LOCUS       sequence-331875-        7883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cyterm-ystagRFP to check for monomericity. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7883)
  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 7883)
  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: Cyterm-ystagRFP pDRF1-GW
FEATURES             Location/Qualifiers
     source          1..7883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      114..1460
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(1571..2374)
                     /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(2375..2590)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2615..2633)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2701..2805
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2806..2874,2875..3666)
                     /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(3024..3043)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3837..4425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4326..4345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4579..4596
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4713..4734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4749..4754,4755..4772,4773..4779)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4787..4803
                     /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     4792..4814
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4811..4827
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4811..4827
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(4857..5312)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4994..5015)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5206..5225
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    6120..6144
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      6344..6353
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(7093..7117)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7179..7198)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7180..7198)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      7346..7533
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(7401..7425)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(7545..7562)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7545..7561)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7554..7576)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gtatttcggt tcctggagaa actattgcat ctattgcata ggtaatcttg cacgtcgcat
       61 ccccggttca ttttctgcgt ttccatcttg cacttcaata gcatatcttt gttaacgaag
      121 catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa
      181 agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta ttttaccaac
      241 gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca
      301 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag cgctatttta
      361 ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag agcgctattt
      421 ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata atgcagtctc
      481 ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt tggtgtctat
      541 tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta ctagcgaagc
      601 tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc gatgtggatt
      661 gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt cagaaaatta
      721 tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt acattttcgt
      781 attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct aaagagtaat
      841 actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca aggagcgaaa
      901 ggtggatggg taggttatat agggatatag cacagagata tatagcaaag agatactttt
      961 gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca gtccggtgcg
     1021 tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag
     1081 ttcctatact ttctagctag agaataggaa cttcggaata ggaacttcaa agcgtttccg
     1141 aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc
     1201 gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt
     1261 gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt
     1321 acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc
     1381 tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc
     1441 atttcctttg atattggatc gatccgatga taagctgtca aacatgagaa ttgggtaata
     1501 actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata
     1561 atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc
     1621 tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa
     1681 taataatgtc agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg
     1741 cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat
     1801 ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg
     1861 caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt
     1921 ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac
     1981 cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta
     2041 ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc
     2101 tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc
     2161 cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac
     2221 ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca
     2281 agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag
     2341 tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcatgttt
     2401 ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata
     2461 tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga
     2521 ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg
     2581 atgaattgaa aagctaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     2641 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     2701 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2761 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2821 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2881 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2941 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3001 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3061 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3121 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3181 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3241 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3301 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3361 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3421 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3481 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3541 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3601 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3661 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3721 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3781 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3841 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3901 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3961 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4021 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4081 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4141 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4201 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4261 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4321 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4381 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4441 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4501 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4561 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     4621 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     4681 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     4741 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     4801 caatttcaca caggaaacag ctatgaccat gattacgcca agcttaccgc atcaggaaat
     4861 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4921 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     4981 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5041 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5101 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5161 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5221 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5281 cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc caagcttcct gaaacggaga
     5341 aacataaaca ggcattgctg ggatcaccca tacatcactc tgttttgcct gaccttttcc
     5401 ggtaatttga aaacaaaccc ggtctcgaag cggagatccg gcgataatta ccgcagaaat
     5461 aaacccatac acgagacgta gaaccagccg cacatggccg gagaaactcc tgcgagaatt
     5521 tcgtaaactc gcgcgcattg catctgtatt tcctaatgcg gcacttccag gcctcgatcg
     5581 agaccgttta tccattgctt ttttgttgtc tttttccctc gttcacagaa agtctgaaga
     5641 agctatagta gaactatgag ctttttttgt ttctgttttc cttttttttt tttttacctc
     5701 tgtggaaatt gttactctca cactctttag ttcgtttgtt tgttttgttt attccaatta
     5761 tgaccggtga cgaaacgtgg tcgatggtgg gtaccgctta tgctcccctc cattagtttc
     5821 gattatataa aaaggccaaa tattgtatta ttttcaaatg tcctatcatt atcgtctaac
     5881 atctaatttc tcttaaattt tttctctttc tttcctataa caccaatagt gaaaatcttt
     5941 ttttcttcta tatctacaaa aacttttttt ttctatcaac ctcgttgata aattttttct
     6001 ttaacaatcg ttaataatta attaattgga aaataaccat tttttctctc ttttatacac
     6061 acattcaaaa gaaagaaaaa aaatataccc cagcctcgac tagtgaattc gcggccatca
     6121 caagtttgta caaaaaagca ggctgctagc gctaccggac tcagatctgg taccccttgc
     6181 agcttccagt tgtcaggaag agaaggctcc catggatcct gtggtggtgc tggggctctg
     6241 tctctcctgt ttgcttctcc tttcactctg gaaacagagc tatgggggag ggaagcttcg
     6301 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tggtatctaa
     6361 aggtgaagag ttgataaagg aaaatatgca catgaaactc tatatggaag gcacagtgaa
     6421 taaccatcat tttaagtgta cttcagaagg agaaggtaag ccttatgagg ggacacaaac
     6481 tatgagaatt aaagttgttg aaggaggtcc actaccattc gcttttgaca tcctggcaac
     6541 gagcttcatg tatggctcca gaacattcat aaaccataca caaggtattc cagatttctt
     6601 caaacaaagt tttccagaag gctttacttg ggagagagtt actacctacg aggacggggg
     6661 tgttttaact gcgacacagg atacatcatt acaagatggc tgtttgatct acaacgtaaa
     6721 aatcagaggt gtcaattttc catctaacgg acctgttatg cagaaaaaga ccttgggttg
     6781 ggaggctaat actgaaatgc tatacccagc agatggagga ttggaaggta ggacagtcat
     6841 ggcattgaaa cttgtaggag gcggtcacct aatttgcaac tttaagacaa cctacagaag
     6901 caaaaagcct gctaaaaatc ttaaaatgcc aggggtgtac tacgtggatc atagacttga
     6961 acgtatcaag gaagctgaca aagagactta cgttgaacaa cacgaagtcg ccgtcgccag
     7021 atactgtgac ttaccttcaa agttaggtca taaactgaat ggtatggatg aattgtataa
     7081 gtaagcggcc gcacccagct ttcttgtaca aagtggtgta gttaattcat gatggccgct
     7141 gcaggtcgac ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg
     7201 cgcggatcca gctttggact tcttcgccag aggtttggtc aagtctccaa tcaaggttgt
     7261 cggcttgtct accttgccag aaatttacga aaagatggaa aagggtcaaa tcgttggtag
     7321 atacgttgtt gacacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     7381 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     7441 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca
     7501 tgaggtcgct cttattgacc acacctctac cggcatgcca attcactggc cgtcgtttta
     7561 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     7621 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     7681 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     7741 atttcacacc gcataatcgg atcgtacttg ttacccatca ttgaattttg aacatccgaa
     7801 cctgggagtt ttccctgaaa cagatagtat atttgaacct gtataataat atatagtcta
     7861 gcgctttacg gaagacaatg tat
//
