LOCUS       sequence-393164-        3549 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 3549)
  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 3549)
  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-mini
FEATURES             Location/Qualifiers
     source          1..3549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(153..221,222..1013)
                     /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(371..390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1184..1772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1673..1692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1926..1943
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2060..2081
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2096..2101,2102..2119,2120..2126)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2134..2150
                     /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     2158..2174
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2158..2174
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2262..2283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2298..2303,2304..2321,2322..2328)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2336..2352
                     /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             2370..2381
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2388..3065
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      3110..3181
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3197..3224
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3262..3279)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3262..3278)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3271..3293)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aaaaaatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
       61 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      121 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      181 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      241 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      301 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      361 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
      421 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      481 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      541 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      601 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      661 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
      721 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
      781 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
      841 ctgataaatc tggagccggt gagcgtggat ctcgcggtat cattgcagca ctggggccag
      901 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
      961 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1021 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1081 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1141 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1201 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1261 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1321 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1381 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1441 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1501 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1561 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1621 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1681 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1741 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     1801 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     1861 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     1921 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     1981 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     2041 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     2101 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     2161 gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggtcgac tctagaggat
     2221 ccccgggtac ccggatccgc tagctgccag agacccgcaa ttaatgtgag ttagctcact
     2281 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2341 agcggataac aatttcacac atactagaga aagaggagaa atactagatg gcttcctccg
     2401 aagacgttat caaagagttc atgcgtttca aagttcgtat ggaaggttcc gttaacggtc
     2461 acgagttcga aatcgaaggt gaaggtgaag gtcgtccgta cgaaggtacc cagaccgcta
     2521 aactgaaagt taccaaaggt ggtccgctgc cgttcgcttg ggacatcctg tccccgcagt
     2581 tccagtacgg ttccaaagct tacgttaaac acccggctga catcccggac tacctgaaac
     2641 tgtccttccc ggaaggtttc aaatgggaac gtgttatgaa cttcgaagac ggtggtgttg
     2701 ttaccgttac ccaggactcc tccctgcaag acggtgagtt catctacaaa gttaaactgc
     2761 gtggtaccaa cttcccgtcc gacggtccgg ttatgcagaa aaaaaccatg ggttgggaag
     2821 cttccaccga acgtatgtac ccggaagacg gtgctctgaa aggtgaaatc aaaatgcgtc
     2881 tgaaactgaa agacggtggt cactacgacg ctgaagttaa aaccacctac atggctaaaa
     2941 aaccggttca gctgccgggt gcttacaaaa ccgacatcaa actggacatc acctcccaca
     3001 acgaagacta caccatcgtt gaacagtacg aacgtgctga aggtcgtcac tccaccggtg
     3061 cttaataacg ctgatagtgc tagtgtagat cgctactaga gccaggcatc aaataaaacg
     3121 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     3181 ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata taggtctctg
     3241 caagctagcg agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3301 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3361 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3421 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taatcgctgg
     3481 gccattctca tgaagaatat cttgaattta ttgtcatatt actagttggt gtggaagtcc
     3541 atatatcgg
//
