LOCUS       sequence-270306-        6229 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 6229)
  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 6229)
  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: pIW070
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             44..460
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     390..412
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     428..444
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      495..504
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(501..503,504..506,507..1217)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(501..503,504..506,507..1217)
                     /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(546..567)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(807..826)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1154..1175
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1263..1283
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1296..1340
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1916..1936)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1921..2250
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2101..2236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2258..2656
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             2825..3294
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3424..3441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3595..4183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3675..3694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4343..5134,5135..5203))
                     /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     4966..4985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5204..5308)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5339..5808
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    5873..6072
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
ORIGIN
        1 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac
       61 tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc acaaccagtc
      121 ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa
      181 cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa
      241 ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga
      301 agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc
      361 tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg
      421 atcctccctt tatccagccc tcactccttc tctaggcgcc ggccggatcc cagtgtggtg
      481 gtacgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      541 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      601 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      661 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      721 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      781 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
      841 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
      901 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
      961 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1021 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1081 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1141 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1201 tggacgagct gtacaagtcc ggaggggatc ggtggagctc caccggtggc ggccgctcta
     1261 gagagaatct ttattttcag ggcgccgcca aattcaaaga aaccgctgct gctaaattcg
     1321 aacgccagca catggacagc ggaggtggag gtactagtct cgagatggca agcagcgacc
     1381 tggagcagct gtgctctcat gttaatgaaa agattggcaa tattaagaaa accttatcat
     1441 taagaaactg tggacaagag cccacactga agaccgtatt aaataaaata ggagatgaga
     1501 tcattgtaat aaatgaactt ctaaataaat tggaattgga aattcagtat caagaacaaa
     1561 ccaacaattc actcaaggaa ctctgtgaat ctcttgaaga agattacaaa gacatagaac
     1621 atcttaaaga aaacgttcct tcccatttgc ctcaagtaac agtaacccag agctgtgtta
     1681 agggatcaga tcttgatcct gaagaaccaa tcaaagttga agaacctgaa cccgtaaaga
     1741 agcctcccaa agagcaatga ccgcgggccc gggatccacc ggatctagat aactgatcat
     1801 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     1861 cctgaacctg aaacataaaa tgaatgcaat tcgccagcac agtggtcgac cctgtggaat
     1921 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1981 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2041 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2101 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2161 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2221 ggcttttttg gaggcctagg cttttgcaaa aagcttcatg gccaagcctt tgtctcaaga
     2281 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     2341 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     2401 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     2461 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     2521 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     2581 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     2641 tgtgtgggag ggctaagatc gataaaataa aagattttat ttagtctcca gaaaaagggg
     2701 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
     2761 gcatggaaaa atacataact gagaatagag aagttcagat caaggtcagg aacagatgga
     2821 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     2881 ccaagaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc
     2941 tgccccggct cagggccaag aacagatggt ccccagatgc ggtccagccc tcagcagttt
     3001 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     3061 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     3121 caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc
     3181 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     3241 tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca
     3301 gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat ggccatagtt
     3361 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4021 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4261 attatcaaaa aggatcttca cctagatcct tttgcggccg caaatcaatc taaagtatat
     4321 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4381 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4441 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4501 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4561 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4621 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4681 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4741 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4801 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4861 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4921 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4981 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5041 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5101 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5161 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5221 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5281 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgcagcct
     5341 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     5401 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     5461 ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag
     5521 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     5581 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     5641 aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta
     5701 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     5761 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg
     5821 tccgggatcg ggagacccct gcccagggac caccgaccca ccaccgggag gcaagctggc
     5881 cagcaactta tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc
     5941 gtcggtacta gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt
     6001 tctgaacacc cggccgcaac cctgggagac gtcccaggga ctttgggggc cgtttttgtg
     6061 gcccgacctg aggaagggag tcgatgtgga atccgacccc gtcaggatat gtggttctgg
     6121 taggagacga gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttgga
     6181 accgaagccg cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtg
//
