LOCUS       sequence-238534-        5859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express tIVR protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5859)
  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 5859)
  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: tIVR
FEATURES             Location/Qualifiers
     source          1..5859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     125..144
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        125..143
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    144..168
                     /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             183..205
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             224..241
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             251..268
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    572..583
                     /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             848..865
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(918..936)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      932..979
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1016..1471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1103..1122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1313..1334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1563..2378)
                     /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(1563..2378)
                     /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     2286..2305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2370..2389
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2500..3088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2989..3008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3242..3259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3274..3413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3339..3358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3499..3521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3515..3706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4515..5597)
                     /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     5559..5578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5598..5675)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
       61 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
      121 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
      181 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
      241 cagcagcggc ctggtgccgc gcggcagcca tatgaagcag gaggtgcagg catgggatgg
      301 ggaagtacgg caggtgtcta agcatgcctt cagcctcaag cagttggaca accctgctcg
      361 aatccctccc tctggctgga agtgctccaa gtgtgacatg agagagaacc tgtggctcaa
      421 cctgactgat ggctccatcc tctgtgggcg acgctacttc gatggcagtg ggggcaacaa
      481 ccacgctgtg gagcactacc gagagacagg ctacccgtta gctgtcaagc tgggcaccat
      541 cacccctgat ggagctgacg tgtactccta tgatgaggat gacatggtcc tggaccccag
      601 cctggctgag cacctgtccc acttcggcat cgacatgctg aagatgcaga agggatcctg
      661 cgcggcagct gaagaagctg agctggattt aaaagctgct attcaggaaa gcttgagaga
      721 agctggtggc ggttccgatc tcctgtgcaa gaaaggatgt ggttactacg gcaaccctgc
      781 ctggcagggt ttctgctcca agtgctggag ggaagagtac cacaaagcca ggcagaagtg
      841 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
      901 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      961 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1021 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1081 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1141 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1201 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1261 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1321 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1381 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1441 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1561 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1621 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1681 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1741 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1801 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1861 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1921 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1981 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2041 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2101 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2161 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2221 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2281 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2341 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2401 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     2461 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2521 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2581 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2641 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2701 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2761 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2821 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2881 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2941 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3001 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3061 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3121 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3181 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3241 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3301 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3361 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3421 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3481 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3541 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3601 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3661 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3721 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3781 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3841 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3901 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3961 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4021 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4081 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4141 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4201 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4261 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4321 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4381 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4441 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4501 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4561 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4621 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4681 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4741 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4801 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4861 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4921 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4981 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5041 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5101 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5161 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5221 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5281 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5341 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5401 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5461 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5521 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5581 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5641 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5701 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5761 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5821 catacccacg ccgaaacaag cgctcatgag cccgaagtg
//
