I am having trouble developing or finding the correct primer

I am having trouble developing or finding the correct primers and restriction enzymes to fit the following DNA coding sequence into a selected vector for cloning. The sequence is as follows with the start and stop codons in bold and coding material italicized. Also attached is the vector diagram. How would I go about developing a working system of primers and enzymes to fit the desired DNA sequence into the primer for cloning and ultimately transfection?

1 gcgggccatg acaagctcca ggctttggtt ttcgctgctg ctggcggcag cgttcgcagg

61 acgggcgacg gccctctggc cctggcctca gaacttccaa acctccgacc agcgctacgt

121 cctttacccg aacaactttc aattccagta cgatgtcagc tcggccgcgc agcccggctg

181 ctcagtcctc gacgaggcct tccagcgcta tcgtgacctg cttttcggtt ccgggtcttg

241 gccccgtcct tacctcacag ggaaacggca tacactggag aagaatgtgt tggttgtctc

301 tgtagtcaca cctggatgta accagcttcc tactttggag tcagtggaga attataccct

361 gaccataaat gatgaccagt gtttactcct ctctgagact gtctggggag ctctccgagg

421 tctggagact tttagccagc ttgtttggaa atctgctgag ggcacattct ttatcaacaa

481 gactgagatt gaggactttc cccgctttcc tcaccggggc ttgctgttgg atacatctcg

541 ccattacctg ccactctcta gcatcctgga cactctggat gtcatggcgt acaataaatt

601 gaacgtgttc cactggcatc tggtagatga tccttccttc ccatatgaga gcttcacttt

661 tccagagctc atgagaaagg ggtcctacaa ccctgtcacc cacatctaca cagcacagga

721 tgtgaaggag gtcattgaat acgcacggct ccggggtatc cgtgtgcttg cagagtttga

781 cactcctggc cacactttgt cctggggacc aggtatccct ggattactga ctccttgcta

841 ctctgggtct gagccctctg gcacctttgg accagtgaat cccagtctca ataataccta

901 tgagttcatg agcacattct tcttagaagt cagctctgtc ttcccagatt tttatcttca

961 tcttggagga gatgaggttg atttcacctg ctggaagtcc aacccagaga tccaggactt

1021 tatgaggaag aaaggcttcg gtgaggactt caagcagctg gagtccttct acatccagac

1081 gctgctggac atcgtctctt cttatggcaa gggctatgtg gtgtggcagg aggtgtttga

1141 taataaagta aagattcagc cagacacaat catacaggtg tggcgagagg atattccagt

1201 gaactatatg aaggagctgg aactggtcac caaggccggc ttccgggccc ttctctctgc

1261 cccctggtac ctgaaccgta tatcctatgg ccctgactgg aaggatttct acgtagtgga

1321 acccctggca tttgaaggta cccctgagca gaaggctctg gtgattggtg gagaggcttg

1381 tatgtgggga gaatatgtgg acaacacaaa cctggtcccc aggctctggc ccagagcagg

1441 ggctgttgcc gaaaggctgt ggagcaacaa gttgacatct gacctgacat ttgcctatga

1501 acgtttgtca cacttccgct gtgagttgct gaggcgaggt gtccaggccc aacccctcaa

1561 tgtaggcttc tgtgagcagg agtttgaaca gacctgagcc ccaggcaccg aggagggtgc

1621 tggctgtagg tgaatggtag tggagccagg cttccactgc atcctggcca ggggacggag

1681 ccccttgcct tcgtgcccct tgcctgcgtg cccctgtgct tggagagaaa ggggccggtg

1741 ctggcgctcg cattcaataa agagtaatgt ggcatttttc tataaaaaaa aaaaaaaaaa

1801 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

Nhe I CMV Afl ll pro Hind Ill Kpn BamHI pcDNA3.1 +/C-(K)-DYK EcoRI 5444bp EcoRV Not I Xho I Xba I Apa I Stop SV40 promoter Hind III Kozak AGC TGG CTA GCG TTT AAA CTT AAG CTT GGT ACC GAG CTC GGA TCC GCC ACC GAA TTC TGC AGA TAT CCA Afl ll Nhe I Kpn I EcoRI EcoR V BamHI DYK tag Not I GCA CAG TGG CGG CCG CTC GAG TCT AGA GGG CCC GAT TAC AAG GAT GAC GAC GAT AAG TGA TAA Xho Xba Apa D Y K D D D D K Stop Stop

Solution

for cloning, few important points are to be taken into consideration.

1. ATG of the gene needs to be in-frame for translation for primer. For this, we can add few extra bases from the back sequence of the gene or ignore the region before start site also, if required. in this case, the start codon is not in-frame, so few bases after addition of RE to the primer sequence can be added as overhangs.

2. Stop codon does not require to be checked for in-frame while designing primers.

3. Forward primer can be designed directly by taking the sequence from gene, whereas for reverse primer, reverse complement of the sequence is to be taken. if the free energy is not in range then try using the back sequence of the gene to make the primers.

4. Enzymes which are absent in the gene and found only in the MCS vector are to be used in the primers. after addition of REs to the primers, a minimum of at least 3 base pairs are to be added as overhangs for allowing the restriction digestion. you can also check the compatibility of the enzymes being used in forward and reverse primers and use them accordingly.

5. After designing the primers, they need to be checked for primer self-dimers and hairpin formations, if the dG values are less than 3.6, then the dimers are easily formed, so the primers cannot participate in the amplification by binding to the template. Both forward and reverse primers together are to be checked for primer dimers.

6. The vector being used here is a mammalian expression vector and has a Kozak sequence in the middle of MCs. if you think the Kozak sequence is essential for your gene expression, then clone the gene using either upstream restriction sites or downstream but not both as it would delete your Kozak sequence.

hope these things would help you in designing your primer.