LOCUS       sequence-165374-        5221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCDF-His6-MBP-Sem1 Sc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5221)
  AUTHORS   Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, 
            Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T,
            Katina C, Pilzer I, Ben-Aroya S, Prag G
  TITLE     A bacterial genetic selection system for ubiquitylation cascade 
            discovery.
  JOURNAL   Nat Methods. 2016 Nov;13(11):945-952. doi: 10.1038/nmeth.4003. Epub 
            2016 Oct 3.
  PUBMED    27694912
REFERENCE   2  (bases 1 to 5221)
  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: 
            "10.1038/nmeth.4003"; journalName: "Nat Methods"; date: "2016-11"; 
            volume: "13"; issue: "11"; pages: "945-952"
COMMENT     Sequence Label: pCOG75-Sem1 Sc
FEATURES             Location/Qualifiers
     source          1..5221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..1216
                     /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"
     primer_bind     1190..1213
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1277..1297
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1534..1545
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        1654..1672
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1673..1697
                     /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)."
     CDS             1806..1850
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1888..1906)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1902..1949
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2120..2911)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2912..3003)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3051..3789)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3999..5081)
                     /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     5043..5062
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5082..5159)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5205..5221,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgaa
      121 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
      181 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
      241 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
      301 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
      361 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
      421 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
      481 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
      541 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
      601 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt
      661 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
      721 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
      781 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
      841 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
      901 gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
      961 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     1021 cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccacaat
     1081 ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta
     1141 tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct
     1201 gaaagacgcg cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct
     1261 cgggatcgat acgaccgaaa acctgtattt tcagggcggg cgcgccatga gtacggacgt
     1321 tgctgctgca caagcccaaa gtaaaataga tttaacaaaa aaaaaaaatg aggaaataaa
     1381 taagaagtct ctagaggaag acgatgaatt tgaagatttc cccattgaca catgggccaa
     1441 tggtgaaaca ataaaaagca atgctgtaac gcaaacaaac atatgggaag aaaactggga
     1501 tgatgttgaa gttgacgacg atttcaccaa tgaattgaag gccgaattgg atagatacaa
     1561 acgggaaaat cagtgagtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
     1621 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
     1681 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
     1741 tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt
     1801 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
     1861 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1921 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
     1981 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
     2041 aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc
     2101 ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
     2161 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
     2221 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
     2281 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
     2341 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
     2401 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
     2461 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
     2521 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     2581 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     2641 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     2701 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     2761 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     2821 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     2881 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     2941 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3001 aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga
     3061 cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa
     3121 cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat
     3181 aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag
     3241 tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct
     3301 tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac
     3361 gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt
     3421 cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc
     3481 ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc
     3541 attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt
     3601 gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc
     3661 agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag
     3721 gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat
     3781 cctttgatct tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta
     3841 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     3901 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3961 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4081 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4141 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4201 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4261 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4321 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4381 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4441 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4501 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4561 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4621 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4681 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4741 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4801 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4861 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4921 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4981 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5041 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5101 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5161 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
     5221 a
//
