LOCUS       sequence-343392-        5047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Scaﬀold design for studying ubiquitin interacting proteins by 
            cryo-EM..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5047)
  AUTHORS   Chiu YH, Ko KT, Yang TJ, Wu KP, Ho MR, Draczkowski P, Hsu SD
  TITLE     Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy
            Aided by a Small Scaffold Protein.
  JOURNAL   Biochemistry. 2021 Apr 13;60(14):1075-1079. doi: 
            10.1021/acs.biochem.0c00961. Epub 2021 Mar 15.
  PUBMED    33719392
REFERENCE   2  (bases 1 to 5047)
  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: 
            "10.1021/acs.biochem.0c00961"; journalName: "Biochemistry"; date: 
            "2021-04-13- 13"; volume: "60"; issue: "14"; pages: "1075-1079"
COMMENT     Sequence Label: pRSFDuet1-His-TEV-OTC_d1_Ub
FEATURES             Location/Qualifiers
     source          1..5047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..28
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    29..53
                     /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)."
     CDS             109..126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             139..159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1162..1389
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     promoter        1458..1476
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1477..1501
                     /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)."
     CDS             1610..1654
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1692..1710)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1706..1753
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1983..2798)
                     /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(1983..2798)
                     /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     2706..2725
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2799..2890)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2906..3655)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2916..2933)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3156..3175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3851..4933)
                     /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     4895..4914
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4934..5011)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ttaggaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctgtag
       61 aaataatttt gtttaacttt aataaggaga tataccatgg gcagcagcca tcaccatcat
      121 caccacggca gcagcggtga aaacctgtat tttcagggat ccatgtccgg gttttatcat
      181 aagcatttcc tgaaattact cgatttcacg ccagctgaac tcaacagcct gctgcagtta
      241 gccgcgaagc tgaaagccga taagaaaagc ggtaaagaag aagccaaact cactggtaaa
      301 aacatcgcgc tcatcttcga aaaagactcg actcgtaccc gatgctcttt cgaagttgcc
      361 gcatatgacc agggtgctcg cgttacttat ctcggcccaa gcggcagcca gattggtcat
      421 aaagagtcga ttaaagacac tgcccgcgtg cttggtcgca tgtatgacgg tattcagtat
      481 cgcggctatg gtcaggagat tgtcgaaaca ctggcggaat acgctagcgt gccggtatgg
      541 aatggcctga ccaatgagtt ccatcccacg cagctgctgg cggatcttct caccatgcag
      601 gagcatttgc ccggcaaagc gttcaacgaa atgacgctgg tctatgcagg tgacgcgcgt
      661 aacaacatgg gcaattcgat gctcgaagct gcggcgctta ccggtctgga tttgcgtctg
      721 gtcgcgccac aagcgtgctg gccggaagct gcgctggtta cggaatgccg cgccctggca
      781 cagcaaaatg gtgggaatat tacgctgact gaagatgtcg cgaagggagt tgaaggtgct
      841 gactttatct ataccgatgt gtgggtgtcg atgggggaag caaaagagaa atgggcggaa
      901 cggattgcat tgctgcgtga atatcaggtg aacagcaaga tgatgcagtt gaccggtaac
      961 ccggaggtca aattcctcca ctgcctgccc gcgtttcatg acgaccaaac gacgcttggc
     1021 aagaaaatgg cggaagaatt tggcctacat ggcggtatgg aagtcactga tgaggtcttc
     1081 gaatctgccg ccagcattgt ttttgatcag gcggaaaacc gtatgcatac tatcaaagcg
     1141 gtgatggtcg cgacgctcag tatgcagatt ttcgtgaaaa cccttacggg gaagaccatc
     1201 accctcgagg ttgaaccctc ggatacgata gaaaatgtaa aggccaagat ccaggataag
     1261 gaaggaattc ctcctgatca gcagagactg atctttgctg gcaagcagct ggaagatgga
     1321 cgtactttgt ctgactacaa tattcaaaag gagtctactc ttcatcttgt gttgagactt
     1381 cgtggtggtt gagcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac
     1441 ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1501 cccatcttag tatattagtt aagtataaga aggagatata catatggcag atctcaattg
     1561 gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta aagaaaccgc
     1621 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     1681 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1741 gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt
     1801 agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     1861 accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg
     1921 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1981 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2041 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2101 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2161 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2221 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2281 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2341 tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc
     2401 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2461 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2521 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2581 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2641 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2701 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2761 ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     2821 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2881 aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg
     2941 gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca
     3001 gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc
     3061 aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3121 ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3181 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3241 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt
     3301 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3361 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3421 ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag
     3481 gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca
     3541 aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt
     3601 atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct
     3661 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     3721 agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3781 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3841 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3901 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3961 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4021 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4081 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     4141 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4201 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4261 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4321 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4381 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4441 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4501 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4561 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4621 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4681 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4741 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4801 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4861 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4921 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4981 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5041 tcctgca
//
