LOCUS       sequence-443806-        7171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Polycistronic bacterial coexpression construct for human 
            His-Sumo-H2A, H2B, H3, and Dual-StrepII-H4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7171)
  TITLE     STAR-protocols--Evaluating the E3 Ligase Activity of BRCA1-BARD1 and
            its Variants by Nucleosomal Histone Ubiquitylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7171)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET29a-human histone 4-in-One
FEATURES             Location/Qualifiers
     source          1..7171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(170..193,194..229,230..253,254..259))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(257..277)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     RBS             609..631
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             1064..1086
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             1487..1509
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(1912..1944)
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     CDS             complement(1954..1974)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(2002..2019)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2026..2043)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2053..2097)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             2108..2130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2145..2169
                     /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     complement(2169..2188)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2170..2188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2276..2295)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2501..2578
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2579..3661
                     /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     complement(2598..2617)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4470..4661
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4655..4677)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4763..4902
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4818..4837
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4917..4934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5088..5676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5168..5187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5787..5806)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5798..6613
                     /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             5798..6613
                     /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     complement(5871..5890)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6705..7160)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6842..6863)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7054..7073
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgcta ttacccgggc ttctcaaatt
      181 gtgggtgtga ccatgctgaa ccacctgagc ctcctccaga gccacctccc ttctcgaact
      241 gtgggtggct ccatgcactt tggaagtaga gattctcctc gaggccgctg ctaccaccga
      301 aaccgtacag ggtacgaccc tgacgtttca gagcgtaaac aacgtccata gcggtaacgg
      361 ttttacgttt agcgtgttcg gtgtaggtaa cagcgtcacg gataacgttt tccaggaaaa
      421 ctttcagaac accgcgggtt tcttcgtaga tcagaccgga gatacgttta acaccaccac
      481 gacgagccag acgacggata gccggcttgg tgataccctg gatgttgtca cgcagaactt
      541 tacggtgacg tttagcacca cctttaccca gacctttacc acctttacca cgaccagaca
      601 tatgtatatc tccttcttaa agttaaacaa aattattgcg gccgcctaag ccctctcgcc
      661 tcggattctg cgggccagct ggatgtcctt gggcatgatg gtgaccctct tggcgtggat
      721 ggcacacagg ttggtgtcct caaagagagc gaccagataa gcctcgctgg cctcctgcag
      781 agccataacg gccgagctct ggaagcgcag gtcggtcttg aagtcctgag cgatctcccg
      841 gaccaggcgc tggaaaggca gtttgcggat gagcagctcg gtggatttct ggtagcggcg
      901 gatctcgcgg agagcgactg tgccgggccg gtaacggtga ggtttcttga ctccgccggt
      961 agcaggagcg gacttcctgg ctgccttggt ggccagctgc ttgcggggag ccttccctcc
     1021 ggtggattta cgggcggtct gcttggtacg ggccatatgt atatctcctt cttaaagtta
     1081 aacaaaatta ttgtcgactt acttagcgct ggtgtacttg gtgacggcct tggtgccctc
     1141 ggacacggcg tgcttggcca actccccggg cagcagcagg cgcacggccg tctggatctc
     1201 cctggaggtg atggtcgagc gcttgttgta atgcgccagg cgggaagcct cacccgcgat
     1261 gcgttcgaag atgtcgttga cgaaggagtt catgattccc atggccttag aggagatgcc
     1321 ggtgtcgggg tggacctgct tcagcacctt gtacacgtat acggagtagc tctccttgcg
     1381 gctgcgcttg cgcttcttgc cgtccttctt ctgcgcctta gtcacggctt tcttcgagcc
     1441 cttcttgggc gcgggagcgg acttcgctgg ttccggcata tgtatatctc cttcttaaag
     1501 ttaaacaaaa ttattgaatt cttatttacc tttcgctttg tggtgagatt cggttttttt
     1561 cggcagcaga accgcctgga tgttcgacag aacaccaccc tgcgcgatgg taactttacc
     1621 cagcagtttg ttcagttctt cgtcgttacg gatcgccagc tgcaggtgac gcgggatgat
     1681 acgggttttt ttgttgtcac gcgccgcgtt acccgccagt tccaggattt ccgcggtcag
     1741 gtattccaga accgccgcca tgtaaaccgg cgcacccgca ccaacacgtt ccgcgtagtt
     1801 acctttacgc agcagacggt gaacacgacc aaccgggaac tgcagacccg cacgagaaga
     1861 acgagatttc gctttcgcac gcgctttacc accctgttta ccacgaccag actttccaag
     1921 acggttcatc tcaatgtctg tgtatcccat ggcgccctga aaatacaggt tttcggtcgt
     1981 tgggatatcg taatcaccca tggaaccgcg tggcaccagg gtaccgtggt ggtggtggtg
     2041 gtgcagatct gggctgtcca tgtgctggcg ttcgaattta gcagcagcgg tttctttcat
     2101 atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     2161 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatcgat ctcgatcctc
     2221 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     2281 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     2341 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     2401 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     2461 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     2521 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     2581 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     2641 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     2701 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     2761 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     2821 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     2881 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     2941 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     3001 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     3061 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     3121 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     3181 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     3241 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     3301 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     3361 gctgcgcgtt ggtgcggaca tctcggtagt gggatacgac gataccgaag acagctcatg
     3421 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     3481 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     3541 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     3601 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3661 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     3721 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     3781 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     3841 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     3901 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     3961 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg
     4021 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     4081 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     4141 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     4201 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     4261 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     4321 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
     4381 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
     4441 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
     4501 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     4561 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     4621 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4681 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4741 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     4801 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt
     4861 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     4921 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4981 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5041 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5101 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5161 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5221 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5281 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5341 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5401 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5461 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5521 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5581 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5641 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5701 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     5761 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     5821 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     5881 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     5941 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     6001 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     6061 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     6121 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     6181 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     6241 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     6301 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     6361 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     6421 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     6481 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     6541 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     6601 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6661 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     6721 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     6781 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     6841 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     6901 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     6961 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     7021 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     7081 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     7141 tgcgccgcta cagggcgcgt cccattcgcc a
//
