LOCUS       sequence-272294-        5177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human Ska1 microtubule binding domain (MTBD)
            mutant.
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: pDK616
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     156..175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        156..174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             248..270
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             290..307
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             311..343
                     /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(347..355,356..370)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             371..394
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(862..880)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      876..923
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1289..1294,1295..1311,1312..1317))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1344..1362)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1430..1534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1535..1603,1604..2395)
                     /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(1753..1772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2566..3154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3055..3074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3308..3325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3340..3479
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3405..3424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3565..3587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3581..3772)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
       61 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
      121 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
      181 acaacggttt ccctctagtg ccggctccgg agagctccaa ttggaattcg ctagctctag
      241 aaataatttt gtttaacttt aagaaggaga tatacgtatg cggggttctc atcatcatca
      301 tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga
      361 tgacgataag ctggaagttc tgttccaggg gccccatatg agaagtatta aggaaatgcc
      421 atttataact tgtgatgagt tcaatggtgt tccttcgtac atgaaatccc gcttaaccta
      481 taatcaaatt aatgatgtta ttaaagaaat caacaaggca gtaattagta aatataaaat
      541 cctacatcag ccaaaaaagt ctatgaattc tgtgaccaga aatctctatc acagatttat
      601 tgatgaagaa acgaaggata ccaaaggtcg ttattttata gtggaagctg acataaagga
      661 gttcacaact ttggccgcgg atgcaaagtt tcacgtgtta ctgaatattt tacgacactg
      721 ccggaggcta tcagaggtcc gagggggagg acttactcgt tatgttataa cctgaggatc
      781 cagatctaag cttggtacca cgcgtgcgcg ctgatccggc tgctaacaaa gcccgaaagg
      841 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
      901 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat ccacaggacg
      961 ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact
     1021 gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc
     1081 aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata
     1141 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg
     1201 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
     1261 tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca
     1321 tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1381 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1441 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1501 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1561 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1621 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1681 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1741 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     1801 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1861 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1921 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1981 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2041 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2101 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2161 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2221 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2281 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2341 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2701 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3361 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3421 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3481 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3541 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3601 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3661 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3721 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3781 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3841 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3901 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3961 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4021 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4081 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4141 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4201 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
     4261 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt
     4321 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga
     4381 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc
     4441 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc
     4501 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga
     4561 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac
     4621 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat
     4681 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc
     4741 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4801 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4861 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4921 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4981 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc
     5041 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5101 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5161 gcctgccacc ataccca
//
