LOCUS       sequence-271348-        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: pFR093
FEATURES             Location/Qualifiers
     source          1..5837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1149..1166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1256..1274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1270..1317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1683..1688,1689..1705,1706..1711))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1738..1756)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1824..1928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1929..1997,1998..2789)
                     /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(2147..2166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2960..3548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3449..3468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3702..3719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3734..3873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3799..3818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3959..3981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3975..4166)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5624..5643
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5727..5746
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5727..5745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             join(5819..5837,1..4)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gagatataca tatggcagac cacaaccctg acagcgattc tacacctcgt acactgctgc
       61 gtcgtgtgct ggatactgct gatcctcgta ctcctcgtcg tcctcgtagc gctcgtgctg
      121 gtgctcgccg tgccctgctg gaaacagcct ctcctcgtaa actgagtggc cagacccgta
      181 ctattgcccg tgggcgttca catggagccc gctcggtcgg tcgtagtgct cacattcaag
      241 cctcgggaca cctggaagaa caagaccctc gtaccctgct gaaaaacatc ctgctgactg
      301 cccctgaaag ctctattctg atgccggaaa gcgtcgttaa accggttcct gctcctcaag
      361 ccgttcaacc ttctcgtcaa gagtcctcct gtggatctct ggagctgcaa ctgcctgaac
      421 tggagcctcc tacaacactg gcaccggggc tgctggcgcc tggtcgtcgt aaacaacgtc
      481 tgcgtctgag cgtgtttcaa caaggtgttg atcagggcct gagtctgtct caagaacctc
      541 aaggcaacgc tgatgcttct agtctgaccc gttctctgaa cctgaccttt gctactccac
      601 tgcaacctca aagcgtccaa cgtcctggac tggctcgccg ccctcctgct cgccgtgccg
      661 ttgatgtggg tgcttttctg cgtgacctgc gtgatacaag cctggcacct ccgaatattg
      721 tgctggagga tacacagccg ttttcacagc ctatggttgg tagccctaac gtttatcact
      781 cactgccgtg tacccctcac actggtgccg aagatgctga acaagctgct ggtcgtaaaa
      841 cccaaagctc tggaccggga ctgcaaaaaa actctccggg gaaacctgcc caattcctgg
      901 cgggtgaagc tgaagaagtc aacgcctttg ctctgggatt tctgtctacc agctcgggtg
      961 ttagcggaga agatgaggtg gaacctctgc atgatggtgt ggaagaagcc gagaagaaaa
     1021 tggaagagga aggggtgtct gtctctgaaa tggaagcgac tggtgctcaa ggaccaagtc
     1081 gtgtcgaaga ggcagaaggc cataccgagg ttaccgaagc cgaaggaagc caagggggtt
     1141 ctggttctca tcatcatcat catcactgaa gatccagatc taagcttggt accacgcgtg
     1201 cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1261 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1321 aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga tcgcgtagtc
     1381 gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc ggtcggacag
     1441 tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc
     1501 atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg gcagtaccgg
     1561 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     1621 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     1681 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
     1741 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     1801 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1861 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1921 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1981 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2041 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2101 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2161 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2221 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2281 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2341 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2401 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2461 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2521 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2581 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2641 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2701 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2761 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2821 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     2881 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3001 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3061 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3121 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3181 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3241 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3301 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3361 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3421 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3481 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3541 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3601 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3661 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3721 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3781 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     3841 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3901 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3961 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4021 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4081 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4141 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4201 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4261 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4321 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4381 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4441 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4501 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4561 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4621 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     4681 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     4741 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     4801 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     4861 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     4921 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     4981 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     5041 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     5101 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     5161 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5221 gcaagattcc gaataccgca agcgacaggc caatcatcgt cgcgctccag cgaaagcggt
     5281 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5341 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5401 ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga
     5461 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5521 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5581 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5641 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5701 ggatcgagat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
     5761 tccctctagt gccggctccg gagagctcca attggaattc gctagctcta gaaataattt
     5821 tgtttaactt taagaag
//
