LOCUS       sequence-367986-        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ki67 (LR)-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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: Ki67 (LR)-mNeonGreen-IRESpuro2
FEATURES             Location/Qualifiers
     source          1..6806
                     /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             1564..2274
                     /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      1819..1828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          2343..2572
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2555..2574
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2644..3218,3219..3221,3222..3230)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2811..2828)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3038..3057
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3250..3849
                     /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(3250..3269)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3746..3766
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3985..4002)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3991..4215
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4262..4281
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4341..4357)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4341..4357)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4354..4376)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4365..4381
                     /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(4389..4395,4396..4413,4414..4419))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4434..4455
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4572..4589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4743..5331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4823..4842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5502..6293,6294..6362))
                     /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     6125..6144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6363..6467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6542..6561)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6733..6806,1..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 gaaatgactg aggaactggc aaatggtgct
      601 gctgatagct ttacaagcgc tccaaagcaa acacctgaca gtggaaaacc tctaaaaata
      661 tccagaagag ttcttcgggc ccctaaagta gaacccgtgg gagacgtggt aagcaccaga
      721 gaccctgtaa aatcacaaag caaaagcaac acttccctgc ccccactgcc cttcaagagg
      781 ggaggtggca aagatggaag cgtcacggga accaagaggt tgcgctgcat gccagcacca
      841 gaggaaattg tggaggagct gccagccagc aagaagcaga gggttgctcc cagggcaaga
      901 ggcaaatcat ccgaacccgt ggtcatcatg aagagaagtt tgaggacttc tgcaaaaaga
      961 attgaacctg cggaagagct gaacagcaac gacatgaaaa ccaacaaaga ggaacacaaa
     1021 ttacaagact cggtccctga aaataaggga atatccctgc gctccagacg ccaaaataag
     1081 actgaggcag aacagcaaat aactgaggtc tttgtattag cagaaagaat agaaataaac
     1141 agaaatgaaa agaagcccat gaagacctcc ccagagatgg acattcagaa tccagatgat
     1201 ggagcccgga aacccatacc tagagacaaa gtcactgaga acaaaaggtg cttgaggtct
     1261 gctagacaga atgagagctc ccagcctaag gtggcagagg agagcggagg gcagaagagt
     1321 gcgaaggttc tcatgcagaa tcagaaaggg aaaggagaag caggaaattc agactccatg
     1381 tgcctgagat caagaaagac aaaaagccag cctgcagcaa gcactttgga gagcaaatct
     1441 gtgcagagag taacgcggag tgtcaagagg tgtgcagaaa atccaaagaa ggctgaggac
     1501 aatgtgtgtg tcaagaaaat aagaaccaga agtcataggg acagtgaaga tatttgggat
     1561 cctatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta
     1621 cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat
     1681 ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc
     1741 ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac
     1801 gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt ccatcgcaca
     1861 atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc
     1921 cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg
     1981 accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa
     2041 accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc
     2101 actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag
     2161 ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag
     2221 gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa gtaagcggcc
     2281 gcatagataa ctgatccagt gtgctggaat taattcgctg tctgcgaggg ccagctgttg
     2341 gggtgagtac tccctctcaa aagcgggcat gacttctgcg ctaagattgt cagtttccaa
     2401 aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg tggccgcgtc
     2461 catctggtca gaaaagacaa tctttttgtt gtcaagcttg aggtgtggca ggcttgagat
     2521 ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac aggtgtccac
     2581 tcccaggtcc aactgcaggt cgagcatgca tctagggcgg ccaattccgc ccctctcccc
     2641 ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     2701 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     2761 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     2821 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     2881 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     2941 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     3001 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     3061 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     3121 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     3181 cggggacgtg gttttccttt gaaaaacacg atgataagct tgccacaacc cacaaggaga
     3241 cgaccttcca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     3301 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     3361 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     3421 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     3481 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     3541 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     3601 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     3661 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     3721 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     3781 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     3841 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
     3901 ggctccgacc gaagccaccc ggggcggccc cgccgacccc gcacccgccc ccgaggccca
     3961 ccgactctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     4021 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     4081 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     4141 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     4201 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgagt gcattctagt
     4261 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4321 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4381 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4441 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4501 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5461 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5521 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5581 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5641 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5701 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5761 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5821 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5881 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5941 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6001 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6061 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6121 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6181 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6241 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6301 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6361 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6421 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6481 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6541 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6601 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     6661 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     6721 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     6781 tagttcatag cccatatatg gagttc
//
