LOCUS       sequence-468015-        7534 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of p300 KAT enzyme with truncated 
            autoinhibitory loop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7534)
  AUTHORS   Dewing SM, Showalter SA
  TITLE     Probing Enzymatic Acetylation Events in Real Time With NMR 
            Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 
            Modification of Histone H4.
  JOURNAL   Proteins. 2025 Jun 1. doi: 10.1002/prot.26848.
  PUBMED    40452297
REFERENCE   2  (bases 1 to 7534)
  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: "Proteins. 
            2025 Jun 1. doi: 10.1002/prot.26848."
COMMENT     Sequence Label: p300dAIL KAT LIC 1M
FEATURES             Location/Qualifiers
     source          1..7534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..1435
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1409..1432
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1448..1468
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      2285..2294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2606..2623
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2676..2694)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2690..2737
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2774..3229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2861..2880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3071..3092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3322..4137)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3322..4137)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4045..4064
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4129..4148
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4259..4847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4748..4767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5001..5018
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5033..5172
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5098..5117)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5258..5280
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5274..5465)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6274..7356)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7318..7337
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7357..7434)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 ttcttctcac catcaccatc accatggttc ttctatgaaa atcgaagaag gtaaactggt
      361 aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga
      421 gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc
      481 acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg
      541 tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa
      601 gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat
      661 cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac
      721 ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat
      781 gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc
      841 gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc
      901 gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac
      961 cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg
     1021 cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc
     1081 gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc
     1141 cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga
     1201 aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga
     1261 ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga
     1321 aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat
     1381 caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaatgg
     1441 gatcgaggaa aacctgtact tccaatccaa tgcaaaagaa aataagtttt ctgctaaaag
     1501 gttgccatct accagacttg gcacctttct agagaatcgt gtgaatgact ttctgaggcg
     1561 acagaatcac cctgagtcag gagaggtcac tgttagagta gttcatgctt ctgacaaaac
     1621 cgtggaagta aaaccaggca tgaaagcaag gtttgtggac agtggagaga tggcagaatc
     1681 ctttccatac cgaaccaaag ccctctttgc ctttgaagaa attgatggtg ttgacctgtg
     1741 cttctttggc atgcatgttc aagagtatgg ctctgactgc cctccaccca accagaggag
     1801 agtatacata tcttacctcg atagtgttca tttcttccgt cctaaatgct tgaggactgc
     1861 agtctatcat gaaatcctaa ttggatattt agaatatgtc aagaaattag gttacacaac
     1921 agggcatatt tgggcatgtc caccaagtga gggagatgat tatatcttcc attgccatcc
     1981 tcctgaccag aagataccca agcccaagcg actgcaggaa tggtacaaaa aaatgcttga
     2041 caaggctgta tcagagcgta ttgtccatga ctacaaggat atttttaaac aagctactga
     2101 agatagatta acaagtgcaa aggaattgcc ttatttcgag ggtaatttct ggcccaatgt
     2161 tctggaagaa agcattaagg aactggaaca gaaaaccagc aaaaataaga gcagcctgag
     2221 taggggcaac aagaagaaac ccgggatgcc caatgtatct aacgacctct cacagaaact
     2281 atatgccacc atggagaagc ataaagaggt cttctttgtg atccgcctca ttgctggccc
     2341 tgctgccaac tccctgcctc ccattgttga tcctgatcct ctcatcccct gcgatctgat
     2401 ggatggtcgg gatgcgtttc tcacgctggc aagggacaag cacctggagt tctcttcact
     2461 ccgaagagcc cagtggtcca ccatgtgcat gctggtggag ctgcacacgc agagccagga
     2521 ccgctttgtc tacacctaat aacattggaa gtggataacg gatccgaatt cgagcgccgt
     2581 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2641 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2701 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2761 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2821 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2881 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2941 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3001 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3061 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3121 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3181 taacaaaaat ttaacgcgaa ttttaacaaa ctagtaacgt ttacaatttc aggtggcact
     3241 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3301 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3361 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3421 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3481 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     3541 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     3601 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3661 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3721 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3781 gttttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3841 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3901 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3961 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     4021 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     4081 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     4141 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     4201 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4261 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4321 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4381 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4441 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4501 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4561 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4621 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4681 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4741 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4801 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4861 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4921 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4981 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     5041 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     5101 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     5161 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5221 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5281 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5341 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5401 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5461 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5521 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5581 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5641 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5701 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5761 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5821 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5881 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     5941 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     6001 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     6061 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     6121 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     6181 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     6241 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6301 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6361 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     6421 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     6481 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6541 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6601 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatac cctcattcag
     6661 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6721 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6781 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6841 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6901 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6961 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     7021 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     7081 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     7141 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     7201 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     7261 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     7321 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     7381 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     7441 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     7501 cgttgagcac cgccgccgca aggaatggtg catg
//
