LOCUS       sequence-104538-        6361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag SKA2 for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6361)
  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 6361)
  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: pIC292
FEATURES             Location/Qualifiers
     source          1..6361
                     /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(2776..2796)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2781..3110
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2961..3096
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3125..3172
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3191..3589
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3642..4235
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4436..4453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4607..5195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4687..4706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5373..6164,6165..6233))
                     /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     5996..6015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6234..6338)
                     /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 cgagatggag gcggaggtcg ataagctgga
     2281 actgatgttc cagaaagctg agtctgatct ggattacatt caatacaggc tggaatatga
     2341 aatcaagact aatcatcctg attcagcaag tgagaaaaat ccagttacac tcttaaagga
     2401 attgtcagtg ataaagtctc gatatcaaac tttgtatgcc cgctttaaac cagttgctgt
     2461 tgagcagaaa gagagtaaga gccgcatttg tgctactgtg aaaaagacta tgaatatgat
     2521 acaaaaacta cagaagcaaa cagacctgga gctgtcacca ctgactaaag aagagaaaac
     2581 tgcggcagag caattcaaat ttcacatgcc agatttatga ccgcgggccc gggatccacc
     2641 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     2701 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tcgccagcac
     2761 agtggtcgac cctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2821 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2941 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3001 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3061 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttgagc
     3121 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3181 gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     3241 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     3301 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     3361 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     3421 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     3481 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     3541 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcatcg
     3601 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     3661 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg
     3721 agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac
     3781 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct
     3841 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     3901 acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt
     3961 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
     4021 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
     4081 ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata
     4141 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg
     4201 agtgattgac tacccgtcag cgggggtctt tcatttccga cttgtggtct cgctgccttg
     4261 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtcttcacat gcagcatgta
     4321 tcaaaattaa tttggttttt tttcttaagt atttacatta aatggccata gttgcattaa
     4381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg gcgctcttcc gcttcctcgc
     4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5041 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5221 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag tttgcggccg caaatcaatc
     5341 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5401 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5461 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5521 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5581 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5641 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5701 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5761 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5821 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5881 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5941 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     6001 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6061 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6121 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6181 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6241 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6301 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6361 c
//
