LOCUS       sequence-271809-        6104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of his-CENP-C 510-end.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6104)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Polo-like kinase 1 licenses CENP-A deposition at centromeres.
  JOURNAL   Cell. 2014 Jul 17;158(2):397-411. doi: 10.1016/j.cell.2014.06.016.
  PUBMED    25036634
REFERENCE   2  (bases 1 to 6104)
  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.cell.2014.06"; journalName: "Cell"; date: "2014-07-17- 
            17"; volume: "158"; issue: "2"; pages: "397-411"
COMMENT     Sequence Label: pKM149
FEATURES             Location/Qualifiers
     source          1..6104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        90..108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             182..204
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             224..241
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             245..277
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(281..289,290..304)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             305..328
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1723..1741)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1737..1784
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2150..2155,2156..2172,2173..2178))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2205..2223)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2291..2395
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2396..2464,2465..3256)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2614..2633)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3427..4015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3916..3935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4169..4186
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4201..4340
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4266..4285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4426..4448
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4442..4633)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     join(6091..6104,1..6)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
       61 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg
      121 gtttccctct agtgccggct ccggagagct ccaattggaa ttcgctagct ctagaaataa
      181 ttttgtttaa ctttaagaag gagatatacg tatgcggggt tctcatcatc atcatcatca
      241 tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
      301 taagctggaa gttctgttcc aggggcccca tatggaagaa gtgacttcaa ccgttaccaa
      361 aagccgccgt attagtcgtc gtcctagcga ttggtgggtt gtgaaaagcg aagaaagccc
      421 ggtgtatagc aattctagcg tccgtaatga gctgcctatg caccataaca gttcccgtaa
      481 aagcaccaaa aaaaccaacc agtctagcaa aaacattcgt aaaaaaacga tccctctgaa
      541 acgccagaaa accgccacaa aaggcaacca acgtgttcag aaattcctga acgccgaagg
      601 atcaggaggc attgttgggc acgatgagat tagtcgctgc tccctgtcag aacctctgga
      661 atccgatgaa gccgatctgg ctaaaaaaaa aaacctggac tgctcccgtt ctacccgtag
      721 cagtaaaaat gaggataaca tcatgaccgc ccagaatgtc ccactgaaac ctcagacaag
      781 cggttatacc tgtaacattc cgaccgaaag caatctggac agcggtgagc acaaaacaag
      841 tgtcctggaa gagtctggtc ctagtcgtct gaataacaat tatctgatgt ccgggaaaaa
      901 tgatgtggac gatgaggaag ttcacggtag tagcgacgat tcaaaacaga gtaaagtcat
      961 ccctaaaaat cgtattcacc ataaactggt gctgccgagt aatactccga acgttcgtcg
     1021 tacaaaacgt acacgcctga aacctctgga atattggcgt ggtgaacgta tcgactacca
     1081 gggtcgtcca tctggtggtt ttgtaatcag cggtgtgctg agtcctgata cgattagcag
     1141 caaacgtaaa gccaaagaga acatcggcaa agtcaacaaa aaatccaaca aaaaacgcat
     1201 ctgcctggat aacgatgagc gtaaaacgaa tctgatggtg aatctgggta ttcctctggg
     1261 agatcctctg cagcctaccc gtgtaaaaga tcctgagaca cgtgagatta tcctgatgga
     1321 cctggttcgt cctcaagaca cctatcagtt tttcgtcaaa cacggcgagc tgaaagtcta
     1381 taaaaccctg gacaccccgt tctttagtac cggcaaactg attctgggtc ctcaggagga
     1441 aaaaggcaaa cagcacgtgg gtcaagacat cctggtcttt tatgtgaatt ttggggacct
     1501 gctgtgtact ctgcatgaaa ccccgtatat tctgtctacc ggtgacagct tctatgtgcc
     1561 gtctggaaac tattataaca tcaaaaacct gcgcaacgaa gaaagtgttc tgctgttcac
     1621 acagatcaaa cgctgaggat ccagatctaa gcttggtacc acgcgtgcgc gctgatccgg
     1681 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1741 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1801 tatccggata tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca
     1861 agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg
     1921 ggtgcgcata gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg
     1981 atgctgtcgg aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct
     2041 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc
     2101 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta
     2161 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
     2221 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2281 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2341 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2401 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2461 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2521 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2581 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2641 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2701 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2761 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2821 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2881 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2941 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3001 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3061 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3121 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3181 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3241 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3301 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3361 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3421 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3481 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3541 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3601 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3661 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3721 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3781 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3841 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3901 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3961 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4021 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4081 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4141 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4201 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4261 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4321 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4381 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4441 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4501 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4561 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4621 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4681 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4741 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4801 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4861 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4921 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4981 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5041 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5101 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     5161 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     5221 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     5281 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     5341 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     5401 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     5461 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     5521 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     5581 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     5641 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5701 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5761 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5821 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5881 caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5941 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     6001 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     6061 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcga
//
