LOCUS       sequence-258014-        6058 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression of CENP-C and KNL2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6058)
  AUTHORS   McKinley KL, Sekulic N, Guo LY, Tsinman T, Black BE, Cheeseman IM
  TITLE     The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork
            of Interactions at the Centromere-Kinetochore Interface.
  JOURNAL   Mol Cell. 2015 Dec 17;60(6):886-98. doi: 
            10.1016/j.molcel.2015.10.027. Epub 2015 Nov 19.
  PUBMED    26698661
REFERENCE   2  (bases 1 to 6058)
  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: 
            "10.1016/j.molcel.2015.10.027"; journalName: "Mol Cell"; date: 
            "2015-12-17- 17"; volume: "60"; issue: "6"; pages: "886-98"
COMMENT     Sequence Label: pKM294
FEATURES             Location/Qualifiers
     source          1..6058
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             989..1696
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1134..1152)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1402..1421
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1742..1762
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1775..1819
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2010..2027)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2016..2240
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2373..2392)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2524..2571
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2579..3601
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3731..3852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3768..3787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3822..3841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3901..3917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3901..3917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3914..3936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3925..3941
                     /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(3949..3955,3956..3973,3974..3979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3994..4015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4132..4149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4303..4891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4383..4402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5062..5853,5854..5922))
                     /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     5685..5704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5923..6027)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt tagctaccgg tcgccaccat ggtgagcaag ggcgaggagg ataacatggc
     1021 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     1081 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1141 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     1201 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     1261 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     1321 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     1381 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     1441 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     1501 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     1561 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     1621 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     1681 ggacgagctg tacaagtccg gaggggatcg gtggagctcc accggtggcg gccgctctag
     1741 agagaatctt tattttcagg gcgccgccaa attcaaagaa accgctgctg ctaaattcga
     1801 acgccagcac atggacagcg gaggtggagg tactagtctc gaggaattct gcagtcgacg
     1861 gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc cataccacat
     1921 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1981 aaatgaatgc aattaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2041 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2101 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2161 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2221 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2281 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2341 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2401 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2461 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     2521 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     2581 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     2641 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     2701 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     2761 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     2821 gttcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     2881 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     2941 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3001 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     3061 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3121 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3181 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3241 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3301 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3361 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3421 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3481 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3541 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3601 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3661 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3721 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3781 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3841 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3901 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
