LOCUS       sequence-199665-        5634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-MBP-tagged mutant LEM-domain (FLAG-tagged) in E.coli 
            (SUMO-cleavable).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5634)
  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 5634)
  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-FLAG-LEM
FEATURES             Location/Qualifiers
     source          1..5634
                     /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             1618..1641
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1839..1856
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1879..1973
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2021..2040)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2032..2847
                     /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             2032..2847
                     /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(2105..2124)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3066..3152
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3245..4327)
                     /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     4289..4308
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4328..4405)
                     /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(4489..4511)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4597..4737
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4652..4671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4752..4769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4923..5511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5003..5022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5602..5620
                     /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 aatcgacgta atgctgcacc agactggtgg cgccgtggac
     1621 tacaaggacg acgatgacaa agatcccccg ggctgcagga attcggacga gaaaatggcg
     1681 gcggcagcag cttcggcgcc tcagcagctc tcggatgagg agcttttctc tcagctccgc
     1741 cgttacggcc tgtctcccgg accagtgacg gagagcaccc gcccggtcta cctcaagaag
     1801 ctgaagaagc ttcgagagga agagtaacag cggccgcaca tcaccatcac catcactaag
     1861 cttaattagc tgagcttgga ctcctgttga tagatccagt aatgacctca gaactccatc
     1921 tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagct
     1981 agccatgaaa ataaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     2041 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     2101 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     2161 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     2221 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     2281 atcaagcatt ttatccgtac tcctgatgat gcttggttac tcacgactgc gatccccggc
     2341 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     2401 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     2461 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     2521 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcac
     2581 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     2641 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     2701 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     2761 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     2821 catttgatgc tcgatgagtt tttctaagaa ttaattcatg ggcaaatatt atacgcaagg
     2881 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     2941 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     3001 atttttttaa ggcagttatt ggtgccctta aacgcctggg gtaatgactc tctagcttga
     3061 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     3121 tgtcggtgaa cgctctcctg agtaggacaa atccgccctc tagattacgt gcagtcgatg
     3181 ataagctgtc aaacatgaga attgtgccta atgagtgagc taacttacat taattgcgtt
     3241 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3301 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3361 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3421 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     3481 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     3541 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3601 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     3661 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3721 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3781 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     3841 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     3901 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     3961 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4021 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4081 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4141 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4201 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     4261 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4321 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4381 aggttttgca ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt
     4441 gcgcatgatc tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     4501 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     4561 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     4621 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4681 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     4741 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4801 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4861 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4921 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4981 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5041 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5101 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5161 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5221 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5281 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5341 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5401 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5461 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5521 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5581 gtcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcac
//
