LOCUS       sequence-226486-        7143 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses yeast Spt2-GFP fusion protein mutated from lysine to 
            arginine at site 166.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7143)
  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 7143)
  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-K166R-GFP
FEATURES             Location/Qualifiers
     source          1..7143
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_feature    37..540
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     581..599
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(637..659)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     759..778
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        802..1017
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1018..1821
                     /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(1952..2407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2089..2110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2301..2320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2533..2555
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2547..2564
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2548..2564
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2571..2590
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2571..2589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2622..2638
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2622..2638
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3959..4669
                     /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(3986..4014)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4609..4629
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4693..4858
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4748..4772)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(4880..4896)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4881..4897)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4925..4943)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4964..4980)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4964..4980)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4977..4999)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4988..5004
                     /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(5012..5018,5019..5036,5037..5042))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5057..5078
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5195..5212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5366..5954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5446..5465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6125..6916,6917..6985))
                     /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     6748..6767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6986..7090)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgggtc cttttcatca cgtgctataa aaataattat
       61 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
      121 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
      181 actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt ttcatcttgt
      241 ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
      301 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
      361 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
      421 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
      481 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
      541 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
      601 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
      661 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
      721 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
      781 gagagtgcac cataccacct tttcaattca tcattttttt tttattcttt tttttgattt
      841 cggtttcctt gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag
      901 gagcacagac ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc
      961 ccagtattct taacccaact gcacagaaca aaaacctgca ggaaacgaag ataaatcatg
     1021 tcgaaagcta catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta
     1081 tttaatatca tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc
     1141 aaggaattac tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat
     1201 gtggatatct tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc
     1261 gccaagtaca attttttact cttcgaagac agaaaatttg ctgacattgg taatacagtc
     1321 aaattgcagt actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca
     1381 cacggtgtgg tgggcccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca
     1441 aaggaaccta gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact
     1501 ggagaatata ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc
     1561 tttattgctc aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca
     1621 cccggtgtgg gtttagatga caagggagac gcattgggtc aacagtatag aaccgtggat
     1681 gatgtggtct ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga
     1741 agggatgcta aggtagaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga
     1801 agatgcggcc agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca
     1861 caaattagag cttcaattta attatatcag ttattaccct atgcggtgtg aaataccgca
     1921 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     1981 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2041 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2101 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2161 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2221 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2281 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2341 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2401 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     2461 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     2521 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     2581 cactataggg cgaattggag ctccaccgcg gtggcggccg ctctagaact agtggatccc
     2641 ccgggctgca gaatgaggct gaaacggttt gaataattag gaaagtatgt ttttaataaa
     2701 gaaattctat gttcaggaat tttgtatata ctttgtaatg aatgagaact tagttggctt
     2761 caaacttttt cgtttaacat gattattttt cttgttcgac taagatattc ccacatggac
     2821 aagtgccaca gattaatata tgaatacaat aaaataacta gtgtaatttg aaaataaaag
     2881 ttgatgagag ggacagggac ttgagtccta ttcaaagtga aatattttag ttatgagttt
     2941 tctttccaaa ctttcccaaa tacgaaaatc aacgactgca tcaaaagccc aagtgcaaga
     3001 tccattaccc aagaagaatg acgaagagta ttccttgtta cccaaaaatt acataagaga
     3061 cgaagatcct gcagtaaaaa gattgaagga gctgaggcgg caggaactgt taaagaatgg
     3121 tgctttggct aaaaaaagtg gtgtaaaacg gaaacgtggc acctcatctg gatctgagaa
     3181 aaagaaaata gaaaggaatg acgatgatga aggtggcctt ggaattaggt ttaagaggtc
     3241 tattggagca agtcatgcgc cactcaagcc agttgtaagg aagaaacctg aacctatcaa
     3301 aaagatgtca tttgaagagc taatgaaaca agcggaaaat aatgagaaac agcccccaaa
     3361 agttaagtca tcggaacccg taactaagga acgcccacat tttaacaagc caggtttcaa
     3421 aagttctaga agaccacaaa agaaagcatc ccctggcgca acattgcgtg gagtatcttc
     3481 tggaggcaat agcataaaat catcagactc acccaagccc gtcaagctca acttgcccac
     3541 aaatggattt gctcaaccta ataggagatt aaaagaaaag ttagaatcta gaaaacagaa
     3601 atcaagatac caggatgact atgatgaaga agataacgat atggatgatt ttatagaaga
     3661 cgatgaagat gaaggttacc acagcaaatc gaaacacagc aatggtcccg gatatgatcg
     3721 tgacgaaatt tgggctatgt tcaatagagg caagaagcgg tcagaatacg attacgatga
     3781 gcttgaggat gatgatatgg aagcaaatga gatggaaatc ttggaagagg aggaaatggc
     3841 aagaaaaatg gcaaggttag aggataaacg tgaggaagct tggttaaaaa agcatgaaga
     3901 ggagaagaga cgccgtaaga agggcatacg cggtcgacgg atccccgggt taattaacag
     3961 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
     4021 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
     4081 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
     4141 tactttcact tatggtgttc aatgcttttc aagataccca gatcatatga aacggcatga
     4201 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
     4261 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
     4321 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
     4381 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
     4441 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
     4501 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     4561 cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga
     4621 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat agggcgcgcc
     4681 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     4741 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     4801 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt
     4861 attgaccaca cctctacatc gataccgtcg acctcgaggg ggggcccggt accagctttt
     4921 gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg
     4981 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5041 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5101 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5161 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5221 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5281 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5341 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5401 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5461 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5521 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5581 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5641 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5701 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5761 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5821 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5881 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5941 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6001 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6061 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6121 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6181 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6241 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6301 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6361 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6421 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6481 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6541 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6601 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6661 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6721 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6781 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6841 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6901 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6961 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7021 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7081 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7141 cga
//
