Materials
On this page, you will find information about the plasmids, strains, primers and DNA sequences we used. For the kits used for our experiments, the references are indicated on the protocol page.
Plasmids
- • pET21a (+): this plasmid was used as a backbone for almost all our constructs, as it allowed us to express all the proteins we needed. This expression is dependent on a T7 promoter. It confers ampicillin resistance. pET21a (+) was a gift from Ambre Jousselin. See the plasmid map on Snapgene here.
- • pET26b pelB-HER2-tev stops: this plasmid was used to express and produce the VHH anti-HER2. It contains a pelB leader sequence to direct the produced protein to the periplasm for further extraction and a Histag. This device confers the kanamycin resistance. This plasmid was a gift from Adilya Dagkesamanskaya.
- • p0579: this plasmid was used to clone and express the following fragment: Nterminal of the T7 RNA polymerase - linker - VH and VL of Pertuzumab antibody. It confers spectinomycin resistance. It originally contains Nterminal of the T7 RNA polymerase - linker - FRB to which we added an SP6 promoter with an RBS upstream and a T7 terminator. p0579 was a gift from Jérôme Bonnet.
- • p0539: this plasmid was used to clone and express the following fragment: VH and VL of Trastuzumab antibody - linker - Cterminal of the T7 RNA polymerase. It originally contains FKBP- linker - Nterminal of the T7 RNA polymerase to which we added an SP6 promoter with an RBS upstream and a T7 terminator. It confers chloramphenicol resistance. p0539 was a gift from Jérôme Bonnet.
Strains
- • E. coli Stellar: (F–, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ–) used for transformation during In-Fusion® cloning (Stellar Competent cells from Takara, ref: 638952).
- • E. coli BL21 (DE3): (E. coli str. B, F–, ompT, gal, dcm, lon, hsdSB(rB–mB–), λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]), [malB+]K-12(λS) used to express and produce DhdR and anti-Her2 nanobodies. The production of the T7 RNA polymerase can be induced by IPTG. This was a gift from Cédric Montanier (TBI, Toulouse).
Primers
gBlocks amplification
Primers used for gBlocks amplification:
| gBlock | Primers | Sequences 5’ to 3’ |
| dhdR_HisTag | dhdR_pET21_F | agcagccggatctcatcatgacgtctgacgcgc |
| dhdR_pET21_F | agcagccggatctcatcatgacgtctgacgcgc | |
| T7promoter_dhdO_typh_T7terminator | T7term-F | agttcctcctttcagcaaaaaacccctcaagaccc |
| T7prom-R | gagatctcgatcccgcgaaattaatacgactcactatagg | |
| T7promoter_dhdO_sfgfp_T7terminator | T7term-F | agttcctcctttcagcaaaaaacccctcaagaccc |
| T7prom-R | gagatctcgatcccgcgaaattaatacgactcactatagg | |
| SP6promoter_NtermT7_linkerJB_Pertuzumab_TerminatorT7 | T7-F | agttcctcctttcagatttaggtgacactataggggagac |
| T7-R | gagatctcgatcccgcaaaaaacccctcaagacccg | |
| SP6promoter_Trastuzumab_linkerJB | T7-F | agttcctcctttcagatttaggtgacactataggggagac |
| Trastuzumab-R1’ | agtagacccttagagagcatgtcagcctcgacaact | |
| CtermT7_T7terminator | Trastuzumab-F1’ | cgaggctgacatgctctctaagggtctactcggtggc |
| T7-R | gagatctcgatcccgcaaaaaacccctcaagacccg | |
| SP6promoter_NtermT7_linkerJB_FRB_T7terminator (2 steps PCR) |
RBS-FRB-F1 | tagaaataattttgtttaactttaagaaggagatataccaatgaacacgattaacatcgc |
| T7term-FRB-R1 | cccgtttagaggccccaaggggttatgctattagccgattttacgaaaga | |
| SP6-F | tacctatttaggtgacactataggggagaccacaacggtttccctctagaaataattttgt | |
| T7term-rap-R2 | cgcacccaaaaaacccctcaagacccgtttagaggccc | |
| SP6promoter_FKBP_linkerJB_CtermT7_T7terminator (2 steps PCR) |
RBS-FRB-F1 | tagaaataattttgtttaactttaagaaggagatataccaatgaacacgattaacatcgc |
| R1-rap-FKBP | cccgtttagaggccccaaggggttatgctattacgcgaacgcgaagtccg | |
| SP6-F | tacctatttaggtgacactataggggagaccacaacggtttccctctagaaataattttgt | |
| T7term-rap-R2 | cgcacccaaaaaacccctcaagacccgtttagaggccc |
Plasmid linearization
Primers used for plasmid linearization:
| Plasmid | Inserts(s) | Primers | Sequence 5' to 3' |
| pET21a(+) | dhdR_HisTag | pET21-R | tgagatccggctgctaaca |
| pET21-F | atgtatatctccttcttaaagttaa | ||
| T7promoter_dhdO_typh_T7terminator
or T7promoter_dhdO_sfgfp_T7terminator or SP6promoter_NtermT7_linkerJB_Pertuzumab_TerminatorT7 or SP6promoter_Trastuzumab_linkerJB + CtermT7_T7terminator |
pET19T7-R | ctgaaaggaggaactatatccgg | |
| pET19T7-F | cgggatcgagatctcgatcctctac | ||
| p0579 | SP6promoter_NtermT7_linkerJB_Pertuzumab_TerminatorT7 | Pertuv2-R | gaaaacgaaaggcccagtctttcgactg |
| p0579-Pertu-F | tggagattttcaacatgctccctc | ||
| p0539 | SP6promoter_Trastuzumab_linkerJB + CtermT7_T7terminator |
p0539-Trastu-R | tcaatgtggacttttctgccg |
| p0539-Trastu-F | acttaattaacggcactcctcagc |
Sequencing
Primers used for sequencing:
| Construction | Primers | Sequence 5' to 3' |
| pET21_dhdR | pET21seq-F | ttaatgcgccgctacagggcgcg |
| pET21seq-R | gttgagcaccgccgccgcaaggaat | |
| dhdRseq-1 | tgagctcctgtacgagaaactacaa | |
| pET21_typh | pET21seq-F | ttaatgcgccgctacagggcgcg |
| pET21seq-R | gttgagcaccgccgccgcaaggaat | |
| TYPH-1 | aggcccggcaccgtccatgcataac | |
| TYPH-2 | cccattaacgactgcggctacaaaa | |
| pET21_sfgfp | pET21seq-F | ttaatgcgccgctacagggcgcg |
| pET21seq-R | gttgagcaccgccgccgcaaggaat | |
| sfgfpseq-1 | ctattaacaagggtatcaccttcaa | |
| p0579_NtermT7_linkerJB_Pertuzumab | P0579-F | ggtttaccagcgccaaagac |
| T7-RNAP-1 | caagaaaaacgttgaggaacaactca | |
| VH pertuzumab-1 | gccatcaaacaccaaggtcg | |
| VL pertuzumab-1 | ctccctcggacgagcag | |
| P0579-R | ggtttaccagcgccaaagac | |
| p0539_Trastuzumab_linkerJB_CtermT7 | P0579-F | ggtttaccagcgccaaagac |
| VH Trastuzumab-1 | aacagtggcgcgctga | |
| VL Trastuzumab-1 | gccaagatgttaacaccgcag | |
| Trastuzumab-F1’ | cgaggctgacatgctctctaagggtctactcggtggc | |
| T7 Cterm-1 | cttacaacatggactggcgc | |
| T7 Cterm-2 | tcaacaagtgctggaagataccat | |
| T7 Cterm-3 | tgtactggctgatttctacgacc | |
| P0539-R | tgtcggcagaatgcttaatgaattac | |
| SP6promoter_FRB_NtermT7_T7terminator: | SP6-seq-F | tacctatttaggtgacactataggg |
| T7Nterm-mid-seq-R | tgctacagcctgaacggttg | |
| rap-T7term-R | cgcacccaaaaaacccctca | |
| SP6promoter_FKBP_CtermT7_T7terminator: | SP6-seq-F | tacctatttaggtgacactataggg |
| T7Cterm -0 | cgaggctgacatgctctctaagggt | |
| T7 Cterm-1 | cttacaacatggactggcgc | |
| T7 Cterm-2 | tcaacaagtgctggaagataccat | |
| T7 Cterm-3 | tgtactggctgatttctacgacc | |
| rap-T7term-R | cgcacccaaaaaacccctca |
DNA sequencies
Plasmids
• pET21 a (+):
atccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatctcagtggtggtggtggtggtgctcgagtgcggccgcaagcttgtcgacggagctcgaattcggatccgcgacccatttgctgtccaccagtcatgctagccatatgtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccgggatctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgcca
| Feature name | Start | End |
| T7 terminator | 26 | 73 |
| His-tag | 140 | 157 |
| MCS | 158 | 203 |
| T7 promoter | 309 | 327 |
| Ampicillin resistance gene | 3985 | 4845 |
• pET26b pelB-Her2-tev stops:
tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggaggtacaactagtagagtcggggggggggctagtacaagctggggggtcgctaaggctatcgtgcgctacttcggggataactttcatgaggtacgctctagggtggtacaggcaatcgcccgggaagcaaagggagatggtagcttcgataaactcgggggggactactaactacgctgactcggtaaaggggaggttcactatatcgagggacaacgctaagaacactgtatacctacaaatgaactcgctaaagcccgaggacactgctgtatactactgcaacgctaggtgggtaaagccccaattcatagacaacaactactgggggcaagggactcaagtaactgtatcgtcgctcgagaacctgtacttccagggtcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg
| Feature name | Start | End |
| T7 promoter | 2495 | 2514 |
| pelB leader | 2586 | 2648 |
| VHH HER2 | 2652 | 3011 |
| TEV site | 3015 | 3035 |
| His-Tag | 3036 | 3053 |
| T7 terminator | 3121 | 3167 |
| Kanamycin resistance gene | 3755 | 4567 |
• p0579:
acggatcgcttcatgtggcaggagaaaaaagactgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgattcgagctcgcccggggatcgaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgatttccggtcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttctaaacaactaacggacaattctacctaggatccctcgaaaggaaaaaaaaaatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgttcagctcgcggacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcccgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaggcgcccgacagccttccagttcctaaaagaaatcaagccggaagccgtagcgtacatcaccattaagacctctctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcggaccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagggtggctctggctctggctcgagcgccgtgttatggcacgaacagtggtatgagggtttggatgacgcatcgcgccagttcttcggagaacataacaccgaaaaaatgtttgcggcactggaacctctttatgagatgcttaaacgtgggcctgaaacgttgcgtgagatttcttttcagaactcatttgggcgtgatttgaacgatgcctacgaatggctgatgaattacaaaaagtcgaaagatgtatctaacctgaatcaagcatgggatatttactataatgtctttcgtaaaatcggctaatggagattttcaacatgctccctcaatcggttgaatgtcgcccttttgtctttggcgctggtaaaccatatgaattttctattgattgtgacaaaataaacttattccgtggtgtctttgcgtttcttttatatgttgccacctttatgtatgtattttctacgtttgctaacatactgcgtaataaggagtcttaatcatgccagttcttttgggtattccgtagaaaaaggaagagtatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaa
| Feature name | Start | End |
| Nterm T7 | 1415 | 1951 |
| Linker | 1952 | 1975 |
| FRB | 1976 | 2257 |
| Spectinomycin resistance gene | 2497 | 3285 |
• p0539:
cactcggtcgctacgctccgggcgtgagactgcggcgggcgctgcggacacatacaaagttacccacagattccgtggataagcaggggactaacatgtgaggcaaaacagcagggccgcgccggtggcgtttttccataggctccgccctcctgccagagttcacataaacagacgcttttccggtgcatctgtgggagccgtgaggctcaaccatgaatctgacagtacgggcgaaacccgacaggacttaaagatccccaccgtttccggcgggtcgctccctcttgcgctctcctgttccgaccctgccgtttaccggatacctgttccgcctttctcccttacgggaagtgtggcgctttctcatagctcacacactggtatctcggctcggtgtaggtcgttcgctccaagctgggctgtaagcaagaactccccgttcagcccgactgctgcgccttatccggtaactgttcacttgagtccaacccggaaaagcacggtaaaacgccactggcagcagccattggtaactgggagttcgcagaggatttgtttagctaaacacgcggttgctcttgaagtgtgcgccaaagtccggctacactggaaggacagatttggttgctgtgctctgcgaaagccagttaccacggttaagcagttccccaactgacttaaccttcgatcaaaccacctccccaggtggttttttcgtttacagggcaaaagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctactgaaccgctctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagcccaggaggaagaggacatccggtcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttagacttagggaccctttatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccaccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaacccaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttttttggacgcgtacaactcaagtctgacataaatgaccgctatgggagttcaggtagagacaattagtcctggggatggccgcacatttccgaaacgcggccaaacctgtgtggtacactatacaggaatgttggaagatggcaaaaagtttgattccagtcgtgatcgcaacaagccttttaaatttatgttgggaaaacaggaggtaattcgcggttgggaggaaggtgtcgcacaaatgtccgtgggtcaacgcgccaagttgacgatctcacctgattacgcttacggagccaccggccatcctggcattattcccccacatgccactttggtctttgatgttgaacttttaaagttagaaacgtcgggtggaagcggtaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaacttaattaacggcactcctcagccaagtcaaaagcctccgaccggaggcttttgactacatgcccatggcgtttacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatagtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacggaactccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagtaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccaagccaaataggccgt
| Feature name | Start | End |
| FKBP | 2166 | 2489 |
| Linker | 2490 | 2507 |
| Cterm T7 | 2508 | 4622 |
| Chloramphenicol resistance gene | 4696 | 5355 |
DNA gBlocks sequencies
• dhdR-HisTag:
tcatgacgtctgacgcgcggctgggcttaaaagatccaagcgaagacgagaagccgcttgttgaagatggcgcgtagcagccaaacgagcgcgatccgggtctcctgctaaaatcgcgtcatacactgctacatgctcttgatgaactaccgcggtcaatccttcctggcgacgactgttggtacgagcagtcgatacagctaaacgtaactgaaggttaagatattgcagcagatcttgatagtagcgattatgagtcgcagcagcaatagctacatgaaaagcaatgtcgtgttccacaccttgttctgggtggtcaagattggcttccaaagctgcaagggcctcagccatggcggccagatctgtagcattgcgacgacgtgctgccaatgccgctgccccaccctccagttccatacgcagttcgtacacggaagccagttgttcacggtcatctaacccggcgcttacaaggaatccttgagctcctgtacgagaaactacaacgctccctgacccttggcgactctggataagcccctgtgcacgcagacgttcggtcgcctcacggattacagcagcactaaccccgtattgctcagccaggacacggccagggggtaacttcgctccgacagagtgaacaccttcggcgatgtcacccgcaatttgacgagcgacttgctctgtaagagttaaactctttgaaagcatcttgtcgtcgtcgtcgatatggccgctgctatgatgatgatgatgatggcccat
| Feature name | Start | End |
| dhdR | 1 | 717 |
| Enterokinase site | 718 | 732 |
| His-tag | 748 | 765 |
| Start codon | 769 | 771 |
• T7promoter_dhdO_typh_T7terminator:
caaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatcctcgagcatatgttactgttgtggtggcaagaccagctcggcgaaaggcgacggcgccgcgaagggggctctgtctgataaaaccaaagcttcctgtagcgcgcgtgattgtggcccgctcagggccggcccatcgcggtgaacacgtaaccacggtgtccctcttctcaatctctgcccaacgtctaccaaaagctcggcgccgacaccaagacgtaagggctcgcccgcgcgggaacgacccgcccccaactcgtgtaacaccagagctaacggaagtgctctgaccaactcaacggttccgtcagccggtgccaacaactcttcttgctcgcgtgcacggggcagtaattgccgtctttcagcaggagagcccgagcataaggctctggctaatcccggatcaactccctgcgcggcaagcatgcgttcaaatctaccaagggctgatccatcatccaatgcagctgcgacccgagctgctccctgcgcctgcgtaccggcgtggcctgacagccaaagaagggctccgcccaacgtcgtaaccaagtcccgaagatcgggaggcccggcaccgtccatgcataacaaagcctcttcgacctcaagggcatgaccgacacatcttccaagtggcttatccattgcggtcagcgcggcagctacgcgcagtcctaaagacgcccccaccccaaccaaagttttggccagttctcttgcttgttcttggttgggaaagacagctgcccccccgaattttacgtctacaaccaaggccgataatccctccaccagtttctttgacaggatgcttgccgttatcagtggcaaactatcaactgtcgcggtgacgtcacgcgccgcgtataatatgccttcagcgggaacaagctgttcgctctgaccgacaatacagcacccagcttgatccagcagaacttgcatctgctcaggcgactggattacattgaatccaggaatcgactcaagcttatcaagggttcccccggtgtgccccaagccacggccagaaatcattggaaccttgcatccgcaggccgctaaagcgggcgccaacaccaatgatactttgtcgccgaccccccctgtgctatgcttatcaacaagttgctggcgccaagcctcaggccactcaagctgctgaccactctgtgccaaagcctgagtcaacacacttgtctcctccagatccatgccacgcaatctgatggccatcaacattgcgccaatttgggcgccttgggctgacccattaacgactgcggctacaaaaccccggatatctgcttctgataatctcccgccgtcccgtttcatgcgtatcagctctggcagctgtttgggttctggagatgggtcaggcagaccttgcgacccctcacctgaaaagtctccgggcgcagggggcgcaccagtacctggagtcattaatgctgccatggtatatctccttcttaaagttaaacaaaattatttctatagggttatctgataaccctatagtgagtcgtattaatttcgc
| Feature name | Start | End |
| T7 terminator | 1 | 48 |
| Thymidine phosphorylase | 124 | 1572 |
| RBS | 1580 | 1584 |
| dhdO | 1616 | 1628 |
| T7 promoter | 1629 | 1647 |
• T7promoter_dhdO_ppnp_T7terminator:
taatacgactcactatagggttatcagataaccctatagaaataattttgtttaactttaagaaggagatataccatgcttcaaagtaatgagtacttttccggcaaagtgaaatcaatcggcttttccagcagcagcactggtcgcgccagcgtgggtgttatggttgaaggcgaatacaccttcagcaccgctgagccggaagagatgacggtaatcagtggcgcgctgaatgtgttactgcctgacgcgaccgactggcaggtgtatgaagccggttcggtgtttaatgttcccggtcacagtgagtttcatctgcaagttgccgaacccacctcttatctgtgccgctatctgtaaggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttg
| Feature name | Start | End |
| T7 promoter | 1 | 19 |
| dhdO | 20 | 32 |
| RBS | 64 | 68 |
| ppnp | 76 | 360 |
| T7 terminator | 419 | 466 |
• T7promoter_dhdO_sfgfp_T7terminator:
caaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatcctcgagcatatgttatttgtatagttcatccatgccatgtgtaattccagcagctgttacaaactcaagaaggaccatgtggtctctcttttcgttgggatctttcgaaagggcagattgtgtggacaggtaatggttgtctggtaaaaggacagggccatcgccaattggagtattttgttgataatggtctgctagttgaacgcttccatcttcaatgttgtgtctaattttgaagttaactttgattccattcttttgtttgtctgccatgatgtatacattgtgtgagttaaagttgtattccaatttgtgtccaagaatgtttccatcttctttaaaatcaataccttttaacacgattctattaacaagggtatcaccttcaaacttgacttcagcacgtgtcttgtagtacccgtcatctttgaaaaatatagttctttcctgtacataaccttcgggcatggcactcttgaaaaagtcatgctgtttcatatgatctgggtatctcgcaaagcattgaagaccatacgcgagagtagtgacgagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtaagttttccgtatgttgcatcaccttcaccctctccactgacagaaaatttgtgcccattaacgtcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttactcatggtatatctccttcttaaagttaaacaaaattatttcttgagggttatcagataaccctatagtgagtcgtattaatttcgc
| Feature name | Start | End |
| T7 terminator | 1 | 48 |
| sfgfp | 124 | 840 |
| RBS | 848 | 852 |
| dhdO | 884 | 896 |
| T7 promoter | 897 | 915 |
• SP6promoter_NtermT7_linkerJB_Pertuzumab_TerminatorT7:
atttaggtgacactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgttcagctcgcggacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcccgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaggcgcccgacagccttccagttcctaaaagaaatcaagccggaagccgtagcgtacatcaccattaagacctctctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcggaccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagggtggctctggctctggctcgagcgaagttcaacttgtagaatctggaggtggattagtgcaacctggcggaagtcttcgtctgtcttgtgcagcctccgggtttacattcactgactatacaatggattgggtccgtcaggcccccggaaaagggttagaatgggttgctgatgttaatccaaattctgggggttccatctacaatcagcgttttaaaggtcgctttacattgtccgtggaccgcagcaagaacacactgtacttacaaatgaactcgctgcgcgctgaggacaccgctgtctattactgtgcgcgcaatctgggaccgtcgttttattttgactattgggggcaaggaacactggttacagtttcctcggcgtccactaagggtccttcggttttccccttggctccgagctccaaatcgacaagtggagggacggcggctcttgggtgtcttgtcaaggactattttcccgaaccggtaactgtaagctggaatagcggtgcccttacgagtggggtccatacgttccctgccgtattgcaaagttcaggattatacagcctgtcaagtgttgtaacagttccaagttcatcactgggcactcaaacatacatttgtaatgttaaccataagccatcaaacaccaaggtcgataagaaggtagaacccaaaagctgtgataaaactcacggtgggggcggaagcggcggagggggatcaggcggaggggggtctgacattcagatgactcaaagcccatcgtcgttaagtgcatcagttggggaccgtgtgaccatcacgtgtaaagcgtcgcaagacgtttctattggtgtagcctggtatcaacaaaaaccgggtaaagctcctaagttacttatttatagtgcaagctaccgttacacgggagtgccttcgcgcttttcgggtagcgggagtggcaccgactttactttgaccatcagttcattacaaccagaagattttgcaacctattactgccaacaatactacatctacccgtatacattcggtcaagggactaaagttgagatcaaacgtaccgtggcggccccttctgtattcatcttccctccctcggacgagcagctgaagagcgggaccgcatctgtagtgtgtttgttgaacaacttttatccccgcgaggccaaggttcagtggaaggttgataacgcgttacagtcgggcaactcgcaggagagtgttactgagcaggattcaaaagattctacttattcgctgagctctaccttaactttatcaaaagctgattatgagaagcataaggtctacgcttgcgaagttactcatcaaggactgtctagtcctgtcactaagagctttaaccgcggcgagtgctaatagcataaccccttggggcctctaaacgggtcttgaggggttttttg
| Feature name | Start | End |
| SP6 promoter | 1 | 18 |
| RBS | 68 | 74 |
| Nterm T7 RNA polymerase | 82 | 618 |
| Linker | 619 | 642 |
| VH pertuzumab | 643 | 1320 |
| Linker (GGGGS)3 | 1321 | 1365 |
| VL pertuzumab | 1366 | 2007 |
| T7 terminator | 2011 | 2057 |
• SP6promoter_Trastuzumab_linkerJB:
atttaggtgacactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggaagtacaattagttgaatcgggtggtggtctggtccaaccgggtggctcgctgcgtctgtcgtgtgcggcgtcgggctttaacatcaaagatacctatattcattgggttcgtcaggcaccgggcaaaggtctggaatgggtcgctcgcatctacccgaccaacggctatacgcgttacgcggattctgtgaaaggtcgctttaccatttccgcggacacctcaaaaaacacggcctatctgcaaatgaacagcctgcgtgcagaagacacggctgtttattactgtagtcgctggggcggtgatggcttttatgccatggactactggggccaaggtaccctggtcaccgtgagctctgcatcgaccaaaggtccgagcgtcttcccgctggctccgagttccaaatcgaccagcggcggtacggcagcactgggttgtctggttaaagattattttccggaaccggttaccgtctcttggaacagtggcgcgctgaccagcggtgtccatacgttcccggcagtgctgcaatcatcgggtctgtatagcctgagctctgtggttaccgtgccgagttcctcactgggtacccaaacgtacatctgcaacgttaatcacaaaccgtccaatacgaaagtggataagaaagttgaaccgccgaaatcatgtgacaaaacccatacctgcccgccgtgtccggcaccggaactgctgggcggtccgagcgtgtttctgttcccgccgaaaccgaaagataccctgatgatctcgcgtaccccggaagttacgtgcgtcgtggttgatgtctctcatgaagacccggaagtgaaattcaactggtatgttgacggtgtcgaagtgcacaatgccaaaaccaaaccgcgtgaagaacagtataactcgacctaccgcgtcgtgagcgttctgacggtcctgcatcaagattggctgaacggcaaagaatacaaatgcaaagttagtaacaaagcactgccggctccgattgaaaagaccatcagcaaagcaaagggtcagccgcgtgaaccgggagggggtgggtcaggcggcggtggaagtgggggtgggggctcggatatacaaatgactcagtccccgagcagcctgagcgccagcgttggtgaccgtgttacgattacctgtcgtgcgagccaagatgttaacaccgcagtcgcttggtatcagcaaaaaccgggcaaagcgccgaaactgctgatttatagcgcctcttttctgtactcgggtgtgccgagccgtttcagtggctcccgctcaggtaccgattttaccctgacgatcagctctctgcaaccggaagacttcgcaacgtattactgccagcaacattacaccacgccgccgacctttggccagggtacgaaagtggaaattaaacgtaccgttgcggccccgtctgtctttatcttcccgccgagcgatgaacagctgaaatctggcaccgcgagtgtggtttgtctgctgaacaatttctatccgcgcgaagcaaaagtccagtggaaagtggacaacgctctgcaatcgggtaatagccaagaaagcgtgaccgaacaagatagtaaagactccacgtactcactgagttccaccctgacgctgtccaaagcggattatgaaaaacataaagtgtacgcctgcgaagttacccaccagggcctgtcatcgccggtcacgaaatcttttaatcgtggcgagtgcacgtcgggtggaagcggtaaagcatttatgcaagttgtcgaggctgacatgct
| Feature name | Start | End |
| SP6 promoter | 1 | 18 |
| RBS | 68 | 74 |
| VH trastuzumab | 82 | 1134 |
| linker (GGGGS)3 | 1135 | 1179 |
| VL trastuzumab | 1180 | 1821 |
| linker | 1822 | 1839 |
| 5’Cterm T7 RNA polymerase | 1840 | 1874 |
• CtermT7_T7terminator:
ctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaatagcataaccccttggggcctctaaacgggtcttgaggggttttttg
| Feature name | Start | End |
| 3’Cterm T7 RNA polymerase | 1 | 2080 |
| T7 terminator | 2081 | 2127 |
• SP6promoter_FRB_NtermT7_T7terminator:
atttaggtgacactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgttcagctcgcggacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcccgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaggcgcccgacagccttccagttcctaaaagaaatcaagccggaagccgtagcgtacatcaccattaagacctctctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcggaccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagggtggctctggctctggctcgagcgccgtgttatggcacgaacagtggtatgagggtttggatgacgcatcgcgccagttcttcggagaacataacaccgaaaaaatgtttgcggcactggaacctctttatgagatgcttaaacgtgggcctgaaacgttgcgtgagatttcttttcagaactcatttgggcgtgatttgaacgatgcctacgaatggctgatgaattacaaaaagtcgaaagatgtatctaacctgaatcaagcatgggatatttactataatgtctttcgtaaaatcggctaatagcataaccccttggggcctctaaacgggtcttgaggggttttttg
| Feature name | Start | End |
| SP6 promoter | 1 | 18 |
| RBS | 68 | 74 |
| NtermT7 | 82 | 618 |
| Linker | 619 | 642 |
| FRB | 643 | 924 |
| T7 Terminator | 925 | 971 |
• SP6promoter_FKBP_CtermT7_T7terminator:
atttaggtgacactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatgggagttcaggtagagacaattagtcctggggatggccgcacatttccgaaacgcggccaaacctgtgtggtacactatacaggaatgttggaagatggcaaaaagtttgattccagtcgtgatcgcaacaagccttttaaatttatgttgggaaaacaggaggtaattcgcggttgggaggaaggtgtcgcacaaatgtccgtgggtcaacgcgccaagttgacgatctcacctgattacgcttacggagccaccggccatcctggcattattcccccacatgccactttggtctttgatgttgaacttttaaagttagaaacgtcgggtggaagcggtaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaatagcataaccccttggggcctctaaacgggtcttgaggggttttttg
| Feature name | Start | End |
| SP6 promoter | 1 | 18 |
| RBS | 68 | 74 |
| FKBP | 82 | 405 |
| Linker | 406 | 423 |
| CtermT7 | 424 | 2538 |
| T7 Terminator | 2539 | 2585 |
