LOCUS       sequence-367987-        6300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H2B-2A-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6300)
  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 6300)
  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-2A-GFP-IRESpuro2
FEATURES             Location/Qualifiers
     source          1..6300
                     /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             992..1045
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1046..1048,1049..1759)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1046..1048,1049..1759)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1088..1109)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1349..1368)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1696..1717
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1837..2066
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2049..2068
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2138..2712,2713..2715,2716..2724)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2305..2322)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2532..2551
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2744..3343
                     /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(2744..2763)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3240..3260
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3479..3496)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3485..3709
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3756..3775
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3835..3851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3835..3851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3848..3870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3859..3875
                     /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(3883..3889,3890..3907,3908..3913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3928..3949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4066..4083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4237..4825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4317..4336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4996..5787,5788..5856))
                     /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     5619..5638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5857..5961)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6036..6055)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6227..6300,1..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 ctggcagtgg agagggcaga ggaagtctgc taacatgcgg
     1021 tgacgtcgag gagaatcctg gcccagtgag caagggcgag gagctgttca ccggggtggt
     1081 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1141 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1201 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1261 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1321 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1381 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1441 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1501 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1561 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1621 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1681 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1741 catggacgag ctgtacaagg aattctaagc ggccgcatag ataactgatc cagtgtgctg
     1801 gaattaattc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg
     1861 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct
     1921 ggcccgcggt gatgcctttg agggtggccg cgtccatctg gtcagaaaag acaatctttt
     1981 tgttgtcaag cttgaggtgt ggcaggcttg agatctggcc atacacttga gtgacaatga
     2041 catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc aggtcgagca
     2101 tgcatctagg gcggccaatt ccgcccctct cccccccccc ctctccctcc ccccccccta
     2161 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     2221 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     2281 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     2341 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     2401 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     2461 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     2521 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     2581 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     2641 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     2701 cacgatgata agcttgccac aacccacaag gagacgacct tccatgaccg agtacaagcc
     2761 cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc
     2821 gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt
     2881 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2941 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3001 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3061 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3121 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3181 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3241 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3301 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc
     3361 gcagcgcccg accgaaagga gcgcacgacc ccatggctcc gaccgaagcc acccggggcg
     3421 gccccgccga ccccgcaccc gcccccgagg cccaccgact ctagagctcg ctgatcagcc
     3481 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3541 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3601 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3661 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3721 gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3781 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3901 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3961 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4021 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4081 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4141 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4201 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4261 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4321 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4381 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4441 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4501 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4561 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4621 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4681 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4741 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4801 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4861 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4921 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4981 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5041 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5101 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5161 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5221 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5281 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5341 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5401 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5461 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5521 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5581 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5641 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5701 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5761 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5821 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5881 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5941 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     6001 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6061 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     6121 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     6181 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     6241 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
//
