Example of a Plasmid Restriction/Ligation Cloning
Visit the full library documentation here
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:
The ase1 gene fragment is first cloned from a portion of the S. pombe genome through PCR:
The pFA6a-kanMX6 cloning vector is then cleaved with AscI and SalI. The ase1 gene fragment is also cleaved with SalI and AscI
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.
# Install pydna (only when running on Colab)
import sys
if 'google.colab' in sys.modules:
%%capture
# Install the current development version of pydna (comment to install pip version)
!pip install git+https://github.com/BjornFJohansson/pydna@dev_bjorn
# Install pip version instead (uncomment to install)
# !pip install pydna
# 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
# 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
//
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
# 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
//
# 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))
# 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
//