LOCUS       sequence-271194-        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: pIW028
FEATURES             Location/Qualifiers
     source          1..5177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     191..210
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        191..209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             283..305
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             325..342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             346..378
                     /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(382..390,391..405)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             406..429
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(897..915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      911..958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1324..1329,1330..1346,1347..1352))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1379..1397)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1465..1569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1570..1638,1639..2430)
                     /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(1788..1807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2601..3189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3090..3109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3343..3360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3375..3514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3440..3459)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3600..3622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3616..3807)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
       61 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
      121 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
      181 cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc
      241 tccggagagc tccaattgga attcgctagc tctagaaata attttgttta actttaagaa
      301 ggagatatac gtatgcgggg ttctcatcat catcatcatc atggtatggc tagcatgact
      361 ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataagctgga agttctgttc
      421 caggggcccc atatgagaag tattaaggaa atgccattta taacttgtga tgagttcaat
      481 ggtgttcctt cgtacatgaa atcccgctta acctataatc aaattaatga tgttattaaa
      541 gaaatcaaca aggcagtaat tagtaaatat aaaatcctac atcagccagc cgcctctatg
      601 aattctgtga ccagaaatct ctatcacaga tttattgatg aagaaacgaa ggataccaaa
      661 ggtcgttatt ttatagtgga agctgacata aaggagttca caactttgaa agctgacaag
      721 aagtttcacg tgttactgaa tattttacga cactgccgga ggctatcaga ggtccgaggg
      781 ggaggactta ctcgttatgt tataacctga ggatccagat ctaagcttgg taccacgcgt
      841 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      901 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      961 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     1021 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     1081 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     1141 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtaccg
     1201 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     1261 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     1321 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     1381 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1441 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1501 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1561 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1621 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1681 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1741 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1801 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1861 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1921 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1981 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2041 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2101 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2161 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2221 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2281 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2341 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2401 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2461 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2521 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2581 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2641 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2941 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3001 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3061 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3121 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3181 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3241 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3301 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3361 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3421 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3481 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3541 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3601 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3661 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3721 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3781 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3841 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3901 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3961 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4021 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4081 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4141 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4201 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4261 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     4321 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     4381 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     4441 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     4501 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     4561 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     4621 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     4681 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     4741 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     4801 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4861 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4921 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4981 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5041 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     5101 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5161 aaggagatgg cgcccaa
//
