LOCUS       Exported                6028 bp ds-DNA     circular SYN 07-JAN-2025
DEFINITION  Plasmid for tagging Sys1 with FRBx2 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with NheI for integration at SYS1.
FEATURES             Location/Qualifiers
     source          1..6028
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(942..1802)
                     /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"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2005..2220
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2221..3024
                     /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"
     primer_bind     3492..3508
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3516..3943
                     /label=5' SYS1
     CDS             3944..4552
                     /codon_start=1
                     /label=SYS1
                     /translation="MVSIRRYLRVPNELKPSQIFKQDSLSPSKIGLQIVLLQIFYYTTA
                     IVLFYCWAKLAGYDLNIKEWLFSWENIDFTNAYGLSISLLWLLDSLICVFFLTVIVGRS
                     KLAWDFAITIHAINFIVVFLYTRKFPSFSWFFLQILSSLILIFLGTWTTRWRELRDTFF
                     EGLVDPNEGEVGLVTPSQQHSNHSELEQSPIQLKDLESQI"
     CDS             4562..4603
                     /codon_start=1
                     /label=GSS linker
                     /translation="GSSGSSGSSGSSAT"
     CDS             4616..4894
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             4901..4942
                     /codon_start=1
                     /label=GSS linker
                     /translation="GSSGSSGSSGSSAT"
     CDS             4955..5233
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             5240..5266
                     /codon_start=1
                     /label=HA tag
                     /translation="YPYDVPDYA"
     misc_feature    5279..5790
                     /label=3' SYS1
     primer_bind     complement(5809..5825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccagcttt tcaattcaat tcatcatttt ttttttattc ttttttttga
     2041 tttcggtttc tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg
     2101 aaggagcaca gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat
     2161 tgcccagtat tcttaaccca actgcacaga acaaaaacat gcaggaaacg aagataaatc
     2221 atgtcgaaag ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag
     2281 ctatttaata tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc
     2341 accaaggaat tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca
     2401 catgtggata tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta
     2461 tccgccaagt acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca
     2521 gtcaaattgc agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat
     2581 gcacacggtg tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta
     2641 acaaaggaac ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct
     2701 actggagaat atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc
     2761 ggctttattg ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg
     2821 acacccggtg tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg
     2881 gatgatgtgg tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag
     2941 ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg
     3001 agaagatgcg gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac
     3061 tcacaaatta gagcttcaat ttaattatat cagttattac ccgccctttc gtctcgcgcg
     3121 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3181 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3241 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     3301 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
     3361 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     3421 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     3481 gtcacgacgt tgtaaaacga cggccagtga attcctggta atatttcagt tgttgactaa
     3541 tatatatagt ggtttttgcc aaaattaaag acttcctttt attaactagg tgaccagata
     3601 taaggaagtg tatttgcttt ctaataactt gtgtacggcc ataacccggc ttttaatcag
     3661 tttatttaac aaggtataca aatcgccaaa cgtaacaaga gataggcgct attattcatt
     3721 taatattgtc gtattcaaaa tactaacggt gaacaatttg ctccattgta acactatagg
     3781 acaaggctaa tcatgaataa atatctcccc acccaaaata ttcgtagctc gcgatattca
     3841 cggtaatatt ctgtaacttc atgttaagag atgatcttta ggaaggctaa ggaagggtac
     3901 acatagaagt gagataaaaa ataggcattt gaattactgt aacatggttt cgataagaag
     3961 gtatttgcga gtgccgaatg agttgaaacc ttcccagatc ttcaaacaag attctctctc
     4021 tccaagtaag attggacttc aaattgttct tttacagatt ttttattaca ccacagcgat
     4081 cgttcttttt tattgttggg cgaagctagc gggatatgat ctcaatatca aggaatggct
     4141 gttttcttgg gagaatatcg actttacgaa cgcatacggt ttatcaatct cactgttatg
     4201 gcttctggat tcattaatat gtgttttttt cttaactgtt atcgttggac ggagtaaact
     4261 cgcatgggat tttgctatca ctatacatgc catcaatttt attgtggtgt tcctctatac
     4321 tcggaaattt ccttcttttt cttggttttt tcttcaaatt ttatcatctt taatactaat
     4381 atttttgggg acatggacaa caagatggag agagcttaga gacacctttt ttgagggctt
     4441 ggttgatcct aatgaaggag aagttggatt ggttacgcct agtcagcagc atagtaatca
     4501 ttcagaattg gagcaatcac caatacaact aaaagactta gaaagccaaa tacgggatcc
     4561 aggttcctct gggtcttccg gttcctcagg gtcgtcggcc accatggctt ctagaatcct
     4621 ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag
     4681 gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc
     4741 ccagactctg aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca
     4801 agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcaccc aagcctggga
     4861 cctctattat catgtgttcc gacgaatctc aaagactagt ggttcctctg ggtcttccgg
     4921 ttcctcaggg tcgtcggcca ccatggcttc tagaatcctc tggcatgaga tgtggcatga
     4981 aggcctggaa gaggcatctc gtttgtactt tggggaaagg aacgtgaaag gcatgtttga
     5041 ggtgctggag cccttgcatg ctatgatgga acggggcccc cagactctga aggaaacatc
     5101 ctttaatcag gcctatggtc gagatttaat ggaggcccaa gagtggtgca ggaagtacat
     5161 gaaatcaggg aatgtcaagg acctcaccca agcctgggac ctctattatc atgtgttccg
     5221 acgaatctca aagactagtt atccgtacga cgtaccagac tacgcataaa gcggccgcga
     5281 ggagatgaaa caacatattc gaggcaattt tggcaatatt atagtcattc aagagaaaac
     5341 agtaaatgta tgaaaaatgg caagaacctt atctctatag gcttaataaa tctgcctttt
     5401 tggccaattt ttcaaaacag atcctatttc ttatttatac acaatctata tttaacataa
     5461 ttaagtacgt gatgacattg aagataataa taagtaaaga gtttacgtac ttaaatttta
     5521 tatagtttta gtatacttgc aatgtgtgaa tggatctagt aagctgaggt acttctctag
     5581 cgtgaatatg aaacgagtaa cagggtggta cataatttac gaacagaact caagttgata
     5641 aatcctttgc gttcttaacc atttgtaaaa gttcgtcaac aacattgaaa atattgcttc
     5701 gagcagatgt tttatcgttt ctgcaaattt ccagtcctga acacctctgt tgtcgtaaat
     5761 gtaataatgc cacactggat tcgattctag aagcttggcg taatcatggt catagctgtt
     5821 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5881 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5941 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6001 ggggagaggc ggtttgcgta ttgggcgc
//
