LOCUS       sequence-272665-        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: pJS249
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     291..310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     394..413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        394..412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             486..508
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             528..545
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             549..581
                     /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(585..593,594..608)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             609..632
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1100..1118)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1114..1161
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1527..1532,1533..1549,1550..1555))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1582..1600)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1668..1772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1773..1841,1842..2633)
                     /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(1991..2010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2804..3392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3293..3312
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3546..3563
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3578..3717
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3643..3662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3803..3825
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3819..4010)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
       61 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
      121 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
      181 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
      241 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      301 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      361 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
      421 aacggtttcc ctctagtgcc ggctccggag agctccaatt ggaattcgct agctctagaa
      481 ataattttgt ttaactttaa gaaggagata tacgtatgcg gggttctcat catcatcatc
      541 atcatggtat ggctagcatg actggtggac agcaaatggg tcgggatctg tacgacgatg
      601 acgataagct ggaagttctg ttccaggggc cccatatgag aagtattaag gaaatgccat
      661 ttataacttg tgatgagttc aatggtgttc cttcgtacat gaaatcccgc ttaacctata
      721 atcaaattaa tgatgttatt aaagaaatca acaaggcagt aattagtaaa tataaaatcc
      781 tacatcagcc aaaaaagtct atgaattctg tgaccagaaa tctctatcac agatttattg
      841 atgaagaaac gaaggatacc aaaggtcgtt attttatagt ggaagctgac ataaaggagt
      901 tcacaacttt gaaagctgac aagaagtttc acgtgttact gaatatttta cgacactgcc
      961 ggaggctatc agaggtccga gggggaggac ttactcgtta tgttataacc tgaggatcca
     1021 gatctaagct tggtaccacg cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa
     1081 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1141 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg
     1201 tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg
     1261 gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa
     1321 cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc
     1381 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     1441 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
     1501 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
     1561 agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1621 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1681 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1741 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1801 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1861 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1921 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1981 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2041 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2101 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2161 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2221 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2281 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2341 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2401 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2461 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2521 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2581 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2641 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2701 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2761 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2821 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2881 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2941 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3001 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3061 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3121 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3181 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3241 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3301 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3361 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3421 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3481 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3541 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3601 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3661 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3721 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3781 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3841 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3901 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3961 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4021 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4081 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4141 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4201 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4261 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4321 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4381 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4441 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     4501 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     4561 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     4621 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     4681 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     4741 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     4801 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     4861 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     4921 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     4981 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5041 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5101 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5161 gtcctacgag ttgcatg
//
