LOCUS       Exported                6763 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Retroviral expression of EGFP-TEV-S-tag SKA1 for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    pIC291
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6763)
  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 6763)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6763
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1391..2107
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1391..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(3178..3198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3183..3512
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3363..3498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3425..3444
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3527..3574
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3593..3991
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             4044..4637
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4838..4855)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5009..5597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5089..5108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5775..6635)
                     /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     6398..6417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6636..6740)
                     /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 cgagatggcc tcgtcagatc tggaacaatt
     2281 atgctctcat gttaatgaaa agattggcaa tattaagaaa accttatcat taagaaactg
     2341 tggccaggaa cctaccttga aaactgtatt aaataaaata ggagatgaga tcattgtaat
     2401 aaatgaactt ctaaataaat tggaattgga aattcagtat caagaacaaa ccaacaattc
     2461 actcaaggaa ctctgtgaat ctcttgaaga agattacaaa gacatagaac atcttaaaga
     2521 aaacgttcct tcccatttgc ctcaagtaac agtaacccag agctgtgtta agggatcaga
     2581 tcttgatcct gaagaaccaa tcaaagttga agaacctgaa cccgtaaaga agcctcccaa
     2641 agagcaaaga agtattaagg aaatgccatt tataacttgt gatgagttca atggtgttcc
     2701 ttcgtacatg aaatcccgct taacctataa tcaaattaat gatgttatta aagaaatcaa
     2761 caaggcagta attagtaaat ataaaatcct acatcagcca aaaaagtcta tgaattctgt
     2821 gaccagaaat ctctatcaca gatttattga tgaagaaacg aaggatacca aaggtcgtta
     2881 ttttatagtg gaagctgaca taaaggagtt cacaactttg aaagctgaca agaagtttca
     2941 cgtgttactg aatattttac gacactgccg gaggctatca gaggtccgag ggggaggact
     3001 tactcgttat gttataacct gaccgcgggc ccgggatcca ccggatctag ataactgatc
     3061 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3121 cccctgaacc tgaaacataa aatgaatgca attcgccagc acagtggtcg accctgtgga
     3181 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3241 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3301 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3361 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3421 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3481 gaggcttttt tggaggccta ggcttttgca aaaagcttga gcacgtgttg acaattaatc
     3541 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa
     3601 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     3661 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     3721 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     3781 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     3841 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     3901 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     3961 gctgccctct ggttatgtgt gggagggcta agcacttcat cgataaaata aaagatttta
     4021 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     4081 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     4141 tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     4201 agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg
     4261 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     4321 cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     4381 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     4441 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt
     4501 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     4561 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     4621 agcgggggtc tttcatttcc gacttgtggt ctcgctgcct tgggagggtc tcctctgagt
     4681 gattgactac ccgtcagcgg gggtcttcac atgcagcatg tatcaaaatt aatttggttt
     4741 tttttcttaa gtatttacat taaatggcca tagttgcatt aatgaatcgg ccaacgcgcg
     4801 gggagaggcg gtttgcgtat tggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4861 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4921 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4981 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5041 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5101 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5161 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5221 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5281 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5341 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5401 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5461 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5521 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5581 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5641 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5701 tccttttaaa ttaaaaatga agtttgcggc cgcaaatcaa tctaaagtat atatgagtaa
     5761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5881 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6061 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6361 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     6421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
