I'm trying to pcr amplify out the promoter region for Car2 in mouse gDNA. The reference sequence is:

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tgaagacctgacagaaagaaaatgcctacagcctctgtaatttgaaaaga

aaatgaatatgagatataatacctgatgctctcttg{START}actgtccaaaggtg

ggcaaggagactccctagcaggagcctggctcccactaaaacgtaccaga

caggatgaggattcccgtagctgcaaccagaattggtggggtctgaagcc

ttgagaagcacttcatcaaaatctgttcttattccacgcacggtagtaat

ggaacaggaaatgggaaacccctagcaaattgtgcatctctagtgtcaca

gatgcaagggactatttgtgaattgcactttgtaatggtcctataaaaca

ttcttcatcaaaaatttcttttgacatcaacctgaagagtcttgtataga

gaagaccataattttctgagtcttagtataagaacaaggagatctacatg

tgcatcatctttgtgtggcactgggtgaaataaaggcacatgatgagtct

gagaacacttaaaatgtagtcagatatttttcattatacttacacataga

ttctaatatataggtaatgtgatgtctaggtgcctccatagaaacacaaa

aaaactgtacccttctcaatttgaattctaaataatatggcctcaggtcc

acaatagattcaatacaaaaaaattgttctcaaagtcaaataagaaaaaa

agtgttaggtgaagagaaatgtttaaataaaaatacacaaaaagaacatt

gaaaggtgtgtccccgggacacccacggtccctgggaagccatacagcta

cctttcttttctattttttaagttttgtgttttttgttttgttttgtttt

ggggggggggggttggtttcggcttgtcaaatacaaattcctttgccagg

aaggggaagttctcatctccttggaagacatttttaaaatccaattccaa

aaagcaaagcgatttttgagaattgttggttatgttacatcaacagttcg

gtgcctagggttaatttgtggagcaagcgggaagctggagggcgccctgg

cggcaaagccgggtcgtcttcccgcagcccggcagcagggggcccccccg

cttcccgctcccggctccaggatccccgctcccggagcctctaggacgct

ccccgcccaaggtcaaactccgcagccgcgtgcgtgcgggccgcggccgg

gtggccggctggcccgccagctctgggaagcggagccccgctgtgcgggc

gggtgaccaggtcggtgctcacaatggggctggggagtaggggcgccggc

ccgcgaggctgcgcagcgccggggcgagctggagagcgcgcagctgctgc

agagcgggtcggacgcagaagcggagagcagccggagggcgccccgcccc

aagcaaggtgcgggggccctcgaacaccgaggcctccgcctgtcacctct

gcctgtcacctccgtccgccacctccacggtctcctccccttgctcaggt

c{STOP}cactcggtccctcccctgggccgcccagagcaccaagttggcgggagcc

tataaaagccggacggtgcgacccgcgacacacactgcaggaccgctaga

I used primer blast to generate the primers:

forward - AGA AAG AAA ATG CCT ACA GCC TC

reverse - TTT ATA GGC TCC CGC CAA CTT

I tried a few others as well, but they all failed except for this one which got amplification at my predicted amplicon size of ~1500bp at 10ng of starting gDNA per pcr reaction at 40 cycles:

Admittedly the banding was faint, but there were several other off-target bands that were predicted by primer blast. So I assumed I got the right amplification. I excised the appropriate band, purified from gel, and retrieved approx. 200ng of DNA. I then put this DNA in an additional PCR reaction at 10ng @ 40 cycles.

This time, I get strong banding at approx. 200bp, and no banding at 1.5Kb. Which is peculiar because I sized selected between 1.2-2Kb on the gel. I blasted my primers against the imputed reference sequence and they both only align once against reference, so I'm not sure what the issue is.

Any ideas?