LOCUS       Exported                5177 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression of human Ska1 microtubule binding domain 
            (MTBD).
ACCESSION   .
VERSION     .
KEYWORDS    pIW039
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5177
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     446..465
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     549..568
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        549..567
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             641..663
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             683..700
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             704..736
                     /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             740..763
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             764..787
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1255..1273)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1269..1316
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(1682..1710)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1737..1755)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1823..1927
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1928..2788
                     /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(2146..2165)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2959..3547
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3448..3467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3701..3718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3733..3872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3798..3817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3958..3980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3974..4165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
       61 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
      121 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
      181 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
      241 gcatcggtcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
      301 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      361 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      421 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      481 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      541 cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta gtgccggctc
      601 cggagagctc caattggaat tcgctagctc tagaaataat tttgtttaac tttaagaagg
      661 agatatacgt atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
      721 tggacagcaa atgggtcggg atctgtacga cgatgacgat aagctggaag ttctgttcca
      781 ggggccccat atgagaagta ttaaggaaat gccatttata acttgtgatg agttcaatgg
      841 tgttccttcg tacatgaaat cccgcttaac ctataatcaa attaatgatg ttattaaaga
      901 aatcaacaag gcagtaatta gtaaatataa aatcctacat cagccaaaaa agtctatgaa
      961 ttctgtgacc agaaatctct atcacagatt tattgatgaa gaaacggccg ataccgccgg
     1021 tcgttatttt atagtggaag ctgacataaa ggagttcaca actttgaaag ctgacaagaa
     1081 gtttcacgtg ttactgaata ttttacgaca ctgccggagg ctatcagagg tccgaggggg
     1141 aggacttact cgttatgtta taacctgagg atccagatct aagcttggta ccacgcgtgc
     1201 gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1261 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1321 aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg
     1381 atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt
     1441 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca
     1501 tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc
     1561 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     1621 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     1681 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     1741 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     1801 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1861 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1921 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1981 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2041 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2101 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2161 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2221 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     2281 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     2341 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2401 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2461 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2521 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2581 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2641 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2701 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2761 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2821 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2881 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2941 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3001 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3061 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4621 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
     4681 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
     4741 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
     4801 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     4861 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
     4921 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
     4981 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
     5041 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
     5101 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
     5161 ctgcttctcg ccgaaac
//
