LOCUS       sequence-270308-        4869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GFP-Ska1 and Ska2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4869)
  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 4869)
  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: pIW090
FEATURES             Location/Qualifiers
     source          1..4869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        96..114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             146..168
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     566..583
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     567..583
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             614..636
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             656..673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             701..721
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(725..727,728..730,731..1441)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(725..727,728..730,731..1441)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(770..791)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1031..1050)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1378..1399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2368..2386)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2382..2429
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2496..2587
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3452..3491
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      3522..4110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4011..4030
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4264..4281
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4296..4435
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4361..4380)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4521..4543
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4537..4728)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
       61 gtaggtgttg gcaccatgca tcgatcccgc gaaattaata cgactcacta tagggagacc
      121 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgga
      181 ggcggaggtc gataagctgg aactgatgtt ccagaaagct gagtctgatc tggattacat
      241 tcaatacagg ctggaatatg aaatcaagac taatcatcct gattcagcaa gtgagaaaaa
      301 tccagttaca ctcttaaagg aattgtcagt gataaagtct cgatatcaaa ctttgtatgc
      361 ccgctttaaa ccagttgctg ttgagcagaa agagagtaag agccgcattt gtgctactgt
      421 gaaaaagact atgaatatga tacaaaaact acagaagcaa acagacctgg agctgtcacc
      481 actgactaaa gaagagaaaa ctgcggcaga gcaattcaaa tttcacatgc cagatttatg
      541 aggatccggg ccctgtacaa gatcctgtaa aacgacggcc agtgaattgg aattcgctag
      601 ctctagaaat aattttgttt aactttaaga aggagatata catatgggca gcagccatca
      661 tcatcatcat cacagcagcg gatctggtgg tggtggtggt gaaaacctgt acttccaggg
      721 atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      781 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      841 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      901 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      961 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1021 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1081 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1141 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1201 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1261 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1321 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1381 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1441 gggtagtggt agtggtagtc atatggcctc gtcagatctg gaacaattat gctctcatgt
     1501 taatgaaaag attggcaata ttaagaaaac cttatcatta agaaactgtg gccaggaacc
     1561 taccttgaaa actgtattaa ataaaatagg agatgagatc attgtaataa atgaacttct
     1621 aaataaattg gaattggaaa ttcagtatca agaacaaacc aacaattcac tcaaggaact
     1681 ctgtgaatct cttgaagaag attacaaaga catagaacat cttaaagaaa acgttccttc
     1741 ccatttgcct caagtaacag taacccagag ctgtgttaag ggatcagatc ttgatcctga
     1801 agaaccaatc aaagttgaag aacctgaacc cgtaaagaag cctcccaaag agcaaagaag
     1861 tattaaggaa atgccattta taacttgtga tgagttcaat ggtgttcctt cgtacatgaa
     1921 atcccgctta acctataatc aaattaatga tgttattaaa gaaatcaaca aggcagtaat
     1981 tagtaaatat aaaatcctac atcagccaaa aaagtctatg aattctgtga ccagaaatct
     2041 ctatcacaga tttattgatg aagaaacgaa ggataccaaa ggtcgttatt ttatagtgga
     2101 agctgacata aaggagttca caactttggc cgcggatgca aagtttcacg tgttactgaa
     2161 tattttacga cactgccgga ggctatcaga ggtccgaggg ggaggactta ctcgttatgt
     2221 tataacctga aagcttcgcc agggttttcc cagtcgagct cgatatcggt accagcggat
     2281 aacaatttca catccggatt cgcgaacgcg tctcgagaga tccggctgct aacaaagccc
     2341 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2401 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc gactagtagc
     2461 ccgcctaatg agcgggcttt tttttaattc ccctatttgt ttatttttct aaatacattc
     2521 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2581 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2641 ccttcctgtt tttgctcacc cagaaacgct cgtgaaagta aaagacgcag aggaccaatt
     2701 gggggcacga gtgggataca tagaactgga cttgaatagc ggtaaaatcc ttgagagttt
     2761 tcgccctgaa gagcgttttc caatgatgag cactttcaaa gttctgctat gtggagcagt
     2821 attatcccgt gtagatgcgg ggcaagagca actcggacga cgaatacact attcgcagaa
     2881 tgacttggtt gaatactccc cagtgacaga aaagcacctt acggacggaa tgacggtaag
     2941 agaattatgt agtgccgcca taacgatgag tgataacact gcggcgaact tacttctgac
     3001 aaccatcggt ggaccgaagg aattaaccgc ttttttgcac aatatgggag accatgtaac
     3061 tcgccttgac cgttgggaac cagaactgaa tgaagccata ccaaacgacg agcgagacac
     3121 cacaatgcct gcggcaatgg caacaacatt acgcaaacta ttaactggcg aactacttac
     3181 tctggcttca cggcaacaat taatagactg gcttgaagcg gataaagttg caggaccact
     3241 actgcgttcg gcacttcctg ctggctggtt tattgctgat aaatctgggg caggagagcg
     3301 tggttcacgg ggtatcattg ccgcacttgg accagatggt aagccttccc gtatcgtagt
     3361 tatctacacg acgggtagtc aggcaactat ggacgaacga aatagacaga ttgctgaaat
     3421 aggggcttca ctgattaagc attggtaaac cgatacaatt aaaggctcct tttggagcct
     3481 ttttttttgg acggaccggt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3541 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3601 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3661 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3721 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3781 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3841 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3901 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3961 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4021 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4081 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4141 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4201 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4261 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4321 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4381 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4441 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4501 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4561 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4621 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4681 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4741 gtaaggggga tttctgttca tggggtaatg ataccgatga aacgagagag gatgctcacg
     4801 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4861 gcggtatgg
//
