LOCUS       Exported                6991 bp ds-DNA     circular SYN 18-JAN-2025
DEFINITION  Plasmid for tagging Vrg4 with FRB-msGFP2 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6991)
  AUTHORS   Adam Krahn
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with MluI for integration at VRG4.
FEATURES             Location/Qualifiers
     source          1..6991
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    234..719
                     /label=5' VRG4
     CDS             732..1010
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1029..1736
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /note="yeast codon-optimized"
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     CDS             1737..2747
                     /codon_start=1
                     /label=VRG4
                     /translation="SELKTGHAGHNPWASVANSGPISILSYCGSSILMTVTNKFVVNLK
                     DFNMNFVMLFVQSLVCTITLIILRILGYAKFRSLNKTDAKNWFPISFLLVLMIYTSSKA
                     LQYLAVPIYTIFKNLTIILIAYGEVLFFGGSVTSMELSSFLLMVLSSVVATWGDQQAVA
                     AKAASLAEGAAGAVASFNPGYFWMFTNCITSALFVLIMRKRIKLTNFKDFDTMFYNNVL
                     ALPILLLFSFCVEDWSSVNLTNNFSNDSLTAMIISGVASVGISYCSGWCVRVTSSTTYS
                     MVGALNKLPIALSGLIFFDAPRNFLSILSIFIGFLSGIIYAVAKQKKQQAQPLRK"
     misc_feature    2748..3478
                     /label=3' VRG4
     primer_bind     complement(3484..3500)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3968..4771)
                     /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(4772..4992)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        5085..5189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5190..6050
                     /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"
     rep_origin      6221..6809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tggcttctag aatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
      781 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
      841 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
      901 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
      961 tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag ggaggttctg
     1021 gaggttccat ggatagcact gagagcttgt tcacaggtgt tgttcctatc ttggtcgaat
     1081 tagacggtga cgtaaacggt cataagttca gtgttagagg tgaaggtgaa ggtgacgcta
     1141 ctaacggtaa attgacattg aagtttattt gtactacagg taaattgcca gttccttggc
     1201 caactttggt tactacattg acatacggtg ttcaatgttt ttcaagatac ccagatcata
     1261 tgaagcaaca tgatttcttt aagtctgcta tgccagaagg ttacgttcaa gaaagaacta
     1321 tcacttttaa agatgatggt acttacaaga caagagcaga ggttaagttc gaaggtgaca
     1381 ctttggttaa cagaatcgaa ttgaagggta tcgattttaa agaagatggt aatattttgg
     1441 gtcataaatt ggaatacaac tacaactcac ataacgttta cattacagct gataaacaaa
     1501 agaatggtat taaagcaaac ttcaagatca gacataatgt tgaagatggt tctgttcaat
     1561 tagcagatca ttaccaacaa aacactccaa ttggtgacgg tccagttttg ttaccagata
     1621 accattactt gtctacacaa tcaaaattgt ctaaagatcc aaacgaaaag agagatcata
     1681 tggttttgtt agaatttgtt actgctgctg gtatcacagg tggcagtggc ggttcttcag
     1741 aattgaaaac aggtcatgca ggccataacc cttgggcttc agttgccaat tccggtccga
     1801 tctctatttt atcctactgt ggttcctcta ttttaatgac ggtgactaac aagttcgtcg
     1861 tcaatttgaa ggatttcaac atgaactttg tcatgctttt cgtgcaatct ttggtttgta
     1921 ctataacctt gattatccta cgtatactgg gctatgcgaa gttccgttca ttaaacaaaa
     1981 cagacgccaa gaactggttc cctatttcct ttttactggt cttgatgatc tacacctctt
     2041 cgaaggcttt acaatacttg gctgttccaa tttacaccat tttcaagaat ttgactatta
     2101 tcttgattgc ttatggtgag gttctctttt ttggtggctc tgtcacctcc atggaattgt
     2161 catcattttt gttgatggtc ctttcttctg tcgttgcaac ttggggtgac cagcaagctg
     2221 tggctgccaa ggctgcttca ttggctgaag gagcagccgg tgctgttgcc tcctttaacc
     2281 caggttattt ctggatgttc accaactgta tcacttctgc attattcgtt cttataatga
     2341 gaaagagaat taagttaact aacttcaagg atttcgacac tatgttttac aacaatgttt
     2401 tggctctacc tattctattg ctgttttctt tctgtgtgga agattggtct tcagttaatt
     2461 tgaccaataa cttttctaac gattcgctaa ctgctatgat catcagtggt gttgcatccg
     2521 tcggtatttc ttactgttcc ggttggtgtg ttcgtgttac ttcgtctact acatattcga
     2581 tggtaggggc tttgaacaag ctgccaattg ccttgtctgg tttgattttc tttgatgctc
     2641 caagaaactt cttatctatt ctctccattt ttattggttt cctatcaggt attatttatg
     2701 ctgttgccaa acaaaagaag caacaagccc aacctttacg taaatgagaa cttacggggg
     2761 gtgcaattta tttttttttt ttggtttatt ttattttata gaggcatcta atgcaagtag
     2821 atttatatac aattatactt aaaattgata taccttagaa caggtcggac acaacgtgtg
     2881 gtaactccaa tatatcacta atgacaatag cgccttctgg tgtttgcccc aaaatttcat
     2941 ttacttcatt ctcaactaaa tgaatacatg ttttcatgcc caatttgata cctgtctcga
     3001 tgtttttccc actgtcatct ataaaataag cattctcgta tcttgccaac ccgctttcct
     3061 tcatggcttt ttcaaatgct ttaacatgtg gtttgcagac cagcgtatct gtcctagagt
     3121 agtcacaata tgtcaatcca tcaaataaat ccgctatacc caacaatctc aggcaacgta
     3181 ttgcgtggtt tttataagcg ttggtgaaaa gccacaattt atcaatcttt cccgattgtc
     3241 tcaatctcaa cagcatattc cttaatggta tatcaggttt caagatatct tgtagtggta
     3301 aggaatcatc cacaagccgg ttatattcca gtgcgttcac tttgtggaac attacaagtc
     3361 cccttatggc aagtccatac tctttgtagt aagagttatt caaaacgtga gcatcttcag
     3421 gtgatagttt caagtgtgtt tgaaagaacc tcagtataga ttgttgcata agatcgtgaa
     3481 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3541 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3601 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3661 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3721 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3781 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3841 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcg ggtaataact
     3901 gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata
     3961 cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt
     4021 aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa
     4081 taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt
     4141 ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc
     4201 ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa
     4261 tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat gacaattctg
     4321 ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc
     4381 taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc
     4441 tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt
     4501 cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct
     4561 ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta
     4621 atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt
     4681 ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag
     4741 cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg catgtttttg
     4801 ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc
     4861 gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta
     4921 ccgaatcaaa aaaatttcaa agaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg
     4981 aattgaattg aaaagctggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5041 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5161 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5221 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5281 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5341 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5401 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5461 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5521 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5581 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5641 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5701 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5761 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5821 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5881 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5941 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6001 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6061 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6121 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6181 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6241 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6301 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6361 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6421 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6481 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6541 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6601 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6661 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6721 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6781 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6841 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6901 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6961 gcagcgagtc agtgagcgag gaagcggaag a
//
