LOCUS       sequence-356892-        6615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of engineered CBX1 with enhanced binding to 
            H3K9me.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6615)
  AUTHORS   Albanese KI, Krone MW, Petell CJ, Parker MM, Strahl BD, Brustad EM, 
            Waters ML
  TITLE     Engineered Reader Proteins for Enhanced Detection of Methylated 
            Lysine on Histones.
  JOURNAL   ACS Chem Biol. 2020 Jan 17;15(1):103-111. doi: 
            10.1021/acschembio.9b00651. Epub 2019 Nov 1.
  PUBMED    31634430
REFERENCE   2  (bases 1 to 6615)
  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/acschembio.9b00651"; journalName: "ACS Chem Biol"; date: 
            "2020-01-17- 17"; volume: "15"; issue: "1"; pages: "103-111"
COMMENT     Sequence Label: pET28a-MBP-CBX1-eReader
FEATURES             Location/Qualifiers
     source          1..6615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             368..1465
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1322..1331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1439..1462
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1481..1501
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1688..1705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1758..1776)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1772..1819
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1856..2311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1943..1962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2153..2174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2403..3218)
                     /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(2403..3218)
                     /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     3126..3145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3210..3229
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3340..3928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3829..3848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4082..4099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4114..4253
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4179..4198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4339..4361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4355..4546)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5355..6437)
                     /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     6399..6418
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6438..6515)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
      361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga
     1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg
     1501 atccgaagaa gaggaagagg aatatgtggt ggaaaaagtt cttgatcggc gagttgtcaa
     1561 gggcaaggtg gaatatcttc taaagtgggc cggtttctca gatgaggaca acacttggga
     1621 gccagaagag aatctgtttt gccctgacct tattgctgag tttctacagt cacagaaaac
     1681 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1741 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1801 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1861 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1921 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1981 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2041 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2101 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2161 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2221 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2281 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2341 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2401 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2461 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2521 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2581 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2641 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2701 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2761 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2821 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2881 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2941 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3001 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3061 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3121 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3181 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3241 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3301 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3361 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3421 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3481 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3541 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3601 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3661 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3721 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3781 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3841 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3901 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3961 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4021 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4081 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4141 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4201 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4261 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4321 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4381 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4441 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4501 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4561 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4621 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4681 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4741 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4801 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4861 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4921 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4981 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     5041 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5101 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5161 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5221 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5281 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5461 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5521 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5581 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5641 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5701 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5761 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5821 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5881 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5941 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6001 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6061 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6121 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6181 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6241 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6301 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6361 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6421 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6481 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6541 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6601 aaggaatggt gcatg
//
