LOCUS Exported 5177 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of human Ska1 microtubule binding domain (MTBD). ACCESSION . VERSION . KEYWORDS pJS278 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5177) 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 5177) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5177 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 79..98 /label=T7 /note="T7 promoter, forward primer" promoter 79..97 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 171..193 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 213..230 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 234..266 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 270..293 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS 294..317 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" primer_bind complement(785..803) /label=T7 Term /note="T7 terminator, reverse primer" terminator 799..846 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(1212..1240) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(1267..1285) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1353..1457 /gene="bla" /label=AmpR promoter CDS 1458..2318 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1676..1695) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2489..3077 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2978..2997 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3231..3248 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3263..3402 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3328..3347) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3488..3510 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3504..3695) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 5153..5172 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag 61 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 121 gtgccggctc cggagagctc caattggaat tcgctagctc tagaaataat tttgtttaac 181 tttaagaagg agatatacgt atgcggggtt ctcatcatca tcatcatcat ggtatggcta 241 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aagctggaag 301 ttctgttcca ggggccccat atgagaagta ttaaggaaat gccatttata acttgtgatg 361 agttcaatgg tgttccttcg tacatgaaat ccgccttaac ctataatcaa attaatgatg 421 ttattaaaga aatcaacaag gcagtaatta gtaaatataa aatcctacat cagccaaaaa 481 agtctatgaa ttctgtgacc agaaatctct atcacagatt tattgatgaa gaaacgaagg 541 ataccaaagg tcgttatttt atagtggaag ctgacataaa ggagttcaca actttgaaag 601 ctgacaagaa gtttcacgtg ttactgaata ttttacgaca ctgccggagg ctatcagagg 661 tccgaggggg aggacttact cgttatgtta taacctgagg atccagatct aagcttggta 721 ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 781 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 841 tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat 901 cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg 961 gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag 1021 cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg 1081 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac 1141 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga 1201 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac 1261 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 1321 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1381 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1441 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1501 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1561 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1621 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1681 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact 1741 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1801 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 1861 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 1921 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 1981 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg 2041 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 2101 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 2161 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 2221 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 2281 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 2341 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2401 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2461 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2521 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2581 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2641 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2701 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2761 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 2821 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 2881 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2941 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3001 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3061 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3121 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 3181 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 3241 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 3301 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3361 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 3421 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 3481 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 3541 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 3601 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 3661 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 3721 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 3781 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 3841 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta 3901 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag 3961 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt 4021 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt 4081 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg 4141 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg 4201 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca 4261 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc 4321 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga 4381 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg 4441 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag 4501 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg 4561 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca 4621 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg 4681 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag 4741 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc 4801 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca 4861 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg 4921 agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct 4981 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg 5041 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca 5101 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt 5161 cggcgatata ggcgcca //