LOCUS       sequence-407613-        8192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of miRFp670nano3-T2A-mtq2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8192)
  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 8192)
  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: pDRF1-GW miRFp670nano3-T2A-mtq2
FEATURES             Location/Qualifiers
     source          1..8192
                     /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             1278..1331
                     /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      1347..1356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1353..1355,1356..1358,1359..2072)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1398..1419)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1659..1678)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2006..2027
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2082..2106)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2168..2187)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2169..2187)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2335..2522
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2390..2414)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2534..2551)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2534..2550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2543..2565)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2986..4332
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(4443..5246)
                     /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(5247..5462)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5487..5505)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5573..5677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5678..5746,5747..6538)
                     /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(5896..5915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6709..7297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7198..7217
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7451..7468
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7585..7606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7621..7626,7627..7644,7645..7651)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7659..7675
                     /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     7664..7686
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7683..7699
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7683..7699
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(7729..8184)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7866..7887)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8078..8097
                     /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 atggcaaacc
      841 tggacaagat gctgaacacc accgtgaccg aggtgcgcaa gttcctgcaa gcggacagag
      901 tgtgcgtgtt caagttcgag gaagattact ccggcaccgt ctcgcacgaa gccgtggacg
      961 acagatggat tagcatcctg aagacccagg tgcaggacag atacttcatg gaaaccagag
     1021 gcgaggaata cgtccacggc agataccagg ccatcgccga catctacaca gccaatctgg
     1081 tcgagtgcta cagagacctg ctgatcgagt ttcaggtgcg ggccattctg gctgtcccca
     1141 tcctgcaagg caagaagctg tggggcctgc tggtggccca ccaactggcc ggccctcggg
     1201 agtggcagac ctgggaaatc gacttcctga aacagcaagc cgtggtgatg ggcatcgcca
     1261 tccagcagag ctccggagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     1321 atcccggccc tggggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca
     1381 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1441 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1501 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgtcc tggggcgtgc
     1561 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1621 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1681 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1741 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac tttagcgaca
     1801 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     1861 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1921 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1981 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2041 tcactctcgg catggacgag ctgtacaagt aaagcggccg cacccagctt tcttgtacaa
     2101 agtggtgtag ttaattcatg atggccgctg caggtcgacc tcgagggggg gcccggtacc
     2161 caattcgccc tatagtgagt cgtattacgc gcggatccag ctttggactt cttcgccaga
     2221 ggtttggtca agtctccaat caaggttgtc ggcttgtcta ccttgccaga aatttacgaa
     2281 aagatggaaa agggtcaaat cgttggtaga tacgttgttg acacttctaa ataagcgaat
     2341 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa
     2401 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg
     2461 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc
     2521 ggcatgccaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2581 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2641 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     2701 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataatcgga tcgtacttgt
     2761 tacccatcat tgaattttga acatccgaac ctgggagttt tccctgaaac agatagtata
     2821 tttgaacctg tataataata tatagtctag cgctttacgg aagacaatgt atgtatttcg
     2881 gttcctggag aaactattgc atctattgca taggtaatct tgcacgtcgc atccccggtt
     2941 cattttctgc gtttccatct tgcacttcaa tagcatatct ttgttaacga agcatctgtg
     3001 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg
     3061 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc
     3121 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga
     3181 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa
     3241 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca
     3301 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac
     3361 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt
     3421 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg
     3481 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac
     3541 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt
     3601 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt
     3661 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga
     3721 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg
     3781 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg
     3841 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt
     3901 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata
     3961 ctttctagct agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag
     4021 cgcttccgaa aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat
     4081 atctgcgtgt tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc
     4141 ttaaatgcgt acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg
     4201 tgatattatc ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg
     4261 ttctatatgc tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt
     4321 tgatattgga tcgatccgat gataagctgt caaacatgag aattgggtaa taactgatat
     4381 aattaaattg aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt
     4441 ttttagtttt gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt
     4501 tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg
     4561 tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc
     4621 ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca
     4681 cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca
     4741 acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac
     4801 atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca
     4861 atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat
     4921 acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg
     4981 aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg
     5041 gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct
     5101 tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt
     5161 tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca
     5221 cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcatgt ttttgttctg
     5281 tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata
     5341 tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa
     5401 tcaaaaaaat ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg
     5461 aaaagctaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5521 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5581 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5641 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5701 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5761 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5821 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5881 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5941 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6001 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6061 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6121 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6181 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6241 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6301 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6361 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6421 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6481 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6541 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6601 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6661 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6721 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6781 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6841 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6901 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6961 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7021 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7081 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7141 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7201 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7261 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7321 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7381 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7441 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     7501 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     7561 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     7621 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     7681 cacaggaaac agctatgacc atgattacgc caagcttacc gcatcaggaa attgtaagcg
     7741 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     7801 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     7861 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     7921 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     7981 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     8041 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     8101 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     8161 ttaatgcgcc gctacagggc gcgtccattc gc
//
