Construction of pYPK0_FBA1_ScTAL1_PDC1 (pMEC1130)

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_FBA1 = read("pYPKa_Z_FBA1.gb")


    

In [6]:

    

# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/9169869', width='100%', height=250)


    

    
Out[6]:





        
        

In [7]:

    

gb = Genbank("bjornjobbb@gmail.com")
gene_template = gb.nucleotide("NM_001182243.1")


    

In [8]:

    

gene_template.list_features()


    

    
Out[8]:





+-----+------------------+-----+-----+-------+------+--------+------+
| Ft# | Label or Note    | Dir | Sta | End   |  Len | type   | orf? |
+-----+------------------+-----+-----+-------+------+--------+------+
|   0 | nd               | --> | 0   | 1008  | 1008 | source | yes  |
|   1 | nd               | --> | <0  | >1008 | 1008 | gene   | yes  |
|   2 | N:Transaldolase, | --> | 0   | 1008  | 1008 | CDS    | yes  |
+-----+------------------+-----+-----+-------+------+--------+------+

In [9]:

    

orf = gene_template.extract_feature(0)


    

In [10]:

    

orf.isorf()


    

    
Out[10]:





True

In [11]:

    

print(len(orf))
orf.seguid()


    

    




1008






    
Out[11]:





GgJ9s-lK11rrZmXZDHjE3AjiVrs

In [12]:

    

print(str(orf.seq.translate()))


    

    




MSEPAQKKQKVANNSLEQLKASGTVVVADTGDFGSIAKFQPQDSTTNPSLILAAAKQPTYAKLIDVAVEYGKKHGKTTEEQVENAVDRLLVEFGKEILKIVPGRVSTEVDARLSFDTQATIEKARHIIKLFEQEGVSKERVLIKIASTWEGIQAAKELEEKDGIHCNLTLLFSFVQAVACAEAQVTLISPFVGRILDWYKSSTGKDYKGEADPGVISVKKIYNYYKKYGYKTIVMGASFRSTDEIKNLAGVDYLTISPALLDKLMNSTEPFPRVLDPVSAKKEAGDKISYISDESKFRFDLNEDAMATEKLSEGIRKFSADIVTLFDLIEKKVTA*

In [13]:

    

pf, pr =parse('''

>TAL1_YPK_rec_fwd 
gtcgaggaacgccaggttgcccactttctcactagtgaAAATGTCTGAACCAGCTCAAAAGAA

>TAL1_YPK_rec_rev 
ATTTAAatcctgatgcgtttgtctgcacagatggcgcgTTAAGCGGTAACTTTCTTTTCAATCA
                        
''')


    

In [14]:

    

g = pcr( pf, pr, orf)


    

In [15]:

    

g.figure()


    

    
Out[15]:





                                        5ATGTCTGAACCAGCTCAAAAGAA...TGATTGAAAAGAAAGTTACCGCTTAA3
                                                                   |||||||||||||||||||||||||| tm 56.8 (dbd) 65.6
                                                                  3ACTAACTTTTCTTTCAATGGCGAATTgcgcggtagacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccactttctcactagtgaAAATGTCTGAACCAGCTCAAAAGAA3
                                         ||||||||||||||||||||||| tm 58.0 (dbd) 65.3
                                        3TACAGACTTGGTCGAGTTTTCTT...ACTAACTTTTCTTTCAATGGCGAATT5

In [16]:

    

terminator_clone = pYPKa_E_PDC1 =read("pYPKa_E_PDC1.gb")


    

In [17]:

    

p  = pcr( p167, p567, promoter_clone)
t  = pcr( p568, p166, terminator_clone)


    

In [18]:

    

pYPK0_E_Z, stuffer = pYPK0.cut((EcoRV, ZraI))
pYPK0_E_Z, stuffer


    

    
Out[18]:





(Dseqrecord(-5681), Dseqrecord(-81))

In [19]:

    

(pYPK0_E_Z, p, g, t)


    

    
Out[19]:





(Dseqrecord(-5681), Amplicon(792), Amplicon(1086), Amplicon(1061))

In [20]:

    

asm =Assembly((pYPK0_E_Z, p, g, t), limit=31)


    

In [21]:

    

asm


    

    
Out[21]:





Assembly
fragments..: 5681bp 792bp 1086bp 1061bp
limit(bp)..: 31
G.nodes....: 8
algorithm..: common_sub_strings

In [22]:

    

candidate = asm.assemble_circular()[0]
candidate.figure()


    

    
Out[22]:





 -|name|98
|       \/
|       /\
|       98|792bp_PCR_prod|38
|                         \/
|                         /\
|                         38|1086bp_PCR_prod|35
|                                            \/
|                                            /\
|                                            35|1061bp_PCR_prod|61
|                                                               \/
|                                                               /\
|                                                               61-
|                                                                  |
 ------------------------------------------------------------------

In [23]:

    

pYPK0_FBA1_ScTAL1_PDC1 = candidate.synced(pYPK0)


    

In [24]:

    

print(len(pYPK0_FBA1_ScTAL1_PDC1))
pYPK0_FBA1_ScTAL1_PDC1.cseguid()


    

    




8374






    
Out[24]:





z56XvyucVzpo2iNPYy5TbXbxWlk

In [25]:

    

pYPK0_FBA1_ScTAL1_PDC1.name="pMEC1130"
#pYPK0_FBA1_ScTAL1_PDC1.description = "pYPK0_FBA1_ScTAL1_PDC1tp"
pYPK0_FBA1_ScTAL1_PDC1.stamp()


    

    
Out[25]:





cSEGUID_z56XvyucVzpo2iNPYy5TbXbxWlk

In [26]:

    

pYPK0_FBA1_ScTAL1_PDC1.write("pYPK0_FBA1_ScTAL1_PDC1.gb")


    

    





pYPK0_FBA1_ScTAL1_PDC1.gb

In [27]:

    

from pydna.all import *


    

In [28]:

    

reloaded = read("pYPK0_FBA1_ScTAL1_PDC1.gb")


    

In [29]:

    

reloaded.cseguid()


    

    
Out[29]:





z56XvyucVzpo2iNPYy5TbXbxWlk

In [30]:

    

reloaded.description


    

    
Out[30]:





'cSEGUID_z56XvyucVzpo2iNPYy5TbXbxWlk_2018-05-15T07:40:46.014321'