# Example of a Plasmid Restriction/Ligation Cloning > Visit the full library documentation [here](https://pydna-group.github.io/pydna/) This example showcases a workflow of modelling molecular cloning with restriction enzymes, PCR, and ligases, to clone gene fragments into plasmids. This example constructs a synthetic plasmid by cloning the ase1 gene, which encodes a microtubule associated protein responsible for mitotic spindle assembly, into the pFA6a-kanMX6 cloning vector: 1. The ase1 gene fragment is first cloned from a portion of the _S. pombe_ genome through PCR: 2. The pFA6a-kanMX6 cloning vector is then cleaved with AscI and SalI. The ase1 gene fragment is also cleaved with SalI and AscI 3. The fragment is ligated with the linearized pFA6a-kanMX6 vector. Source files can be found alongside this notebook, if you would like to follow along. Annotations are made alongside the code to describe key steps. Open In Colab ```python %%capture # Install pydna (only when running on Colab) import sys if 'google.colab' in sys.modules: %pip install pydna ``` ```python # Importing all necessary classes and methods from pydna.parsers import parse from pydna.tm import tm_default from pydna.amplify import pcr from pydna.dseqrecord import Dseqrecord from Bio.Restriction import SalI, AscI ``` ```python # Parsing the files pFA6akanMX6_path = "./pFA6a-kanMX6.gb" ase1_path = "./CU329670.gb" vector = parse(pFA6akanMX6_path)[0] pombe_chromosome_I = parse(ase1_path)[0] # Printing the parsed files print(vector.format("gb")) print(pombe_chromosome_I.format("gb")) ``` LOCUS pFA6a-kanMX6 3938 bp ds-DNA circular SYN 16-JUN-2022 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS pFA6a-kanMX6. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 3938) AUTHORS Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR TITLE Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. JOURNAL Yeast. 1998 Jul;14(10):943-51. PUBMED 9717240 REFERENCE 2 (bases 1 to 3938) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 16, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3938 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(35..52) /label="L4440" /note="L4440 vector, forward primer" rep_origin complement(206..794) /direction=LEFT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(286..305) /label="pBR322ori-F" /note="pBR322 origin, forward primer" CDS complement(965..1825) /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 1588..1607 /label="Amp-R" /note="Ampicillin resistance gene, reverse primer" promoter complement(1826..1930) /gene="bla" /label="AmpR promoter" primer_bind 1998..2016 /label="pBRforEco" /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2054..2076) /label="pGEX 3'" /note="pGEX vectors, reverse primer" primer_bind 2176..2195 /label="pRS-marker" /note="pRS vectors, use to sequence yeast selectable marker" promoter 2276..2294 /label="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 2276..2293 /label="SP6" /note="SP6 promoter, forward primer" gene 2407..3763 /label="kanMX" /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 2407..2750 /label="TEF promoter" /note="Ashbya gossypii TEF promoter" CDS 2751..3560 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label="KanR" /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(2818..2837) /label="Kan-R" /note="Kanamycin resistance gene, reverse primer" terminator 3566..3763 /label="TEF terminator" /note="Ashbya gossypii TEF terminator" primer_bind complement(3867..3886) /label="T7" /note="T7 promoter, forward primer" promoter complement(3868..3886) /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 61 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 1021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 1081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 1141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 1201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 1261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 1321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 1381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 1441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 1501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 1561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 1621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 1681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 1741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 1801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 1861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 1921 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 1981 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg 2041 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 2101 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 2161 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat 2221 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta 2281 ggtgacacta tagaacgcgg ccgccagctg aagcttcgta cgctgcaggt cgacggatcc 2341 ccgggttaat taaggcgcgc cagatctgtt tagcttgcct cgtccccgcc gggtcacccg 2401 gccagcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg 2461 gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca 2521 tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc 2581 tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg 2641 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt 2701 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaagg 2761 aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt 2821 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga 2881 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta 2941 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc 3001 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag 3061 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag 3121 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg 3181 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt 3241 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt 3301 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt 3361 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat 3421 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac 3481 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga 3541 tgctcgatga gtttttctaa tcagtactga caataaaaag attcttgttt tcaagaactt 3601 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat 3661 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt 3721 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta 3781 acgccgccat ccagtttaaa cgagctcgaa ttcatcgatg atatcagatc cactagtggc 3841 ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca 3901 ggttaacctg cattaatgaa tcggccaacg cgcgggga // LOCUS CU329670 4538 bp DNA linear PLN 26-APR-2024 DEFINITION Schizosaccharomyces pombe strain 972h- genome assembly, chromosome: I. ACCESSION CU329670 VERSION CU329670.1 DBLINK BioProject: PRJNA13836 BioSample: SAMEA3138176 KEYWORDS . SOURCE Schizosaccharomyces pombe (fission yeast) ORGANISM Schizosaccharomyces pombe Eukaryota; Fungi; Dikarya; Ascomycota; Taphrinomycotina; Schizosaccharomycetes; Schizosaccharomycetales; Schizosaccharomycetaceae; Schizosaccharomyces. REFERENCE 1 (bases 1 to 4538) AUTHORS Lang,B.F. TITLE The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans JOURNAL EMBO J 3 (9), 2129-2136 (1984) PUBMED 6092057 REFERENCE 2 (bases 1 to 4538) AUTHORS Lang,B.F., Ahne,F. and Bonen,L. TITLE The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene JOURNAL J Mol Biol 184 (3), 353-366 (1985) PUBMED 4046021 REFERENCE 3 (bases 1 to 4538) AUTHORS Lang,B.F., Cedergren,R. and Gray,M.W. TITLE The mitochondrial genome of the fission yeast, Schizosaccharomyces pombe. Sequence of the large-subunit ribosomal RNA gene, comparison of potential secondary structure in fungal mitochondrial large-subunit rRNAs and evolutionary considerations JOURNAL Eur J Biochem 169 (3), 527-537 (1987) PUBMED 2446871 REFERENCE 4 (bases 1 to 4538) AUTHORS Trinkl,H., Lang,B.F. and Wolf,K. TITLE Nucleotide sequence of the gene encoding the small ribosomal RNA in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe JOURNAL Nucleic Acids Res 17 (16), 6730 (1989) PUBMED 2780299 REFERENCE 5 (bases 1 to 4538) AUTHORS Wood,V., Gwilliam,R., Rajandream,M.A., Lyne,M., Lyne,R., Stewart,A., Sgouros,J., Peat,N., Hayles,J., Baker,S., Basham,D., Bowman,S., Brooks,K., Brown,D., Brown,S., Chillingworth,T., Churcher,C., Collins,M., Connor,R., Cronin,A., Davis,P., Feltwell,T., Fraser,A., Gentles,S., Goble,A., Hamlin,N., Harris,D., Hidalgo,J., Hodgson,G., Holroyd,S., Hornsby,T., Howarth,S., Huckle,E.J., Hunt,S., Jagels,K., James,K., Jones,L., Jones,M., Leather,S., McDonald,S., McLean,J., Mooney,P., Moule,S., Mungall,K., Murphy,L., Niblett,D., Odell,C., Oliver,K., O'Neil,S., Pearson,D., Quail,M.A., Rabbinowitsch,E., Rutherford,K., Rutter,S., Saunders,D., Seeger,K., Sharp,S., Skelton,J., Simmonds,M., Squares,R., Squares,S., Stevens,K., Taylor,K., Taylor,R.G., Tivey,A., Walsh,S., Warren,T., Whitehead,S., Woodward,J., Volckaert,G., Aert,R., Robben,J., Grymonprez,B., Weltjens,I., Vanstreels,E., Rieger,M., Schafer,M., Muller-Auer,S., Gabel,C., Fuchs,M., Dusterhoft,A., Fritzc,C., Holzer,E., Moestl,D., Hilbert,H., Borzym,K., Langer,I., Beck,A., Lehrach,H., Reinhardt,R., Pohl,T.M., Eger,P., Zimmermann,W., Wedler,H., Wambutt,R., Purnelle,B., Goffeau,A., Cadieu,E., Dreano,S., Gloux,S., Lelaure,V., Mottier,S., Galibert,F., Aves,S.J., Xiang,Z., Hunt,C., Moore,K., Hurst,S.M., Lucas,M., Rochet,M., Gaillardin,C., Tallada,V.A., Garzon,A., Thode,G., Daga,R.R., Cruzado,L., Jimenez,J., Sanchez,M., del Rey,F., Benito,J., Dominguez,A., Revuelta,J.L., Moreno,S., Armstrong,J., Forsburg,S.L., Cerutti,L., Lowe,T., McCombie,W.R., Paulsen,I., Potashkin,J., Shpakovski,G.V., Ussery,D., Barrell,B.G. and Nurse,P. TITLE The genome sequence of Schizosaccharomyces pombe JOURNAL Nature 415 (6874), 871-880 (2002) PUBMED 11859360 REMARK Erratum:[Nature 2003 Jan 2;421(6918):94. Cerrutti L [corrected to Cerutti L]] REFERENCE 6 AUTHORS Wood,V., Gwilliam,R., Rajandream,M.A., Lyne,M., Lyne,R., Stewart,A., Sgouros,J., Peat,N., Hayles,J., Baker,S., Basham,D., Bowman,S., Brooks,K., Brown,D., Brown,S., Chillingworth,T., Churcher,C., Collins,M., Connor,R., Cronin,A., Davis,P., Feltwell,T., Fraser,A., Gentles,S., Goble,A., Hamlin,N., Harris,D., Hidalgo,J., Hodgson,G., Holroyd,S., Hornsby,T., Howarth,S., Huckle,E.J., Hunt,S., Jagels,K., James,K., Jones,L., Jones,M., Leather,S., McDonald,S., McLean,J., Mooney,P., Moule,S., Mungall,K., Murphy,L., Niblett,D., Odell,C., Oliver,K., O'Neil,S., Pearson,D., Quail,M.A., Rabbinowitsch,E., Rutherford,K., Rutter,S., Saunders,D., Seeger,K., Sharp,S., Skelton,J., Simmonds,M., Squares,R., Squares,S., Stevens,K., Taylor,K., Taylor,R.G., Tivey,A., Walsh,S., Warren,T., Whitehead,S., Woodward,J., Volckaert,G., Aert,R., Robben,J., Grymonprez,B., Weltjens,I., Vanstreels,E., Rieger,M., Schafer,M., Muller-Auer,S., Gabel,C., Fuchs,M., Dusterhoft,A., Fritzc,C., Holzer,E., Moestl,D., Hilbert,H., Borzym,K., Langer,I., Beck,A., Lehrach,H., Reinhardt,R., Pohl,T.M., Eger,P., Zimmermann,W., Wedler,H., Wambutt,R., Purnelle,B., Goffeau,A., Cadieu,E., Dreano,S., Gloux,S., Lelaure,V., Mottier,S., Galibert,F., Aves,S.J., Xiang,Z., Hunt,C., Moore,K., Hurst,S.M., Lucas,M., Rochet,M., Gaillardin,C., Tallada,V.A., Garzon,A., Thode,G., Daga,R.R., Cruzado,L., Jimenez,J., Sanchez,M., del Rey,F., Benito,J., Dominguez,A., Revuelta,J.L., Moreno,S., Armstrong,J., Forsburg,S.L., Cerutti,L., Lowe,T., McCombie,W.R., Paulsen,I., Potashkin,J., Shpakovski,G.V., Ussery,D., Barrell,B.G. and Nurse,P. TITLE The genome sequence of Schizosaccharomyces pombe JOURNAL Nature 415 (6874), 871-880 (2002) PUBMED 11859360 REMARK Erratum:[Nature 2003 Jan 2;421(6918):94. Cerrutti L [corrected to Cerutti L]] REFERENCE 7 (bases 1 to 4538) AUTHORS Schafer,B., Hansen,M. and Lang,B.F. TITLE Transcription and RNA-processing in fission yeast mitochondria JOURNAL RNA 11 (5), 785-795 (2005) PUBMED 15811919 REFERENCE 8 AUTHORS Wood,V. CONSRTM The Schizosaccharomyces pombe Genome Sequencing Consortium TITLE Direct Submission JOURNAL Submitted (29-JUN-2007) European Schizosaccharomyces genome sequencing project, Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA REFERENCE 9 AUTHORS Wood,V. and Rutherford,K. CONSRTM PomBase TITLE Direct Submission JOURNAL Submitted (13-MAR-2024) University of Cambridge, PomBase, Hopkins building, Tennis Court Rd, Cambridge, United Kingdom COMMENT On or before Jan 26, 2012 this sequence version replaced AL672256.4, AL009197.1, AL009227.1, AL021046.4, AL021809.4, AL021813.1, AL021817.2, AL031180.3, AL034486.1, AL034565.1, AL034583.1, AL035064.1, AL035248.2, AL035254.1, AL035439.1, AL096845.1, AL109734.1, AL109738.1, AL109739.1, AL109770.1, AL109820.1, AL109831.1, AL109832.1, AL109951.1, AL109988.1, AL110469.1, AL110509.2, AL117210.1, AL117212.1, AL117213.1, AL117390.1, AL121732.1, AL121741.1, AL121745.2, AL121764.1, AL121765.1, AL121770.1, AL122032.1, AL132667.1, AL132675.1, AL132714.1, AL132769.1, AL132779.2, AL132798.2, AL132828.1, AL132839.1, AL132983.1, AL132984.1, AL133154.2, AL133156.1, AL133157.1, AL133225.2, AL133302.1, AL133357.1, AL133359.1, AL133360.1, AL133361.1, AL133442.1, AL133498.1, AL133521.1, AL133522.1, AL135751.1, AL136078.1, AL136235.1, AL136499.1, AL136521.2, AL136538.1, AL137130.1, AL138666.2, AL138854.1, AL139315.1, AL157734.1, AL157811.1, AL157872.1, AL157917.1, AL157993.1, AL157994.1, AL158056.1, AL159180.1, AL159951.1, AL162531.1, AL162631.1, AL163031.1, AL163071.1, AL163191.2, AL163481.1, AL163529.1, AL353014.1, AL353860.2, AL355012.1, AL355013.1, AL355252.1, AL355452.1, AL355632.1, AL355652.1, AL355653.1, AL356333.1, AL356335.1, AL357232.1, AL358272.1, AL360054.1, AL360094.1, AL390095.1, AL390274.1, AL390814.1, AL391713.1, AL391744.1, AL391746.2, AL391783.1, AL441621.1, AL441624.1, AL512486.1, AL512487.1, AL512491.1, AL512493.1, AL512496.1, AL512549.1, AL512562.1, AL583902.1, AL590562.1, AL590582.1, AL590602.1, AL590605.1, AL590902.2, AL590903.1, AL691401.1, AL691402.1, AL691405.1, Z49811.1, Z50112.1, Z50113.1, Z50142.1, Z50728.2, Z54096.1, Z54142.2, Z54285.2, Z54308.1, Z54328.1, Z54354.1, Z54366.1, Z56276.2, Z64354.1, Z66568.2, Z67757.1, Z67961.2, Z67998.1, Z67999.1, Z68136.2, Z68144.1, Z68166.1, Z68197.2, Z68198.1, Z68887.1, Z69086.1, Z69239.1, Z69240.1, Z69368.1, Z69369.1, Z69380.1, Z69725.1, Z69726.1, Z69727.1, Z69728.1, Z69729.1, Z69730.1, Z69731.1, Z69795.1, Z69796.1, Z69944.1, Z70043.1, Z70690.1, Z70691.1, Z70721.1, Z73099.2, Z73100.2, Z81312.1, Z81317.1, Z94864.1, Z95334.1, Z95395.1, Z95396.2, Z97185.1, Z97208.1, Z97209.1, Z97210.2, Z98056.2, Z98529.1, Z98530.2, Z98531.2, Z98532.1, Z98533.1, Z98559.1, Z98560.1, Z98595.1, Z98596.1, Z98597.1, Z98598.1, Z98600.1, Z98601.1, Z98602.1, Z98603.1, Z98762.1, Z98763.1, Z98849.1, Z98944.1, Z98974.2, Z98975.1, Z98977.4, Z98978.1, Z98979.1, Z98980.1, Z98981.3, Z99091.2, Z99126.1, Z99161.1, Z99162.1, Z99163.2, Z99164.2, Z99165.1, Z99166.1, Z99167.1, Z99168.1, Z99258.1, Z99259.1, Z99260.2, Z99261.1, Z99262.1, Z99292.1, Z99295.1, Z99296.2, Z99531.1, Z99532.2, Z99568.2, Z99753.1. FEATURES Location/Qualifiers source 1..4538 /organism="Schizosaccharomyces pombe" /mol_type="genomic DNA" /strain="972h-" /db_xref="taxon:4896" /chromosome="I" gene <1..676 /locus_tag="SPOM_SPAPB1A10.08" CDS <1..393 /locus_tag="SPOM_SPAPB1A10.08" /codon_start=1 /product="conserved fungal protein" /protein_id="CAC21481.2" /translation="MMTRMELRPLEIGFSKALTEVAPVTCQCECWDHNLCSSQASEMDL IYQSQDTHSCASKQDAVFQLLSETKIPVPNRYRKISHRLSTLSNKKTLKSQLDRFLSSS KKLHNDDVNRGDYCFLLSTPVECSASTNSHSYDCLWNFSCNSFPEYSSYSASETSSVAS YSYYSGPNPATPSSSSCNLVNANSLDIYLNINNLKKSKSVPRLRGQFMEPVEHNHPLSK SLEEQSSFLEQSKDASSNLTACNRSGSSLSSNFYSSRLSKKTSLASLNKSRASLQHKIM SLSRNIIRRVFHKPEVHLDPSASILNLSSSHGESNLTNGLLCQNFKLFQDDWLMEDCAP DANFTLYTPLQPWEKRSVKPEIRRPRLNPNFFRVFVLEAQMRRAGKLSANTAGRAQLIY LPKPAVTFSTSPLHVEL" gene complement(<1..1972) /locus_tag="SPOM_SPNCRNA.2846" ncRNA complement(<1..1972) /ncRNA_class="lncRNA" /locus_tag="SPOM_SPNCRNA.2846" /product="non-coding RNA" 3'UTR 394..676 /locus_tag="SPOM_SPAPB1A10.08" gene 1001..3538 /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" 5'UTR 1001..1173 /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" CDS join(1174..1597,1645..3416) /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" /codon_start=1 /product="antiparallel microtubule cross-linking factor Ase1" /protein_id="CAC21482.1" /translation="MQTVMMDDIQSTDSIAEKDNHSNNESNFTWKAFREQVEKHFSKIE RLHQVLGTDGDNSSLFELFTTAMNAQLHEMEQCQKKLEDDCQQRIDSIRFLVSSLKLTD DTSSLKIESPLIQCLNRLSMVEGQYMAQYDQKLSTIKEMYHKLESYCNRLGSPFVLPDF ENSFLSDVSDAFTESLRGRINEAEKEIDARLEVINSFEEEILGLWSELGVEPADVPQYE QLLESHTNRPNDVYVTQELIDQLCKQKEVFSAEKEKRSDHLKSIQSEVSNLWNKLQVSP NEQSQFGDSSNINQENISLWETELEKLHQLKKEHLPIFLEDCRQQILQLWDSLFYSEEQ RKSFTPMYEDIITEQVLTAHENYIKQLEAEVSANKSFLSLINRYASLIEGKKELEASSN DASRLTQRGRRDPGLLLREEKIRKRLSRELPKVQSLLIPEITAWEERNGRTFLFYDEPL LKICQEATQPKSLYRSASAAANRPKTATTTDSVNRTPSQRGRVAVPSTPSVRSASRAMT SPRTPLPRVKNTQNPSRSISAEPPSATSTANRRHPTANRIDINARLNSASRSRSANMIR QGANGSDSNMSSSPVSGNSNTPFNKFPNSVSRNTHFESKSPHPNYSRTPHETYSKASSK NVPLSPPKQRVVNEHALNIMSEKLQRTNLKEQTPEMDIENSSQNLPFSPMKISPIRASP VKTIPSSPSPTTNIFSAPLNNITNCTPMEDEWGEEGF" 3'UTR 3417..3538 /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" gene complement(3510..>4538) /gene="ypt71" /locus_tag="SPOM_SPAPB1A10.10C" 3'UTR complement(3510..3690) /gene="ypt71" /locus_tag="SPOM_SPAPB1A10.10C" CDS complement(join(3691..4137,4192..>4290)) /gene="ypt71" /locus_tag="SPOM_SPAPB1A10.10C" /codon_start=1 /product="GTPase Ypt71" /protein_id="CAC21483.1" /translation="MSAQKRVFLKVVILGDSGVGKTCLMNQFVNQKFSREYKATIGADF LTKDVVVDDKLVTLQLWDTAGQERFQSLGMAFYRGADCCVIVYNVNNSKSFDSVENWRQ EFLYQTSQDECAFPFIIVGNQIDKDASKRAVSLHRALDYCKSKHGSNMIHFEASAKENT NVTDLFETVSRLALENESSRDDFVNDFSEPLLLSKPLNNTSSCNC" gene 4049..>4538 /locus_tag="SPOM_SPNCRNA.2847" ncRNA 4049..>4538 /ncRNA_class="lncRNA" /locus_tag="SPOM_SPNCRNA.2847" /product="non-coding RNA" ORIGIN 1 atcatcagac gtgtatttca caagccagaa gtgcatttgg atccaagtgc ctccatttta 61 aatctctcat cttcgcatgg cgaaagcaac ctgacaaatg gtttgctttg tcaaaatttc 121 aagctttttc aggatgattg gttgatggag gattgtgcgc cagatgccaa tttcactttg 181 tacaccccgc ttcaaccctg ggaaaagcga agtgtgaaac ctgaaatcag acgtcctcga 241 ttaaatccta attttttccg agtatttgtt ttagaagctc aaatgcgacg agctggaaag 301 ctatcagcaa acactgctgg ccgagcccag ttaatttacc tcccaaagcc tgccgttacc 361 ttctccacta gccctttgca tgttgaattg taaaaattta acgcatgact tatatacatt 421 tgcattcttc caagctggtt atatttattt tcattttttt ctcacccaat acttttttat 481 ccctactgtc tttatggaca atcgactcac aattgtttct ttttgttgta tatgattttt 541 tttttaaagg aaatgggttt cgcgatactg ggttgaatcc caattgcggt taatattaca 601 taaaataatt ctcccatagt cctagatcct gtctttgaat atgagcaaat aaaagaattg 661 aacaaatcat gaatgctttt ctctcttaga tgatattttg tatgcataag tctaattata 721 ttgattacga taagacttaa aaagtaagcc tttgtatcct tttaagcagt atttgaattt 781 tcttgtatca tattttaggt agagcaaaag ataccagttt gtagaacttt atgtgcttcc 841 ttacattggt atatttcagg cacataaata ttcttcaact tacaattcta agtattttgt 901 ttatactaaa aggagctgaa taacgtttat acagtgctga cattgaaatc tatttgcttt 961 ctttggaata taagcgcatg ctgagttact ttcgcaggcc aagccatatc caaccaccat 1021 ttttgtgcca agcttttatg caaggttaat tccttgtact gcttgttatg ttataatata 1081 tcaacatctt aacagttttc atatcttcct ttatattcta ttaattgaat ttcaaacatc 1141 gttttattga gctcatttac atcaaccggt tcaatgcaaa cagtaatgat ggatgacatt 1201 caaagcactg attctattgc tgaaaaagat aatcactcta ataatgaatc taactttact 1261 tggaaagcgt ttcgtgaaca agtggaaaag catttttcta aaattgaaag gcttcaccaa 1321 gtccttggaa cagatggaga caattcatca ttatttgagt tgtttacaac ggcaatgaat 1381 gcccagcttc atgaaatgga acagtgccag aaaaaacttg aagatgactg tcagcaaaga 1441 attgattcaa tcagattttt ggtttcctca ttaaagttaa cggatgatac ttctagtctc 1501 aaaattgagt ctcctttaat tcagtgtttg aatcgtttgt caatggtaga aggacaatat 1561 atggcacagt atgatcaaaa gttaagtacg attaaaggta tgtaatcgtc tttaatttag 1621 acttgtgttt taactgatgt atagaaatgt atcacaaatt ggagtcatat tgtaaccgct 1681 taggaagtcc gttcgtttta cctgattttg agaattcatt tttatctgat gtatccgatg 1741 cttttactga atctttgaga ggacgcatca acgaagccga aaaggagatt gatgcgagat 1801 tagaggttat taattccttt gaagaagaaa ttttgggttt gtggtctgaa ctcggtgttg 1861 agcccgctga tgttccacaa tacgaacaat tgcttgaatc ccatactaat cgaccaaatg 1921 atgtttatgt tactcaagaa cttatcgacc aactttgcaa gcaaaaagaa gttttttccg 1981 ctgaaaaaga aaagagaagt gatcatttaa aaagtataca atcagaagtt agcaacttgt 2041 ggaataagct tcaagtttct cccaatgaac aaagtcaatt tggcgattca tcaaacatta 2101 atcaagaaaa tatttcatta tgggaaactg aacttgaaaa acttcatcag ttaaaaaagg 2161 agcatttacc cattttttta gaagactgtc gtcaacaaat tcttcagctt tgggattctc 2221 tgttttattc agaagaacaa agaaagtcct ttacacctat gtatgaagac attattacag 2281 agcaggttct tacggcccat gaaaactata taaagcaact agaggccgaa gtttctgcta 2341 ataagtcctt tttaagctta attaatcgct atgcctcttt aatagaagga aagaaagagc 2401 ttgaagctag ttctaatgat gcctctcgtc taacacaacg gggacgccgg gacccaggtt 2461 tacttctacg tgaagagaaa atccgtaagc gactttctag agaacttcct aaggttcagt 2521 cgctgcttat accagagatt acagcatggg aagaaagaaa tggaaggacg ttcctttttt 2581 atgatgaacc acttctcaag atttgccaag aggccactca accaaaatca ttatatagaa 2641 gtgcaagtgc tgccgcaaac cgcccgaaaa cagcaactac aacggactct gttaatagaa 2701 caccttctca acgagggcgt gtagctgtac cttcaacacc aagtgttagg tccgcttctc 2761 gagctatgac gagtccaagg acaccgcttc ctagagtaaa aaacactcaa aatccaagtc 2821 gttccattag tgcagaaccg ccatcagcaa ccagtaccgc caatagaaga caccccactg 2881 ctaatcgaat tgatataaac gctagattaa acagtgctag tcggtctcga agcgcgaaca 2941 tgataagaca aggggcaaat ggtagtgaca gcaatatgtc ttcttcaccc gtttctggaa 3001 attccaatac cccttttaac aagtttccaa attctgtatc tcgcaataca cattttgaat 3061 ccaagtcacc gcacccaaat tactctcgaa ctcctcatga aacgtattca aaggcttcat 3121 ctaagaacgt cccattaagt cctccaaagc agcgtgtagt taatgaacac gctttaaata 3181 ttatgtcgga aaaattgcaa agaactaatc tgaaagaaca aacacccgag atggacattg 3241 aaaacagctc gcagaacctt cctttttctc ctatgaagat atcccccata agagcatcac 3301 ccgtaaagac aattccatca tcaccgtccc ccactaccaa cattttttct gctccactca 3361 acaatattac aaattgtaca ccgatggagg atgaatgggg agaagaaggc ttttaagctt 3421 cttatttacc taatcgatca aatttaaata tacatatttt tgcatatgaa tacagcatat 3481 agataattca taaaagttta ttaactgagg tcataattaa aagactattt acacctaaaa 3541 aaaaacgtgt atcaatagag ggaaaagaga agaattaaga acagaaagta accatagttt 3601 tgttaaaata gcaatgtaaa aaaatattat gaaaagaaaa cgtatagcac attttgaaat 3661 gtaaaagaat ctgagagagc gtgtgaatat ctagcaatta caagaagatg tattattcaa 3721 aggctttgaa agaagcaaag gttcagagaa gtcattaaca aagtcatctc tcgagctttc 3781 attttctaaa gctaaacgac tgactgtttc gaaaaggtca gtaacgtttg tattttcttt 3841 tgcactagct tcaaaatgaa tcatatttga tccatgtttg gatttgcaat agtcaagagc 3901 tcgatgaaga gatacggctc gtttagacgc gtctttgtcg atttgatttc caacgataat 3961 gaaagggaat gcacattcat cttgtgaagt ttgatataaa aattcttgcc tccagttttc 4021 tactgagtca aaagacttcg agttattcac attataaaca attacacaac aatcggcccc 4081 tctgtaaaaa gccattccca ggctttgaaa tcgttcttga ccagcagtat cccaaagctg 4141 tttataatta gcaaacgaat ttagatgggc ggaacttata ttggaactta cctgtaatgt 4201 gaccaatttg tcgtcaacca caacgtcctt ggttaaaaaa tcagcaccga tggtagcttt 4261 atattcgcga ctaaactttt gattgacgaa ctaaaatgac gatgttaaca aattgccaaa 4321 gcaatactca tagagaagct gatgtaaaga tcgttaacca tatttgagct agtatttaat 4381 aacaaagtga ataaatttta aaagcaatca ccttgtagcg acaaataaca acttatcgac 4441 ataaaatcaa tgggaaattg cagtattgga ttttacagct caatacaaaa accaaaaaga 4501 aaaatatact gaacgtataa aatttaacgc ttcaattg // ```python from pydna.design import primer_design #Finding the feature containing the CDS with ase1 as a type qualifier gene = next(f for f in pombe_chromosome_I.features if f.type == "CDS" and "gene" in f.qualifiers and "ase1" in f.qualifiers["gene"]) # Using the primer_design function to design primers to amplify the CDS # `min` and `max` can be used on a SeqFeature to get the start (leftmost) and end (rightmost) positions # this works both on feature with SimpleLocation and CompoundLocation amplicon = primer_design(pombe_chromosome_I[min(gene):max(gene)], target_tm=55) fwd_align, rvs_align = amplicon.primers() fwd_primer_ase1 = Dseqrecord("ACCATGTCGAC") + fwd_align # Adding a SalI cut site rvs_primer_ase1 = Dseqrecord("GGCGCGCCAT") + rvs_align # Adding a AscI cut site # Printing out the primers print(fwd_primer_ase1.seq, ', Tm: ', tm_default(fwd_align)) print(rvs_primer_ase1.seq, ', Tm: ', tm_default(rvs_align)) ``` ACCATGTCGACATGCAAACAGTAATGATGGA , Tm: 57.24061148156318 GGCGCGCCATTAAAAGCCTTCTTCTCCC , Tm: 56.64459495003314 ```python # Performing a PCR to check that the primers are specific. An error message is returned if otherwise. pcr_product = pcr(fwd_primer_ase1, rvs_primer_ase1, pombe_chromosome_I) # Printing out the PCR results print(pcr_product.format("gb")) ``` LOCUS 2263bp_PCR_prod 2263 bp DNA linear UNK 01-JAN-1980 DEFINITION pcr_product_description_description. ACCESSION 2263bp VERSION 2263bp DBLINK BioProject: PRJNA13836 BioSample: SAMEA3138176 KEYWORDS . SOURCE . ORGANISM . . FEATURES Location/Qualifiers CDS join(12..435,483..2254) /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" /codon_start=1 /product="antiparallel microtubule cross-linking factor Ase1" /protein_id="CAC21482.1" /translation="MQTVMMDDIQSTDSIAEKDNHSNNESNFTWKAFREQVEKHFSKIE RLHQVLGTDGDNSSLFELFTTAMNAQLHEMEQCQKKLEDDCQQRIDSIRFLVSSLKLTD DTSSLKIESPLIQCLNRLSMVEGQYMAQYDQKLSTIKEMYHKLESYCNRLGSPFVLPDF ENSFLSDVSDAFTESLRGRINEAEKEIDARLEVINSFEEEILGLWSELGVEPADVPQYE QLLESHTNRPNDVYVTQELIDQLCKQKEVFSAEKEKRSDHLKSIQSEVSNLWNKLQVSP NEQSQFGDSSNINQENISLWETELEKLHQLKKEHLPIFLEDCRQQILQLWDSLFYSEEQ RKSFTPMYEDIITEQVLTAHENYIKQLEAEVSANKSFLSLINRYASLIEGKKELEASSN DASRLTQRGRRDPGLLLREEKIRKRLSRELPKVQSLLIPEITAWEERNGRTFLFYDEPL LKICQEATQPKSLYRSASAAANRPKTATTTDSVNRTPSQRGRVAVPSTPSVRSASRAMT SPRTPLPRVKNTQNPSRSISAEPPSATSTANRRHPTANRIDINARLNSASRSRSANMIR QGANGSDSNMSSSPVSGNSNTPFNKFPNSVSRNTHFESKSPHPNYSRTPHETYSKASSK NVPLSPPKQRVVNEHALNIMSEKLQRTNLKEQTPEMDIENSSQNLPFSPMKISPIRASP VKTIPSSPSPTTNIFSAPLNNITNCTPMEDEWGEEGF" primer_bind 12..31 /label="name" /PCR_conditions="primer sequence:ACCATGTCGACATGCAAACAGTAATGATGGA" /ApEinfo_fwdcolor="#baffa3" /ApEinfo_revcolor="#ffbaba" primer_bind complement(2236..2254) /label="name" /PCR_conditions="primer sequence:GGCGCGCCATTAAAAGCCTTCTTCTCCC" /ApEinfo_fwdcolor="#baffa3" /ApEinfo_revcolor="#ffbaba" ORIGIN 1 accatgtcga catgcaaaca gtaatgatgg atgacattca aagcactgat tctattgctg 61 aaaaagataa tcactctaat aatgaatcta actttacttg gaaagcgttt cgtgaacaag 121 tggaaaagca tttttctaaa attgaaaggc ttcaccaagt ccttggaaca gatggagaca 181 attcatcatt atttgagttg tttacaacgg caatgaatgc ccagcttcat gaaatggaac 241 agtgccagaa aaaacttgaa gatgactgtc agcaaagaat tgattcaatc agatttttgg 301 tttcctcatt aaagttaacg gatgatactt ctagtctcaa aattgagtct cctttaattc 361 agtgtttgaa tcgtttgtca atggtagaag gacaatatat ggcacagtat gatcaaaagt 421 taagtacgat taaaggtatg taatcgtctt taatttagac ttgtgtttta actgatgtat 481 agaaatgtat cacaaattgg agtcatattg taaccgctta ggaagtccgt tcgttttacc 541 tgattttgag aattcatttt tatctgatgt atccgatgct tttactgaat ctttgagagg 601 acgcatcaac gaagccgaaa aggagattga tgcgagatta gaggttatta attcctttga 661 agaagaaatt ttgggtttgt ggtctgaact cggtgttgag cccgctgatg ttccacaata 721 cgaacaattg cttgaatccc atactaatcg accaaatgat gtttatgtta ctcaagaact 781 tatcgaccaa ctttgcaagc aaaaagaagt tttttccgct gaaaaagaaa agagaagtga 841 tcatttaaaa agtatacaat cagaagttag caacttgtgg aataagcttc aagtttctcc 901 caatgaacaa agtcaatttg gcgattcatc aaacattaat caagaaaata tttcattatg 961 ggaaactgaa cttgaaaaac ttcatcagtt aaaaaaggag catttaccca tttttttaga 1021 agactgtcgt caacaaattc ttcagctttg ggattctctg ttttattcag aagaacaaag 1081 aaagtccttt acacctatgt atgaagacat tattacagag caggttctta cggcccatga 1141 aaactatata aagcaactag aggccgaagt ttctgctaat aagtcctttt taagcttaat 1201 taatcgctat gcctctttaa tagaaggaaa gaaagagctt gaagctagtt ctaatgatgc 1261 ctctcgtcta acacaacggg gacgccggga cccaggttta cttctacgtg aagagaaaat 1321 ccgtaagcga ctttctagag aacttcctaa ggttcagtcg ctgcttatac cagagattac 1381 agcatgggaa gaaagaaatg gaaggacgtt ccttttttat gatgaaccac ttctcaagat 1441 ttgccaagag gccactcaac caaaatcatt atatagaagt gcaagtgctg ccgcaaaccg 1501 cccgaaaaca gcaactacaa cggactctgt taatagaaca ccttctcaac gagggcgtgt 1561 agctgtacct tcaacaccaa gtgttaggtc cgcttctcga gctatgacga gtccaaggac 1621 accgcttcct agagtaaaaa acactcaaaa tccaagtcgt tccattagtg cagaaccgcc 1681 atcagcaacc agtaccgcca atagaagaca ccccactgct aatcgaattg atataaacgc 1741 tagattaaac agtgctagtc ggtctcgaag cgcgaacatg ataagacaag gggcaaatgg 1801 tagtgacagc aatatgtctt cttcacccgt ttctggaaat tccaataccc cttttaacaa 1861 gtttccaaat tctgtatctc gcaatacaca ttttgaatcc aagtcaccgc acccaaatta 1921 ctctcgaact cctcatgaaa cgtattcaaa ggcttcatct aagaacgtcc cattaagtcc 1981 tccaaagcag cgtgtagtta atgaacacgc tttaaatatt atgtcggaaa aattgcaaag 2041 aactaatctg aaagaacaaa cacccgagat ggacattgaa aacagctcgc agaaccttcc 2101 tttttctcct atgaagatat cccccataag agcatcaccc gtaaagacaa ttccatcatc 2161 accgtccccc actaccaaca ttttttctgc tccactcaac aatattacaa attgtacacc 2221 gatggaggat gaatggggag aagaaggctt ttaatggcgc gcc // ```python # Cleaving the cloning vector with restriction enzymes plasmid_digests = vector.cut(SalI, AscI) # Cleaving the gene fragment with restriction enzymes gene_digests = Dseqrecord(pcr_product).cut(SalI, AscI) # Printing out the digests print(plasmid_digests) print(gene_digests) ``` (Dseqrecord(-30), Dseqrecord(-3916)) (Dseqrecord(-10), Dseqrecord(-2255), Dseqrecord(-6)) ```python # Ligating, then circularising the synthetic plasmid synthetic_vector = plasmid_digests[1] + gene_digests [1] synthetic_vector = synthetic_vector.looped() # Printing out the completed cloning vector print(synthetic_vector.format("gb")) ``` LOCUS name 6163 bp DNA circular UNK 01-JAN-1980 DEFINITION description. ACCESSION id VERSION id KEYWORDS . SOURCE . ORGANISM . . FEATURES Location/Qualifiers gene 52..1408 /label="kanMX" /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 52..395 /label="TEF promoter" /note="Ashbya gossypii TEF promoter" CDS 396..1205 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label="KanR" /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(463..482) /label="Kan-R" /note="Kanamycin resistance gene, reverse primer" terminator 1211..1408 /label="TEF terminator" /note="Ashbya gossypii TEF terminator" primer_bind complement(1512..1531) /label="T7" /note="T7 promoter, forward primer" promoter complement(1513..1531) /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1618..1635) /label="L4440" /note="L4440 vector, forward primer" rep_origin complement(1789..2377) /direction=LEFT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1869..1888) /label="pBR322ori-F" /note="pBR322 origin, forward primer" CDS complement(2548..3408) /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 3171..3190 /label="Amp-R" /note="Ampicillin resistance gene, reverse primer" promoter complement(3409..3513) /gene="bla" /label="AmpR promoter" primer_bind 3581..3599 /label="pBRforEco" /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3637..3659) /label="pGEX 3'" /note="pGEX vectors, reverse primer" primer_bind 3759..3778 /label="pRS-marker" /note="pRS vectors, use to sequence yeast selectable marker" promoter 3859..3877 /label="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3859..3876 /label="SP6" /note="SP6 promoter, forward primer" CDS join(3918..4341,4389..6160) /gene="ase1" /locus_tag="SPOM_SPAPB1A10.09" /codon_start=1 /product="antiparallel microtubule cross-linking factor Ase1" /protein_id="CAC21482.1" /translation="MQTVMMDDIQSTDSIAEKDNHSNNESNFTWKAFREQVEKHFSKIE RLHQVLGTDGDNSSLFELFTTAMNAQLHEMEQCQKKLEDDCQQRIDSIRFLVSSLKLTD DTSSLKIESPLIQCLNRLSMVEGQYMAQYDQKLSTIKEMYHKLESYCNRLGSPFVLPDF ENSFLSDVSDAFTESLRGRINEAEKEIDARLEVINSFEEEILGLWSELGVEPADVPQYE QLLESHTNRPNDVYVTQELIDQLCKQKEVFSAEKEKRSDHLKSIQSEVSNLWNKLQVSP NEQSQFGDSSNINQENISLWETELEKLHQLKKEHLPIFLEDCRQQILQLWDSLFYSEEQ RKSFTPMYEDIITEQVLTAHENYIKQLEAEVSANKSFLSLINRYASLIEGKKELEASSN DASRLTQRGRRDPGLLLREEKIRKRLSRELPKVQSLLIPEITAWEERNGRTFLFYDEPL LKICQEATQPKSLYRSASAAANRPKTATTTDSVNRTPSQRGRVAVPSTPSVRSASRAMT SPRTPLPRVKNTQNPSRSISAEPPSATSTANRRHPTANRIDINARLNSASRSRSANMIR QGANGSDSNMSSSPVSGNSNTPFNKFPNSVSRNTHFESKSPHPNYSRTPHETYSKASSK NVPLSPPKQRVVNEHALNIMSEKLQRTNLKEQTPEMDIENSSQNLPFSPMKISPIRASP VKTIPSSPSPTTNIFSAPLNNITNCTPMEDEWGEEGF" primer_bind 3918..3937 /label="name" /PCR_conditions="primer sequence:ACCATGTCGACATGCAAACAGTAATGATGGA" /ApEinfo_fwdcolor="#baffa3" /ApEinfo_revcolor="#ffbaba" primer_bind complement(6142..6160) /label="name" /PCR_conditions="primer sequence:GGCGCGCCATTAAAAGCCTTCTTCTCCC" /ApEinfo_fwdcolor="#baffa3" /ApEinfo_revcolor="#ffbaba" ORIGIN 1 cgcgccagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag 61 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt 121 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat 181 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac 241 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa 301 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga 361 tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt 421 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 481 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag 541 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca 601 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 661 ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag 721 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 781 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc 841 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta 901 atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg 961 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 1021 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 1081 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 1141 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt 1201 tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt 1261 tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg 1321 cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa 1381 tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt 1441 ttaaacgagc tcgaattcat cgatgatatc agatccacta gtggcctatg cggccgcgga 1501 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta 1561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 1621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 1981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 2041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 2101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 2161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 2221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 2281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 2341 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 2401 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 2461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 2521 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 2581 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 2641 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 2701 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 2761 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 2821 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 2881 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 2941 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 3001 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 3061 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 3121 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 3181 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 3241 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 3301 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 3361 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 3421 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 3481 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 3541 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 3601 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 3661 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 3721 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact 3781 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt 3841 atgtatcata cacatacgat ttaggtgaca ctatagaacg cggccgccag ctgaagcttc 3901 gtacgctgca ggtcgacatg caaacagtaa tgatggatga cattcaaagc actgattcta 3961 ttgctgaaaa agataatcac tctaataatg aatctaactt tacttggaaa gcgtttcgtg 4021 aacaagtgga aaagcatttt tctaaaattg aaaggcttca ccaagtcctt ggaacagatg 4081 gagacaattc atcattattt gagttgttta caacggcaat gaatgcccag cttcatgaaa 4141 tggaacagtg ccagaaaaaa cttgaagatg actgtcagca aagaattgat tcaatcagat 4201 ttttggtttc ctcattaaag ttaacggatg atacttctag tctcaaaatt gagtctcctt 4261 taattcagtg tttgaatcgt ttgtcaatgg tagaaggaca atatatggca cagtatgatc 4321 aaaagttaag tacgattaaa ggtatgtaat cgtctttaat ttagacttgt gttttaactg 4381 atgtatagaa atgtatcaca aattggagtc atattgtaac cgcttaggaa gtccgttcgt 4441 tttacctgat tttgagaatt catttttatc tgatgtatcc gatgctttta ctgaatcttt 4501 gagaggacgc atcaacgaag ccgaaaagga gattgatgcg agattagagg ttattaattc 4561 ctttgaagaa gaaattttgg gtttgtggtc tgaactcggt gttgagcccg ctgatgttcc 4621 acaatacgaa caattgcttg aatcccatac taatcgacca aatgatgttt atgttactca 4681 agaacttatc gaccaacttt gcaagcaaaa agaagttttt tccgctgaaa aagaaaagag 4741 aagtgatcat ttaaaaagta tacaatcaga agttagcaac ttgtggaata agcttcaagt 4801 ttctcccaat gaacaaagtc aatttggcga ttcatcaaac attaatcaag aaaatatttc 4861 attatgggaa actgaacttg aaaaacttca tcagttaaaa aaggagcatt tacccatttt 4921 tttagaagac tgtcgtcaac aaattcttca gctttgggat tctctgtttt attcagaaga 4981 acaaagaaag tcctttacac ctatgtatga agacattatt acagagcagg ttcttacggc 5041 ccatgaaaac tatataaagc aactagaggc cgaagtttct gctaataagt cctttttaag 5101 cttaattaat cgctatgcct ctttaataga aggaaagaaa gagcttgaag ctagttctaa 5161 tgatgcctct cgtctaacac aacggggacg ccgggaccca ggtttacttc tacgtgaaga 5221 gaaaatccgt aagcgacttt ctagagaact tcctaaggtt cagtcgctgc ttataccaga 5281 gattacagca tgggaagaaa gaaatggaag gacgttcctt ttttatgatg aaccacttct 5341 caagatttgc caagaggcca ctcaaccaaa atcattatat agaagtgcaa gtgctgccgc 5401 aaaccgcccg aaaacagcaa ctacaacgga ctctgttaat agaacacctt ctcaacgagg 5461 gcgtgtagct gtaccttcaa caccaagtgt taggtccgct tctcgagcta tgacgagtcc 5521 aaggacaccg cttcctagag taaaaaacac tcaaaatcca agtcgttcca ttagtgcaga 5581 accgccatca gcaaccagta ccgccaatag aagacacccc actgctaatc gaattgatat 5641 aaacgctaga ttaaacagtg ctagtcggtc tcgaagcgcg aacatgataa gacaaggggc 5701 aaatggtagt gacagcaata tgtcttcttc acccgtttct ggaaattcca ataccccttt 5761 taacaagttt ccaaattctg tatctcgcaa tacacatttt gaatccaagt caccgcaccc 5821 aaattactct cgaactcctc atgaaacgta ttcaaaggct tcatctaaga acgtcccatt 5881 aagtcctcca aagcagcgtg tagttaatga acacgcttta aatattatgt cggaaaaatt 5941 gcaaagaact aatctgaaag aacaaacacc cgagatggac attgaaaaca gctcgcagaa 6001 ccttcctttt tctcctatga agatatcccc cataagagca tcacccgtaa agacaattcc 6061 atcatcaccg tcccccacta ccaacatttt ttctgctcca ctcaacaata ttacaaattg 6121 tacaccgatg gaggatgaat ggggagaaga aggcttttaa tgg //