LOCUS       sequence-451631-        4842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast episomal expression vector with type '234' GFP 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 4842)
  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 4842)
  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_GFP
FEATURES             Location/Qualifiers
     source          1..4842
                     /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..2951
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2963..3034
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3050..3077
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             3445..4248
                     /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    4331..4834
                     /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 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc
     2281 tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag
     2341 gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt aaactgccgg
     2401 ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt gctcgttatc
     2461 cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc tatgtgcagg
     2521 aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg
     2581 aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa gaggacggca
     2641 atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac atcaccgccg
     2701 ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca
     2761 gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt cctgttctgc
     2821 tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg aacgagaaac
     2881 gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat ggtatggatg
     2941 aactgtacaa atgaccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3001 tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc
     3061 gggtgggcct ttctgcgttt ataggtctca gctgagcatg agacggaaat ctgctcgtca
     3121 gtggtgctca cactgacgaa tcatgtacag atcataccga tgactgcctg gcgactcaca
     3181 actaagcaag acagccggaa ccagcgccgg cgaacaccac tgcatatatg gcatatcaca
     3241 acagtccaac tagtgcactg cagtacacgg tttccttgaa atttttttga ttcggtaatc
     3301 tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata tatacgcata
     3361 tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca cagaacaaaa
     3421 acgtgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg tgctgctact
     3481 catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca aacaaacttg
     3541 tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga agcattaggt
     3601 cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc catggagggc
     3661 acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt cgaggacaga
     3721 aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt atacagaata
     3781 gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt
     3841 ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat gttagcagaa
     3901 ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt tgacattgcg
     3961 aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg tggaagagat
     4021 gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa gggagatgca
     4081 ttgggtcaac agtatagaac cgtggatgat gtggtttcta caggatctga cattattatt
     4141 gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga
     4201 aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta
     4261 taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt atatcagtta
     4321 ttacccgagt atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata
     4381 taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa
     4441 gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc
     4501 tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaaaaccac acacgaaaat
     4561 cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc
     4621 ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt
     4681 attttttttt cttcattccg taactcttct accttcttta tttactttct aaaatccaaa
     4741 tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag
     4801 atacgaggcg cgtgtaagtt acaggcaagc gatccgtccc ga
//
