LOCUS       Exported                6283 bp ds-DNA     circular SYN 18-JAN-2025
DEFINITION  Plasmid for tagging Vrg4 with FRB by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6283)
  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..6283
                     /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..2039
                     /codon_start=1
                     /label=VRG4
                     /translation="SELKTGHAGHNPWASVANSGPISILSYCGSSILMTVTNKFVVNLK
                     DFNMNFVMLFVQSLVCTITLIILRILGYAKFRSLNKTDAKNWFPISFLLVLMIYTSSKA
                     LQYLAVPIYTIFKNLTIILIAYGEVLFFGGSVTSMELSSFLLMVLSSVVATWGDQQAVA
                     AKAASLAEGAAGAVASFNPGYFWMFTNCITSALFVLIMRKRIKLTNFKDFDTMFYNNVL
                     ALPILLLFSFCVEDWSSVNLTNNFSNDSLTAMIISGVASVGISYCSGWCVRVTSSTTYS
                     MVGALNKLPIALSGLIFFDAPRNFLSILSIFIGFLSGIIYAVAKQKKQQAQPLRK"
     misc_feature    2040..2770
                     /label=3' VRG4
     primer_bind     complement(2776..2792)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3260..4063)
                     /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(4064..4284)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        4377..4481
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4482..5342
                     /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      5513..6101
                     /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 ggtggcagtg
     1021 gcggttcttc agaattgaaa acaggtcatg caggccataa cccttgggct tcagttgcca
     1081 attccggtcc gatctctatt ttatcctact gtggttcctc tattttaatg acggtgacta
     1141 acaagttcgt cgtcaatttg aaggatttca acatgaactt tgtcatgctt ttcgtgcaat
     1201 ctttggtttg tactataacc ttgattatcc tacgtatact gggctatgcg aagttccgtt
     1261 cattaaacaa aacagacgcc aagaactggt tccctatttc ctttttactg gtcttgatga
     1321 tctacacctc ttcgaaggct ttacaatact tggctgttcc aatttacacc attttcaaga
     1381 atttgactat tatcttgatt gcttatggtg aggttctctt ttttggtggc tctgtcacct
     1441 ccatggaatt gtcatcattt ttgttgatgg tcctttcttc tgtcgttgca acttggggtg
     1501 accagcaagc tgtggctgcc aaggctgctt cattggctga aggagcagcc ggtgctgttg
     1561 cctcctttaa cccaggttat ttctggatgt tcaccaactg tatcacttct gcattattcg
     1621 ttcttataat gagaaagaga attaagttaa ctaacttcaa ggatttcgac actatgtttt
     1681 acaacaatgt tttggctcta cctattctat tgctgttttc tttctgtgtg gaagattggt
     1741 cttcagttaa tttgaccaat aacttttcta acgattcgct aactgctatg atcatcagtg
     1801 gtgttgcatc cgtcggtatt tcttactgtt ccggttggtg tgttcgtgtt acttcgtcta
     1861 ctacatattc gatggtaggg gctttgaaca agctgccaat tgccttgtct ggtttgattt
     1921 tctttgatgc tccaagaaac ttcttatcta ttctctccat ttttattggt ttcctatcag
     1981 gtattattta tgctgttgcc aaacaaaaga agcaacaagc ccaaccttta cgtaaatgag
     2041 aacttacggg gggtgcaatt tatttttttt ttttggttta ttttatttta tagaggcatc
     2101 taatgcaagt agatttatat acaattatac ttaaaattga tataccttag aacaggtcgg
     2161 acacaacgtg tggtaactcc aatatatcac taatgacaat agcgccttct ggtgtttgcc
     2221 ccaaaatttc atttacttca ttctcaacta aatgaataca tgttttcatg cccaatttga
     2281 tacctgtctc gatgtttttc ccactgtcat ctataaaata agcattctcg tatcttgcca
     2341 acccgctttc cttcatggct ttttcaaatg ctttaacatg tggtttgcag accagcgtat
     2401 ctgtcctaga gtagtcacaa tatgtcaatc catcaaataa atccgctata cccaacaatc
     2461 tcaggcaacg tattgcgtgg tttttataag cgttggtgaa aagccacaat ttatcaatct
     2521 ttcccgattg tctcaatctc aacagcatat tccttaatgg tatatcaggt ttcaagatat
     2581 cttgtagtgg taaggaatca tccacaagcc ggttatattc cagtgcgttc actttgtgga
     2641 acattacaag tccccttatg gcaagtccat actctttgta gtaagagtta ttcaaaacgt
     2701 gagcatcttc aggtgatagt ttcaagtgtg tttgaaagaa cctcagtata gattgttgca
     2761 taagatcgtg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2821 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2881 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     2941 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     3001 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3061 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3121 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3181 cgggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat
     3241 ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc
     3301 ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc
     3361 ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt
     3421 gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt
     3481 aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt
     3541 cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc
     3601 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct
     3661 gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc
     3721 attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag
     3781 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct
     3841 taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa
     3901 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg
     3961 aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac
     4021 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc
     4081 tgcatgtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa
     4141 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct
     4201 gttcggagat taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa
     4261 aaaaaaatga tgaattgaat tgaaaagctg gcctcgtgat acgcctattt ttataggtta
     4321 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4381 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4441 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4501 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4561 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4621 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4681 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4741 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4801 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4861 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4921 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4981 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5041 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5101 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5161 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5221 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5281 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5341 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5401 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5461 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5521 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5581 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5641 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5701 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5761 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5821 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5881 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5941 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6001 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6061 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6121 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6181 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6241 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aga
//
