LOCUS Exported 5177 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of human Ska1 microtubule binding domain (MTBD) mutant. ACCESSION . VERSION . KEYWORDS pDK616 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5177) TITLE Cheeseman Lab Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 5177) AUTHORS .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 53..72 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 156..175 /label=T7 /note="T7 promoter, forward primer" promoter 156..174 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 248..270 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 290..307 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 311..343 /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 347..370 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS 371..394 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" primer_bind complement(862..880) /label=T7 Term /note="T7 terminator, reverse primer" terminator 876..923 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(1289..1317) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(1344..1362) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1430..1534 /gene="bla" /label=AmpR promoter CDS 1535..2395 /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(1753..1772) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2566..3154 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3055..3074 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3308..3325 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3340..3479 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3405..3424) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3565..3587 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3581..3772) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt 61 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc 121 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc 181 acaacggttt ccctctagtg ccggctccgg agagctccaa ttggaattcg ctagctctag 241 aaataatttt gtttaacttt aagaaggaga tatacgtatg cggggttctc atcatcatca 301 tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga 361 tgacgataag ctggaagttc tgttccaggg gccccatatg agaagtatta aggaaatgcc 421 atttataact tgtgatgagt tcaatggtgt tccttcgtac atgaaatccc gcttaaccta 481 taatcaaatt aatgatgtta ttaaagaaat caacaaggca gtaattagta aatataaaat 541 cctacatcag ccaaaaaagt ctatgaattc tgtgaccaga aatctctatc acagatttat 601 tgatgaagaa acgaaggata ccaaaggtcg ttattttata gtggaagctg acataaagga 661 gttcacaact ttggccgcgg atgcaaagtt tcacgtgtta ctgaatattt tacgacactg 721 ccggaggcta tcagaggtcc gagggggagg acttactcgt tatgttataa cctgaggatc 781 cagatctaag cttggtacca cgcgtgcgcg ctgatccggc tgctaacaaa gcccgaaagg 841 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta 901 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat ccacaggacg 961 ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact 1021 gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc 1081 aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata 1141 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg 1201 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt 1261 tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca 1321 tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 1381 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 1441 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 1501 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 1561 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 1621 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 1681 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 1741 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 1801 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 1861 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 1921 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 1981 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 2041 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg 2101 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 2161 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 2221 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 2281 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 2341 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 2401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 2461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 2521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 2581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 2641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 2701 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 2761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 2821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 2881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 2941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 3241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 3301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 3361 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat 3421 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 3481 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 3541 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 3601 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac 3661 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct 3721 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct 3781 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc 3841 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac 3901 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct 3961 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc 4021 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac 4081 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg 4141 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc 4201 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg 4261 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt 4321 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga 4381 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc 4441 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc 4501 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga 4561 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac 4621 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat 4681 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc 4741 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc 4801 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat 4861 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac 4921 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc 4981 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc 5041 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac 5101 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg 5161 gcctgccacc ataccca //