LOCUS       sequence-270288-        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: pIW069
FEATURES             Location/Qualifiers
     source          1..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    153..352
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             553..969
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     899..921
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     937..953
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1004..1013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1010..1012,1013..1015,1016..1726)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1010..1012,1013..1015,1016..1726)
                     /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(1055..1076)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1316..1335)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1663..1684
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1772..1792
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1805..1849
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2797..2817)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2802..3131
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2982..3117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3139..3537
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3706..4175
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4305..4322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4476..5064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4556..4575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5224..6015,6016..6084))
                     /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     5847..5866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6085..6189)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             join(6220..6601,1..88)
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
ORIGIN
        1 tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga
       61 ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg ggagacccct
      121 gcccagggac caccgaccca ccaccgggag gcaagctggc cagcaactta tctgtgtctg
      181 tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta gttagctaac
      241 tagctctgta tctggcggac ccgtggtgga actgacgagt tctgaacacc cggccgcaac
      301 cctgggagac gtcccaggga ctttgggggc cgtttttgtg gcccgacctg aggaagggag
      361 tcgatgtgga atccgacccc gtcaggatat gtggttctgg taggagacga gaacctaaaa
      421 cagttcccgc ctccgtctga atttttgctt tcggtttgga accgaagccg cgcgtcttgt
      481 ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg
      541 tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg tcactggaaa
      601 gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg ttgggttacc
      661 ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac
      721 cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca
      781 gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc
      841 aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc
      901 cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct
      961 ctaggcgccg gccggatccc agtgtggtgg tacgctaccg gtcgccacca tggtgagcaa
     1021 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1081 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1141 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1201 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1261 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1321 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1381 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1441 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     1501 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     1561 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     1621 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1681 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg gaggggatcg
     1741 gtggagctcc accggtggcg gccgctctag agagaatctt tattttcagg gcgccgccaa
     1801 attcaaagaa accgctgctg ctaaattcga acgccagcac atggacagcg gaggtggagg
     1861 tactagtctc gagatggcaa gcagcgacct ggagcagctg tgctctcatg ttaatgaaaa
     1921 gattggcaat attaagaaaa ccttatcatt aagaaactgt ggacaagagc ccacactgaa
     1981 gaccgtatta aataaaatag gagatgagat cattgtaata aatgaacttc taaataaatt
     2041 ggaattggaa attcagtatc aagaacaaac caacaattca ctcaaggaac tctgtgaatc
     2101 tcttgaagaa gattacaaag acatagaaca tcttaaagaa aacgttcctt cccatttgcc
     2161 tcaagtaaca gtaacccaga gctgtgttaa gggatcagat cttgatcctg aagaaccaat
     2221 caaagttgaa gaacctgaac ccgtaaagaa gcctcccaaa gagcaaagaa gtattaagga
     2281 aatgccattt ataacttgtg atgagtttaa cggggtgcca agttacatga aatcgcgact
     2341 gacatacaac cagattaatg atgttattaa agaaatcaac aaggcagtaa ttagtaaata
     2401 taaaatccta catcagccaa aaaagtctat gaactctgtg accagaaatc tctatcacag
     2461 atttattgat gaagaaacga aggataccaa aggtcgttat ttcatcgtag aggcagatat
     2521 taaggagttc acaactttga aagctgacaa gaagtttcac gtgttactga atattttacg
     2581 acactgccgg aggctatcag aggtccgagg gggaggttta acacgatacg taataacctg
     2641 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt
     2701 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2761 atgaatgcaa ttcgccagca cagtggtcga ccctgtggaa tgtgtgtcag ttagggtgtg
     2821 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2881 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2941 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3001 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3061 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3121 gcttttgcaa aaagcttcat ggccaagcct ttgtctcaag aagaatccac cctcattgaa
     3181 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
     3241 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
     3301 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
     3361 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
     3421 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
     3481 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagat
     3541 cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     3601 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac
     3661 tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa
     3721 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag
     3781 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     3841 gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt
     3901 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
     3961 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
     4021 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa
     4081 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     4141 tgagtgattg actacccgtc agcgggggtc tttcacatgc agcatgtatc aaaattaatt
     4201 tggttttttt tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa
     4261 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4321 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4381 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4441 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4501 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4561 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4621 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4681 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4741 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4801 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4861 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4921 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4981 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5041 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5101 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5161 acctagatcc ttttgcggcc gcaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5221 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5281 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5341 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5401 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5461 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5521 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5581 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5641 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5701 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5761 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5821 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5881 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5941 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6001 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6061 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6121 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6181 ggttccgcgc acatttcccc gaaaagtgcc acctgcagcc tgaatatggg ccaaacagga
     6241 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga acagctgaat
     6301 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     6361 atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag atgtttccag
     6421 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     6481 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     6541 ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc
     6601 c
//
