Gene: 1127 Name: Cacng4 Family: Channel Subfamily: Calcium Accession: NM_019431 GI: 9506454
Gene: 1127

Gene 1127
BLASTx Output


BLASTx Output. The query sequence is the Deltagen target gene.  The top "hits" are shown with
accession number, BLAST score and probability.  The alignment of the best hit is also shown.

BLAST DATE : 3/13/2003

 

                                                                         Score    E
 
 
gi|9506455|ref|NP_062304.1|  voltage-dependent calcium chann...   549   e-156
gi|21687010|ref|NP_542423.1|  voltage-dependent calcium chan...   548   e-156
gi|7656948|ref|NP_055220.1|  voltage-dependent calcium chann...   540   e-153
gi|16758072|ref|NP_445803.1|  calcium channelvoltage-depen...   306   7e-83
 
>gi|9506455|ref|NP_062304.1| voltage-dependent calcium channel gamma-4 subunit [Mus musculus]
 gi|13124021|sp|Q9JJV4|CCG4_MOUSE Voltage-dependent calcium channel gamma-4 subunit (Neuronal
           voltage-gated calcium channel gamma-4 subunit)
 gi|7452996|emb|CAB86386.1| calcium channel gamma 4 subunit [Mus musculus]
          Length = 327
 
 Score =  549 bits (1415)Expect = e-156
 
 Identities = 275/327 (84%)Positives = 275/327 (84%)
 Frame = +1
 
Query: 1   MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGD 180
           MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGD
Sbjct: 1   MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGD 60
 
Query: 181 LTHSGLWRVCCIEGIYRGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPXXXXXXXXX 360
           LTHSGLWRVCCIEGIYRGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFP         
Sbjct: 61  LTHSGLWRVCCIEGIYRGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLL 120
 
Query: 361 XXXXXXXXXXYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYN 540
                     YSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYN
Sbjct: 121 GGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYN 180
 
Query: 541 YGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKXXXXXXXXXXXXXXXXX 720
           YGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLK                 
Sbjct: 181 YGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRR 240
 
Query: 721 XXXXXXXXXXXXXXXXDASPVGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQDAGF 900
                           DASPVGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQDAGF
Sbjct: 241 RRSRSSSRSTEASPSRDASPVGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQDAGF 300
 
Query: 901 LQMHDFFQQDLKEGFHVSMLNRRTTPV 981
           LQMHDFFQQDLKEGFHVSMLNRRTTPV
Sbjct: 301 LQMHDFFQQDLKEGFHVSMLNRRTTPV 327
 
 
 

Construct

Gene:

1127

 

GI Number(s):

9506454

ES Cell Line:

3430

 

 

Accession Number:

NM_019431

 

 

Family:

Channel

 

 

Subfamily:

Calcium

 

 

Species:

Mouse

 

 

 

Sequence Type:

full length cDNA

 

 

 

 

 


Sequence Length:

984 bp

 

red underlined text = deleted in gene sequence

Sequence Deleted:

from base 526 to base 639

 

black text = gene sequence flanking deleted sequence



ATGGTGCGATGCGACCGCGGGCTGCAGATGCTGCTGACCACGGCCGGAGCCTTCGCCGCC
TTCTCGCTCATGGCCATCGCCATCGGCACCGACTACTGGCTGTACTCCAGCGCGCACATC
TGCAACGGCACCAACCTGACCATGGACGACGGGCCCCCGCCCCGCCGCGCTCGCGGCGAC
CTCACCCATTCGGGACTCTGGCGGGTGTGTTGCATCGAAGGCATCTATAGAGGGCACTGC
TTCCGGATCAACCACTTCCCAGAGGACAATGATTACGACCACGACAGCTCCGAGTACCTC
CTCCGCATTGTGCGAGCCTCCAGTGTCTTTCCCATCCTCAGCACCATTCTGCTCCTGCTC
GGAGGGCTCTGCATCGGCGCTGGGAGGATCTACAGCCGCAAGAACAATATTGTCCTCAGC
GCGGGAATCCTCTTTGTGGCGGCAGGCCTCAGTAATATCATCGGTATCATCGTCTACATT
TCCAGCAACACGGGCGACCCCAGTGACAAACGTGACGAAGACAAA
AAGAACCATTACAAC
TACGGCTGGTCTTTTTACTTTGGAGCCCTGTCGTTTATTGTGGCGGAGACCGTGGGCGTC
CTGGCTGTAAACATTTACATTGAGAAAAATAAAGAGTTG
AGGTTTAAGACCAAGCGGGAG
TTCCTTAAGGCCTCTTCCTCCTCTCCTTACGCCAGGATGCCGAGCTACAGGTACCGGCGA
CGGCGGTCCAGGTCCAGTTCAAGGTCCACGGAGGCCTCACCCTCCAGGGATGCATCTCCC
GTGGGCCTGAAGATCACCGGAGCCATTCCCATGGGTGAGCTGTCCATGTACACGCTATCC
AGAGAACCCCTTAAGGTGACCACAGCTGCGAGCTACAGTCCGGACCAGGACGCTGGCTTC
CTGCAGATGCATGACTTCTTCCAACAGGACCTAAAGGAAGGTTTCCATGTCAGCATGCTG
AACCGGCGGACGACTCCCGTGTGA




Targeting Vector* (genomic sequence)

    LacZ-Neo

Construct Number:  6698

Arm Length:
5':  5.5  kb
3':  1.3  kb


  * Not drawn to scale



 

5 ' PCR Reconfirmation

Gene:

1127

ES Line:

3430

Gene Specific Oligo (GS):
GCCACAGGGCCTGTGTCTCCCAGGC

25007



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

25007 + NEO1 (11193)

5025

3

25007 + NEO2 (11192)

5035

4

25007 + NEO3 (2416)

5600

5

DNA Sample Control

5167

6

Positive Control

4900

 


 

3 ' PCR Reconfirmation

Gene:

1127

ES Line:

3430

Gene Specific Oligo (GS):
GCTTGTACATCTGTGTCAGAACACC

25926



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

25926 + NEO1 (1431)

2300

3

25926 + NEO2 (1432)

2250

4

25926 + NEO3 (1170)

1550

5

DNA Sample Control

5167

6

Positive Control

1300

 


 

Legend : Genomic DNA from the recombinant ES line was assayed for homologous recombination using polymerase chain reactions (PCRs). Amplified DNA fragments were visualized by ethidium bromide staining following agarose gel electrophoresis, as shown in the gel image on the left. The test PCRs (a set of three lanes) employed the gene-specific (GS) primer, which lies outside of and adjacent to the targeting vector arm, paired in succession with one of three primers in the insertion fragment. The "DNA sample control" employed a primer pair intended to amplify a fragment from a non-targeted genomic locus. The "positive control" employed the GS primer paired with a primer at the other end of the arm. The table below the gel image lists the primers (numbered) used in each PCR and the expected product sizes, in base pairs (bp).  The diagrams to the right depict the anticipated gel image as well as the relative positions of the PCR primers.

 

 

 

3 ' Southern Blot Confirmation

Gene:

1127

ES Line:

3430

Construct:

6698

Blot Number:

3091

Gene Side:

3'

 

 

MW Marker

Lane

ES Line

Enzyme

 

1

Parent 1

Xba I

 

2

Parent 2

Xba I

 

3

Additional 1

Xba I

 

4

3430

Xba I

 

5

Parent 1

Eco RI

 

6

Parent 2

Eco RI

 

7

Additional 1

Eco RI

 

8

3430

Eco RI

 

Legend : Genomic DNA isolated from the indicated ES lines was digested with the indicated restriction enzymes (determined to cut outside of the construct arms).  The DNA was analyzed by Southern hybridization, probing with a radiolabeled DNA fragment that hybridizes outside of and adjacent to the construct arm.  The parent ES lines (negative controls) showed bands representing the endogenous (wild-type) Gene #1127 allele.  In contrast, the ES#3430  line showed an additional band representing the targeted allele from the expected homologous recombination event.   "Additional" denotes a secondary line that was derived from the same targeting vector but was not used to generate mice.
Note: All lanes represent data from the same blot.

 

Genotyping: Gene 1127 (F2 Mice)



 

W = wild-type

H = heterozygote

K = homozygote



A) Scheme:

B) Expected Result:

 

C) Oligo Information:

Type

ID

Sequence

Multiplex Conc.

GS(E,T)

30959

CTGTAGCTCGGCATCCTGGCGTAAG

400

NEO(T)

3196

GGGTGGGATTAGATAAATGCCTGCTCT

400

GS(E1)

30958

CTACATTTCCAGCAACACGGGCGAC

400

 

D) Reaction Conditions:

Allele

Expected Size

Oligos to use

Targeted

470

oligo 3196 + oligo 30959

Endogenous

236

oligo 30958 + oligo 30959