LOCUS       sequence-243687-        7097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To transform BL21-Gold(DE3) and induced by IPTG for 
            His-proteinA-MNase fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7097)
  AUTHORS   Ku WL, Pan L, Cao Y, Gao W, Zhao K
  TITLE     Profiling single-cell histone modifications using indexing chromatin
            immunocleavage sequencing.
  JOURNAL   Genome Res. 2021 Apr 14. pii: gr.260893.120. doi: 
            10.1101/gr.260893.120.
  PUBMED    33853847
REFERENCE   2  (bases 1 to 7097)
  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: "Genome Res.
            2021 Apr 14. pii: gr.260893.120. doi: 10.1101/gr.260893.120."
COMMENT     Sequence Label: pET15b-pA-Mnase
FEATURES             Location/Qualifiers
     source          1..7097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             543..968
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
     CDS             1041..1487
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVAYVYKPNNTHEQLLRKSEAQAKKEKLNIWSEDNADSGQ"
     primer_bind     complement(1541..1559)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1555..1602
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1777..1782,1783..1799,1800..1805))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1832..1850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1918..2022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2023..2091,2092..2883)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2241..2260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3054..3642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3543..3562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3796..3813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3828..3967
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3893..3912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4053..4075
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4069..4260)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6615..6634
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6654..6731)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6937..6956
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7040..7059
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7040..7058
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7059..7083
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tgcatgcgca acacgatgaa gctcaacaaa
      121 atgcttttta tcaagtctta aatatgccta acttaaatgc tgatcaacgc aatggtttta
      181 tccaaagcct taaagatgat ccaagccaaa gtgctaacgt tttaggtgaa gctaaaaaac
      241 ttaatgactc tcaagctcca aaagctgatg cgcaacaaaa taacttcaac aaagatcaac
      301 aaagcgcctt ctatgaaatt ttgaacatgc ctaacttaaa cgaagcgcaa cgcaatggtt
      361 tcattcaaag tcttaaagac gatccaagcc aaagcactaa cgttttaggt gaagctaaaa
      421 aattaaacga atctcaagca ccgaaagctg acaacaattt caacaaagaa caacaaaatg
      481 ctttctatga aatcttgaac atgcctaact tgaacgaaga acaacgcaat ggtttcatcc
      541 aaagcttaaa agatgaccca agtcaaagtg ctaacctatt gtcagaagct aaaaagttaa
      601 atgaatctca agcaccgaaa gcggataaca aattcaacaa agaacaacaa aatgctttct
      661 atgaaatctt acatttacct aacttaaacg aagaacaacg caatggtttc atccaaagct
      721 taaaagatga cccaagccaa agcgctaacc ttttagcaga agctaaaaag ctaaatgatg
      781 cacaagcacc aaaagctgac aacaaattca acaaagaaca acaaaatgct ttctatgaaa
      841 ttttacattt acctaactta actgaagaac aacgtaacgg cttcatccaa agccttaaag
      901 acgatccttc agtgagcaaa gaaattttag cagaagctaa aaagctaaac gatgctcaag
      961 caccaaaagg tggcggtgaa ttctcacaaa cagataacgg cgtaaataga agtggttctg
     1021 aagatccaac agtatatagt gcaacttcaa ctaaaaaatt acataaagaa cctgcgacat
     1081 taattaaagc gattgatggt gatacggtta aattaatgta caaaggtcaa ccaatgacat
     1141 tcagactatt attggttgat acacctgaaa caaagcatcc taaaaaaggt gtagagaaat
     1201 atggtcctga agcaagtgca tttacgaaaa aaatggtaga aaatgcaaag aaaattgaag
     1261 tcgagtttga caaaggtcaa agaactgata aatatggacg tggcttagcg tatatttatg
     1321 ctgatggaaa aatggtaaac gaagctttag ttcgtcaagg cttggctaaa gttgcttatg
     1381 tttataaacc taacaataca catgaacaac ttttaagaaa aagtgaagca caagcaaaaa
     1441 aagagaaatt aaatatttgg agcgaagaca acgctgattc aggtcaataa ggatccggct
     1501 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1561 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1621 tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     1681 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     1741 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
     1801 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     1861 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     1921 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1981 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2041 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2101 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2161 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     2221 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     2281 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2341 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2401 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2461 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2521 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     2581 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2641 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2701 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2761 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2821 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2881 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2941 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3001 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3061 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3181 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3841 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3901 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3961 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4021 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4081 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4141 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4201 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4261 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4321 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4381 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4441 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4501 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4561 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4621 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4681 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     4741 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     4801 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     4861 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     4921 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     4981 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     5041 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     5101 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     5161 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     5221 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5281 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5341 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     5401 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     5461 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5521 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5581 gctttccagt cgggaaactg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5641 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5701 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5761 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5821 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     5881 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5941 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     6001 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     6061 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     6121 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     6181 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     6241 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     6301 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     6361 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     6421 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     6481 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     6541 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6601 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6661 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6721 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     6781 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     6841 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     6901 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6961 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     7021 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     7081 tcccctctag aaataat
//
