LOCUS       sequence-80732-e        7254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag SKA3 for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7254)
  AUTHORS   Welburn JP, Grishchuk EL, Backer CB, Wilson-Kubalek EM, Yates JR 
            3rd, Cheeseman IM
  TITLE     The human kinetochore Ska1 complex facilitates microtubule 
            depolymerization-coupled motility.
  JOURNAL   Dev Cell. 2009 Mar;16(3):374-85. doi: 10.1016/j.devcel.2009.01.011.
  PUBMED    19289083
REFERENCE   2  (bases 1 to 7254)
  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.devcel.2009.01"; journalName: "Dev Cell"; date: 
            "2009-03"; volume: "16"; issue: "3"; pages: "374-85"
COMMENT     Sequence Label: pIC306
FEATURES             Location/Qualifiers
     source          1..7254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             934..1350
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1318..1334
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1385..1394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1391..1393,1394..1396,1397..2107)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1391..1393,1394..1396,1397..2107)
                     /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(1436..1457)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1697..1716)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2044..2065
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2153..2173
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2186..2230
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3669..3689)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3674..4003
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3854..3989
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4018..4065
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4084..4482
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             4535..5128
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5329..5346)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5500..6088)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5580..5599)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6266..7057,7058..7126))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6889..6908
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7127..7231)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt
      901 ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt
      961 ttgaccttag atcactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc
     1021 aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg
     1081 ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca
     1141 cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct
     1201 tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttctt
     1261 ccatccgcgc cgtctctccc ccttgaacct cctctttcga ccccgcctca atcctccctt
     1321 tatccagccc tcactccttc tctaggcgcc ggccggatcc cagtgtggtg gtacgctacc
     1381 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1441 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1501 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1561 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1621 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1681 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1741 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1801 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1861 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1921 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1981 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2041 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2101 gtacaagtcc ggaggggatc ggtggagctc caccggtggc ggccgctcta gagagaatct
     2161 ttattttcag ggcgccgcca aattcaaaga aaccgctgct gctaaattcg aacgccagca
     2221 catggacagc ggaggtggag gtactagtct cgagatggac cctatccgga gcttctgcgg
     2281 gaagctgcgg tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct
     2341 ggacggagag gaaagcgact ttgaagatta tccaatgaga attttatatg accttcattc
     2401 agaagttcag actctaaagg atgatattaa tattcttctt gataaagcaa gattggaaaa
     2461 tcaagaaggc attgatttca taaaggcaac aaaagtacta atggaaaaaa attcaatgga
     2521 tattatgaaa ataagagagt atttccagaa gtatggatat agtccacgtg tcaagaaaaa
     2581 ttcagtacac gagcaagaag ccattaactc tgacccagag ttgtctaatt gtgaaaattt
     2641 tcagaagact gatgtgaaag atgatctgtc tgatcctcct gttgcaagca gttgtatttc
     2701 tgagaagtct ccacgtagtc cacaactttc agattttgga cttgagcggt acatcgtatc
     2761 ccaagttcta ccaaaccctc cacaggcagt gaacaactat aaggaagagc ccgtaattgt
     2821 aaccccacct accaaacaat cactagtaaa agtactaaaa actccaaaat gtgcactaaa
     2881 aatggatgat tttgagtgtg taactcctaa attagaacac tttggtatct ctgaatatac
     2941 tatgtgttta aatgaagatt acacaatggg acttaaaaat gcgaggaata ataaaagtga
     3001 ggaggccata gatacagaat ccaggctcaa tgataatgtt tttgccactc ccagccccat
     3061 catccagcag ttggaaaaaa gtgatgccga atataccaac tctcctttgg tacctacatt
     3121 ctgtactcct ggtttgaaaa ttccatctac aaagaacagc atagctttgg tatccacaaa
     3181 ttacccatta tcaaaaacaa atagttcatc aaatgatttg gaagttgaag atcgtacttc
     3241 gttggtttta aattcagaca catgctttga gaatttaaca gatccctctt cacctacgat
     3301 ttcttcttat gagaatctgc tcagaacacc tacacctcca gaagtaacta aaattccaga
     3361 agatattctc cagcttttat caaaatacaa ctcaaaccta gctactccaa tagcaattaa
     3421 agcagtgcca cccagtaaaa ggttccttaa acatggacag aacatccgag atgtcagcaa
     3481 caaagaaaac tgagaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta
     3541 gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     3601 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattcgccag cacagtggtc
     3661 gaccctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3721 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3781 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3841 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3901 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3961 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg agcacgtgtt
     4021 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     4081 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     4141 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     4201 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     4261 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     4321 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     4381 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     4441 attcgtgaat tgctgccctc tggttatgtg tgggagggct aagcacttca tcgataaaat
     4501 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg
     4561 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag
     4621 agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat
     4681 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg
     4741 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     4801 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     4861 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     4921 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     4981 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc
     5041 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     5101 gactacccgt cagcgggggt ctttcatttc cgacttgtgg tctcgctgcc ttgggagggt
     5161 ctcctctgag tgattgacta cccgtcagcg ggggtcttca catgcagcat gtatcaaaat
     5221 taatttggtt ttttttctta agtatttaca ttaaatggcc atagttgcat taatgaatcg
     5281 gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct tccgcttcct cgctcactga
     5341 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5401 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5461 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5521 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5581 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5641 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5701 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5761 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5821 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5881 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5941 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6001 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6061 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6121 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6181 cttcacctag atccttttaa attaaaaatg aagtttgcgg ccgcaaatca atctaaagta
     6241 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6301 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6361 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6421 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6481 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6541 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6601 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6661 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6721 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6781 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6841 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     6901 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6961 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7021 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7081 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7141 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7201 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
