LOCUS Exported 6601 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Retroviral expression of GFP-Ska1. ACCESSION . VERSION . KEYWORDS pIW069 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6601 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1010..1726 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1010..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" primer_bind 3044..3063 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" 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(5224..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 6220..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 //