LOCUS       sequence-419982-        8149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SMASh tag fused to YFP at C terminus in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8149)
  AUTHORS   Chung HK, Jacobs CL, Huo Y, Yang J, Krumm SA, Plemper RK, Tsien RY, 
            Lin MZ
  TITLE     Tunable and reversible drug control of protein production via a 
            self-excising degron.
  JOURNAL   Nat Chem Biol. 2015 Jul 27. doi: 10.1038/nchembio.1869.
  PUBMED    26214256
REFERENCE   2  (bases 1 to 8149)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2015 Jul 27. doi: 10.1038/nchembio.1869."
COMMENT     Sequence Label: pAG426GPD-ccdB-YFP-yeastSMASh
FEATURES             Location/Qualifiers
     source          1..8149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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"
     primer_bind     complement(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3450..3471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3486..3491,3492..3509,3510..3516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3628..4271
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     4165..4188
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     4277..4293
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4277..4293
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(4295..4297,4298..4300,4301..5011)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4340..4361)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4601..4620)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4948..4969
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5072..5095
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      5900..6147
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5916..5934)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6165..6184)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6166..6184)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6194..6211)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6194..6210)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6203..6225)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6351..6806
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6438..6457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6648..6669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6937..7740)
                     /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(7741..7956)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7980..7999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8099..8121
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg
     3661 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat
     3721 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg
     3781 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt
     3841 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac
     3901 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca
     3961 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag
     4021 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct
     4081 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct
     4141 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc
     4201 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa
     4261 cttagtttcg acggattcta gaactagtgg atccatggtg agcaagggcg aggagctgtt
     4321 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     4381 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agctgatctg
     4441 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg gctacggcct
     4501 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4561 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     4621 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     4681 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     4741 caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg
     4801 ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat
     4861 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag
     4921 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     4981 gatcactctc ggcatggacg agctgtacaa ggacgaaatg gaagaatgta gtaagcactt
     5041 acctggtgca ggctcatctg gcgatatcat ggactacaag gacgacgatg ataagggttc
     5101 ttctggaaca ggttcaggtt caggtacaag tgcacccatt actgcttatg ctcagcaaac
     5161 aagaggccta ttgggttgta ttattactag tttgacgggc agagacaaaa accaggtaga
     5221 aggtgaagtg cagatcgtat ccacggctac tcagacattc ttggctactt gtattaatgg
     5281 tgtgtgttgg gcagtttacc acggtgcggg aactagaact attgcctctc ctaagggtcc
     5341 agtgatccaa atgtatacta acgttgatca agatttggtc ggctggccag ctccacaagg
     5401 gtcaagaagc ctaactcctt gcacctgcgg ttcttcagat ctgtacttag tgacacgtca
     5461 tgcagatgtt ataccagtta gacgtcgtgg ggacagtaga ggttcactgc tgtcaccacg
     5521 tccaattagt taccttaaag gtagttctgg cggtcccctg ttgtgtcctg caggtcatgc
     5581 agtaggattg tttagagccg ccgtctgcac cagaggggtt gcaaaagctg tggattttat
     5641 tcctgttgaa aatcttgaaa cgactatgag atcacctgtc tttaccgata atagttcccc
     5701 accagctgtg actcttacac atccgatcac caaaatcgat actaaataca ttatgacctg
     5761 tatgtctgcc gatttagaag tagtaacatc tacatgggtt ttagtcggtg gggttttagc
     5821 tgccctagcc gcatattgct tgtccacagg ttgcgttgtt atagttggca ggatcgtttt
     5881 gagcggaaaa taactcgagt catgtaatta gttatgtcac gcttacattc acgccctccc
     5941 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
     6001 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     6061 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
     6121 ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt
     6181 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     6241 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     6301 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     6361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     6421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     6481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     6541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     6601 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     6661 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     6721 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     6781 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     6841 ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc taatttgtga
     6901 gtttagtata catgcattta cttataatac agttttttag ttttgctggc cgcatcttct
     6961 caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc
     7021 ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc
     7081 cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt
     7141 cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc
     7201 gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt
     7261 agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt
     7321 tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc
     7381 attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac
     7441 tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga
     7501 taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg
     7561 tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt
     7621 ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag
     7681 cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat
     7741 gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca
     7801 atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt
     7861 ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa
     7921 tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt
     7981 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     8041 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     8101 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
