Assembly of a pTAx vectors by in-vivo homologous recombination
The pTA7 vector is a circular plasmid assembled from five different linear PCR products. Table 2 shows the five PCR products with templates and primer numbers. The first column in Table 2 contains the designation for each of the five PCR products. The Template column contain the designation of the template sequence. Sequence files corresponding to this column (with “.gb” file endings can be found in the same folder as this file. The Primer#1 and #2 columns contain two numbers corresponding to the first column in Table 4, where you can find the primer sequences.
Table 1: Partial lSEGUIDS for PCR products
| PCR product | Partial lSEGUID |
|---|---|
| ampR | |
| pBR | |
| 2µ | |
| TRP1 | |
| ΔCRP |
Use the information you have together with the WebPCR web service to generate the sequence of each of the PCR products in Table 1.
Table 2: Genetic elements for plasmid pTA7
| PCR product | Template | Primer#1 | Primer#2 |
|---|---|---|---|
| ampR | pBR322 | 1113 | 987 |
| pBR | pBR322 | 1196 | 1195 |
| 2µ | YEplac195 | 984 | 983 |
| TRP1 | pTA8 | 1748 | 1742 |
| ΔCRP | pYPKpw | 978 | 977 |
Table 3: Assembly sequences
| Symbol | Designation | Sequence (30bp) |
|---|---|---|
| ❶ | s1 | AATCCAATCAGCGTAAGGTGTAGACTTTCT |
| ❷ | s2 | ATCGTATCTGCTGCGTAAATAGTAGTCAAC |
| ❸ | s3 | TAAAATCTCGTAAAGGAACTGTCTGCTCTG |
| ❹ | s4 | AACTGTAAAATCAGGTATCTCGTAGTCCGT |
| ❺ | s5 | GAAAAGCGTTTACCTCGGAACTCTATTGTA |
Table 4: Primer sequences
| 1750_s1 | AATCCAATCAGCGTAAG |
|---|---|
| 1749_s2 | ATCGTATCTGCTGCGT |
| 1748_s3 | TAAAATCTCGTAAAGGAACT |
| 1747_s4 | AACTGTAAAATCAGGTATCT |
| 1746_s5 | GAAAAGCGTTTACCTCG |
| 1745_s1r | AGAAAGTCTACACCTTAC |
| 1744_s2r | GTTGACTACTATTTACGCA |
| 1743_s3r | CAGAGCAGACAGTTCC |
| 1742_s4r | ACGGACTACGAGATAC |
| 1741_s5r | TACAATAGAGTTCCGAG |
| 1352_HIS3fp_pTA | TAAAATCTCGTAAAGGAACTGTCTGCTCTGAACACAGTCCTTTCC |
| 1351_HIS3rp_pTA | ACGGACTACGAGATACCTGATTTTACAGTTTTTTTTCTCGAGTTCAAGA |
| 1350_KanMX4fp_pTA | TAAAATCTCGTAAAGGAACTGTCTGCTCTGGACATGGAGGCCC |
| 1349_KanMX4rp_pTA | ACGGACTACGAGATACCTGATTTTACAGTTCAGTATAGCGACCAGC |
| 1348_TRP1fp_pTA | TAAAATCTCGTAAAGGAACTGTCTGCTCTGTTTGCCGATTAAGAATTCG |
| 1347_TRP1rp_pTA | ACGGACTACGAGATACCTGATTTTACAGTTGATCTTTTATGCTTGCTTTTC |
| 1346_URA3fp_pTA | TAAAATCTCGTAAAGGAACTGTCTGCTCTGGATTCCGGTTTCTTTGAAA |
| 1345_URA3rp_pTA | ACGGACTACGAGATACCTGATTTTACAGTTGGTAATAACTGATATAATTAAATTGAA |
| 1344_CEN_ARSfp_pTA | ATCGTATCTGCTGCGTAAATAGTAGTCAACACGGATCGCTTGC |
| 1343_CEN_ARSrp_pTA | CAGAGCAGACAGTTCCTTTACGAGATTTTACTTTTCATCACGTGCTATA |
| 1196_Pbr.FW | AATCCAATCAGCGTAAGGTGTAGACTTTCTCTGTCAGACCAAGTTTACT |
| 1195_Pbr.REV | GTTGACTACTATTTACGCAGCAGATACGATCTCGTTTCATCGGT |
| 1113_Amp.fw.nw | GAAAAGCGTTTACCTCGGAACTCTATTGTAGAACCCCTATTTGTTTATTTTTCTA |
| 988_Amp.FW | GAAAAGCGTTTACCTCGGAACTCTATTGTAAACCCTGATAAATGCTTC |
| 987_Amp.REV | AGAAAGTCTACACCTTACGCTGATTGGATTTGAAGTTTTAAATCAATCTAAA |
| 986_Pbr.FW | AATCCAATCAGCGTAAGGTGTAGACTTTCTCTCGTTTCATCGGTAT |
| 985_Pbr.REV | GTTGACTACTATTTACGCAGCAGATACGATCTGTCAGACCAAGTTTACT |
| 984_Micron.FW | ATCGTATCTGCTGCGTAAATAGTAGTCAACGATCGTACTTGTTACCCAT |
| 983_Micron.REV | CAGAGCAGACAGTTCCTTTACGAGATTTTAGATCCAATATCAAAGGAA |
| 982_dLeu.FW | TAAAATCTCGTAAAGGAACTGTCTGCTCTTATATATATTTCAAGGATATACCATT |
| 981_dLeu.REV | ACGGACTACGAGATACCTGATTTTACAGTTTAAGGCCGTTTCTGAC |
| 980_Leu2.FW | TAAAATCTCGTAAAGGAACTGTCTGCTCTGTTAACTGTGGGAATACTC |
| 979_Leu2.REV | ACGGACTACGAGATACCTGATTTTACAGTTTCTACCCTATGAACATATTC |
| 978_Crp.FW | AACTGTAAAATCAGGTATCTCGTAGTCCGTGTTCTGATCCTCGAGC |
| 977_Crp.REV | TACAATAGAGTTCCGAGGTAAACGCTTTTCGTTCTTGTCTCATTGCC |
© Björn Johansson 2022