Gene: 356 Name: LOC215944 Family: GPCR Subfamily: Orphan GPCR Accession: XM_137063 GI: 20858532
Gene 356

 

Gene 356
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|20858533|ref|XP_137063.1|  similar to probable G protein-...   582   e-166
gi|13929142|ref|NP_113994.1|  putative G protein-coupled rec...   578   e-164
gi|4885341|ref|NP_005275.1|  G protein-coupled receptor 6 [H...   550   e-156
gi|6680065|ref|NP_032180.1|  G-protein coupled receptor 3 [M...   352   1e-96
 
>gi|20858533|ref|XP_137063.1| similar to probable G protein-coupled receptor protein - rat [Mus
            musculus]
          Length = 363
 
 Score =  582 bits (1500)Expect = e-166
 
 Identities = 297/363 (81%)Positives = 297/363 (81%)
 Frame = +1
 
Query: 1    MNASAAALNESQXXXXXXXXXXXXXXXXXXPDTGEWXXXXXXXXXXXXXXXXXXXXXXXQ 180
            MNASAAALNESQ                  PDTGEW                       Q
Sbjct: 1    MNASAAALNESQVVAVAAEGAAAAATAAGAPDTGEWGPPAASAALGGGGGPNGSLELSSQ 60
 
Query: 181  LPAGPSGLLLSAVNPWDVLLCVSGTVIAGENALVVALIASTPALRTPMFVLVGSLATADL 360
            LPAGPSGLLLSAVNPWDVLLCVSGTVIAGENALVVALIASTPALRTPMFVLVGSLATADL
Sbjct: 61   LPAGPSGLLLSAVNPWDVLLCVSGTVIAGENALVVALIASTPALRTPMFVLVGSLATADL 120
 
Query: 361  LAGCGLILHFVFQYVVPSETVSLLMVGXXXXXXXXXXXXXXXITVDRYLSLYNALTYYSR 540
            LAGCGLILHFVFQYVVPSETVSLLMVG               ITVDRYLSLYNALTYYSR
Sbjct: 121  LAGCGLILHFVFQYVVPSETVSLLMVGFLVASFAASVSSLLAITVDRYLSLYNALTYYSR 180
 
Query: 541  RTLLGVHLLLAATWTVSXXXXXXXXXXWNCLADRTSCSVVRPLTRSHVALLSTSFFVVFG 720
            RTLLGVHLLLAATWTVS          WNCLADRTSCSVVRPLTRSHVALLSTSFFVVFG
Sbjct: 181  RTLLGVHLLLAATWTVSLGLGLLPVLGWNCLADRTSCSVVRPLTRSHVALLSTSFFVVFG 240
 
Query: 721  IMLHLYVRICQVVWRHAHQIALQQHCLAPPHLAATRKGVGTLAVVLGTFGASWLPFAIYC 900
            IMLHLYVRICQVVWRHAHQIALQQHCLAPPHLAATRKGVGTLAVVLGTFGASWLPFAIYC
Sbjct: 241  IMLHLYVRICQVVWRHAHQIALQQHCLAPPHLAATRKGVGTLAVVLGTFGASWLPFAIYC 300
 
Query: 901  VVGSQEDPAIYTYATLLPATYNSMINPIIYAFRNQEIQRALWLLFCGCFQSKVPFRSRSP 1080
            VVGSQEDPAIYTYATLLPATYNSMINPIIYAFRNQEIQRALWLLFCGCFQSKVPFRSRSP
Sbjct: 301  VVGSQEDPAIYTYATLLPATYNSMINPIIYAFRNQEIQRALWLLFCGCFQSKVPFRSRSP 360
 
Query: 1081 SEV 1089
            SEV
Sbjct: 361  SEV 363
 
 
 


Construct

Gene:

356

 

GI Number(s):

20858532

Family:

GPCR

 

 

Subfamily:

Orphan GPCR

 

 

Species:

Mouse

 

 

Sequence Type:

CDS

 

 

 

 

Sequence Length:

1092 bp

 

red underlined text = deleted in gene sequence

Sequence Deleted:

from base 45 to base 482

 

black text = gene sequence flanking deleted sequence



ATGAACGCTAGCGCCGCCGCGCTCAACGAGTCCCAAGTGGTGGCAGTAGCGGCCGAGGGA
GCGGCAGCTGCGGCTACAGCAGCAGGGGCACCGGACACCGGCGAATGGGGACCCCCAGCA
GCATCCGCAGCGCTGGGAGGCGGCGGCGGACCTAACGGGTCACTGGAGCTGTCTTCTCAG
CTGCCAGCAGGGCCCTCGGGACTACTGCTTTCGGCAGTGAATCCCTGGGATGTGCTGCTG
TGCGTGTCGGGAACTGTGATCGCAGGCGAAAATGCGCTGGTGGTGGCGCTCATCGCGTCC
ACTCCCGCGCTGCGCACGCCCATGTTTGTGCTCGTGGGTAGTCTGGCCACTGCTGACCTG
TTGGCGGGCTGTGGCCTCATCTTACACTTCGTGTTCCAGTACGTGGTGCCCTCGGAGACT
GTGAGCCTGCTCATGGTGGGCTTCCTGGTGGCGTCCTTTGCCGCCTCAGTCAGCAGCCTG
CTTGCTATCACAGTTGACCGTTACCTGTCTTTGTACAACGCGCTCACCTACTACTCGCGC
CGGACCCTGTTGGGCGTGCACCTCTTGCTAGCAGCCACCTGGACAGTGTCCCTCGGGTTG
GGGTTGCTGCCTGTTCTAGGCTGGAACTGCTTGGCCGACCGCACGTCCTGCAGCGTGGTG
CGCCCCCTGACACGCAGCCATGTGGCGTTGCTCTCTACTTCCTTCTTCGTGGTCTTTGGC
ATCATGCTGCACTTGTACGTGCGCATCTGCCAGGTGGTCTGGCGCCACGCCCACCAGATC
GCTTTGCAACAGCATTGCCTAGCACCGCCCCACTTAGCAGCCACCAGAAAGGGAGTGGGA
ACTTTGGCCGTGGTGCTAGGCACTTTTGGGGCCAGCTGGCTGCCCTTCGCCATCTATTGT
GTGGTGGGTAGCCAAGAGGACCCGGCCATCTACACTTATGCCACCCTGTTGCCTGCCACC
TATAACTCCATGATCAATCCCATCATCTATGCCTTCCGCAACCAGGAGATCCAGCGTGCC
TTGTGGCTCCTGTTCTGTGGCTGTTTCCAATCCAAAGTGCCCTTCCGCTCCAGGTCCCCC
AGTGAGGTCTGA



Targeting Vector* (genomic sequence)

    LacZ-Neo

Construct Number:  10684

Arm Length:
5':  5  kb
3':  1  kb


  * Not drawn to scale



 

5 ' PCR Reconfirmation

Gene:

356

ES Line:

6421

Gene Specific Oligo (GS):
CAAGTGTCAGCCTTCAGCATGGTAC

42742



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

4625bp

4635bp

5200bp

5167bp

4500bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

42742 + NEO1 (11193)

4625

3

42742 + NEO2 (11192)

4635

4

42742 + NEO3 (2416)

5200

5

DNA Sample Control

5167

6

Positive Control

4500

 

 

3 ' PCR Reconfirmation

Gene:

356

ES Line:

6421

Gene Specific Oligo (GS):
GCTGGATCTCCTGGTTGCGGAAGGC

40482



Gel

Expected Results and Diagram

 11000bp

 10000bp

 9000bp

 8000bp

 7000bp

 6000bp

 5000bp

 4000bp

 3000bp

 2000bp

 1600bp

 1000bp

 500bp

1500bp

1450bp

750bp

5167bp

500bp

Lane Number

Label

Expected Size (in bp)

1

1kB Ladder

 

2

40482 + NEO1 (1431)

1500

3

40482 + NEO2 (1432)

1450

4

40482 + NEO3 (1170)

750

5

DNA Sample Control

5167

6

Positive Control

500

 

 

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:

356

ES Line:

6421

Gene Side:

3'

 

 

MW Marker

Lane

ES Line

Enzyme

Yes

1

 

 

 

2

Parent 1

Hind III

 

3

Parent 2

Hind III

 

4

6421

Hind III

 

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 #356 allele.  In contrast, the ES#6421  line showed an additional band representing the targeted allele from the expected homologous recombination event.  
Note: All lanes represent data from the same blot.

 

Genotyping: Gene 356 (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)

54515

CACCTGGCAGATGCGCACGTACAAG

400

NEO(T)

3195

GGGCCAGCTCATTCCTCCCACTCAT

400

GS(E1)

54514

CTTGGGCAGTGCACCTGACAATCTG

400

 

D) Reaction Conditions:

Allele

Expected Size

Oligos to use

Targeted

563

oligo 3195 + oligo 54515

Endogenous

817

oligo 54514 + oligo 54515