LOCUS       sequence-451630-        4816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast episomal expression vector with type '234' RFP drop out site. 
            For use in assembling protein expression cassettes via golden gate 
            assembly. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4816)
  AUTHORS   Cleaver A, Luo R, Smith OB, Murphy L, Schwessinger B, Brock J
  TITLE     High-throughput optimisation of protein secretion in yeast via an 
            engineered biosensor.
  JOURNAL   Trends Biotechnol. 2025 Apr;43(4):838-867. doi: 
            10.1016/j.tibtech.2024.11.010. Epub 2024 Dec 13.
  PUBMED    39674781
REFERENCE   2  (bases 1 to 4816)
  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: 
            "10.1016/j.tibtech.2024.11.010"; journalName: "Trends Biotechnol"; 
            date: "2025-04"; volume: "43"; issue: "4"; pages: "838-867"
COMMENT     Sequence Label: YEE_RFP
FEATURES             Location/Qualifiers
     source          1..4816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(103..894,895..963))
                     /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     726..745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(964..1068)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1171..1759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1660..1679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             2220..2231
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2238..2915
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2933..3004
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3020..3047
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             3419..4222
                     /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"
     misc_feature    4305..4808
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 gcggccgcga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
       61 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
      181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
      241 gcgggaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
      301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
      361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
      421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
      481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
      541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
      601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
      661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
      721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
      781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
      841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
      901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
      961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1081 aaaagtgcca cctgtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1801 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtgc
     1861 ggccgcccct gaattcgcat ctagatggta gagccacaaa cagccggtac aagcaacgat
     1921 ctccaggacc atctgaatca tgcgcggatg acacgaactc acgacggcga tcacagacat
     1981 taacccacag tacagacact gcgacaacgt ggcaattcgt cgcaataccg tctcactgaa
     2041 ctggccgata attgcagacg aacgtgagac cgaaagtgaa acgtgatttc atgcgtcatt
     2101 ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt aatttatgaa
     2161 tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag ctactagaga
     2221 aagaggagaa atactagatg gcgagtagcg aagacgttat caaagagttc atgcgtttca
     2281 aagttcgtat ggaaggttcc gttaacggtc acgagttcga aatcgaaggt gaaggtgaag
     2341 gtcgtccgta cgaaggtact cagaccgcta aactgaaagt taccaaaggt ggtccgctgc
     2401 cgttcgcttg ggacatcctg tccccgcagt tccagtacgg ttccaaagct tacgttaaac
     2461 acccggctga catcccggac tacctgaaac tgtccttccc ggaaggtttc aaatgggaac
     2521 gtgttatgaa cttcgaagac ggtggtgttg ttaccgttac ccaggactcc tccctgcaag
     2581 acggtgagtt catctacaaa gttaaactgc gtggtactaa cttcccgtcc gacggtccgg
     2641 ttatgcagaa aaaaaccatg ggttgggaag cttccaccga acgtatgtac ccggaagacg
     2701 gtgctctgaa aggtgaaatc aaaatgcgtc tgaaactgaa agacggtggt cactacgacg
     2761 ctgaagttaa aaccacctac atggctaaaa aaccggttca gctgccgggt gcttacaaaa
     2821 ccgacatcaa actggacatc acctcccaca acgaagacta caccatcgtt gaacagtacg
     2881 aacgtgctga aggtcgtcac tccaccggtg cttaataagg atctccaggc atcaaataaa
     2941 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     3001 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt ataagtcggt
     3061 ctcagctgag catgagacgg aaatctgctc gtcagtggtg ctcacactga cgaatcatgt
     3121 acagatcata ccgatgactg cctggcgact cacaactaag caagacagcc ggaaccagcg
     3181 ccggcgaaca ccactgcata tatggcatat cacaacagtc caactagtgc actgcagtac
     3241 acggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa
     3301 ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg
     3361 cccagtattc ttaacccaac tgcacagaac aaaaacgtgc aggaaacgaa gataaatcat
     3421 gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct
     3481 atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac
     3541 caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca
     3601 tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc
     3661 cgccaagtac aattttttac tcttcgagga cagaaaattt gctgacattg gtaatacagt
     3721 caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc
     3781 acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac
     3841 aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac
     3901 tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg
     3961 ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac
     4021 acccggtgtg ggtttagatg acaagggaga tgcattgggt caacagtata gaaccgtgga
     4081 tgatgtggtt tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg
     4141 aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag
     4201 aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc
     4261 acaaattaga gcttcaattt aattatatca gttattaccc gagtatcacg tgctataaaa
     4321 ataattataa tttaaatttt ttaatataaa tatataaatt aaaaatagaa agtaaaaaaa
     4381 gaaattaaag aaaaaatagt ttttgttttc cgaagatgta aaagactcta gggggatcgc
     4441 caacaaatac taccttttat cttgctcttc ctgctctcag gtattaatgc cgaattgttt
     4501 catcttgtct gtgtagaaaa ccacacacga aaatcctgtg attttacatt ttacttatcg
     4561 ttaatcgaat gtatatctat ttaatctgct tttcttgtct aataaatata tatgtaaagt
     4621 acgctttttg ttgaaatttt ttaaaccttt gtttattttt ttttcttcat tccgtaactc
     4681 ttctaccttc tttatttact ttctaaaatc caaatacaaa acataaaaat aaataaacac
     4741 agagtaaatt cccaaattat tccatcatta aaagatacga ggcgcgtgta agttacaggc
     4801 aagcgatccg tcccga
//
