LOCUS       Exported                7773 bp ds-DNA     circular SYN 18-JAN-2025
DEFINITION  Plasmid for tagging Kex2 with FRBx2-msGFPx3 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  AUTHORS   Adam Krahn
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with BstXI for integration at KEX2.
FEATURES             Location/Qualifiers
     source          1..7773
                     /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"
     CDS             244..1013
                     /codon_start=3
                     /label=KEX2
                     /translation="HWGENGVGDWKIKVKTTENGHRIDFHSWRLKLFGESIDSSKTETF
                     VFGNDKEEVEPAATESTVSQYSASSTSISISATSTSSISIGVETSAIPQTTTASTDPDS
                     DPNTPKKLSSPRQAMHYFLTIFLIGATFLVLYFMFFMKSRRRIRRSRAETYEFDIIDTD
                     SEYDSTLDNGTSGITEPEEVEDFDFDLSDEDHLASLSSSENGDAEHTIDSVLTNENPFS
                     DPIKQKFPNDANAESASNKLQELQPDVPPSSGRS"
     misc_feature    1023..1064
                     /label=GSS linker
     CDS             1077..1355
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1362..1403
                     /codon_start=1
                     /label=GSS linker
                     /translation="GSSGSSGSSGSSAT"
     CDS             1416..1694
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1701..1727
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1734..1769
                     /codon_start=1
                     /label=GSS linker
                     /translation="GSSGSSGSSGAS"
     CDS             1770..2483
                     /codon_start=1
                     /product="monomeric superfolder GFP"
                     /label=msGFP
                     /note="mammalian codon-optimized"
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGSSGSSG"
     CDS             2490..3203
                     /codon_start=1
                     /product="monomeric superfolder GFP"
                     /label=msGFP
                     /note="mammalian codon-optimized"
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGSSGSSG"
     CDS             3210..3926
                     /codon_start=1
                     /product="monomeric superfolder GFP"
                     /label=msGFP
                     /note="mammalian codon-optimized"
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGSSGSSG"
     3'UTR           3936..4249
                     /label=3' KEX2
     primer_bind     complement(4266..4282)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4750..5553)
                     /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(5554..5774)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        5867..5971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5972..6832
                     /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      7003..7591
                     /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 ccaagcttgc
      241 atgcacattg gggtgagaac ggcgtaggtg attggaaaat caaggttaag acaacagaaa
      301 atggacacag gattgacttc cacagttgga ggctgaagct ctttggggaa tccattgatt
      361 catctaaaac agaaactttc gtctttggaa acgataaaga ggaggttgaa ccagctgcta
      421 cagaaagtac cgtatcacaa tattctgcca gttcaacttc tatttccatc agcgctactt
      481 ctacatcttc tatctcaatt ggtgtggaaa cgtcggccat tccccaaacg actactgcga
      541 gtaccgatcc tgattctgat ccaaacactc ctaaaaaact ttcctctcct aggcaagcca
      601 tgcattattt tttaacaata tttttgattg gcgccacatt tttggtgtta tacttcatgt
      661 tttttatgaa atcaaggaga aggatcagaa ggtcaagagc ggaaacgtat gaattcgata
      721 tcattgatac agactctgag tacgattcta ctttggacaa tggaacttcc ggaattactg
      781 agcccgaaga ggttgaggac ttcgattttg atttgtccga tgaagaccat cttgcaagtt
      841 tgtcttcatc agaaaacggt gatgctgaac atacaattga tagtgtacta acaaacgaaa
      901 atccatttag tgaccctata aagcaaaagt tcccaaatga cgccaacgca gaatctgctt
      961 ccaataaatt acaagaatta cagcctgatg ttcctccatc ttccggacga tcgacggatc
     1021 caggttcctc tgggtcttcc ggttcctcag ggtcgtcggc caccatggct tctagaatcc
     1081 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
     1141 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
     1201 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
     1261 aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcacc caagcctggg
     1321 acctctatta tcatgtgttc cgacgaatct caaagactag tggttcctct gggtcttccg
     1381 gttcctcagg gtcgtcggcc accatggctt ctagaatcct ctggcatgag atgtggcatg
     1441 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     1501 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat
     1561 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     1621 tgaaatcagg gaatgtcaag gacctcaccc aagcctggga cctctattat catgtgttcc
     1681 gacgaatctc aaagactagt tatccgtacg acgtaccaga ctacgcagat ccaggaagtt
     1741 cggggtccag cggttcttca ggcgctagca tggatagcac tgagagcctg ttcaccgggg
     1801 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     1861 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     1921 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1981 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2041 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     2101 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2161 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     2221 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     2281 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2341 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     2401 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2461 acggcagctc cggctccagc gggtctagca tggatagcac tgagagcctg ttcaccgggg
     2521 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     2581 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     2641 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2701 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2761 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     2821 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2881 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     2941 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     3001 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3061 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3121 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3181 acggcagctc cggctccagc gggtctagca tggatagcac tgagagcctg ttcaccgggg
     3241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     3301 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     3361 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3421 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3481 gctacgtcca ggagcgcacc atcaccttca aggacgacgg cacctacaag acccgcgccg
     3541 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3601 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     3661 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaacg
     3721 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3781 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3841 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3901 acggcagctc cggctccagc gggtaaagcg gccgcttcga tatgtacaga aagcttcaaa
     3961 ttacaaaata gcattttttt cttatagatt ataatactct ctcatacgta tacgtatatg
     4021 tgtatatgat atataaacaa acattaatat cctattcctt ccgtttgaaa tccctatgat
     4081 gtactttgca ttgtttgcac ccgcgaataa aatgaaaact ccgaaccgat atatcaagca
     4141 cataaaaggg gagggtccaa ttaatgcata tttaagacca cagctgaata actttaaaac
     4201 ggcagacaaa acaaaaaata ggtcgaataa accttacctg cctagaaggt accgagctcg
     4261 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4321 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4381 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     4441 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4501 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4561 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4621 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cgggtaataa
     4681 ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa
     4741 tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct
     4801 gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat
     4861 aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc
     4921 gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc
     4981 tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc
     5041 aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc
     5101 tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc
     5161 gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat
     5221 tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct
     5281 gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc
     5341 ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc
     5401 taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa
     5461 gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt
     5521 agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc tgcatgtttt
     5581 tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat
     5641 gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat
     5701 taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa aaaaaaatga
     5761 tgaattgaat tgaaaagctg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5821 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5881 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5941 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6001 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6061 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6121 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6181 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6241 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6301 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6361 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6421 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6481 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6541 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6601 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6661 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6721 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6781 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6841 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6901 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6961 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7021 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7081 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7141 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     7201 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7261 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     7321 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7381 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7441 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7501 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7561 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7621 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     7681 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     7741 gcgcagcgag tcagtgagcg aggaagcgga aga
//
