LOCUS       sequence-270295-        6601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of GFP-Ska1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6601)
  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 6601)
  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: pIW085
FEATURES             Location/Qualifiers
     source          1..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             110..526
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     456..478
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     494..510
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      561..570
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(567..569,570..572,573..1283)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(567..569,570..572,573..1283)
                     /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(612..633)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(873..892)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1220..1241
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1329..1349
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1362..1406
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2354..2374)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2359..2688
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2539..2674
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2696..3094
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3263..3732
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3862..3879)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4033..4621)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4113..4132)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4781..5572,5573..5641))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5404..5423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5642..5746)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5777..6246
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6311..6510
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
ORIGIN
        1 tttgctttcg gtttggaacc gaagccgcgc gtcttgtctg ctgcagcgct gcagcatcgt
       61 tctgtgttgt ctctgtctga ctgtgtttct gtatttgtct gaaaattagg gccagactgt
      121 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa
      181 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac
      241 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa
      301 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac
      361 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc
      421 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc
      481 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccggcc ggatcccagt
      541 gtggtggtac gctaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
      601 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      661 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      721 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      781 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      841 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      901 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      961 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1021 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1081 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1141 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1201 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1261 tcggcatgga cgagctgtac aagtccggag gggatcggtg gagctccacc ggtggcggcc
     1321 gctctagaga gaatctttat tttcagggcg ccgccaaatt caaagaaacc gctgctgcta
     1381 aattcgaacg ccagcacatg gacagcggag gtggaggtac tagtctcgag atggcaagca
     1441 gcgacctgga gcagctgtgc tctcatgtta atgaaaagat tggcaatatt aagaaaacct
     1501 tatcattaag aaactgtgga caagagccca cactgaagac cgtattaaat aaaataggag
     1561 atgagatcat tgtaataaat gaacttctaa ataaattgga attggaaatt cagtatcaag
     1621 aacaaaccaa caattcactc aaggaactct gtgaatctct tgaagaagat tacaaagaca
     1681 tagaacatct taaagaaaac gttccttccc atttgcctca agtaacagta acccagagct
     1741 gtgttaaggg atcagatctt gatcctgaag aaccaatcaa agttgaagaa cctgaacccg
     1801 taaagaagcc tcccaaagag caaagaagta ttaaggaaat gccatttata acttgtgatg
     1861 agtttaacgg ggtgccaagt tacatgaaat cgcgactgac atacaaccag attaatgatg
     1921 ttattaaaga aatcaacaag gcagtaatta gtaaatataa aatcctacat cagccagccg
     1981 cctctatgaa ctctgtgacc agaaatctct atcacagatt tattgatgaa gaaacgaagg
     2041 ataccaaagg tcgttatttc atcgtagagg cagatattaa ggagttcaca actttgaaag
     2101 ctgacaagaa gtttcacgtg ttactgaata ttttacgaca ctgccggagg ctatcagagg
     2161 tccgaggggg aggtttaaca cgatacgtaa taacctgacc gcgggcccgg gatccaccgg
     2221 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2281 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattc gccagcacag
     2341 tggtcgaccc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2401 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2461 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2521 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2581 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2641 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttcatggc
     2701 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     2761 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     2821 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     2881 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     2941 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     3001 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     3061 attgctgccc tctggttatg tgtgggaggg ctaagatcga taaaataaaa gattttattt
     3121 agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa
     3181 gtaacgccat tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca
     3241 aggtcaggaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
     3301 tcctgccccg gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata
     3361 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     3421 tccagccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     3481 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     3541 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct
     3601 ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc
     3661 gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc
     3721 gggggtcttt cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta
     3781 cattaaatgg ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3841 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3901 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3961 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4021 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4081 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4141 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4201 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4261 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4321 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4381 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4441 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4501 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4561 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4621 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4681 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tgcggccgca
     4741 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4801 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4861 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4921 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4981 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5041 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5101 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5161 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5221 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5281 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5341 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5401 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5461 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5521 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5581 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5641 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5701 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5761 aagtgccacc tgcagcctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     5821 cccggctcag ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg
     5881 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc
     5941 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     6001 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     6061 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt
     6121 gactgagtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     6181 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     6241 ctttcatttg ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc
     6301 accgggaggc aagctggcca gcaacttatc tgtgtctgtc cgattgtcta gtgtctatga
     6361 ctgattttat gcgcctgcgt cggtactagt tagctaacta gctctgtatc tggcggaccc
     6421 gtggtggaac tgacgagttc tgaacacccg gccgcaaccc tgggagacgt cccagggact
     6481 ttgggggccg tttttgtggc ccgacctgag gaagggagtc gatgtggaat ccgaccccgt
     6541 caggatatgt ggttctggta ggagacgaga acctaaaaca gttcccgcct ccgtctgaat
     6601 t
//
