LOCUS       Exported                6287 bp ds-DNA     circular SYN 18-JAN-2025
DEFINITION  Plasmid for replacing Shs1 with Shs1-FKBPx2-mScarlet-2I by gene 
            replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6287)
  AUTHORS   Adam Krahn
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with BglII for integration at SHS1.
FEATURES             Location/Qualifiers
     source          1..6287
                     /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             238..839
                     /codon_start=3
                     /label=Shs1
                     /translation="KRQSNAPSLSNFASLISTGQFNSSQTLANNLRADTPRNQVSGNFK
                     ENEYEDNGEHDSAENEQEMSPVRQLGREIKQENENLIRSIKTESSPKFLNSPDLPERTK
                     LRNISETVPYVLRHERILARQQKLEELEAQSAKELQKRIQELERKAHELKLREKLINQN
                     KLNGSSSSINSLQQSTRSQIKKNDTYTDLASIASGRD"
     CDS             858..1178
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1185..1505
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /note="synthetic gene with alternative codons"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1524..2210
                     /codon_start=1
                     /product="mScarlet-I derivative in which the N- and 
                     C-termini have been modified to reduce cytotoxicity 
                     (Valbuena et al., 2020)"
                     /label=mScarlet2-I
                     /note="yeast codon-optimized"
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGSGAS"
     3'UTR           2211..2747
                     /label=3' SHS1
     primer_bind     complement(2780..2796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3264..4067)
                     /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(4068..4283)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        4381..4485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4486..5346
                     /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      5517..6105
                     /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 ccaagctcaa
      241 agagacagtc aaatgctcca agtttaagca actttgcctc tttgataagc actggtcaat
      301 tcaattcttc tcaaactctt gcaaacaatt tgagagcgga cacaccaaga aaccaagtaa
      361 gtggaaactt taaggaaaac gaatacgaag acaatggcga acatgattca gcagaaaatg
      421 aacaggaaat gtctcccgtg agacagttgg gtagagaaat aaaacaagaa aatgaaaatt
      481 tgataagatc tatcaaaaca gaatcttcac caaaattctt gaactctccg gacttaccag
      541 agcgtaccaa gttaagaaat atttcagaaa ccgttccata tgtcttgaga catgaaagaa
      601 ttttagcaag acaacaaaaa ctggaagagt tagaggccca gtcagctaaa gaattacaaa
      661 aaagaattca agaattagaa agaaaagcac acgaattgaa attgagggaa aaactaataa
      721 atcagaataa actaaacggt tcatcatctt caatcaattc tctacaacag agcacaagga
      781 gccaaattaa aaaaaatgac acgtatactg atttagcctc tattgcatcg ggtagagatg
      841 gaggttctgg aggttccgga gtgcaggtgg aaaccatctc cccaggagac gggcgcacct
      901 tccccaagcg cggccagacc tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga
      961 aatttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg
     1021 tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga
     1081 ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg
     1141 ccactctcgt cttcgatgtg gagcttctaa aactggaaac tagaggcgtg caggtcgaga
     1201 ccatcagccc tggagatggt aggacctttc ccaagagagg tcagacttgc gtggtccact
     1261 acaccggcat gctggaggac ggcaagaagt tcgacagcag ccgcgaccgc aacaagccct
     1321 tcaagttcat gctgggcaaa caggaagtga tccgtggttg ggaggaagga gtagctcaaa
     1381 tgagtgtggg acagagagct aagctgacca tcagccccga ctatgcctac ggcgccaccg
     1441 gccaccctgg catcattcca cctcacgcca cactggtgtt cgacgtggag ctgctgaagc
     1501 tggagggtgg cagtggcggt tctatggata gtacagaagc agttataaag gaattcatga
     1561 gattcaaagt ccacatggaa ggtagtatga acggtcacga attcgaaata gaaggtgaag
     1621 gtgaaggtag accatatgaa ggtactcaaa cagctaaatt gaaggttact aaaggtggtc
     1681 cattgccatt ttcttgggat atcttgtctc cacaattcat gtacggttca agagctttta
     1741 ttaaacatcc agcagatatt ccagattact acaagcaatc atttccagaa ggttttaaat
     1801 gggaaagagt tatgaacttc gaagatggtg gtgcagttac tgttacacaa gatacttctt
     1861 tggaagatgg tacattgatc tataaggtta aattgagagg tacaaatttt ccaccagatg
     1921 gtccagttat gcaaaagaaa actatgggtt gggaagcatc tacagaaaga ttgtacccag
     1981 aagatggtgt tttgaagggt gacatcaaga tggcattgag attgaaggat ggtggtagat
     2041 atttggctga tttcaagact acatacaagg ctaagaaacc agttcaaatg ccaggtgctt
     2101 acaacgttga tagaaaattg gatatcactt ctcataacga agattacaca gttgttgaac
     2161 aatacgaaag aagtgaaggt agacatagta caggtggtag tggtgcatca tgagttgtat
     2221 ctgtacaaaa tccaaagctg agcaaataaa taaataaata aatgtataag ttaccgaacg
     2281 ggggtatttt tacttttgat caaaaattta tgtaccaact acaaagtttc ctcagcacag
     2341 ccttcaagaa gggaacacac atacaaacag tgtcaaataa ttgtagggat aaatttaaat
     2401 atggcataaa ctaaataagt agagcatgaa aaaactgcaa aatccaaaaa gtaaaaacga
     2461 aggtcagaaa gtaaagcaaa agaaaattaa taaagcaata ctaaatctat catgatttcc
     2521 cgtaacttcc attaaagctg taaccagatt tactcctact gtttgagcct ctaacgccta
     2581 atggattttt agagaagctc aacctgatac ctcctttgtt gttgagggaa gggcgggggt
     2641 gaggtagttg actaccatat aattctgcca atgctctagt ggcaaagcta acatcctcaa
     2701 attctacaaa acaaattgga ccatggccat gaccgttgcc atgaccgcta gaggatcccc
     2761 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2821 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2881 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     3001 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3061 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3121 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3181 agggcgggta ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat
     3241 gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc
     3301 cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt
     3361 cctcttccaa caataataat gtcagatcct gtagagacca catcatccac ggttctatac
     3421 tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa
     3481 tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct
     3541 ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc
     3601 ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc
     3661 gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct
     3721 gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg
     3781 tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc
     3841 ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa
     3901 caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc
     3961 aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca
     4021 ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt
     4081 ttcctgcatg tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct
     4141 tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc
     4201 ttctgttcgg agattaccga atcaaaaaaa tttcaaagaa accgaaatca aaaaaaagaa
     4261 taaaaaaaaa atgatgaatt gaattgaaaa gctggcctcg tgatacgcct atttttatag
     4321 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4381 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4441 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4501 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4561 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4621 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4681 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4741 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4801 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4861 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4921 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4981 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5041 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5101 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5161 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5221 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5281 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5341 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5401 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5461 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5521 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5581 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5641 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5701 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5761 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5821 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5881 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5941 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6001 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6061 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6121 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6181 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6241 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaga
//
