LOCUS       sequence-343391-        5050 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 5050)
  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 5050)
  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_Ub
FEATURES             Location/Qualifiers
     source          1..5050
                     /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             1165..1392
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     promoter        1461..1479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1480..1504
                     /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             1613..1657
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1695..1713)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1709..1756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1986..2801)
                     /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(1986..2801)
                     /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     2709..2728
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2802..2893)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2909..3658)
                     /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(2919..2936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3159..3178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3854..4936)
                     /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     4898..4917
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4937..5014)
                     /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 taaaatgcag attttcgtga aaacccttac ggggaagacc
     1201 atcaccctcg aggttgaacc ctcggatacg atagaaaatg taaaggccaa gatccaggat
     1261 aaggaaggaa ttcctcctga tcagcagaga ctgatctttg ctggcaagca gctggaagat
     1321 ggacgtactt tgtctgacta caatattcaa aaggagtcta ctcttcatct tgtgttgaga
     1381 cttcgtggtg gttgagcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta
     1441 cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1501 ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg cagatctcaa
     1561 ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac
     1621 cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac
     1681 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1741 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     1801 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     1861 tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc
     1921 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1981 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2041 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2101 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2161 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2221 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2281 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2341 gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt acaaacagga
     2401 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2461 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2521 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2581 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2641 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2701 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2761 cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct ttttcaatat
     2821 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2881 aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga ctcgctacgc
     2941 tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat acggttatcc
     3001 acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc accggaagaa
     3061 gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3121 acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt atctcagttc
     3301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3421 actggcagca gccattggta actgatttag aggactttgt cttgaagtta tgcacctgtt
     3481 aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac ttaccttggt
     3541 tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg gtggtttttc
     3601 tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct attccgttac
     3661 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     3721 ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3781 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3961 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4021 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4081 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     4141 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4201 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4261 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4321 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4381 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4441 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4501 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4561 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4621 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4681 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4741 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4801 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4861 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4921 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4981 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5041 gactcctgca
//
