5078)
                     /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     5040..5059
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5079..5156)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5362..5381
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5465..5484
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5465..5483
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5484..5508
                     /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             5523..5545
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5558..5575
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(5576..6062,1..167)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
ORIGIN
        1 tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta
       61 ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt
      121 tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctgagca
      181 gcggtctgga agttctgttt cagggcccgg cagacgaagg gttttgggat tgtagcgtct
      241 gcaccttcag aaacagtgct gaagccttta aatgcagcat ctgcgatgtg aggaaaggca
      301 cctcctaaag ctttctagac catttaaaca ccaccaccac caccactgag atccggctgc
      361 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      421 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      481 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
      541 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
      601 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
      661 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
      721 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
      781 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
      841 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
      901 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
      961 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1021 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1081 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1141 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1201 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1261 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1321 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1381 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     1441 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1501 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     1561 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1621 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     1681 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     1741 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     1801 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     1861 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     1921 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1981 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2041 agcggtggtt tgtttgccgg atcaagagct aacaactctt tttccgaagg taactggctt
     2101 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2161 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2221 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2281 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2341 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2401 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2461 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2521 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2581 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2641 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2701 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     2761 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     2821 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2881 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2941 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3001 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3061 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3121 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3181 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3241 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3301 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3361 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3421 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3481 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3541 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3601 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3661 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3721 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3781 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3841 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3901 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3961 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4021 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4081 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4141 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4201 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4261 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4321 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4381 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4441 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4501 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4561 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4621 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4681 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4741 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4801 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4861 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4921 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4981 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5041 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5101 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5161 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5221 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5281 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5341 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5401 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     5461 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5521 attttgttta actttaagaa ggagatatac catggcacat caccatcacc atcacatgtc
     5581 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
     5641 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
     5701 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
     5761 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
     5821 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
     5881 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
     5941 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
     6001 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
     6061 tt
//
