LOCUS       sequence-393305-        3916 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 3916)
  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 3916)
  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.H05R
FEATURES             Location/Qualifiers
     source          1..3916
                     /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(664..682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(665..682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(696..713)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(696..712)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(705..727)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(853..1308)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(990..1011)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1202..1221
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1335..1439
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1658..1677)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2020..2039)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2031..2846
                     /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             2031..2846
                     /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(2104..2123)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2931..3519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3420..3439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3673..3690
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3807..3828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3843..3848,3849..3866,3867..3873)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3881..3897
                     /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     3886..3908
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(3905..3916,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3905..3916,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag ctctaatacg actcactata gggaaagctc ggtaccggtc
       61 tccttactta cgtggattga gccagcaata cagatcatta ttaaattgtt ttgtacatga
      121 tgttagtata taatcgtaaa gcttttctaa tatgtatacc ttatacatgg aactccacag
      181 aacttgcaaa cataccaaaa atcccttatt cttgttcact cattttacat caaaaaataa
      241 tatttcagtt attaaggaaa ataaaaaaat agattagaga agcattttga agaaatagta
      301 tattctttta ttgaacctaa gagcgtgata tttttactcg aaataaaata cgaaaaatct
      361 atgcactcat ctttccgact actattggct cctgctcaaa aaaagaggga aaaaaagctc
      421 caaaattcta tcttttccta tcgctcctgt cctatcctta ttacgttcat tactatttta
      481 atactatcca ttcttttatt ttcagtctaa aaaaaacatt tctcataacg ggaaaagcaa
      541 aaaaatgtca agcttataca tcaaaacacc actgcatgca ttatctgctg gtccggattc
      601 tcagcaagga gaccgctagc tctagaccac acgtgtgggg gcccgagctc gcggccgctg
      661 tattctatag tgtcacctaa atggccgcac aattcactgg ccgtcgtttt acaacgtcgt
      721 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      781 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      841 aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
      901 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
      961 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1021 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     1081 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     1141 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     1201 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     1261 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     1321 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1381 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1441 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1501 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1561 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1621 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1681 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1741 ttgagtcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1801 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1861 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1921 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1981 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     2041 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     2101 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     2161 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     2221 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     2281 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     2341 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     2401 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     2461 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     2521 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     2581 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     2641 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     2701 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     2761 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     2821 atttgatgct cgatgagttt ttctaagaat taattcatga ccaaaatccc ttaacgtgag
     2881 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2941 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3001 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3061 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3121 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3181 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3241 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3301 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3361 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3421 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3481 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3541 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3601 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3661 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     3721 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3781 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     3841 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     3901 cacacaggaa acagct
//
