LOCUS       sequence-270311-        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: pIW095
FEATURES             Location/Qualifiers
     source          1..4869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     201..218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    233..372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(298..317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     458..480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(474..665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     902..921
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        902..920
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             952..974
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     1372..1389
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1373..1389
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             1420..1442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1462..1479
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1507..1527
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1531..1533,1534..1536,1537..2247)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1531..1533,1534..1536,1537..2247)
                     /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(1576..1597)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1837..1856)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2184..2205
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3174..3192)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3188..3235
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3302..3393
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4258..4297
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      join(4328..4869,1..47)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4817..4836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
       61 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
      241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
      301 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
      361 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      421 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      481 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
      541 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
      601 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
      661 gtcactgatg cctccgtgta agggggattt ctgttcatgg ggtaatgata ccgatgaaac
      721 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
      781 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
      841 aatgccagcg cttcgttaat acagatgtag gtgttggcac catgcatcga tcccgcgaaa
      901 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagaaataa ttttgtttaa
      961 ctttaagaag gagatataca tatggaggcg gaggtcgata agctggaact gatgttccag
     1021 aaagctgagt ctgatctgga ttacattcaa tacaggctgg aatatgaaat caagactaat
     1081 catcctgatt cagcaagtga gaaaaatcca gttacactct taaaggaatt gtcagtgata
     1141 aagtctcgat atcaaacttt gtatgcccgc tttaaaccag ttgctgttga gcagaaagag
     1201 agtaagagcc gcatttgtgc tactgtgaaa aagactatga atatgataca aaaactacag
     1261 aagcaaacag acctggagct gtcaccactg actaaagaag agaaaactgc ggcagagcaa
     1321 ttcaaatttc acatgccaga tttatgagga tccgggccct gtacaagatc ctgtaaaacg
     1381 acggccagtg aattggaatt cgctagctct agaaataatt ttgtttaact ttaagaagga
     1441 gatatacata tgggcagcag ccatcatcat catcatcaca gcagcggatc tggtggtggt
     1501 ggtggtgaaa acctgtactt ccagggatcc atggtgagca agggcgagga gctgttcacc
     1561 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1621 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1681 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1741 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1801 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1861 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1921 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1981 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2041 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2101 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2161 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2221 actctcggca tggacgagct gtacaagggt agtggtagtg gtagtcatat ggcctcgtca
     2281 gatctggaac aattatgctc tcatgttaat gaaaagattg gcaatattaa gaaaacctta
     2341 tcattaagaa actgtggcca ggaacctacc ttgaaaactg tattaaataa aataggagat
     2401 gagatcattg taataaatga acttctaaat aaattggaat tggaaattca gtatcaagaa
     2461 caaaccaaca attcactcaa ggaactctgt gaatctcttg aagaagatta caaagacata
     2521 gaacatctta aagaaaacgt tccttcccat ttgcctcaag taacagtaac ccagagctgt
     2581 gttaagggat cagatcttga tcctgaagaa ccaatcaaag ttgaagaacc tgaacccgta
     2641 aagaagcctc ccaaagagca aagaagtatt aaggaaatgc catttataac ttgtgatgag
     2701 ttcaatggtg ttccttcgta catgaaatcc gccttaacct ataatcaaat taatgatgtt
     2761 attaaagaaa tcaacaaggc agtaattagt aaatataaaa tcctacatca gccaaaaaag
     2821 tctatgaatt ctgtgaccag aaatctctat cacagattta ttgatgaaga aacgaaggat
     2881 accaaaggtc gttattttat agtggaagct gacataaagg agttcacaac tttgaaagct
     2941 gacaagaagt ttcacgtgtt actgaatatt ttacgacact gccggaggct atcagaggtc
     3001 cgagggggag gacttactcg ttatgttata acctgaaagc ttcgccaggg ttttcccagt
     3061 cgagctcgat atcggtacca gcggataaca atttcacatc cggattcgcg aacgcgtctc
     3121 gagagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     3181 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3241 aggaggaact atatccgact agtagcccgc ctaatgagcg ggcttttttt taattcccct
     3301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctcgtg
     3481 aaagtaaaag acgcagagga ccaattgggg gcacgagtgg gatacataga actggacttg
     3541 aatagcggta aaatccttga gagttttcgc cctgaagagc gttttccaat gatgagcact
     3601 ttcaaagttc tgctatgtgg agcagtatta tcccgtgtag atgcggggca agagcaactc
     3661 ggacgacgaa tacactattc gcagaatgac ttggttgaat actccccagt gacagaaaag
     3721 caccttacgg acggaatgac ggtaagagaa ttatgtagtg ccgccataac gatgagtgat
     3781 aacactgcgg cgaacttact tctgacaacc atcggtggac cgaaggaatt aaccgctttt
     3841 ttgcacaata tgggagacca tgtaactcgc cttgaccgtt gggaaccaga actgaatgaa
     3901 gccataccaa acgacgagcg agacaccaca atgcctgcgg caatggcaac aacattacgc
     3961 aaactattaa ctggcgaact acttactctg gcttcacggc aacaattaat agactggctt
     4021 gaagcggata aagttgcagg accactactg cgttcggcac ttcctgctgg ctggtttatt
     4081 gctgataaat ctggggcagg agagcgtggt tcacggggta tcattgccgc acttggacca
     4141 gatggtaagc cttcccgtat cgtagttatc tacacgacgg gtagtcaggc aactatggac
     4201 gaacgaaata gacagattgc tgaaataggg gcttcactga ttaagcattg gtaaaccgat
     4261 acaattaaag gctccttttg gagccttttt ttttggacgg accggtagaa aagatcaaag
     4321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4441 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4861 tctgacttg
//
