LOCUS       sequence-271249-        5177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human Ska1 microtubule binding domain 
            (MTBD).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5177)
  AUTHORS   Monda JK, Whitney IP, Tarasovetc EV, Wilson-Kubalek E, Milligan RA, 
            Grishchuk EL, Cheeseman IM
  TITLE     Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1
            Requires Diverse Tubulin-Interacting Surfaces.
  JOURNAL   Curr Biol. 2017 Dec 4;27(23):3666-3675.e6. doi: 
            10.1016/j.cub.2017.10.018. Epub 2017 Nov 16.
  PUBMED    29153323
REFERENCE   2  (bases 1 to 5177)
  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.cub.2017.10.018"; journalName: "Curr Biol"; date: 
            "2017-12-4- 4"; volume: "27"; issue: "23"; pages: "3666-3675.e6"
COMMENT     Sequence Label: pJS278
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        79..97
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             171..193
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             213..230
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             234..266
                     /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(270..278,279..293)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             294..317
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(785..803)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      799..846
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1212..1217,1218..1234,1235..1240))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1267..1285)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1353..1457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1458..1526,1527..2318)
                     /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(1676..1695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2489..3077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2978..2997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3231..3248
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3263..3402
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3328..3347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3488..3510
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3504..3695)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5153..5172
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
       61 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
      121 gtgccggctc cggagagctc caattggaat tcgctagctc tagaaataat tttgtttaac
      181 tttaagaagg agatatacgt atgcggggtt ctcatcatca tcatcatcat ggtatggcta
      241 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aagctggaag
      301 ttctgttcca ggggccccat atgagaagta ttaaggaaat gccatttata acttgtgatg
      361 agttcaatgg tgttccttcg tacatgaaat ccgccttaac ctataatcaa attaatgatg
      421 ttattaaaga aatcaacaag gcagtaatta gtaaatataa aatcctacat cagccaaaaa
      481 agtctatgaa ttctgtgacc agaaatctct atcacagatt tattgatgaa gaaacgaagg
      541 ataccaaagg tcgttatttt atagtggaag ctgacataaa ggagttcaca actttgaaag
      601 ctgacaagaa gtttcacgtg ttactgaata ttttacgaca ctgccggagg ctatcagagg
      661 tccgaggggg aggacttact cgttatgtta taacctgagg atccagatct aagcttggta
      721 ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      781 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      841 tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat
      901 cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg
      961 gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag
     1021 cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg
     1081 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     1141 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     1201 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1261 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1321 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1381 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1441 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1501 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1561 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1621 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1681 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1741 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1801 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1861 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1921 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1981 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2041 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2101 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2161 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2221 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2281 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2341 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2401 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2461 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2521 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2581 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2641 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2701 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2761 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2821 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2881 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2941 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3001 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3061 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3121 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3181 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3241 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3301 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3361 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3421 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3481 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3541 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3601 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3661 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3721 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3781 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3841 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3901 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3961 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4021 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4081 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4141 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     4201 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     4261 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     4321 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4381 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4441 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4501 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4561 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4621 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4681 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4741 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4801 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4861 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4921 agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct
     4981 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5041 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5101 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5161 cggcgatata ggcgcca
//
