LOCUS       sequence-367981-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H2B-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  AUTHORS   Cuylen S, Blaukopf C, Politi AZ, Muller-Reichert T, Neumann B, Poser
            I, Ellenberg J, Hyman AA, Gerlich DW
  TITLE     Ki-67 acts as a biological surfactant to disperse mitotic 
            chromosomes.
  JOURNAL   Nature. 2016 Jun 29;535(7611):308-12. doi: 10.1038/nature18610.
  PUBMED    27362226
REFERENCE   2  (bases 1 to 6225)
  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"; journalName: 
            "Nature"; date: "2016-06-29- 29"; volume: "535"; issue: "7611"; 
            pages: "308-12"
COMMENT     Sequence Label: H2B-mNeonGreen-IRESpuro2
FEATURES             Location/Qualifiers
     source          1..6225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        307..510
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     461..481
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             599..976
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             983..1693
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1238..1247
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          1762..1991
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1974..1993
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2063..2637,2638..2640,2641..2649)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2230..2247)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2457..2476
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2669..3268
                     /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(2669..2688)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3165..3185
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3404..3421)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3410..3634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3681..3700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3760..3776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3760..3776)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3773..3795)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3784..3800
                     /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(3808..3814,3815..3832,3833..3838))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3853..3874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3991..4008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4162..4750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4242..4261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4921..5712,5713..5781))
                     /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     5544..5563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5782..5886)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5961..5980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6152..6225,1..306)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
       61 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      121 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      181 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      241 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      301 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      361 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      421 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      481 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      541 taccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgccaccat
      601 gccagagcca gcgaagtctg ctcccgcccc gaaaaagggc tccaagaagg cggtgactaa
      661 ggcgcagaag aaaggcggca agaagcgcaa gcgcagccgc aaggagagct attccatcta
      721 tgtgtacaag gttctgaagc aggtccaccc tgacaccggc atttcgtcca aggccatggg
      781 catcatgaat tcgtttgtga acgacatttt cgagcgcatc gcaggtgagg cttcccgcct
      841 ggcgcattac aacaagcgct cgaccatcac ctccagggag atccagacgg ccgtgcgcct
      901 gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag ggtactaagg ccatcaccaa
      961 gtacaccagc gctaaggatc ctatggtgag caagggcgag gaggataaca tggcctctct
     1021 cccagcgaca catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt
     1081 gggtcagggc accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa
     1141 gggtgacctc cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca
     1201 gtacctgccc taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg
     1261 ataccaagtc catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg
     1321 ctacacctac gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc
     1381 tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa
     1441 gacttacccc aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa
     1501 tggcaagcgc taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc
     1561 taactatctg aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa
     1621 gaccgagctc aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga
     1681 gctgtacaag taagcggccg catagataac tgatccagtg tgctggaatt aattcgctgt
     1741 ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg acttctgcgc
     1801 taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc gcggtgatgc
     1861 ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg tcaagcttga
     1921 ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc actttgcctt
     1981 tctctccaca ggtgtccact cccaggtcca actgcaggtc gagcatgcat ctagggcggc
     2041 caattccgcc cctctccccc cccccctctc cctccccccc ccctaacgtt actggccgaa
     2101 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     2161 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     2221 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     2281 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     2341 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     2401 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     2461 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     2521 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
     2581 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt
     2641 gccacaaccc acaaggagac gaccttccat gaccgagtac aagcccacgg tgcgcctcgc
     2701 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2761 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     2821 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     2881 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     2941 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3001 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     3061 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3121 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     3181 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3241 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     3301 aaggagcgca cgaccccatg gctccgaccg aagccacccg gggcggcccc gccgaccccg
     3361 cacccgcccc cgaggcccac cgactctaga gctcgctgat cagcctcgac tgtgccttct
     3421 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3481 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3541 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3601 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3661 ggctcgagtg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3721 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3781 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3841 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3901 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3961 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4021 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4081 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4141 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4201 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4261 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4321 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4381 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4441 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4501 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4561 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4621 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4681 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4741 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4801 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4861 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4921 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4981 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5041 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5101 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5161 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5221 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5281 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5341 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5401 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5461 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5521 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5581 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5641 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5701 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5761 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5821 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5881 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     5941 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     6001 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     6061 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     6121 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     6181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttc
//
