LOCUS       sequence-437597-        8393 bp    DNA     circular SYN 15-APR-2025
DEFINITION  equimolar expression of Staygold-E138D and mCherry in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8393)
  AUTHORS   Teusink B
  TITLE     Expression of FPs in budding yeast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8393)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pDRF1 StayGold-E138D-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..8393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    800..824
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             831..1481
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             1488..1541
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1557..1566
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1563..2273
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1708..1726)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1976..1995
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2283..2307)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2369..2388)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2370..2388)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2536..2723
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2591..2615)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2735..2752)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2735..2751)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2744..2766)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3187..4533
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4644..5447)
                     /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(5448..5663)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5688..5706)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5774..5878
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5879..5947,5948..6739)
                     /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(6097..6116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6910..7498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7399..7418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7652..7669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7786..7807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7822..7827,7828..7845,7846..7852)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7860..7876
                     /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     7865..7887
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7884..7900
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7884..7900
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7930..8385)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8067..8088)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8279..8298
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caagcttcct gaaacggaga aacataaaca ggcattgctg ggatcaccca tacatcactc
       61 tgttttgcct gaccttttcc ggtaatttga aaacaaaccc ggtctcgaag cggagatccg
      121 gcgataatta ccgcagaaat aaacccatac acgagacgta gaaccagccg cacatggccg
      181 gagaaactcc tgcgagaatt tcgtaaactc gcgcgcattg catctgtatt tcctaatgcg
      241 gcacttccag gcctcgatcg agaccgttta tccattgctt ttttgttgtc tttttccctc
      301 gttcacagaa agtctgaaga agctatagta gaactatgag ctttttttgt ttctgttttc
      361 cttttttttt tttttacctc tgtggaaatt gttactctca cactctttag ttcgtttgtt
      421 tgttttgttt attccaatta tgaccggtga cgaaacgtgg tcgatggtgg gtaccgctta
      481 tgctcccctc cattagtttc gattatataa aaaggccaaa tattgtatta ttttcaaatg
      541 tcctatcatt atcgtctaac atctaatttc tcttaaattt tttctctttc tttcctataa
      601 caccaatagt gaaaatcttt ttttcttcta tatctacaaa aacttttttt ttctatcaac
      661 ctcgttgata aattttttct ttaacaatcg ttaataatta attaattgga aaataaccat
      721 tttttctctc ttttatacac acattcaaaa gaaagaaaaa aaatataccc cagcctcgac
      781 tagtgaattc gcggccatca caagtttgta caaaaaagca ggctgctagc atggccagca
      841 cgccgtttaa gttccagtta aaggggacaa ttaacgggaa aagctttaca gtcgagggtg
      901 aaggggaagg gaactcgcat gaaggtagtc ataaaggtaa gtatgtgtgt acgagcggaa
      961 agttgccgat gtcttgggcg gctcttggta cttcgtttgg gtacggaatg aaatactata
     1021 caaaataccc gtcgggcctg aaaaattggt tccatgaggt gatgcccgag gggttcacgt
     1081 atgatcgcca catccagtac aaaggtgatg gctcaatcca cgcaaagcac caacacttca
     1141 tgaagaatgg gacttatcat aacattgtcg agtttactgg ccaggacttc aaggaaaaca
     1201 gccccgtatt gacaggtgac atgaatgtga gcttgccgaa cgacgtgcag catattccac
     1261 gtgacgacgg agttgaatgt cccgtcacat tgttataccc cttgcttagc gataaatcaa
     1321 agtgtgtcga agcacatcaa aacactatct gcaaacctct tcataaccaa cctgcgccag
     1381 atgtaccgta ccattggatt cgcaaacaat atacacagtc taaggatgat acggaggaac
     1441 gcgatcatat ctgtcagtcc gagacgttag aagctcatct ttccggagag ggcagaggaa
     1501 gtcttctaac atgcggtgac gtggaggaga atcccggccc tggggatcca ccggtcgcca
     1561 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     1621 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     1681 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1741 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     1801 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     1861 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     1921 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     1981 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2041 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2101 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2161 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2221 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taaagcggcc
     2281 gcacccagct ttcttgtaca aagtggtgta gttaattcat gatggccgct gcaggtcgac
     2341 ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcggatcca
     2401 gctttggact tcttcgccag aggtttggtc aagtctccaa tcaaggttgt cggcttgtct
     2461 accttgccag aaatttacga aaagatggaa aagggtcaaa tcgttggtag atacgttgtt
     2521 gacacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
     2581 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     2641 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct
     2701 cttattgacc acacctctac cggcatgcca attcactggc cgtcgtttta caacgtcgtg
     2761 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2821 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2881 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2941 gcataatcgg atcgtacttg ttacccatca ttgaattttg aacatccgaa cctgggagtt
     3001 ttccctgaaa cagatagtat atttgaacct gtataataat atatagtcta gcgctttacg
     3061 gaagacaatg tatgtatttc ggttcctgga gaaactattg catctattgc ataggtaatc
     3121 ttgcacgtcg catccccggt tcattttctg cgtttccatc ttgcacttca atagcatatc
     3181 tttgttaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta
     3241 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg
     3301 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag
     3361 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
     3421 gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc
     3481 gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct
     3541 ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta
     3601 ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga
     3661 ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat tatattctat
     3721 accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt
     3781 ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg
     3841 tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg
     3901 tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt
     3961 tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca
     4021 aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt
     4081 acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa
     4141 aagcgctctg aagttcctat actttctagc tagagaatag gaacttcgga ataggaactt
     4201 caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc acatacagct
     4261 cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac atgagaagaa
     4321 cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta tgtaggatga
     4381 aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc gtatgcttcc
     4441 ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat tagtctcatc
     4501 cttcaatgct atcatttcct ttgatattgg atcgatccga tgataagctg tcaaacatga
     4561 gaattgggta ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat
     4621 gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc
     4681 cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt
     4741 cctcttccaa caataataat gtcagatcct gtagagacca catcatccac ggttctatac
     4801 tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa
     4861 tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct
     4921 ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc
     4981 ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc
     5041 gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct
     5101 gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg
     5161 tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc
     5221 ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa
     5281 caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc
     5341 aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca
     5401 ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt
     5461 ttcctgcatg tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct
     5521 tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc
     5581 ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca aaaaaaagaa
     5641 taaaaaaaaa atgatgaatt gaaaagctaa ttcttgaaga cgaaagggcc tcgtgatacg
     5701 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5761 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5821 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5881 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5941 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6001 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6061 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7621 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     7681 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     7741 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     7801 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     7861 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttac
     7921 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     7981 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     8041 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     8101 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     8161 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     8221 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     8281 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     8341 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgc
//
