LOCUS       sequence-238510-        5835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express tUI-HA protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5835)
  AUTHORS   Choi YS, Bollinger SA, Prada LF, Scavone F, Yao T, Cohen RE
  TITLE     High-affinity free ubiquitin sensors for quantifying ubiquitin 
            homeostasis and deubiquitination.
  JOURNAL   Nat Methods. 2019 Jul 15. pii: 10.1038/s41592-019-0469-9. doi: 
            10.1038/s41592-019-0469-9.
  PUBMED    31308549
REFERENCE   2  (bases 1 to 5835)
  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: "Nat 
            Methods. 2019 Jul 15. pii: 10.1038/s41592-019-0469-9. doi: 
            10.1038/s41592-019-0469-9."
COMMENT     Sequence Label: tUI-HA
FEATURES             Location/Qualifiers
     source          1..5835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..113
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        94..112
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    113..137
                     /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             152..174
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             193..210
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             220..237
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    655..666
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     CDS             754..780
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             793..810
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(863..881)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      877..924
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      961..1416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1048..1067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1258..1279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1508..2323)
                     /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(1508..2323)
                     /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     2231..2250
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2315..2334
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2445..3033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2934..2953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3187..3204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3219..3358
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3284..3303)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3444..3466
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3460..3651)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4460..5542)
                     /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     5504..5523
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5543..5620)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5826..5835,1..10)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
       61 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
      121 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
      181 atgcccagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
      241 atgggttcta ctgtcagatt tcagcaacaa ctggaacaac tcagtgcaat gggatttttg
      301 aaccgtgaag caaacttgca agctctaata gcaacttgtg gagatattaa tgcagctatt
      361 gaaaggttac tgggctccgg atccgaagta cggcaggtgt ctaagcatgc cttcagcctc
      421 aagcagttgg acaaccctgc tcgaatccct ccctctggct ggaagtgctc caagtgtgac
      481 atgagagaga acctgtggct caacctgact gatggctcca tcctctgtgg gcgacgctac
      541 ttcgatggca gtgggggcaa caaccacgct gtggagcact accgagagac aggctacccg
      601 ttagctgtca agctgggcac catcacccct gatggagctg acgtgtactc ctatgatgag
      661 gatgacatgg tcctggaccc cagcctggct gagcacctgt cccacttcgg catcgacatg
      721 ctgaagatgc agaagtgcgg gtccggtagc ggttacccat acgatgttcc agattacgct
      781 agctaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      841 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      901 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
      961 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1021 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1081 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1141 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1201 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1261 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1321 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1381 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1441 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1501 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1561 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1621 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1681 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1741 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     1801 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     1861 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     1921 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1981 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2041 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2101 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     2161 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     2221 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2281 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     2341 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     2401 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2461 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2521 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2581 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2641 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2701 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2761 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2821 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2881 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2941 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3001 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3061 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3121 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3181 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3241 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3301 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3361 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3421 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3481 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3541 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3601 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3661 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3721 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     3781 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     3841 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     3901 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3961 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4021 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4081 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4141 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4201 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4261 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4321 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4381 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4441 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4501 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4561 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4621 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4681 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4741 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     4801 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     4861 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     4921 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4981 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5041 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5101 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5161 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5221 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5281 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5341 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5401 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5461 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5521 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5581 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5641 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5701 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     5761 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     5821 ccatcggtga tgtcg
//
