Construction of pYPK0_TEF1_PsXYL1_TDH3 (pMEC1126)

pYPKa_Z_TEF1

pYPKa_E_TDH3


In [1]:
from pydna.all import *

In [2]:
p567,p577,p468,p467,p568,p578,p775,p778,p167,p166 = parse("yeast_pahtway_kit_standard_primers.txt")

In [3]:
from Bio.Restriction import ZraI, AjiI, EcoRV

In [4]:
pYPK0 = read("pYPK0.gb")

In [5]:
promoter_clone = pYPKa_Z_TEF1 = read("pYPKa_Z_TEF1.gb")

Genbank record


In [6]:
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/1756986', width='100%', height=250)


Out[6]:

In [7]:
gb =Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("X59465.1")

In [8]:
gene_template.list_features()


Out[8]:
+-----+------------------+-----+------+------+------+--------------+------+
| Ft# | Label or Note    | Dir | Sta  | End  |  Len | type         | orf? |
+-----+------------------+-----+------+------+------+--------------+------+
|   0 | nd               | --> | 0    | 2052 | 2052 | source       |  no  |
|   1 | nd               | --> | 0    | 355  |  355 | regulatory   |  no  |
|   2 | nd               | --> | 192  | 199  |    7 | regulatory   |  no  |
|   3 | nd               | --> | 272  | 281  |    9 | regulatory   |  no  |
|   4 | nd               | --> | 287  | 301  |   14 | stem_loop    |  no  |
|   5 | N:label:xyl1_cDN | --> | 342  | 1366 | 1024 | misc_feature |  no  |
|   6 | nd               | --> | 355  | 1312 |  957 | gene         | yes  |
|   7 | nd               | --> | 355  | 1312 |  957 | CDS          | yes  |
|   8 | nd               | --> | 1334 | 1339 |    5 | polyA_site   |  no  |
+-----+------------------+-----+------+------+------+--------------+------+

In [9]:
orf = gene_template.extract_feature(7)

In [10]:
orf.isorf()


Out[10]:
True

In [11]:
print(orf.seq)


ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTTCGGCTGTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTGCTATCAAGACCGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGTCAAGCGTGAAGACTTGTTCCTTACCTCCAAGTTGTGGAACAACTACCACCACCCAGACAACGTCGAAAAGGCCTTGAACAGAACCCTTTCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCTACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATCGAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCTTTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACTATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCAAGATTGTTGGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAACAAGATTTCGCTGACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCTATCTTCGTCTAA

In [12]:
print(len(orf))
orf.seguid()


957
Out[12]:
lGWKlZ3EDAyY2V-7tTQoJRqgW6w

In [13]:
print(str(orf.seq.translate()))


MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLDINLRFNDPWDWDKIPIFV*

In [14]:
pf, pr =parse('''

>XR_YPK_rec_fwd 
gtcgaggaacgccaggttgcccactttctcactagtgaAAATGCCTTCTATTAAGTTGAACTCTGG
>XR_YPK_rec_rev 
ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTAGACGAAGATAGGAATCTTGTCCC

''')

In [15]:
g =pcr( pf, pr, orf)

In [16]:
g.figure()


Out[16]:
                                        5ATGCCTTCTATTAAGTTGAACTCTGG...GGGACAAGATTCCTATCTTCGTCTAA3
                                                                      |||||||||||||||||||||||||| tm 58.7 (dbd) 66.4
                                                                     3CCCTGTTCTAAGGATAGAAGCAGATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGCCTTCTATTAAGTTGAACTCTGG3
                                         |||||||||||||||||||||||||| tm 57.9 (dbd) 64.7
                                        3TACGGAAGATAATTCAACTTGAGACC...CCCTGTTCTAAGGATAGAAGCAGATT5

In [17]:
terminator_clone = pYPKa_E_TDH3 =read("pYPKa_E_TDH3.gb")

In [18]:
p  = pcr( p167, p567, promoter_clone)
t  = pcr( p568, p166, terminator_clone)

In [19]:
pYPK0_E_Z, stuffer = pYPK0.cut((EcoRV, ZraI))
pYPK0_E_Z, stuffer


Out[19]:
(Dseqrecord(-5681), Dseqrecord(-81))

In [20]:
(pYPK0_E_Z, p, g, t)


Out[20]:
(Dseqrecord(-5681), Amplicon(741), Amplicon(1035), Amplicon(804))

In [21]:
asm =Assembly((pYPK0_E_Z, p, g, t), limit=29)

In [22]:
asm


Out[22]:
Assembly
fragments..: 5681bp 741bp 1035bp 804bp
limit(bp)..: 29
G.nodes....: 8
algorithm..: common_sub_strings

In [23]:
candidate = asm.assemble_circular()[0]
candidate.figure()


Out[23]:
 -|name|98
|       \/
|       /\
|       98|741bp_PCR_prod|38
|                         \/
|                         /\
|                         38|1035bp_PCR_prod|35
|                                            \/
|                                            /\
|                                            35|804bp_PCR_prod|61
|                                                              \/
|                                                              /\
|                                                              61-
|                                                                 |
 -----------------------------------------------------------------

In [24]:
pYPK0_TEF1_PsXYL1_TDH3 = candidate.synced(pYPK0)

The pYPK0_TEF1_PsXYL1_TDH3 has cseguid ak608NPPLpMmn_YIHGwQPcQzee0 and 8015 bp


In [25]:
print(len(pYPK0_TEF1_PsXYL1_TDH3))
pYPK0_TEF1_PsXYL1_TDH3.cseguid()


8015
Out[25]:
ak608NPPLpMmn_YIHGwQPcQzee0

In [26]:
pYPK0_TEF1_PsXYL1_TDH3.name="pMEC1126"
#pYPK0_TEF1_PsXYL1_TDH3.description = "pYPK0_TEF1_PsXYL1_TDH3tp"
pYPK0_TEF1_PsXYL1_TDH3.stamp()


Out[26]:
cSEGUID_ak608NPPLpMmn_YIHGwQPcQzee0

In [27]:
pYPK0_TEF1_PsXYL1_TDH3.write("pYPK0_TEF1_PsXYL1_TDH3.gb")




Download sequence

pYPK0_TEF1_PsXYL1_TDH3


In [28]:
reloaded =read("pYPK0_TEF1_PsXYL1_TDH3.gb")

In [29]:
reloaded.cseguid()


Out[29]:
ak608NPPLpMmn_YIHGwQPcQzee0

In [30]: