LOCUS       sequence-199654-        5780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-MBP-tagged mutant BAF (G47E) in E.coli 
            (SUMO-cleavable).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5780)
  AUTHORS   Samwer M, Schneider MWG, Hoefler R, Schmalhorst PS, Jude JG, Zuber 
            J, Gerlich DW
  TITLE     DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic 
            Chromosomes.
  JOURNAL   Cell. 2017 Aug 24;170(5):956-972.e23. doi: 
            10.1016/j.cell.2017.07.038.
  PUBMED    28841419
REFERENCE   2  (bases 1 to 5780)
  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: 
            "10.1016/j.cell.2017.07"; journalName: "Cell"; date: "2017-08-24- 
            24"; volume: "170"; issue: "5"; pages: "956-972.e23"
COMMENT     Sequence Label: H14-MBP-SUMO-Cys-Linker-BAF_G47E
FEATURES             Location/Qualifiers
     source          1..5780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             193..1290
                     /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="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1147..1156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1264..1287
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1985..2002
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2025..2119
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2167..2186)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2178..2993
                     /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             2178..2993
                     /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     complement(2251..2270)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3212..3298
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3391..4473)
                     /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     4435..4454
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4474..4551)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4635..4657)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4743..4883
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4798..4817
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4898..4915)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5069..5657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5149..5168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5748..5766
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgagc
      121 aagcatcacc atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat
      181 caccataccg ggaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      241 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      301 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      361 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      421 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      481 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      541 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      601 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      661 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      721 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      781 gacctgatta aaaacaaaca catgaatgca gacactgatt actccatcgc agaagctgcc
      841 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      901 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      961 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1021 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1081 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1141 attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1201 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1261 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct caggtgaaga tgaaagtgga
     1321 ggctctgctg cgggtggcga agaagataag aaaccggcag gtggcgaagg tggcggtgcc
     1381 catatcaacc tgaaagtgaa aggtcaagac ggcaacgaag tctttttccg catcaaacgt
     1441 tctacccagc tgaaaaagct gatgaacgca tactgtgacc gtcagtctgt agacatgacc
     1501 gcaattgctt tcctgtttga tggtcgtcgc ctgcgtgcgg aacagacccc ggatgaactg
     1561 gagatggaag atggcgacga aatcgacgca atgctgcacc agactggtgg cgccgtttgt
     1621 ggaagtggca gtactggttc cggcagctcc ggcagcggcg gcgggagtag cggatcatct
     1681 acagggacca ccaccggtat gacaacctcc caaaagcacc gagacttcgt ggcagagccc
     1741 atgggggaga agccagtggg gagcctggct gggattggtg aagtcctggg caagaagctg
     1801 gaggaaaggg gttttgacaa ggcctatgtt gtccttgaac agtttctggt gctaaagaaa
     1861 gatgaagacc tcttccggga atggctgaaa gacacttgtg gcgccaacgc caagcagtcc
     1921 cgggactgct tcggatgcct tcgagagtgg tgtgatgcct tcttgtaggg atccagcggc
     1981 cgcacatcac catcaccatc actaagctta attagctgag cttggactcc tgttgataga
     2041 tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc
     2101 gttttttatt ggtgagaatc caagctagcc atgaaaataa actgtctgct tacataaaca
     2161 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
     2221 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     2281 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     2341 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     2401 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgctt
     2461 ggttactcac gactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
     2521 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     2581 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     2641 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2701 ttgaacaagt ctggaaagaa atgcacaaac ttttgccatt ctcaccggat tcagtcgtca
     2761 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2821 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2881 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2941 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     3001 ttcatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     3061 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac
     3121 tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg cccttaaacg
     3181 cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc agtcgaaaga
     3241 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     3301 gccctctaga ttacgtgcag tcgatgataa gctgtcaaac atgagaattg tgcctaatga
     3361 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3421 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3481 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3541 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3601 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3661 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3721 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3781 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3841 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     3901 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     3961 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4021 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4081 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4141 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4201 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4261 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4321 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4381 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4441 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4501 cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cggaatttcg
     4561 ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga gctgcctcgc gcgtttcggt
     4621 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4681 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4741 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     4801 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     4861 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4921 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4981 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5701 acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca
     5761 cgaggccctt tcgtcttcac
//
