LOCUS       sequence-163610-        7475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGREG600-His6-Sem1Sc, Expresses the His6-Sem1 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7475)
  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 7475)
  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: pSc-His6-Sem1Sc
FEATURES             Location/Qualifiers
     source          1..7475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..98,99..105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    113..129
                     /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)."
     primer_bind     118..140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     137..153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     137..153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     172..192
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        174..192
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        232..673
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    232..349
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     644..667
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             770..787
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1015..1026
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      1067..1314
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1083..1101)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(1327..1360)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(1418..2774)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(1418..1615)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1621..2430)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2344..2363
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2431..2774)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(2829..2862)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2898..2917)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2899..2917)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2927..2944)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2927..2943)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2936..2958)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3088..3543
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3175..3194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3385..3406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3674..4477)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4478..4698)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4724..4743)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4843..4865
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4903..4921)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4963..5466
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5503..5607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5792..6169
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     complement(5826..5845)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6638..7226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7127..7146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7380..7397
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat
       61 taggcacccc aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc
      121 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      181 cctcactaaa gggaacaaaa gctggagctc gtttaaacgg cgcgcctagt acggattaga
      241 agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc
      301 accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga acaataaaga
      361 ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac
      421 aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga ttagtttttt
      481 agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat
      541 ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc ggtttgtatt
      601 acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac ctctatactt
      661 taacgtcaag gagaaaaacc ccggattctg aagcggccgc actagtggat cccccgggct
      721 gcaggaattc gatatcaagc ttatcgatac cgtcgacatg ggcagcagcc atcaccatca
      781 tcaccacatg agtacggacg ttgctgctgc acaagcccaa agtaaaatag atttaacaaa
      841 aaaaaaaaat gaggaaataa ataagaagtc tctagaggaa gacgatgaat ttgaagattt
      901 ccccattgac acatgggcca atggtgaaac aataaaaagc aatgctgtaa cgcaaacaaa
      961 catatgggaa gaaaactggg atgatgttga agttgacgac gatttcacca atgaattgaa
     1021 ggccgaattg gatagataca aacgggaaaa tcagtgataa ctcgagtcat gtaattagtt
     1081 atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt
     1141 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     1201 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     1261 atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg
     1321 tacctaataa cttcgtatag catacattat acgaagttat attaagggtt ctcgacgttt
     1381 tcgacactgg atggcggcgt tagtatcgaa tcgacagcag tatagcgacc agcattcaca
     1441 tacgattgac gcatgatatt actttctgcg cacttaactt cgcatctggg cagatgatgt
     1501 cgaggcgaaa aaaaatataa atcacgctaa catttgatta aaatagaaca actacaatat
     1561 aaaaaaacta tacaaatgac aagttcttga aaacaagaat ctttttattg tcagtactga
     1621 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1681 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1741 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1801 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1861 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     1921 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1981 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2041 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2101 ttcttctaat acctggaatg ctgttttgcc ggggatcgca gtggtgagta accatgcatc
     2161 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2221 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2281 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2341 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2401 cctcgaaacg tgagtctttt ccttacccat ggttgtttat gttcggatgt gatgtgagaa
     2461 ctgtatccta gcaagatttt aaaaggaagt atatgaaaga agaacctcag tggcaaatcc
     2521 taacctttta tatttctcta caggggcgcg gcgtggggac aattcaacgc gtctgtgagg
     2581 ggagcgtttc cctgctcgca ggtctgcagc gaggagccgt aatttttgct tcgcgccgtg
     2641 cggccatcaa aatgtatgga tgcaaatgat tatacatggg gatgtatggg ctaaatgtac
     2701 gggcgacagt cacatcatgc ccctgagctg cgcacgtcaa gactgtcaag gagggtattc
     2761 tgggcctcca tgtcgctggc cgggtgaccc ggcggggacg aggcaagcta aacagatctc
     2821 tagacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg ttgtcttaat
     2881 taagggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     2941 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3001 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3061 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg
     3121 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3181 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3241 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3301 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3361 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3421 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3481 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3541 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagggta
     3601 ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt
     3661 ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt
     3721 ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa
     3781 caataataat gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca
     3841 atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt
     3901 catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct
     3961 tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca
     4021 attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca
     4081 aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg
     4141 ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt
     4201 ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg
     4261 tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg
     4321 gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac
     4381 acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat
     4441 gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg
     4501 tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac
     4561 atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg
     4621 agattaccga atcaaaaaaa tttcaaagaa accgaaatca aaaaaaagaa taaaaaaaaa
     4681 atgatgaatt gaattgaaaa gctgtggtat ggtgcactct cagtacaatc tgctctgatg
     4741 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4801 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4861 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4921 ttttataggt taatgtcatg ataataatgg tttcttagga cggatcgctt gcctgtaact
     4981 tacacgcgcc tcgtatcttt taatgatgga ataatttggg aatttactct gtgtttattt
     5041 atttttatgt tttgtatttg gattttagaa agtaaataaa gaaggtagaa gagttacgga
     5101 atgaagaaaa aaaaataaac aaaggtttaa aaaatttcaa caaaaagcgt actttacata
     5161 tatatttatt agacaagaaa agcagattaa atagatatac attcgattaa cgataagtaa
     5221 aatgtaaaat cacaggattt tcgtgtgtgg tcttctacac agacaagatg aaacaattcg
     5281 gcattaatac ctgagagcag gaagagcaag ataaaaggta gtatttgttg gcgatccccc
     5341 tagagtcttt tacatcttcg gaaaacaaaa actatttttt ctttaatttc tttttttact
     5401 ttctattttt aatttatata tttatattaa aaaatttaaa ttataattat ttttatagca
     5461 cgtgatgaaa aggacccagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5521 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5581 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5641 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5701 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5761 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5821 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5881 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5941 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6001 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6061 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctaat gaagccatac
     6121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6781 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7261 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7321 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7381 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     7441 gttggccgat tcattaatgc agctggcacg acagg
//
