LOCUS       sequence-393303-        3956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An Easy Modular Integrative fuSion-ready Expression toolkit 
            (Easy-MISE toolkit) for fast engineering of heterologous productions
            in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3956)
  AUTHORS   Maestroni L, Butti P, Milanesi R, Pagliari S, Campone L, Serra I, 
            Branduardi P
  TITLE     Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit
            for Fast Engineering of Heterologous Productions in Saccharomyces 
            cerevisiae.
  JOURNAL   ACS Synth Biol. 2023 Apr 14. doi: 10.1021/acssynbio.3c00015.
  PUBMED    37058502
REFERENCE   2  (bases 1 to 3956)
  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"; journalName: "ACS Synth 
            Biol. 2023 Apr 14. doi: 10.1021/acssynbio.3c00015."
COMMENT     Sequence Label: pEM.H05L
FEATURES             Location/Qualifiers
     source          1..3956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(704..722)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(705..722)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(736..753)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(736..752)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(745..767)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(893..1348)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1030..1051)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1242..1261
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1375..1479
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1698..1717)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2060..2079)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2071..2886
                     /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             2071..2886
                     /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(2144..2163)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2971..3559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3460..3479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3713..3730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3847..3868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3883..3888,3889..3906,3907..3913)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3921..3937
                     /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     3926..3948
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(3945..3956,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3945..3956,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag ctctaatacg actcactata gggaaagctc ggtaccggtc
       61 tcctgcctag ttacttgctc tatgcgtttg cgcatcctct ttttactttt tttttttcag
      121 taaagcctaa gcataaatcg ttttatacgt acgacacgtt caacttttct tggttagtag
      181 tggcaatctc tgcaatacat acagggagtc atggtctatc atcttgtcca atcaaagaag
      241 catcggttca gatcgagcaa actgtaggga gaaaggaaag tagaaatgca gagtgtgcta
      301 tatgtccaat ctcggttttg tagtttggat gtcattagag atctaccacc caaccggctg
      361 ctttcatgtg gaacagaaaa gaaatcgggg cgcttcctct tctgtattcc tttagttaac
      421 gtttttattc agccatctaa ccatcatacc cccatacggt aacaaaacct cttctaagaa
      481 aagaagtctc tgctcctccg ccatcttatt tttattcgct gcgcgcgttt attgtcgcat
      541 cgctggccag caaaaaattg gttgcctttt tttacctaaa aaagacacat ctaactgatt
      601 agttttccgt tttaggatat tgacgccaag cgtgcgtctg attactagga gaccgctagc
      661 tctagaccac acgtgtgggg gcccgagctc gcggccgctg tattctatag tgtcacctaa
      721 atggccgcac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
      781 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
      841 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
      901 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
      961 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1021 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1081 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1141 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1201 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1261 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1321 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1381 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1441 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1501 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1561 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1621 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1681 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1741 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtcggt atcattgcag
     1801 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     1861 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     1921 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     1981 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaacaat aaaactgtct
     2041 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     2101 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     2161 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     2221 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     2281 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     2341 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
     2401 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     2461 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     2521 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     2581 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     2641 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     2701 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     2761 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     2821 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     2881 ttctaagaat taattcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2941 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3001 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3061 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3121 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3181 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3241 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3301 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3361 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3421 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3481 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3541 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3601 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3661 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3721 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3781 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3841 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     3901 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagct
//
