LOCUS       sequence-271198-        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: pIW040
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..35
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     119..138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        119..137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             211..233
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             253..270
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             274..306
                     /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(310..318,319..333)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             334..357
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(825..843)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      839..886
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1252..1257,1258..1274,1275..1280))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1307..1325)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1393..1497
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1498..1566,1567..2358)
                     /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(1716..1735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2529..3117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3018..3037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3271..3288
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3303..3442
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3368..3387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3528..3550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3544..3735)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
       61 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
      121 atacgactca ctatagggag accacaacgg tttccctcta gtgccggctc cggagagctc
      181 caattggaat tcgctagctc tagaaataat tttgtttaac tttaagaagg agatatacgt
      241 atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
      301 atgggtcggg atctgtacga cgatgacgat aagctggaag ttctgttcca ggggccccat
      361 atgagaagta ttaaggaaat gccatttata acttgtgatg agttcaatgg tgttccttcg
      421 tacatgaaat cccgcttaac ctataatcaa attaatgatg ttattaaaga aatcaacaag
      481 gcagtaatta gtaaatataa aatcctacat cagccaaaaa agtctatgaa ttctgtgacc
      541 agaaatctct atcacagatt tattgatgaa gaaacgaagg ataccaaagg tcgttatttt
      601 atagtggaag ctgacataaa ggagttcaca actttgaaag ctgacaagaa gtttcacgtg
      661 ttactgaata ttttagccca ctgccggagg ctatcagagg tccgaggggg aggacttact
      721 cgttatgtta taacctgagg atccagatct aagcttggta ccacgcgtgc gcgctgatcc
      781 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
      841 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
      901 tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc
      961 caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa
     1021 cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg
     1081 cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     1141 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     1201 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     1261 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     1321 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1381 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1441 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1501 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1561 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1621 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1681 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1741 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1801 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1861 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1921 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1981 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2041 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2101 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2161 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2221 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2281 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2341 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2401 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2641 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3241 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3301 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3361 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3421 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3481 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3541 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3601 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3661 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3721 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3781 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3841 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3901 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3961 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4021 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4081 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4141 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4201 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     4261 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     4321 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     4381 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     4441 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     4501 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     4561 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     4621 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     4681 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     4741 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4801 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4861 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4921 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4981 ctcaagggca tcggtcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5041 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5101 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5161 agcccgaagt ggcgagc
//
