LOCUS       sequence-270298-        6601 bp    DNA     circular SYN 15-APR-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 Apr 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: pIW088
FEATURES             Location/Qualifiers
     source          1..6601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             140..556
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     486..508
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     524..540
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      591..600
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(597..599,600..602,603..1313)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(597..599,600..602,603..1313)
                     /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(642..663)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(903..922)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1250..1271
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1359..1379
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1392..1436
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2384..2404)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2389..2718
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2569..2704
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2726..3124
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3293..3762
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3892..3909)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4063..4651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4143..4162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4811..5602,5603..5671))
                     /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     5434..5453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5672..5776)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5807..6276
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    6341..6540
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
ORIGIN
        1 cctaaaacag ttcccgcctc cgtctgaatt tttgctttcg gtttggaacc gaagccgcgc
       61 gtcttgtctg ctgcagcgct gcagcatcgt tctgtgttgt ctctgtctga ctgtgtttct
      121 gtatttgtct gaaaattagg gccagactgt taccactccc ttaagtttga ccttaggtca
      181 ctggaaagat gtcgagcgga tcgctcacaa ccagtcggta gatgtcaaga agagacgttg
      241 ggttaccttc tgctctgcag aatggccaac ctttaacgtc ggatggccgc gagacggcac
      301 ctttaaccga gacctcatca cccaggttaa gatcaaggtc ttttcacctg gcccgcatgg
      361 acacccagac caggtcccct acatcgtgac ctgggaagcc ttggcttttg acccccctcc
      421 ctgggtcaag ccctttgtac accctaagcc tccgcctcct cttcctccat ccgccccgtc
      481 tctccccctt gaacctcctc gttcgacccc gcctcgatcc tccctttatc cagccctcac
      541 tccttctcta ggcgccggcc ggatcccagt gtggtggtac gctaccggtc gccaccatgg
      601 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      661 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      721 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      781 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      841 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      901 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      961 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1021 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1081 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1141 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1201 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1261 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggag
     1321 gggatcggtg gagctccacc ggtggcggcc gctctagaga gaatctttat tttcagggcg
     1381 ccgccaaatt caaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagcggag
     1441 gtggaggtac tagtctcgag atggcaagca gcgacctgga gcagctgtgc tctcatgtta
     1501 atgaaaagat tggcaatatt aagaaaacct tatcattaag aaactgtgga caagagccca
     1561 cactgaagac cgtattaaat aaaataggag atgagatcat tgtaataaat gaacttctaa
     1621 ataaattgga attggaaatt cagtatcaag aacaaaccaa caattcactc aaggaactct
     1681 gtgaatctct tgaagaagat tacaaagaca tagaacatct taaagaaaac gttccttccc
     1741 atttgcctca agtaacagta acccagagct gtgttaaggg atcagatctt gatcctgaag
     1801 aaccaatcaa agttgaagaa cctgaacccg taaagaagcc tcccaaagag caaagaagta
     1861 ttaaggaaat gccatttata acttgtgatg agtttaacgg ggtgccaagt tacatgaaat
     1921 cgcgactgac atacaaccag attaatgatg ttattaaaga aatcaacaag gcagtaatta
     1981 gtaaatataa aatcctacat cagccaaaaa agtctatgaa ctctgtgacc agaaatctct
     2041 atcacagatt tattgatgaa gaaacgaagg ataccaaagg tcgttatttc atcgtagagg
     2101 ctgacataaa ggagttcaca actttggccg cggatgcaaa gtttcacgtg ttactgaata
     2161 ttttacgaca ctgccggagg ctatcagagg tccgaggggg aggtttaaca cgatacgtaa
     2221 taacctgacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     2281 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2341 acataaaatg aatgcaattc gccagcacag tggtcgaccc tgtggaatgt gtgtcagtta
     2401 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2461 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2521 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2581 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2641 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2701 ggcctaggct tttgcaaaaa gcttcatggc caagcctttg tctcaagaag aatccaccct
     2761 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     2821 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     2881 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     2941 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     3001 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     3061 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     3121 ctaagatcga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     3181 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat
     3241 acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat
     3301 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     3361 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     3421 gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat
     3481 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     3541 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     3601 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg
     3661 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     3721 tctcctctga gtgattgact acccgtcagc gggggtcttt cacatgcagc atgtatcaaa
     3781 attaatttgg ttttttttct taagtattta cattaaatgg ccatagttgc attaatgaat
     3841 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3901 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3961 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4021 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4081 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4141 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4201 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4261 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4321 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4381 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4441 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4501 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4561 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4621 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4681 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4741 gatcttcacc tagatccttt tgcggccgca aatcaatcta aagtatatat gagtaaactt
     4801 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4861 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4921 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4981 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5041 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5101 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5161 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5221 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5281 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5341 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5401 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5461 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5521 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5581 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5641 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5701 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5761 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgcagcctga atatgggcca
     5821 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca
     5881 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     5941 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg
     6001 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     6061 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     6121 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
     6181 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     6241 ctgagtgatt gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg
     6301 agacccctgc ccagggacca ccgacccacc accgggaggc aagctggcca gcaacttatc
     6361 tgtgtctgtc cgattgtcta gtgtctatga ctgattttat gcgcctgcgt cggtactagt
     6421 tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg
     6481 gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag
     6541 gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga
     6601 a
//
