LOCUS       sequence-226297-        7090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses yeast Spt2-GFP fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  AUTHORS   Rossl A, Denoncourt A, Lin MS, Downey M
  TITLE     A synthetic non-histone substrate to study substrate targeting by 
            the Gcn5 HAT and sirtuin HDACs.
  JOURNAL   J Biol Chem. 2019 Apr 19;294(16):6227-6239. doi: 
            10.1074/jbc.RA118.006051. Epub 2019 Feb 25.
  PUBMED    30804216
REFERENCE   2  (bases 1 to 7090)
  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"; doi: 
            "10.1074/jbc.RA118.006051"; journalName: "J Biol Chem"; date: 
            "2019-04-19- 19"; volume: "294"; issue: "16"; pages: "6227-6239"
COMMENT     Sequence Label: Spt2-GFP
FEATURES             Location/Qualifiers
     source          1..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1027..1737
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1054..1082)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1677..1697
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1761..1926
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1816..1840)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(1948..1964)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1949..1965)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1993..2011)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2032..2048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2032..2048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2045..2067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2056..2072
                     /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)."
     promoter        complement(join(2080..2086,2087..2104,2105..2110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2125..2146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2263..2280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2434..3022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2514..2533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3193..3984,3985..4053))
                     /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     3816..3835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4054..4158)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4195..4698
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4739..4757
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4795..4817)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4917..4936
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4960..5175
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             5176..5979
                     /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"
     rep_origin      complement(6110..6565)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6247..6268)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6459..6478
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6691..6713
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6705..6722
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6706..6722
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6729..6748
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6729..6747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atgagttttc tttccaaact ttcccaaata cgaaaatcaa cgactgcatc aaaagcccaa
       61 gtgcaagatc cattacccaa gaagaatgac gaagagtatt ccttgttacc caaaaattac
      121 ataagagacg aagatcctgc agtaaaaaga ttgaaggagc tgaggcggca ggaactgtta
      181 aagaatggtg ctttggctaa aaaaagtggt gtaaaacgga aacgtggcac ctcatctgga
      241 tctgagaaaa agaaaataga aaggaatgac gatgatgaag gtggccttgg aattaggttt
      301 aagaggtcta ttggagcaag tcatgcgcca ctcaagccag ttgtaaggaa gaaacctgaa
      361 cctatcaaaa agatgtcatt tgaagagcta atgaaacaag cggaaaataa tgagaaacag
      421 cccccaaaag ttaagtcatc ggaacccgta actaaggaac gcccacattt taacaagcca
      481 ggtttcaaaa gttcaaaaag accacaaaag aaagcatccc ctggcgcaac attgcgtgga
      541 gtatcttctg gaggcaatag cataaaatca tcagactcac ccaagcccgt caagctcaac
      601 ttgcccacaa atggatttgc tcaacctaat aggagattaa aagaaaagtt agaatctaga
      661 aaacagaaat caagatacca ggatgactat gatgaagaag ataacgatat ggatgatttt
      721 atagaagacg atgaagatga aggttaccac agcaaatcga aacacagcaa tggtcccgga
      781 tatgatcgtg acgaaatttg ggctatgttc aatagaggca agaagcggtc agaatacgat
      841 tacgatgagc ttgaggatga tgatatggaa gcaaatgaga tggaaatctt ggaagaggag
      901 gaaatggcaa gaaaaatggc aaggttagag gataaacgtg aggaagcttg gttaaaaaag
      961 catgaagagg agaagagacg ccgtaagaag ggcatacgcg gtcgacggat ccccgggtta
     1021 attaacagta aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat
     1081 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac
     1141 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca
     1201 cttgtcacta ctttcactta tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa
     1261 cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt
     1321 ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt
     1381 gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac
     1441 aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat
     1501 ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca
     1561 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat
     1621 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc
     1681 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaatag
     1741 ggcgcgccac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
     1801 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     1861 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     1921 tcgctcttat tgaccacacc tctacatcga taccgtcgac ctcgaggggg ggcccggtac
     1981 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     2041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2161 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2221 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2281 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2341 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2401 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2461 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2521 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2581 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2641 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2701 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2761 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2821 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2881 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2941 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3001 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3061 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3121 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3181 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3241 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3301 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3361 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3421 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3481 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3541 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3601 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3661 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3721 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3781 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3841 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3901 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3961 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4021 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4081 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcct tttcatcacg
     4201 tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt aaaaatagaa
     4261 agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta aaagactcta
     4321 gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag gtattaatgc
     4381 cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg attttacatt
     4441 ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct aataaatata
     4501 tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt ttttcttcat
     4561 tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa acataaaaat
     4621 aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga ggcgcgtgta
     4681 agttacaggc aagcgatccg tctaagaaac cattattatc atgacattaa cctataaaaa
     4741 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     4801 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4861 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     4921 atcagagcag attgtactga gagtgcacca taccaccttt tcaattcatc attttttttt
     4981 tattcttttt tttgatttcg gtttccttga aatttttttg attcggtaat ctccgaacag
     5041 aaggaagaac gaaggaagga gcacagactt agattggtat atatacgcat atgtagtgtt
     5101 gaagaaacat gaaattgccc agtattctta acccaactgc acagaacaaa aacctgcagg
     5161 aaacgaagat aaatcatgtc gaaagctaca tataaggaac gtgctgctac tcatcctagt
     5221 cctgttgctg ccaagctatt taatatcatg cacgaaaagc aaacaaactt gtgtgcttca
     5281 ttggatgttc gtaccaccaa ggaattactg gagttagttg aagcattagg tcccaaaatt
     5341 tgtttactaa aaacacatgt ggatatcttg actgattttt ccatggaggg cacagttaag
     5401 ccgctaaagg cattatccgc caagtacaat tttttactct tcgaagacag aaaatttgct
     5461 gacattggta atacagtcaa attgcagtac tctgcgggtg tatacagaat agcagaatgg
     5521 gcagacatta cgaatgcaca cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag
     5581 gcggcagaag aagtaacaaa ggaacctaga ggccttttga tgttagcaga attgtcatgc
     5641 aagggctccc tatctactgg agaatatact aagggtactg ttgacattgc gaagagcgac
     5701 aaagattttg ttatcggctt tattgctcaa agagacatgg gtggaagaga tgaaggttac
     5761 gattggttga ttatgacacc cggtgtgggt ttagatgaca agggagacgc attgggtcaa
     5821 cagtatagaa ccgtggatga tgtggtctct acaggatctg acattattat tgttggaaga
     5881 ggactatttg caaagggaag ggatgctaag gtagagggtg aacgttacag aaaagcaggc
     5941 tgggaagcat atttgagaag atgcggccag caaaactaaa aaactgtatt ataagtaaat
     6001 gcatgtatac taaactcaca aattagagct tcaatttaat tatatcagtt attaccctat
     6061 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     6121 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     6181 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     6241 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     6301 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     6361 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     6421 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6481 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6541 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     6601 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6661 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6721 gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct
     6781 ctagaactat tggatccccc gggctgcaga atgaggctga aacggtttga ataattagga
     6841 aagtatgttt ttaataaaga aattctatgt tcaggaattt tgtatatact ttgtaatgaa
     6901 tgagaactta gttggcttca aactttttcg tttaacatga ttatttttct tgttcgacta
     6961 agatattccc acatggacaa gtgccacaga ttaatatatg aatacaataa aataactagt
     7021 gtaatttgaa aataaaagtt gatgagaggg acagggactt gagtcctatt caaagtgaaa
     7081 tattttagtt
//
