LOCUS       sequence-367984-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expesses H2B-mNeonGreen_FRB(DmrC). Can be used to target FKBP(DmrA) 
            tagged proteins to H2B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Cuylen-Haering S, Petrovic M, Hernandez-Armendariz A, Schneider MWG,
            Samwer M, Blaukopf C, Holt LJ, Gerlich DW
  TITLE     Chromosome clustering by Ki-67 excludes cytoplasm during nuclear 
            assembly.
  JOURNAL   Nature. 2020 Nov;587(7833):285-290. doi: 10.1038/s41586-020-2672-3. 
            Epub 2020 Sep 2.
  PUBMED    32879492
REFERENCE   2  (bases 1 to 6540)
  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"; doi: 
            "10.1038/s41586-020-2672-3"; journalName: "Nature"; date: "2020-11";
            volume: "587"; issue: "7833"; pages: "285-290"
COMMENT     Sequence Label: H2B-mNeonGreen_FRB(DmrC)_IRESpuro2
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             13..390
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             397..1101
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      649..658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1141..1419
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     intron          1491..1720
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1703..1722
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1792..2366,2367..2369,2370..2378)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1959..1976)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2186..2205
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2398..2997
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2398..2417)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2894..2914
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3133..3150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3139..3363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3410..3429
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3489..3505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3489..3505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3502..3524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3513..3529
                     /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)."
     promoter        complement(join(3537..3543,3544..3561,3562..3567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3582..3603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3720..3737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3891..4479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3971..3990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4650..5441,5442..5510))
                     /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     5273..5292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5511..5615)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5690..5709)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5881..6260
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6261..6464
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6415..6435
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 ggtaccgcca ccatgccaga gccagcgaag tctgctcccg ccccgaaaaa gggctccaag
       61 aaggcggtga ctaaggcgca gaagaaaggc ggcaagaagc gcaagcgcag ccgcaaggag
      121 agctattcca tctatgtgta caaggttctg aagcaggtcc accctgacac cggcatttcg
      181 tccaaggcca tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg catcgcaggt
      241 gaggcttccc gcctggcgca ttacaacaag cgctcgacca tcacctccag ggagatccag
      301 acggccgtgc gcctgctgct gcctggggag ttggccaagc acgccgtgtc cgagggtact
      361 aaggccatca ccaagtacac cagcgctaag gatccagtga gcaagggcga ggaggataac
      421 atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac
      481 tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg
      541 aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat
      601 ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta
      661 gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact
      721 gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg
      781 actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc
      841 aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac
      901 accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag
      961 ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc
     1021 aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg
     1081 ggcatggacg agctgtacaa gaccggtggc agctccggca gcggcggcat ggcttctaga
     1141 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg
     1201 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg
     1261 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag
     1321 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc
     1381 tgggacctct attatcatgt gttccgacga atctcaaagt aagcggccgc atagataact
     1441 gatccagtgt gctggaatta attcgctgtc tgcgagggcc agctgttggg gtgagtactc
     1501 cctctcaaaa gcgggcatga cttctgcgct aagattgtca gtttccaaaa acgaggagga
     1561 tttgatattc acctggcccg cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga
     1621 aaagacaatc tttttgttgt caagcttgag gtgtggcagg cttgagatct ggccatacac
     1681 ttgagtgaca atgacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa
     1741 ctgcaggtcg agcatgcatc tagggcggcc aattccgccc ctctcccccc ccccctctcc
     1801 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     1861 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     1921 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     1981 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     2041 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     2101 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     2161 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     2221 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     2281 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     2341 tttcctttga aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg
     2401 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     2461 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
     2521 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2581 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2641 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2701 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     2761 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     2821 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2881 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     2941 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     3001 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga
     3061 agccacccgg ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagag
     3121 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3181 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3241 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3301 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3361 tggcttctga ggcggaaaga accagctggg gctcgagtgc attctagttg tggtttgtcc
     3421 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3481 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3541 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3601 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3721 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3781 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3841 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3901 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3961 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4081 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4321 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4561 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4621 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4681 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4741 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4801 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4861 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4921 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4981 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5041 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5101 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5161 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5221 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5281 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5341 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5401 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5461 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5521 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5581 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5641 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5701 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     5761 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     5821 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     5881 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5941 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6001 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     6061 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     6121 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     6181 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6241 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     6301 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     6361 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     6421 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     6481 agatccgcta gcgctaccgg actcagatct cgagctcaag cttcgaattc tgcagtcgac
//
