LOCUS       sequence-271346-        5837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CENP-T.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5837)
  AUTHORS   Nishino T, Rago F, Hori T, Tomii K, Cheeseman IM, Fukagawa T
  TITLE     CENP-T provides a structural platform for outer kinetochore 
            assembly.
  JOURNAL   EMBO J. 2013 Feb 6;32(3):424-36. doi: 10.1038/emboj.2012.348. Epub 
            2013 Jan 18.
  PUBMED    23334297
REFERENCE   2  (bases 1 to 5837)
  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: 
            "10.1038/emboj.2012.348"; journalName: "EMBO J"; date: "2013-02-6- 
            6"; volume: "32"; issue: "3"; pages: "424-36"
COMMENT     Sequence Label: pFR092
FEATURES             Location/Qualifiers
     source          1..5837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..89
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        70..88
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             162..184
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1329..1346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1436..1454)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1450..1497
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1863..1868,1869..1885,1886..1891))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1918..1936)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2004..2108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2109..2177,2178..2969)
                     /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(2327..2346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3140..3728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3629..3648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3882..3899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3914..4053
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3979..3998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4139..4161
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4155..4346)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5804..5823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
       61 ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct agtgccggct
      121 ccggagagct ccaattggaa ttcgctagct ctagaaataa ttttgtttaa ctttaagaag
      181 gagatataca tatggcagac cacaaccctg acagcgattc tacacctcgt acactgctgc
      241 gtcgtgtgct ggatactgct gatcctcgta ctcctcgtcg tcctcgtagc gctcgtgctg
      301 gtgctcgccg tgccctgctg gaaacagcct ctcctcgtaa actgagtggc cagacccgta
      361 ctattgcccg tgggcgttca catggagccc gctcggtcgg tcgtagtgct cacattcaag
      421 cctcgggaca cctggaagaa caaacccctc gtaccctgct gaaaaacatc ctgctgactg
      481 cccctgaaag ctctattctg atgccggaaa gcgtcgttaa accggttcct gctcctcaag
      541 ccgttcaacc ttctcgtcaa gagtcctcct gtggatctct ggagctgcaa ctgcctgaac
      601 tggagcctcc tacaacactg gcaccggggc tgctggcgcc tggtcgtcgt aaacaacgtc
      661 tgcgtctgag cgtgtttcaa caaggtgttg atcagggcct gagtctgtct caagaacctc
      721 aaggcaacgc tgatgcttct agtctgaccc gttctctgaa cctgaccttt gctactccac
      781 tgcaacctca aagcgtccaa cgtcctggac tggctcgccg ccctcctgct cgccgtgccg
      841 ttgatgtggg tgcttttctg cgtgacctgc gtgatacaag cctggcacct ccgaatattg
      901 tgctggagga tacacagccg ttttcacagc ctatggttgg tagccctaac gtttatcact
      961 cactgccgtg tacccctcac actggtgccg aagatgctga acaagctgct ggtcgtaaaa
     1021 cccaaagctc tggaccggga ctgcaaaaaa actctccggg gaaacctgcc caattcctgg
     1081 cgggtgaagc tgaagaagtc aacgcctttg ctctgggatt tctgtctacc agctcgggtg
     1141 ttagcggaga agatgaggtg gaacctctgc atgatggtgt ggaagaagcc gagaagaaaa
     1201 tggaagagga aggggtgtct gtctctgaaa tggaagcgac tggtgctcaa ggaccaagtc
     1261 gtgtcgaaga ggcagaaggc cataccgagg ttaccgaagc cgaaggaagc caagggggtt
     1321 ctggttctca tcatcatcat catcactgaa gatccagatc taagcttggt accacgcgtg
     1381 cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1441 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1501 aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga tcgcgtagtc
     1561 gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc ggtcggacag
     1621 tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc
     1681 atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg gcagtaccgg
     1741 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     1801 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     1861 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
     1921 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     1981 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2041 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2101 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2161 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2221 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2281 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2341 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2401 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2461 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2521 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2581 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2641 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2701 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2761 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2821 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2881 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2941 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3001 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3061 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3121 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3181 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3241 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3301 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3361 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3421 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3481 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3541 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3601 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3661 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3721 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3781 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3841 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3901 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3961 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4021 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4081 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4141 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4201 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4261 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4321 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4381 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4441 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4501 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4561 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4621 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4681 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4741 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4801 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     4861 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     4921 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     4981 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     5041 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     5101 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     5161 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     5221 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     5281 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     5341 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5401 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5461 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5521 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5581 ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga
     5641 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5701 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5761 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5821 aggcgccagc aaccgca
//
