LOCUS       sequence-393160-        6613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Easy-MISE toolkit Level 1 destination plasmid for cassette cloning 
            with BsaI-based Golden Gate Assembly; allowing fast postitive clones
            test with mRFP1 fluorescent protein-based red/white screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6613)
  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 6613)
  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: pGA-red-maxi
FEATURES             Location/Qualifiers
     source          1..6613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1046..1849)
                     /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(1850..2064)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2089..2107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2175..2279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2280..2348,2349..3140)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2498..2517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3311..3899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3800..3819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4053..4070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4187..4208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4223..4228,4229..4246,4247..4253)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4261..4277
                     /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     4285..4301
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4285..4301
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4389..4410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4425..4430,4431..4448,4449..4455)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4463..4479
                     /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             4497..4508
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4515..5192
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      5237..5308
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5324..5351
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5389..5406)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5389..5405)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5398..5420)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    join(6162..6613,1..711)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN4 centromere fused to the 
                     autonomously replicating sequence ARS1/ARS416"
     rep_origin      join(6487..6613,1..711)
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1/ARS416"
ORIGIN
        1 ttatctatct gcatgaacac gaaatcttta cttgacgact tgaggctgat ggtgtttatg
       61 caaagaaacc actgtgttta atatgtgtca ctgtttgata ttactgtcag cgtagaagat
      121 aatagtaaaa gcggttaata agtgtatttg agataagtgt gataaagttt ttacagcgaa
      181 aagacgataa atacaagaaa atgattacga ggatacggag agaggtatgt acatgtgtat
      241 ttatatacta agctgccggc ggttgtttgc aagaccgaga aaaggctagc aagaatcggg
      301 tcattgtagc gtatgcgcct gtgaacattc tcttcaacaa gtttgattcc attgcggtga
      361 aatggtaaaa gtcaaccccc tgcgatgtat attttcctgt acaatcaatc aaaaagccaa
      421 atgatttagc attatcttta catcttgtta ttttacagat tttatgttta gatcttttat
      481 gcttgctttt caaaaggctt gcaggcaagt gcacaaacaa tacttaaata aatactactc
      541 agtaataacc tatttcttag catttttgac gaaatttgct attttgttag agtcttttac
      601 accatttgtc tccacacctc cgcttacatc aacaccaata acgccattta atctaagcgc
      661 atcaccaaca ttttctggcg tcagtccacc agctaacata aaatgtaagc tctcggggct
      721 ctcttgcctt ccaacccagt cagaaatcga gttccaatcc aaaagttcac ctgtcccacc
      781 tgcttctgaa tcaaacaagg gaataaacga atgaggtttc tgtgaagctg cactgagtag
      841 tatgttgcag tcttttggaa atacgagtct tttaataact ggcaaaccga ggaactcttg
      901 gtattcttgc cacgactcat ctccatgcag ttggacgatc gatgataagc tgtcaaacat
      961 gagaattggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac
     1021 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt
     1081 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata
     1141 gtcctcttcc aacaataata atgtcagatc ctgtagagac tacatcatcc acggttctat
     1201 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc
     1261 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat
     1321 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc
     1381 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg
     1441 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt
     1501 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa
     1561 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta
     1621 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta
     1681 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat
     1741 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa
     1801 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc
     1861 gtttcctgca tgtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt
     1921 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt
     1981 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag
     2041 aataaaaaaa aatgatgaat tgaaaagcta attcttgaag acgaaagggc ctcgtgatac
     2101 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2221 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2281 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2341 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2401 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2461 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2521 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2581 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2641 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2701 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2761 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2821 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2881 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2941 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggatctc
     3001 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3061 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3121 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3181 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3241 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3301 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3361 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3421 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     3481 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3541 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3601 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3661 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3721 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3781 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3841 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3901 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3961 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4021 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4081 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     4141 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     4201 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     4261 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
     4321 catgcctgca ggtcgactct agaggatccc cgggtacccg gatccgctag ctgccagaga
     4381 cccgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     4441 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacata ctagagaaag
     4501 aggagaaata ctagatggct tcctccgaag acgttatcaa agagttcatg cgtttcaaag
     4561 ttcgtatgga aggttccgtt aacggtcacg agttcgaaat cgaaggtgaa ggtgaaggtc
     4621 gtccgtacga aggtacccag accgctaaac tgaaagttac caaaggtggt ccgctgccgt
     4681 tcgcttggga catcctgtcc ccgcagttcc agtacggttc caaagcttac gttaaacacc
     4741 cggctgacat cccggactac ctgaaactgt ccttcccgga aggtttcaaa tgggaacgtg
     4801 ttatgaactt cgaagacggt ggtgttgtta ccgttaccca ggactcctcc ctgcaagacg
     4861 gtgagttcat ctacaaagtt aaactgcgtg gtaccaactt cccgtccgac ggtccggtta
     4921 tgcagaaaaa aaccatgggt tgggaagctt ccaccgaacg tatgtacccg gaagacggtg
     4981 ctctgaaagg tgaaatcaaa atgcgtctga aactgaaaga cggtggtcac tacgacgctg
     5041 aagttaaaac cacctacatg gctaaaaaac cggttcagct gccgggtgct tacaaaaccg
     5101 acatcaaact ggacatcacc tcccacaacg aagactacac catcgttgaa cagtacgaac
     5161 gtgctgaagg tcgtcactcc accggtgctt aataacgctg atagtgctag tgtagatcgc
     5221 tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     5281 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     5341 gcctttctgc gtttatatag gtctctgcaa gctagcgagc tcgaattcac tggccgtcgt
     5401 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5461 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5521 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     5581 cggtatttca caccgcataa tcgctgggcc attctcatga agaatatctt gaatttattg
     5641 tcatattact agttggtgtg gaagtcctaa tatcggtgat caatatagtg gttgacatgc
     5701 tggctagtca acattgagcc ttttgatcat gcaaatatat tacggtattt tacaatcaaa
     5761 tatcaaactt aactattgac tttataactt atttaggtgg taacattctt ataaaaaaga
     5821 aaaaaattac tgcaaaacag tactagcttt taacttgtat cctaggttat ctatgctgtc
     5881 tcaccataga gaatattacc tatttcagaa tgtatgtcca tgattcgccg ggtaaataca
     5941 tataatacac aaatctggct taataaagtc tataatatat ctcataaaga agtgctaaat
     6001 tggctagtgc tatatatttt taagaaaatt tcttttgact aagtccatat cgactttgta
     6061 aaagttcaca ttagcataca tatattacac gagccagaaa tagtaacttt tgcctaaatc
     6121 acaaattgca aaatttaatt gcttgcaaaa ggtcacatgc ttataatcaa cttttttaaa
     6181 aatttaaaat acttttttat tttttatttt taaacataaa tgaaataatt tatttattgt
     6241 ttatgattac cgaaacataa aacctgctca agaaaaagaa actgttttgt ccttggaaaa
     6301 aaagcactac ctaggagcgg ccaaaatgcc gaggctttca tagcttaaac tctttacaga
     6361 aaataggcat tatagatcag ttcgagtttt cttattcttc cttccggttt tatcgtcaca
     6421 gttttacagt aaataagtat cacctcttag agttcgatga taagctgtca aacatgagaa
     6481 ttaattccac atgttaaaat agtgaaggag catgttcggc acacagtgga ccgaacgtgg
     6541 ggtaagtgca ctagggtccg gttaaacgga tctcgcattg atgaggcaac gctaattatc
     6601 aacatataga ttg
//
