LOCUS       sequence-368215-        5702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the C-terminal tagging of H2B histones with 
            mRuby2 in human cells using CRISPR/Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5702)
  AUTHORS   Husser MC, Ozugergin I, Resta T, Martin VJJ, Piekny AJ
  TITLE     Cytokinetic diversity in mammalian cells is revealed by the 
            characterization of endogenous anillin, Ect2 and RhoA.
  JOURNAL   Open Biol. 2022 Nov;12(11):220247. doi: 10.1098/rsob.220247. Epub 
            2022 Nov 23.
  PUBMED    36416720
REFERENCE   2  (bases 1 to 5702)
  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: "Open 
            Biol."; date: "2022-11"; volume: "12(11):220247. doi"; pages: " 
            10.1098/rsob.220247. Epub 2022 Nov 23"
COMMENT     Sequence Label: pODN-H2BC11-mR2
FEATURES             Location/Qualifiers
     source          1..5702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    880..901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(916..921,922..939,940..946)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2003..2380
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2393..2402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2399..3109
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(4156..4175)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4157..4175)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4481..4499)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4567..4671
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4672..4740,4741..5532)
                     /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     complement(4890..4909)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcaattg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      901 ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg gaattatgag
      961 cggataataa tttcacacag gaggtttaaa ctttaaacat gtcaaaagag acgtcttttg
     1021 ttaagaatgc tgaggaactt gcaaagcaaa aaatggatgc tattaaccct gaactttctt
     1081 caaaatttaa atttttaata aaattcctgt ctcagtttcc tgaagcttgc tctaaacctc
     1141 gttcaaaaaa aatgcagaat aaagttggtc aagaggaaca tattgaatat ttagctcgta
     1201 gttttcatga gagtcgattg ccaagaaaac ccacgccacc tacaacggtt cctgatgagg
     1261 tggttagcat agttcttaat ataagtttta atatacagcc tgaaaatctt gagagaataa
     1321 aagaagaaca tcgattttcc atggcagctg agaatattgt aggagatctt ctagaaagat
     1381 ctttgcccag cagcttgttg agctcctcgt cgttgcggat ggccagttgg agatgacgcg
     1441 ggatgatgcg ggtcttcttg ttgtcgcggg ccgcgttgcc cgccagttcc aggatctcgg
     1501 cggtcaggta ctccagcacc gctgccagat acaccggcgc gccggccccg acccgctcgg
     1561 catagttgcc tttgcggagc aggcggtgca ctcggcccac ggggaactgg agaccggccc
     1621 tagaagagcg agtcttggcc ttagcgcggg ctttgcctcc ctgcttgcca cgtccagaca
     1681 tagcgagcgc aactcactac gagcaaccac aaagtgaacg ggaaaggcgg cgctttttat
     1741 aaacactatt gggcgcgaaa aagaagacgt gttgttggtt agggctgcag tttaatttca
     1801 accaatagta gtgcgtcttc tggatttgcg aatcctgatt gggcagacct gacctctgac
     1861 gttaccctga ataactacca atcagacaca agacttcaac tcttcacctt atttgcataa
     1921 gcgattctat ataaaagcgc cttgtcatac cctgctcacg ctgtttttcc ttttcgttgg
     1981 cgctttatag ctacacagtg ctatgccaga gccagcgaag tctgctcccg ccccgaaaaa
     2041 gggctccaag aaggcggtga ctaaggcgca gaagaaagac ggcaagaagc gcaagcgcag
     2101 ccgcaaggag agctattcca tctatgtgta caaggttctg aagcaggtcc accctgacac
     2161 cggcatttcg tccaaggcca tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg
     2221 catcgcaggt gaggcttccc gcctggcgca ttacaacaag cgctcgacca tcacctccag
     2281 ggagatccag acggccgtgc gcctgctgct gcctggggag ttggccaagc acgccgtgtc
     2341 cgagggtact aaggccgtca ccaagtacac cagcgctaag gaccctcctg tggccaccat
     2401 ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg tcatggaagg
     2461 ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc cgtacatggg
     2521 aactcaaacc atgaggatca aagtcatcga gggaggaccc ctgccatttg cctttgacat
     2581 tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga aaggcattcc
     2641 tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta cgagatacga
     2701 agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct gtctcgttta
     2761 ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc agaagaaaac
     2821 caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc tgaggggata
     2881 cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt tcgtaacaac
     2941 ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg ccgttgatca
     3001 ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac gcgaacacgc
     3061 agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagt aaacagtgag
     3121 ttggttgcaa actctcaatc ctaacgcctc ttttaagagc cacccatgtt ctcaaagaaa
     3181 gagctggtgc ttgtattcct cctctgctgg ccactgacaa acccttgtaa cttgctactg
     3241 tgttttttgg tctgaagtag agcagttatt taactaatcc ttagtgactt ttttttttag
     3301 atctgccatt ctaatcttag agttaagtaa ggagatggga aattttctat tataagttcg
     3361 aaaccaatta aaatacgtta gaaaccaatt aaaatactcg tcggtccccc gtcggttagt
     3421 gatttggaac agtgccaagt tgcagcggtt gtcagtttga atttgcccgg gcaacgcccg
     3481 cccttcctct gcatcctgat tggttgttgt atccaggaag tccacggtcg gcttgggtgc
     3541 gccaaaggaa tccaaacccc gcctttattt ggccttcctt acaaactcag taaactgtca
     3601 gtttctcatt ctttagtatt ttatcgcttt cgggaaggag atttatttct aattagtttt
     3661 cgttggatag agcgtttttt tttttaaagg gaccctttat ggcaacataa tcagtttttt
     3721 tttttttgag aaggagactt cctctgtcgc ccagactgga gtgcggtggc gcgatctcag
     3781 ctcactgcag cctccgcctc ccgggttcca gcgattctcc tgcttcaccc ttccgtgtag
     3841 ctgggattac aggcatgcgc caccatgccc ggataatttt tgtattttta gtagagatgg
     3901 ggtttcacca tgttggctag gctggtctcg aactgacttc aggtgatcca ctagcctcag
     3961 ccttaaagtg ctaggattac aggcatgagc catcaagccc ggctacaacg gaatttgaat
     4021 gctgtagttt tcagcctgag aaagatcaat gctttatttc tgattagaat actattttcc
     4081 ctttatggta tatctatata agcaaaggat cttgctgaaa aactcgagcc atccggaaga
     4141 tctggcggcc gctctcccta tagtgagtcg tattacgccg gatggatatg gtgttcaggc
     4201 acaagtgtta aagcagttga ttttattcac tatgatgaaa aaaacaatga atggaacctg
     4261 ctccaagtta aaaatagaga taataccgaa aactcatcga gtagtaagat tagagataat
     4321 acaacaataa aaaaatggtt tagaacttac tcacagcgtg atgctactaa ttgggacaat
     4381 tttccagatg aagtatcatc taagaattta aatgaagaag acttcagagc ttttgttaaa
     4441 aattatttgg caaaaataat ataattcggc tgcaggggcg gcctcgtgat acgcctattt
     4501 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4561 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4621 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4681 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4741 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4801 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4861 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4921 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4981 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5041 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5101 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5161 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5221 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5281 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5341 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5401 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5461 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5521 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5581 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5641 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5701 tc
//
