LOCUS       sequence-270316-        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: pIW084
FEATURES             Location/Qualifiers
     source          1..4869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..82
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        63..81
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             113..135
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     533..550
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     534..550
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             581..603
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             623..640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             668..688
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(692..694,695..697,698..1408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(692..694,695..697,698..1408)
                     /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(737..758)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(998..1017)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1345..1366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2335..2353)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2349..2396
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2463..2554
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3419..3458
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      3489..4077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3978..3997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4231..4248
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4263..4402
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4328..4347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4488..4510
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4504..4695)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 caatgccagc gcttcgttaa tacagatgta ggtgttggca ccatgcatcg atcccgcgaa
       61 attaatacga ctcactatag ggagaccaca acggtttccc tctagaaata attttgttta
      121 actttaagaa ggagatatac atatggaggc ggaggtcgat aagctggaac tgatgttcca
      181 gaaagctgag tctgatctgg attacattca atacaggctg gaatatgaaa tcaagactaa
      241 tcatcctgat tcagcaagtg agaaaaatcc agttacactc ttaaaggaat tgtcagtgat
      301 aaagtctcga tatcaaactt tgtatgcccg ctttaaacca gttgctgttg agcagaaaga
      361 gagtaagagc cgcatttgtg ctactgtgaa aaagactatg aatatgatac aaaaactaca
      421 gaagcaaaca gacctggagc tgtcaccact gactaaagaa gagaaaactg cggcagagca
      481 attcaaattt cacatgccag atttatgagg atccgggccc tgtacaagat cctgtaaaac
      541 gacggccagt gaattggaat tcgctagctc tagaaataat tttgtttaac tttaagaagg
      601 agatatacat atgggcagca gccatcatca tcatcatcac agcagcggat ctggtggtgg
      661 tggtggtgaa aacctgtact tccagggatc catggtgagc aagggcgagg agctgttcac
      721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      781 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      901 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      961 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1141 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1201 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1381 cactctcggc atggacgagc tgtacaaggg tagtggtagt ggtagtcata tggcctcgtc
     1441 agatctggaa caattatgct ctcatgttaa tgaaaagatt ggcaatatta agaaaacctt
     1501 atcattaaga aactgtggcc aggaacctac cttgaaaact gtattaaata aaataggaga
     1561 tgagatcatt gtaataaatg aacttctaaa taaattggaa ttggaaattc agtatcaaga
     1621 acaaaccaac aattcactca aggaactctg tgaatctctt gaagaagatt acaaagacat
     1681 agaacatctt aaagaaaacg ttccttccca tttgcctcaa gtaacagtaa cccagagctg
     1741 tgttaaggga tcagatcttg atcctgaaga accaatcaaa gttgaagaac ctgaacccgt
     1801 aaagaagcct cccaaagagc aaagaagtat taaggaaatg ccatttataa cttgtgatga
     1861 gttcaatggt gttccttcgt acatgaaatc ccgcttaacc tataatcaaa ttaatgatgt
     1921 tattaaagaa atcaacaagg cagtaattag taaatataaa atcctacatc agccaaaaaa
     1981 gtctatgaat tctgtgacca gaaatctcta tcacagattt attgatgaag aaacgaagga
     2041 taccaaaggt cgttatttta tagtggaagc tgacataaag gagttcacaa ctttgaaagc
     2101 tgacaagaag tttcacgtgt tactgaatat tttagcccac tgccggaggc tatcagaggt
     2161 ccgaggggga ggacttactc gttatgttat aacctgaaag cttcgccagg gttttcccag
     2221 tcgagctcga tatcggtacc agcggataac aatttcacat ccggattcgc gaacgcgtct
     2281 cgagagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2341 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2401 aaggaggaac tatatccgac tagtagcccg cctaatgagc gggctttttt ttaattcccc
     2461 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2521 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2581 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctcgt
     2641 gaaagtaaaa gacgcagagg accaattggg ggcacgagtg ggatacatag aactggactt
     2701 gaatagcggt aaaatccttg agagttttcg ccctgaagag cgttttccaa tgatgagcac
     2761 tttcaaagtt ctgctatgtg gagcagtatt atcccgtgta gatgcggggc aagagcaact
     2821 cggacgacga atacactatt cgcagaatga cttggttgaa tactccccag tgacagaaaa
     2881 gcaccttacg gacggaatga cggtaagaga attatgtagt gccgccataa cgatgagtga
     2941 taacactgcg gcgaacttac ttctgacaac catcggtgga ccgaaggaat taaccgcttt
     3001 tttgcacaat atgggagacc atgtaactcg ccttgaccgt tgggaaccag aactgaatga
     3061 agccatacca aacgacgagc gagacaccac aatgcctgcg gcaatggcaa caacattacg
     3121 caaactatta actggcgaac tacttactct ggcttcacgg caacaattaa tagactggct
     3181 tgaagcggat aaagttgcag gaccactact gcgttcggca cttcctgctg gctggtttat
     3241 tgctgataaa tctggggcag gagagcgtgg ttcacggggt atcattgccg cacttggacc
     3301 agatggtaag ccttcccgta tcgtagttat ctacacgacg ggtagtcagg caactatgga
     3361 cgaacgaaat agacagattg ctgaaatagg ggcttcactg attaagcatt ggtaaaccga
     3421 tacaattaaa ggctcctttt ggagcctttt tttttggacg gaccggtaga aaagatcaaa
     3481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3601 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4141 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4201 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4261 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4321 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4381 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4441 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4501 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4561 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4621 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4681 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg ggtaatgata
     4741 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4801 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4861 actcagggt
//
