LOCUS       sequence-327180-        6100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Characterisation of 2A-mediated bicistronic expression of Y-FAST and
            yEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6100)
  AUTHORS   Lu Z, Peng B, Ebert BE, Dumsday G, Vickers CE
  TITLE     Auxin-mediated protein depletion for metabolic engineering in 
            terpene-producing yeast.
  JOURNAL   Nat Commun. 2021 Feb 16;12(1):1051. doi: 10.1038/s41467-021-21313-1.
  PUBMED    33594068
REFERENCE   2  (bases 1 to 6100)
  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: "Nat 
            Commun."; date: "2021-02-16"; volume: "12(1):1051. doi"; pages: " 
            10.1038/s41467-021-21313-1"
COMMENT     Sequence Label: pILGFPY2A
FEATURES             Location/Qualifiers
     source          1..6100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             422..796
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKVHMKKALSGDSYWVFVKRV"
     CDS             857..1567
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      1577..1847
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(2112..2128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2112..2128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2125..2147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2136..2152
                     /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)."
     promoter        complement(join(2160..2166,2167..2184,2185..2190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2205..2226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2343..2360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2514..3102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2594..2613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3273..4064,4065..4133))
                     /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     3896..3915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4134..4238)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4306..4324
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4362..4384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4484..4503
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4697..4719
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4711..4728
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4712..4728
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5842..5875)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(6095..6100,1..408)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 ccacacacca tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga
       61 ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca gcatactaaa tttcccctct
      121 ttcttcctct agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac
      181 cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt
      241 cttgaaaatt tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt
      301 taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt
      361 tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaagtt ttaattacaa
      421 aatggaacac gttgctttcg gttctgaaga tattgaaaac actttggcta aaatggatga
      481 tggtcaatta gacggtttag ctttcggtgc tattcaattg gacggtgatg gtaacatttt
      541 gcaatacaat gctgctgaag gtgatattac tggtagagat ccaaaacaag ttattggtaa
      601 gaacttcttt aaggacgttg ctccaggtac tgactctcca gagttctatg gtaagtttaa
      661 ggaaggtgtt gcttctggta acttaaacac tatgtttgaa tggatgatcc caacttccag
      721 aggtccaact aaggttaaag ttcacatgaa aaaggcttta tctggtgact cttactgggt
      781 tttcgttaaa agagttggtg ctactaactt ctctttgttg aagttggctg gtgacgttga
      841 acttaaccca ggtccatcta aaggtgaaga attattcact ggtgttgtcc caattttggt
      901 tgaattagat ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga
      961 tgctacttac ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc
     1021 atggccaacc ttagtcacta ctttcggtta tggtgttcaa tgttttgcga gatacccaga
     1081 tcatatgaaa caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag
     1141 aactattttt ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg
     1201 tgatacctta gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat
     1261 tttaggtcac aaattggaat acaactataa ctctcacaat gtttacatca tggctgacaa
     1321 acaaaagaat ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt
     1381 tcaattagct gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc
     1441 agacaaccat tacttatcca ctcaatctgc cttatccaaa gatccaaacg aaaagagaga
     1501 ccacatggtc ttgttagaat ttgttactgc tgctggtatt acccatggta tggatgaatt
     1561 gtacaaaaga tcttaaattg aattgaattg aaatcgatag atcaattttt ttcttttctc
     1621 tttccccatc ctttacgcta aaataatagt ttattttatt ttttgaatat tttttattta
     1681 tatacgtata tatagactat tatttatctt ttaatgatta ttaagatttt tattaaaaaa
     1741 aaattcgctc ctcttttaat gcctttatgc agtttttttt tcccattcga tatttctatg
     1801 ttcgggttca gcgtatttta agtttaataa ctcgaaaatt ctgcgttcgt taaagctttc
     1861 gagaaggata ttatttacta gtaggactat ttgcaaaggg aagggatgct aaggtagagg
     1921 gtgaacgtta cagaaaagca ggctgggaag catatttgag aagatgcggc cagcaaaact
     1981 aaaaaactgt attataagta aatgcatgta tactaaactc acaaattaga gcttcaattt
     2041 aattatatca gttattaccc gggaatctcg gtcgtaatga tttaaatgca tgcaagcttg
     2101 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2161 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2341 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2401 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2461 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2521 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2581 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2641 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2701 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2761 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2821 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2881 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2941 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3001 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3061 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3121 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3181 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3241 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3301 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3361 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3421 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3481 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3541 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3601 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3661 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3721 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3781 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3841 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3901 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3961 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4021 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4081 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4141 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4201 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4261 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     4321 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4381 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4441 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     4501 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4561 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4621 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4681 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcatttaa
     4741 atttttttga ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta
     4801 gattggtata tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa
     4861 cccaactgca cagaacaaaa acctgcagga aacgaagata aaacatgtcc acaaaatcat
     4921 ataccagtag agctgagact catgcaagtc cggttgcatc gaaactttta cgtttaatgg
     4981 atgaaaagaa gaccaatttg tgtgcttctc ttgacgttcg ttcgactgat gagctattga
     5041 aacttgttga aacgttgggt ccatacattt gccttttgaa aacacacgtt gatatcttgg
     5101 atgatttcag ttatgagggt actgtcgttc cattgaaagc attggcagag aaatacaagt
     5161 tcttgatatt tgaggacaga aaattcgccg atatcggtaa cacagtcaaa ttacaatata
     5221 catcgggcgt ttaccgtatc gcagaatggt ctgatatcac caacgcccac ggggttactg
     5281 gtgctggtat tgttgctggc ttgaaacaag gtgcgcaaga ggtcaccaaa gaaccaaggg
     5341 gattattgat gcttgctgaa ttgtcttcca agggttctct agcacacggt gaatatacta
     5401 agggtaccgt tgatattgca aagagtgata aagatttcgt tattgggttc attgctcaga
     5461 acgatatggg aggaagagaa gaagggtttg attggctaat catgacccca ggtgtaggtt
     5521 tagacgacaa aggcgatgca ttgggtcagc agtacagaac cgtcgacgaa gttgtaagtg
     5581 gtggatcaga tatcatcatt gttggcagag gacttttcgc caagggtaga gatcctaagg
     5641 ttgaaggtga aagatacaga aatgctggat gggaagcgta ccaaaagaga atcagcgctc
     5701 cccattaatt atacaggaaa cttaatagaa caaatcacat atttaatcta atagccacct
     5761 gcattggcac ggtgcaacac tacttcaact tcatcttaca aaaagatcac gtgatctgtt
     5821 gtattgggat ctctagacct aataacttcg tatagcatac attatacgaa gttatattaa
     5881 gggttgctcg agaaagagct ccagaaagcg accacccaac tttggctgat aatagcgtat
     5941 aaacaatgca tactttgtac gttcaaaata caatgcagta gatatattta tgcatattac
     6001 atataataca tatcacatag gaagcaacag gcgcgttgga cttttaattt tcgaggaccg
     6061 cgaatcctta catcacaccc aatcccccac aagtgatccc
//
