LOCUS       sequence-271206-        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: pJS183
FEATURES             Location/Qualifiers
     source          1..4869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..196
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     180..196
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             227..249
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             269..286
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             314..334
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(338..340,341..343,344..1054)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(338..340,341..343,344..1054)
                     /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(383..404)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(644..663)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     991..1012
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1981..1999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1995..2042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2109..2200
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3065..3104
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      3135..3723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3624..3643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3877..3894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3909..4048
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3974..3993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4134..4156
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4150..4341)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4578..4597
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4578..4596
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4628..4650
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gaaagagagt aagagccgca tttgtgctac tgtgaaaaag actatgaata tgatacaaaa
       61 actacagaag caaacagacc tggagctgtc accactgact aaagaagaga aaactgcggc
      121 agagcaattc aaatttcaca tgccagattt atgaggatcc gggccctgta caagatcctg
      181 taaaacgacg gccagtgaat tggaattcgc tagctctaga aataattttg tttaacttta
      241 agaaggagat atacatatgg gcagcagcca tcatcatcat catcacagca gcggatctgg
      301 tggtggtggt ggtgaaaacc tgtacttcca gggatccatg gtgagcaagg gcgaggagct
      361 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      421 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      481 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      541 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      601 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      661 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
      721 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
      781 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
      841 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
      901 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
      961 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1021 cgggatcact ctcggcatgg acgagctgta caagggtagt ggtagtggta gtcatatggc
     1081 ctcgtcagat ctggaacaat tatgctctca tgttaatgaa aagattggca atattaagaa
     1141 aaccttatca ttaagaaact gtggccagga acctaccttg aaaactgtat taaataaaat
     1201 aggagatgag atcattgtaa taaatgaact tctaaataaa ttggaattgg aaattcagta
     1261 tcaagaacaa accaacaatt cactcaagga actctgtgaa tctcttgaag aagattacaa
     1321 agacatagaa catcttaaag aaaacgttcc ttcccatttg cctcaagtaa cagtaaccca
     1381 gagctgtgtt aagggatcag atcttgatcc tgaagaacca atcaaagttg aagaacctga
     1441 acccgtaaag aagcctccca aagagcaaag aagtattaag gaaatgccat ttataacttg
     1501 tgatgagttc aatggtgttc cttcgtacat gaaatcccgc ttaacctata atcaaattaa
     1561 tgatgttatt aaagaaatca acaaggcagt aattagtaaa tataaaatcc tacatcagcc
     1621 aaaaaagtct atgaattctg tgaccagaaa tctctatcac agatttattg atgaagaaac
     1681 gaaggatacc aaaggtcgtt attttatagt ggaagctgac ataaaggagt tcacaacttt
     1741 gaaagctgac aagaagtttc acgtgttact gaatatttta cgacactgcc ggaggctatc
     1801 agaggtccga gggggaggac ttactcgtta tgttataacc tgaaagcttc gccagggttt
     1861 tcccagtcga gctcgatatc ggtaccagcg gataacaatt tcacatccgg attcgcgaac
     1921 gcgtctcgag agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1981 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     2041 tgctgaaagg aggaactata tccgactagt agcccgccta atgagcgggc ttttttttaa
     2101 ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2161 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2221 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2281 gctcgtgaaa gtaaaagacg cagaggacca attgggggca cgagtgggat acatagaact
     2341 ggacttgaat agcggtaaaa tccttgagag ttttcgccct gaagagcgtt ttccaatgat
     2401 gagcactttc aaagttctgc tatgtggagc agtattatcc cgtgtagatg cggggcaaga
     2461 gcaactcgga cgacgaatac actattcgca gaatgacttg gttgaatact ccccagtgac
     2521 agaaaagcac cttacggacg gaatgacggt aagagaatta tgtagtgccg ccataacgat
     2581 gagtgataac actgcggcga acttacttct gacaaccatc ggtggaccga aggaattaac
     2641 cgcttttttg cacaatatgg gagaccatgt aactcgcctt gaccgttggg aaccagaact
     2701 gaatgaagcc ataccaaacg acgagcgaga caccacaatg cctgcggcaa tggcaacaac
     2761 attacgcaaa ctattaactg gcgaactact tactctggct tcacggcaac aattaataga
     2821 ctggcttgaa gcggataaag ttgcaggacc actactgcgt tcggcacttc ctgctggctg
     2881 gtttattgct gataaatctg gggcaggaga gcgtggttca cggggtatca ttgccgcact
     2941 tggaccagat ggtaagcctt cccgtatcgt agttatctac acgacgggta gtcaggcaac
     3001 tatggacgaa cgaaatagac agattgctga aataggggct tcactgatta agcattggta
     3061 aaccgataca attaaaggct ccttttggag cctttttttt tggacggacc ggtagaaaag
     3121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3241 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3781 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3841 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3901 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3961 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4021 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4081 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4141 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4201 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4261 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4321 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatggggta
     4381 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4441 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4501 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tggcaccatg
     4561 catcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag
     4621 aaataatttt gtttaacttt aagaaggaga tatacatatg gaggcggagg tcgataagct
     4681 ggaactgatg ttccagaaag ctgagtctga tctggattac attcaataca ggctggaata
     4741 tgaaatcaag actaatcatc ctgattcagc aagtgagaaa aatccagtta cactcttaaa
     4801 ggaattgtca gtgataaagt ctcgatatca aactttgtat gcccgcttta aaccagttgc
     4861 tgttgagca
//
