LOCUS       sequence-332147-        8438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar expression of AausFP1 and mCherry for use in 
            yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8438)
  AUTHORS   Botman D, de Groot DH, Wehrens M, van Rossum K, Yska S, van der 
            Kieft B, Albert V, Minghetti N, Goedhart J, Teusink B
  TITLE     Side-by-side systematic characterization of novel FPs in budding 
            yeast
  JOURNAL   bioRxiv 2025.06.27.661975; doi: 
            https://doi.org/10.1101/2025.06.27.661975
REFERENCE   2  (bases 1 to 8438)
  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: "bioRxiv 
            2025.06.27.661975; doi: https://doi.org/10.1101/2025.06.27.661975"
COMMENT     Sequence Label: AausFP1-T2A-mCherry pdrf1-GW
FEATURES             Location/Qualifiers
     source          1..8438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(196..784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(276..295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(955..1746,1747..1815))
                     /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     1578..1597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1816..1920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1988..2006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2031..2246
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2247..3050
                     /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"
     rep_origin      3161..4507
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     4928..4950
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4942..4959
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4943..4959
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(4971..5158)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5079..5103
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     5306..5325
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5306..5324
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5387..5411
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5421..6131)
                     /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(5699..5718)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5968..5986
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6128..6137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(6153..6206)
                     /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"
     protein_bind    complement(6915..6939)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      7747..8202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7834..7853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8044..8065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8232..8248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8232..8248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8245..8267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8256..8272
                     /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(8280..8286,8287..8304,8305..8310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8325..8346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
       61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1921 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     1981 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattagctt ttcaattcat
     2041 catttttttt ttattctttt ttttgatttc ggtttccttg aaattttttt gattcggtaa
     2101 tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca
     2161 tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa
     2221 aaacatgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta
     2281 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact
     2341 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag
     2401 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg
     2461 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca
     2521 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa
     2581 tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt attgttagcg
     2641 gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg atgttagcag
     2701 aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact gttgacattg
     2761 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggaagag
     2821 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg
     2881 cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct gacattatta
     2941 ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt gaacgttaca
     3001 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat
     3061 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt
     3121 tattacccaa ttctcatgtt tgacagctta tcatcggatc gatccaatat caaaggaaat
     3181 gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa
     3241 gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt
     3301 actagactac ctttcatcct acataaatag acgcatataa gtacgcattt aagcataaac
     3361 acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag atataggtgc
     3421 gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt
     3481 cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagctagaa agtataggaa
     3541 cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa
     3601 cgcaccggac tgtaacgagc tactaaaata ttgcgaatac cgcttccaca aacattgctc
     3661 aaaagtatct ctttgctata tatctctgtg ctatatccct atataaccta cccatccacc
     3721 tttcgctcct tgaacttgca tctaaactcg acctctacat tttttatgtt tatctctagt
     3781 attactcttt agacaaaaaa attgtagtaa gaactattca tagagtgaat cgaaaacaat
     3841 acgaaaatgt aaacatttcc tatacgtagt atatagagac aaaatagaag aaaccgttca
     3901 taattttctg accaatgaag aatcatcaac gctatcactt tctgttcaca aagtatgcgc
     3961 aatccacatc ggtatagaat ataatcgggg atgcctttat cttgaaaaaa tgcacccgca
     4021 gcttcgctag taatcagtaa acgcgggaag tggagtcagg ctttttttat ggaagagaaa
     4081 atagacacca aagtagcctt cttctaacct taacggacct acagtgcaaa aagttatcaa
     4141 gagactgcat tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa
     4201 aaatagcgct ctcgggatgc atttttgtag aacaaaaaag aagtatagat tctttgttgg
     4261 taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt
     4321 tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa aaatgaagca cagattcttc
     4381 gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct
     4441 ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc tacaaaatga agcacagatg
     4501 cttcgttaac aaagatatgc tattgaagtg caagatggaa acgcagaaaa tgaaccgggg
     4561 atgcgacgtg caagattacc tatgcaatag atgcaatagt ttctccagga accgaaatac
     4621 atacattgtc ttccgtaaag cgctagacta tatattatta tacaggttca aatatactat
     4681 ctgtttcagg gaaaactccc aggttcggat gttcaaaatt caatgatggg taacaagtac
     4741 gatccgatta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     4861 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     4921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattggcatg ccggtagagg
     4981 tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc tacaggaaag
     5041 agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa aatttgtata
     5101 cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag aaattcgctt
     5161 atttagaagt gtcaacaacg tatctaccaa cgatttgacc cttttccatc ttttcgtaaa
     5221 tttttggcaa ggtagacaag ccgacaacct tgattggaga cttgaccaaa cctctggcga
     5281 agaagtccaa agctggatcc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
     5341 gccccccctc gaggtcgacc tgcagcggcc atcatgaatt aactacacca ctttgtacaa
     5401 gaaagctggg tgcggccgct ttacttgtac agctcgtcca tgccgccggt ggagtggcgg
     5461 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     5521 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
     5581 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
     5641 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
     5701 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
     5761 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     5821 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
     5881 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
     5941 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     6001 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     6061 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
     6121 ttgctcacca tggtggcgac cggtggatcc ccagggccgg gattctcctc cacgtcaccg
     6181 catgttagaa gacttcctct gccctctccg gatgcatatg cggttttgca atcgatagct
     6241 ctgaacacct ctactacgtt catatgatct cttgtttctt tgggatcttt agaaagtatt
     6301 gtgtgcgctt gtatatgatg gtagtgtgga atgtcgactg gtccaccgcc aagaggagta
     6361 tttaattgtt ggtgcgcagc aattatttgt cctcctccaa ttacgtcatg aacaatgttg
     6421 atgtagcatt tgattccatt attttgtaca tctggaagaa catagatgca atgagggttg
     6481 tagttaaatt ccaatttttt gcccaagatg ttaccgcctt ttttaaatcc tgaaccgttc
     6541 aacgtaactc ggttataaac tgatcctttc tcgtacgtga cttcagcgcg tgtcttataa
     6601 acgccgtcgt tttcaaaagt gattgttcgc tcttgaacgt aaccttctgg catagcactt
     6661 tttgggtagt ctttgatatc atttggatat tttgcaaaac actgtgcacc gtatgtgaca
     6721 gttgttagaa tgcttgacca aggtactggt agttcaccag ttgtgcagat gaaactggct
     6781 tcaattttac cagtgttagc atcaccatgt cctttacctc ttactgaaaa cttcattccc
     6841 tcaacatcac cttccatctc aacaacatag gggatttttt ctctgaacaa aagtgctccg
     6901 taactcatgc tagcagcctg cttttttgta caaacttgtg atggccgcga attcactagt
     6961 cgaggctggg gtatattttt tttctttctt ttgaatgtgt gtataaaaga gagaaaaaat
     7021 ggttattttc caattaatta attattaacg attgttaaag aaaaaattta tcaacgaggt
     7081 tgatagaaaa aaaaagtttt tgtagatata gaagaaaaaa agattttcac tattggtgtt
     7141 ataggaaaga aagagaaaaa atttaagaga aattagatgt tagacgataa tgataggaca
     7201 tttgaaaata atacaatatt tggccttttt atataatcga aactaatgga ggggagcata
     7261 agcggtaccc accatcgacc acgtttcgtc accggtcata attggaataa acaaaacaaa
     7321 caaacgaact aaagagtgtg agagtaacaa tttccacaga ggtaaaaaaa aaaaaaagga
     7381 aaacagaaac aaaaaaagct catagttcta ctatagcttc ttcagacttt ctgtgaacga
     7441 gggaaaaaga caacaaaaaa gcaatggata aacggtctcg atcgaggcct ggaagtgccg
     7501 cattaggaaa tacagatgca atgcgcgcga gtttacgaaa ttctcgcagg agtttctccg
     7561 gccatgtgcg gctggttcta cgtctcgtgt atgggtttat ttctgcggta attatcgccg
     7621 gatctccgct tcgagaccgg gtttgttttc aaattaccgg aaaaggtcag gcaaaacaga
     7681 gtgatgtatg ggtgatccca gcaatgcctg tttatgtttc tccgtttcag gaagcttggc
     7741 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     7801 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     7861 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     7921 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     7981 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     8041 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     8101 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     8161 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtaagcttg
     8221 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     8281 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     8341 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     8401 cattaatgaa tcggccaacg cgcggggaga ggcggttt
//
