Typically, the first ~50 bases or so will not give a high-quality read. But the easy fix is to pick primers from the pGEM plasmid backbone that are at least 50 bp away from the insert.

The minimum size is whatever length of DNA you'd need that would be the entire tRNA. I'd go bigger than 40 bp. That's barely enough to get ~20 bp of the rRNA (plus ~20 bp of your primers). Why not design primers that would amplify the entire tRNA coding region?

The minimum size of a PCR product that needs to be cloned and sequenced in order to obtain accurate results depends on several factors, including the sequencing technology used, the quality of the PCR product, and the complexity of the sample. However, in general, the minimum size of a PCR product that can be reliably cloned and sequenced is typically around 100-150 base pairs.

tRNAs are relatively small molecules, typically around 70-90 nucleotides in length, and they can be difficult to amplify by PCR due to their secondary structure. However, if a tRNA PCR product can be successfully amplified, it can potentially be cloned and sequenced using standard molecular biology techniques.

It's worth noting that sequencing tRNAs can be challenging due to their high degree of sequence similarity, especially within the anticodon loop region. Therefore, multiple clones may need to be sequenced and aligned to obtain accurate sequence information. Additionally, careful attention to sequencing quality control measures, such as checking for sequencing errors and ensuring sufficient read depth, can help to improve the accuracy of the sequencing results.

For accurate Sanger sequencing, it is recommended to have a minimum of 100-150 base pairs in the cloned PCR product. However, this may vary depending on the quality and purity of the PCR product. It is also important to ensure that the PCR product is free from any secondary structures or contaminants that could interfere with sequencing. In your case, since you are expecting a PCR product of only 40 base pairs, it may be challenging to obtain reliable Sanger sequencing results. Consider optimizing your PCR conditions to increase the yield of the PCR product or consider alternative sequencing methods such as Next-Generation Sequencing (NGS) which may be more suitable for small fragment sizes.

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https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

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