LOCUS       sequence-271199-        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: pIW087
FEATURES             Location/Qualifiers
     source          1..4869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     369..386
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     370..386
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             417..439
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             459..476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             504..524
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(528..530,531..533,534..1244)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(528..530,531..533,534..1244)
                     /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(573..594)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(834..853)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1181..1202
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2171..2189)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2185..2232
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2299..2390
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3255..3294
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      3325..3913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3814..3833
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4067..4084
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4099..4238
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4164..4183)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4324..4346
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4340..4531)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4768..4787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4768..4786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4818..4840
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tggaactgat gttccagaaa gctgagtctg atctggatta cattcaatac aggctggaat
       61 atgaaatcaa gactaatcat cctgattcag caagtgagaa aaatccagtt acactcttaa
      121 aggaattgtc agtgataaag tctcgatatc aaactttgta tgcccgcttt aaaccagttg
      181 ctgttgagca gaaagagagt aagagccgca tttgtgctac tgtgaaaaag actatgaata
      241 tgatacaaaa actacagaag caaacagacc tggagctgtc accactgact aaagaagaga
      301 aaactgcggc agagcaattc aaatttcaca tgccagattt atgaggatcc gggccctgta
      361 caagatcctg taaaacgacg gccagtgaat tggaattcgc tagctctaga aataattttg
      421 tttaacttta agaaggagat atacatatgg gcagcagcca tcatcatcat catcacagca
      481 gcggatctgg tggtggtggt ggtgaaaacc tgtacttcca gggatccatg gtgagcaagg
      541 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      601 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      661 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      721 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      781 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      841 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
      901 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
      961 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1021 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1081 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1141 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1201 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtagt ggtagtggta
     1261 gtcatatggc ctcgtcagat ctggaacaat tatgctctca tgttaatgaa aagattggca
     1321 atattaagaa aaccttatca ttaagaaact gtggccagga acctaccttg aaaactgtat
     1381 taaataaaat aggagatgag atcattgtaa taaatgaact tctaaataaa ttggaattgg
     1441 aaattcagta tcaagaacaa accaacaatt cactcaagga actctgtgaa tctcttgaag
     1501 aagattacaa agacatagaa catcttaaag aaaacgttcc ttcccatttg cctcaagtaa
     1561 cagtaaccca gagctgtgtt aagggatcag atcttgatcc tgaagaacca atcaaagttg
     1621 aagaacctga acccgtaaag aagcctccca aagagcaaag aagtattaag gaaatgccat
     1681 ttataacttg tgatgagttc aatggtgttc cttcgtacat gaaatcccgc ttaacctata
     1741 atcaaattaa tgatgttatt aaagaaatca acaaggcagt aattagtaaa tataaaatcc
     1801 tacatcagcc agccgcctct atgaattctg tgaccagaaa tctctatcac agatttattg
     1861 atgaagaaac gaaggatacc aaaggtcgtt attttatagt ggaagctgac ataaaggagt
     1921 tcacaacttt gaaagctgac aagaagtttc acgtgttact gaatatttta cgacactgcc
     1981 ggaggctatc agaggtccga gggggaggac ttactcgtta tgttataacc tgaaagcttc
     2041 gccagggttt tcccagtcga gctcgatatc ggtaccagcg gataacaatt tcacatccgg
     2101 attcgcgaac gcgtctcgag agatccggct gctaacaaag cccgaaagga agctgagttg
     2161 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2221 aggggttttt tgctgaaagg aggaactata tccgactagt agcccgccta atgagcgggc
     2281 ttttttttaa ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2461 acccagaaac gctcgtgaaa gtaaaagacg cagaggacca attgggggca cgagtgggat
     2521 acatagaact ggacttgaat agcggtaaaa tccttgagag ttttcgccct gaagagcgtt
     2581 ttccaatgat gagcactttc aaagttctgc tatgtggagc agtattatcc cgtgtagatg
     2641 cggggcaaga gcaactcgga cgacgaatac actattcgca gaatgacttg gttgaatact
     2701 ccccagtgac agaaaagcac cttacggacg gaatgacggt aagagaatta tgtagtgccg
     2761 ccataacgat gagtgataac actgcggcga acttacttct gacaaccatc ggtggaccga
     2821 aggaattaac cgcttttttg cacaatatgg gagaccatgt aactcgcctt gaccgttggg
     2881 aaccagaact gaatgaagcc ataccaaacg acgagcgaga caccacaatg cctgcggcaa
     2941 tggcaacaac attacgcaaa ctattaactg gcgaactact tactctggct tcacggcaac
     3001 aattaataga ctggcttgaa gcggataaag ttgcaggacc actactgcgt tcggcacttc
     3061 ctgctggctg gtttattgct gataaatctg gggcaggaga gcgtggttca cggggtatca
     3121 ttgccgcact tggaccagat ggtaagcctt cccgtatcgt agttatctac acgacgggta
     3181 gtcaggcaac tatggacgaa cgaaatagac agattgctga aataggggct tcactgatta
     3241 agcattggta aaccgataca attaaaggct ccttttggag cctttttttt tggacggacc
     3301 ggtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3361 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3421 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3481 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3541 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3601 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3661 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3721 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3781 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3841 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3901 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3961 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4021 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4081 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4141 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4201 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4261 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4321 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4381 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4441 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4501 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4561 tcatggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4621 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4681 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4741 tggcaccatg catcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt
     4801 ttccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg gaggcggagg
     4861 tcgataagc
//
