LOCUS       sequence-468013-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of p300 KAT enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  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 7630)
  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: p300 KAT LIC 1M
FEATURES             Location/Qualifiers
     source          1..7630
                     /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      2381..2390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2702..2719
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2772..2790)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2786..2833
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2870..3325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2957..2976)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3167..3188
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3418..4233)
                     /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(3418..4233)
                     /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     4141..4160
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4225..4244
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4355..4943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4844..4863
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5097..5114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5129..5268
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5194..5213)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5354..5376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5370..5561)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6370..7452)
                     /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     7414..7433
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7453..7530)
                     /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 ggaggaagaa gagagaaaac gagaggaaaa
     2221 caccagcaat gaaagcacag atgtgaccaa gggagacagc aaaaatgcta aaaagaagaa
     2281 taataagaaa accagcaaaa ataagagcag cctgagtagg ggcaacaaga agaaacccgg
     2341 gatgcccaat gtatctaacg acctctcaca gaaactatat gccaccatgg agaagcataa
     2401 agaggtcttc tttgtgatcc gcctcattgc tggccctgct gccaactccc tgcctcccat
     2461 tgttgatcct gatcctctca tcccctgcga tctgatggat ggtcgggatg cgtttctcac
     2521 gctggcaagg gacaagcacc tggagttctc ttcactccga agagcccagt ggtccaccat
     2581 gtgcatgctg gtggagctgc acacgcagag ccaggaccgc tttgtctaca cctaataaca
     2641 ttggaagtgg ataacggatc cgaattcgag cgccgtcgac aagcttgcgg ccgcactcga
     2701 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     2761 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     2821 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     2881 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2941 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3001 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3061 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3121 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3181 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3241 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3301 aacaaactag taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3361 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     3421 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     3481 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     3541 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     3601 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     3661 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     3721 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     3781 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     3841 caaccggcgc aggaacactg ccagcgcatc aacaatgttt tcacctgaat caggatattc
     3901 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     3961 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4021 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     4081 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     4141 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     4201 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     4261 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4501 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4981 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5041 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5101 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5161 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5221 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5281 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5341 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5401 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5461 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5521 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5581 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5641 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5701 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5761 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5821 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5881 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5941 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6001 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     6061 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6121 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6181 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6241 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6301 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     6361 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6421 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     6481 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     6541 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6601 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     6661 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6721 aaccagcatc gcagtgggaa cgataccctc attcagcatt tgcatggttt gttgaaaacc
     6781 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6841 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6901 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6961 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     7021 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     7081 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     7141 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     7201 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     7261 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     7321 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     7381 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     7441 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     7501 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     7561 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7621 atggtgcatg
//
