Team:NCTU Formosa/Notebook/test6

9/4

Cultivation of:

Pcons+RBS+AseI PCR product-1,2,3,4,7,8 Ar

XhoI+PSB1K3 PCR product-1,2 Kr

PSB1A3 Ar

Cultivation plate test:

Ar plate and Kr plate

Digestion of:

[PBAN-HindIII] Kr*2 (EP), [PBAN-NheI]Kr*2 (EP), [PBAN-KpnI-HF]Kr*2 (EP)

[23101+B0034+PBAN (HindIII)]Ar(ES),[23101+B0034+PBAN (NheI)]Ar(ES)

PCR of:

[J23101+B0034+KpnI-HF] Ar*3

Ligation of:

[J23101+B0034+KpnI-HF+PSB1C3]

Electrophoresis of:

[PBAN-HindIII] Kr*2 (EP), [PBAN-NheI]Kr*2 (EP), [PBAN-KpnI-HF]Kr*2 (EP),[23101+B0034+PBAN (HindIII)]Ar(ES),[23101+B0034+PBAN (NheI)]Ar(ES),[23101+B0034+PBAN (HindIII)]Ar(EP)*2,[23101+B0034+PBAN (NheI)]Ar(EP),[23101+B0034+PBAN (KpnI-HF)]Ar(EP)*2,[23101+B0034+PBAN (KpnI-HF)]Ar PCR*3

9/5

Electrophoresis:

Well 1 Pcons+RBS+AseI--1 (EP) Ar Well 2 Pcons+RBS+AseI--2 (EP) Ar Well 3 Pcons+RBS+AseI--3 (EP) Ar Well 4 Pcons+RBS+AseI--4 (EP) Ar Well 5 Pcons+RBS+AseI--7 (EP) Ar （DNA sequencing） Well 6 Pcons+RBS+AseI--8 (EP) Ar Well 7 Pcons+RBS+NcoI--2 (EP) Cr （DNA sequencing） Well 8 Pcons+RBS+Ndel--2 (EP) Cr （DNA sequencing） Well 9 1 Kb marker Well 10 Pcons+RBS+Ndel--13 (EP) Cr Well 11 SacI--3 (XP) Kr Well 12 SacI--4.1 (XP) Kr Well 13 SacI--4.2 (XP) Kr Well 14 XhoI--7 (EP) Kr （DNA sequencing） Well 15 XhoI--8 (EP) Kr（DNA sequencing） Well 16 SalI--3 (XP) Kr

Mini prep of:

Pcons+RBS+KpnI-HF-7 Pcons+RBS+KpnI-HF-8 Pcons+RBS+KpnI-HF-9

Digestion of:

Pcons+RBS+KpnI-HF-7 (EP) Pcons+RBS+KpnI-HF-8 (EP) Pcons+RBS+KpnI-HF-9 (EP)

Cultivation of:

Pcons+RBS+KpnI-HF Ar PCR product-7,8,9 Ar Ligation of: [J23101+B0034+HindIII+B0034+PSB1K3] Kr [J23101+B0034+HindIII+J61048+PSB1K3] Kr [J23101+B0034+NheI+B0034+PSB1K3] Kr [J23101+B0034+NheI+J61048+PSB1K3] Kr Electrophoresis of digested product: [Pcons+RBS+KpnI-HF]Ar (EP)*3

2014/9/9 Cultivation of: Pcons+RBS+SacI PCR product-16 Ar Pcons+RBS+SaII PCR product-17 Ar RBS+BFP+Ter PCR product-16,17,29,30 Ar Digestion of: XhoI+PSB1K3 (EP)

2014/9/10 Mini prep of: RBS+BFP+Ter-16 Ar RBS+BFP+Ter-17 Ar RBS+BFP+Ter-29 Ar RBS+BFP+Ter-30 Ar Pcons+RBS+Sac-16 Ar Pcons+RBS+Sal-17 Ar Digestion of: RBS+BFP+Ter-16 Ar(EP) RBS+BFP+Ter-17 Ar(EP) RBS+BFP+Ter-29 Ar(EP) RBS+BFP+Ter-30 Ar(EP) Pcons+RBS+SacI-16 Ar(EP) Pcons+RBS+SalI-17 Ar(EP) Electrophoresis: Well1：Marker Well2：Pcons+RBS+SacI-16 dig(EP) Well3：Pcons+RBS+SalI-17 dig(EP) Well4：RBS+BFP+Ter-16 dig(EP) Well5：RBS+BFP+Ter-17 dig(EP) Well6：RBS+BFP+Ter-29 dig(EP) Well7：RBS+BFP+Ter-30 dig(EP) Well8：XhoI-7 dig(XP) Kr

2014/9/11 Digestion of: Pcons+RBS+PBAN(ES)(8 kind except of XhoI) RBS+BFP+Ter(XP) Ligation of: Pcons+RBS+XhoI-7 Ar Cultivation of: Pcons+RBS+AseI-7 Ar Pcons+RBS+SacI-16 Ar Pcons+RBS+SalI-17 Ar Pcons+RBS+NcoI-2 Ar Pcons+RBS+NdeI-2 Ar RBS+BFP+Ter-2 Ar RBS+BFP+Ter-2 Ar SalI-3 Kr Ligation of: Pcons+RBS+XhoI-7 Ar DNA sequencing of: Pcons+RBS+SaeI Ar 8ul VF2 Pcons+RBS+SalI Ar 8ul VF2 RBS+BFP+Ter-29 Ar 8ul VF2 + 8ul VR RBS+BFP+Ter-30 Ar 5ul VF2 + 5ul VR Digestion of: Pcons+RBS+HindIII dig(EP) Pcons+RBS+NheI dig(EP) Pcons+RBS+NcoI dig(EP)-->9/11 mini Pcons+RBS+NdeI dig(EP) Pcons+RBS+SacI dig(EP) Pcons+RBS+SalI dig(EP) Pcons+RBS+AseI dig(EP) Pcons+RBS+KpnI dig(EP) Ligation of: Pcons+RBS+BFP+Ter-30 Kr

2014/9/12 Transformation of: Pcons+RBS+NheI+RBS+BFP+Ter Kr Pcons+RBS+NdeI+RBS+BFP+Ter Kr Pcons+RBS+HindIII+RBS+BFP+Ter Kr Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr Pcons+RBS+AseI+RBS+BFP+Ter Kr Pcons+RBS+SacI+RBS+BFP+Ter Kr Pcons+RBS+NcoI+RBS+BFP+Ter Kr Pcons+RBS+SalI+RBS+BFP+Ter Kr Pcons+RBS+BFP+Ter Kr Pcons+RBS+XhoI Ar Electrophoresis: Well1：Marker Well2：Pcons+RBS+NdeI dig(EP) Cr Well3：Pcons+RBS+NcoI dig(EP) Cr Well4：Pcons+RBS+SacI dig(EP) Ar Well5：Pcons+RBS+SalI dig(EP) Ar Well6：Pcons+RBS+AseI dig(EP) Ar Well7：Pcons+RBS+HindIII dig(EP) Ar Well8：Pcons+RBS+NheI dig(EP) Ar Well9：Pcons+RBS+KpnI dig(EP) Ar

2014/9/13 Ligation of: Pcons+RBS+AseI Kr Pcons+RBS+NdeI Kr Pcons+RBS+SalI Kr Pcons+RBS+SacI Kr PCR of: Pcons+RBS+HindIII+RBS+BFP+Ter Kr x8 Pcons+RBS+NheI+RBS+BFP+Ter Kr x8 Pcons+RBS+NdeI+RBS+BFP+Ter Kr x8 Pcons+RBS+NcoI+RBS+BFP+Ter Kr x8 Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr x8 Pcons+RBS+AseI+RBS+BFP+Ter Kr x8 Pcons+RBS+Xhol Kr x8 Pcons+RBS+SalI+RBS+BFP+Ter Kr x16 Pcons+RBS+SacI+RBS+BFP+Ter Kr x16 Electrophoresis: Gel 1 Pcons+RBS+SalI+RBS+BFP+Ter Kr x16 Gel 2 Pcons+RBS+SacI+RBS+BFP+Ter Kr x16 Gel 3 Pcons+RBS+HindIII+RBS+BFP+Ter Kr x8 Pcons+RBS+NheI+RBS+BFP+Ter Kr x8 Gel 4 Pcons+RBS+NdeI+RBS+BFP+Ter Kr x8 Pcons+RBS+NcoI+RBS+BFP+Ter Kr x8 Gel 5 Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr x8 Pcons+RBS+AseI+RBS+BFP+Ter Kr x8 Gel 6 Pcons+RBS+Xhol Kr x8 psb1K3 dig(EP)

2014/9/16 Transformation of psb3K3 *2 psb3C3 Digestion of: psb3K3 dig(EP) psb1C3 dig(EP) Cultivation of: Pcons+RBS+PBAN+RBS+BFP+Ter x8

2014/9/17 Mini prep of: PBAN：XhoI SalI SacI KpnI Ndel AseI NcoI NheI HindIII (fail) Pcons+RBS+PBAN+RBS+BFP+Ter：AseI-4 Ndel-8 HindIII-4 (fail) SacI-9(fail) KpnI-1 NcoI-2 NheI-1 Digestion of: PBAN：XhoI SalI SacI KpnI Ndel AseI NcoI AseI Pcons+RBS+PBAN+RBS+BFP+Ter：AseI-4 Ndel-8 KpnI-1 NcoI-2 NheI-1 Electrophoresis: Pcons+RBS+PBAN+RBS+BFP+Ter (Ndel) Pcons+RBS+PBAN+RBS+BFP+Ter (NcoI) Pcons+RBS+PBAN+RBS+BFP+Ter (SacI) Pcons+RBS+PBAN+RBS+BFP+Ter (HindIII) Pcons+RBS+PBAN+RBS+BFP+Ter (SalI) Pcons+RBS+PBAN+RBS+BFP+Ter (NheI) Pcons+RBS+PBAN+RBS+BFP+Ter (KpnI) Pcons+RBS+PBAN+RBS+BFP+Ter (AseI) PBAN AseI PBAN Ndel PBAN KpnI PBAN SacI PBAN SalI PBAN XhoI Pcons+RBS+XhoI-1 dig(EP) Pcons+RBS+Xhol-1 dig(ES) psb1C3 dig(ES) Cultivation of: Pcons+RBS+PBAN+RBS+BFP+Ter (SacI)-4.12 Pcons+RBS+PBAN+RBS+BFP+Ter (HindIII)-6 Pcons+RBS+PBAN+RBS+BFP+Ter (SalI)-9 Digestion of: B0034 Kr dig(ES) Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP) Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP) NcoI Kr dig(EP) Ndel Kr dig(EP) SalI Kr dig(EP)

2014/9/18 Electrophoresis: Well1：Marker Well2：B0034 Kr dig(ES) Well3：Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP) Well4：Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP) Well5：NcoI Kr dig(EP) Well6：Ndel Kr dig(EP) Well7：SalI Kr dig(EP) Electrophoresis: Well1：Marker Well2：B0034 Kr dig(ES) Well3：Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP) Well4：Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP) Well5：NcoI Kr dig(EP) Well6：Ndel Kr dig(EP) Well7：SalI Kr dig(EP) Mini prep: Pcons+RBS+HindIII+RBS+BFP+Ter-6 Pcons+RBS+SalI+RBS+BFP+Ter-9 Pcons+RBS+SacI+RBS+BFP+Ter-4 Pcons+RBS+SacI+RBS+BFP+Ter-11 Ligation of: Pcons+RBS+AseI+RBS+BFP+Ter Cr Pcons+RBS+NdeI+RBS+BFP+Ter Cr NheI Cr NcoI Cr

2014/9/20 Transformation of: AseI mini Cr Electrophoresis: Well1：Marker Well2：HindIII-2 Kr dig(EP) Well3：SacI Kr dig(EP) Well4：Pcons+RBS+HindIII-6+RBS+BFP+Ter Kr dig(EP) Well5：Pcons+RBS+SacI-11+RBS+BFP+Ter Kr dig(EP) Well6：Pcons+RBS+SacI-9+RBS+BFP+Ter Kr dig(EP) Well7：RBS+BFP+Ter Ar dig(XP) Well8：Pcons+RBS+NheI+RBS+BFP+Ter Kr dig(EP)

2014/9/21 Electrophoresis of PCR products: Gel 1 Well1：Marker Well2：3H1 Well3：3H2 Well4：3H3 Well5：3H4 Well6：3H5 Well7：3H6 Well8：Marker Well9：6K1 Well10：6K2 Well11：6K3 Well12：6K4 Well13：6K5 Well14：6K6 Well15：3Sl1 Well16：3Sl2 Well17：3Sl3 Gel 2 Well1：Marker Well2：6Sl4 Well3：6Sl5 Well4：3Nh1 Well5：3Nh2 Well6：3Nh3 Well7：3Nh4 Well8：Marker Well9：3Nh5 Well10：3Nh6 Well11：6X1 Well12：6X2 Well13：6X3 Well14：6X4 Well15：6X5 Well16：6X6 Well17：1K1 Gel 3 Well1：Marker Well2：1K2 Well3：1K3 Well4：1K4 Well5：1K5 Well6：1K6 Well7：1X1 Well8：Marker Well9：1X2 Well10：1X3 Well11：1X4 Well12：1X5 Well13：1X6 Well14：3K1 Well15：3K2 Well16：3K3 Well17：3K4 Gel 4 Well1：Marker Well2：3K5 Well3：3K6 Well4：3Sc1 Well5：3Sc2 Well6：3Sc3 Well7：3Sc4 Well8：Marker Well9：3Sc5 Well10：3Sc6 Well11：1Sc1 Well12：1Sc2 Well13：1Sc3 Well14：1Sc4 Well15：1Sc5 Well16：1Sc6 Well17：1Nc1 Gel 5 Well1：Marker Well2：1Nc2 Well3：1Nc3 Well4：1Nc4 Well5：1Nc5 Well6：1Nc6 Well7：1A1 Well8：Marker Well9：1A2 Well10：1A3 Well11：1A4 Well12：1A5 Well13：1A6 Well14：1Nd1 Well15：1Nd2 Well16：1Nd3 Well17：1Nd4 Gel 6 Well1：Marker Well2：1Nd5 Well3：1Nd6 Well4：3X1 Well5：3X2 Well6：3X3 Well7：3X4 Well8：Marker Well9：3X5 Well10：3X6 Well11：3A1 Well12：3A2 Well13：3A3 Well14：3A4 Well15：3A5 Well16：3A6 Cultivation of: KpnI-1 NdeI-1 XhoI-1 SacI-1 AseI-1 Pcons+RBS+HindIII-1 Pcons+RBS+KpnI-4 Pcons+RBS+NheI-4 Pcons+RBS+SalI-5 Pcons+RBS+AseI-5 Pcons+RBS+XhoI-1 Pcons+RBS+SacI-1 Pcons+RBS+KpnI-1+RBS+BFP+Ter Pcons+RBS+XhoI-6+RBS+BFP+Ter Digestion of: SalI Kr dig(EP) Ligation of: NdeI dig(EP)+C backbone

2014/9/22 Transformation of: NheI Cr Pcons+RBS+NheI+RBS+BFP+Ter Cr Pcons+RBS+NcoI+RBS+BFP+Ter Cr Pcons+RBS+Ndel+RBS+BFP+Ter Cr Pcons+RBS+AseI+RBS+BFP+Ter Cr Digestion of: HindIII(EP) Cultivation of: SacI-3 Kr NcoI-7 Cr NcoI-8 Cr Pcons+RBS+SacI-2 Pcons+RBS+XhoI-5+RBS+BFP+Ter pbs3C5 Ligation of: Pcons+RBS+NcoI+RBS+BFP+Ter Cr Pcons+RBS+HindIII+RBS+BFP+Ter Cr Pcons+RBS+SacI+RBS+BFP+Ter Cr Pcons+RBS+SalI+RBS+BFP+Ter Cr

2014/9/23 Electrophoresis: Well1：Marker Well2：KpnI-1 Cr dig(EP) Well3：NdeI-1 Cr dig(EP) Well4：XhoI-1 Cr dig(EP) Well5：SacI-1 Cr dig(EP) Well6：AseI-1 Cr dig(EP) Well7：Pcons+RBS+HindIII-1 dig(EP) Well8：Pcons+RBS+KpnI-4 dig(EP) Well9：Pcons+RBS+NheI-4 dig(EP) Well10：Pcons+RBS+AseI-5 dig(EP) Well11：Pcons+RBS+XhoI-1 dig(EP) Well12：Pcons+RBS+SacI-1 dig(EP) Well13：Pcons+RBS+KpnI-7+RBS+BFP+Ter dig(EP) Well14：SalI Kr dig(EP) 9/21 Well15：SalI Kr dig(EP) 9/19 DNA sequencing of: KpnI-1 Cr dig(EP) NdeI-1 Cr dig(EP) XhoI-1 Cr dig(EP) SacI-1 Cr dig(EP) AseI-1 Cr dig(EP) Pcons+RBS+HindIII-1 dig(EP) Pcons+RBS+KpnI-4 dig(EP) Pcons+RBS+NheI-4 dig(EP)<--錯誤變成KpnI Pcons+RBS+AseI-5 dig(EP) Pcons+RBS+XhoI-1 dig(EP) Pcons+RBS+SacI-1 dig(EP) Pcons+RBS+KpnI-7+RBS+BFP+Ter dig(EP) Transformation of: Pcons+RBS+HindIII+RBS+BFP+Ter Pcons+RBS+NcoI+RBS+BFP+Ter Pcons+RBS+SalI+RBS+BFP+Ter Pcons+RBS+SacI+RBS+BFP+Ter

2014/9/24 Cultivation of: Pcons+RBS+NheI+RBS+BFP+Ter Cr Pcons+RBS+AseI+RBS+BFP+Ter Cr Pcons+RBS+NcoI+RBS+BFP+Ter Cr Pcons+RBS+NdeI+RBS+BFP+Ter Cr NheI Cr psb3C5 Cr Cultivation of: Pcons+RBS+NheI+RBS+BFP+Ter Cr Pcons+RBS+AseI+RBS+BFP+Ter Cr Pcons+RBS+NcoI+RBS+BFP+Ter Cr Pcons+RBS+NdeI+RBS+BFP+Ter Cr NheI Cr psb3C5 Cr Electrophoresis: Well1：Marker Well2：SalI Kr dig(EP) Well3：Pcons+RBS+XhoI+RBS+BFP+Ter Cr dig(EP) Well4：Pcons+RBS+SacI Cr dig(EP) Well5：NcoI Cr dig(EP) Digestion of: Pcons+RBS+XhoI+RBS+BFP+Ter Cr dig(EP) Pcons+RBS+SacI-2 Cr dig(EP) NcoI-8 Cr dig(EP) SalI-3 Kr dig(EP) Ligation of: HindIII+psb1C3 PCR of: SacI x8 SalI x5 HindIII x2 NheI x8 Mini prep and digestion of: Pcons+RBS+AseI+RBS+BFP+Ter Pcons+RBS+NheI+RBS+BFP+Ter Pcons+RBS+NdeI+RBS+BFP+Ter Pcons+RBS+NcoI+RBS+BFP+Ter psb3C5 NheI

2014/9/25 Electrophoresis and DNAsequencing: Well1：Marker Well2：NheI (fail) Well3：Pcons+RBS+AseI+RBS+BFP+Ter Well4：Pcons+RBS+NcoI+RBS+BFP+Ter Well5：Pcons+RBS+NdeI+RBS+BFP+Ter (fail) Transformation of: HindIII SalI Pcons+RBS+SacI+RBS+BFP+Ter Pcons+RBS+SalI+RBS+BFP+Ter Pcons+RBS+HindIII+RBS+BFP+Ter PCR of: NcoI x5(9.10.11.12.13) Pcons+RBS+SacI x5(7.8.9.10.11) Cultivation of: NheI Pcons+RBS+SacI-2(9/22) Pcons+RBS+NcoI-8(9/22) Pcons+RBS+NheI-4 Pcons+RBS+NdeI+RBS+BFP+Ter x2 Pcons+RBS+NheI+RBS+BFP+Ter x2

2014/9/26 Mini prep and digestion(EP) of: NcoI NheI Pcons+RBS+SacI-2 Pcons+RBS+NheI-4 Pcons+RBS+NdeI-1+RBS+BFP+Ter Pcons+RBS+NdeI-2+RBS+BFP+Ter Electrophoresis: Well1：Marker Well2：Pcons+RBS+NdeI-1+RBS+BFP+Ter Well3：Pcons+RBS+NdeI-2+RBS+BFP+Ter Well4：NcoI Well5：NheI Well6：Pcons+RBS+NheI-4 Well7：Pcons+RBS+SacI-2 Cultivation of: psb3C5 x2 HindIII-3 HindIII-4 SalI-3 SalI-4 Pcons+RBS+HindIII-3+RBS+BFP+Ter Pcons+RBS+HindIII-4+RBS+BFP+Ter Pcons+RBS+SalI-3+RBS+BFP+Ter Pcons+RBS+SalI-4+RBS+BFP+Ter Pcons+RBS+SacI-3+RBS+BFP+Ter Pcons+RBS+SacI-4+RBS+BFP+Ter Pcons+RBS+NcoI-3+RBS+BFP+Ter Pcons+RBS+NcoI-4+RBS+BFP+Ter

2014/9/28 Electrophoresis: Well1：HindIII-3 Well2：HindIII-4 Well3：SalI-3 Well4：SalI-4 Well5：Pcons+RBS+SacI-3+RBS+BFP+Ter Well6：Marker Well7：Pcons+RBS+NcoI-3+RBS+BFP+Ter Well8：Pcons+RBS+NcoI-4+RBS+BFP+Ter Well9：Pcons+RBS+SalI-3+RBS+BFP+Ter Well10：Pcons+RBS+SalI-4+RBS+BFP+Ter Well11：psb3C5

2014/9/29 Electrophoresis: Well1：Marker Well2：Pcons+RBS+HindIII+RBS+BFP+Ter Well3：Pcons+RBS+HindIII+RBS+BFP+Ter Well4：Pcons+RBS+HindIII+RBS+BFP+Ter Well5：Pcons+RBS+HindIII+RBS+BFP+Ter Well6：Pcons+RBS+HindIII+RBS+BFP+Ter Well7：Pcons+RBS+HindIII+RBS+BFP+Ter Well8：Pcons+RBS+NcoI+RBS+BFP+Ter Well9：Pcons+RBS+NcoI+RBS+BFP+Ter Well10：Pcons+RBS+NcoI+RBS+BFP+Ter Well11：Pcons+RBS+NcoI+RBS+BFP+Ter Well12：Pcons+RBS+NcoI+RBS+BFP+Ter Well13：Pcons+RBS+NcoI+RBS+BFP+Ter Well14：Pcons+RBS+SalI+RBS+BFP+Ter Well15：Pcons+RBS+SalI+RBS+BFP+Ter Well16：Pcons+RBS+SalI+RBS+BFP+Ter Well17：Pcons+RBS+SalI+RBS+BFP+Ter Well18：Pcons+RBS+SalI+RBS+BFP+Ter Well19：Pcons+RBS+SalI+RBS+BFP+Ter Well20：psb3C5 dig Well21：Marker Ligation of: Pcons+RBS+KpnI + pbs3C5 Pcons+RBS+SacI + pbs3C5 Pcons+RBS+AseI + pbs3C5 Pcons+RBS+HindIII + pbs3C5 Pcons+RBS+NheI + pbs3C5 Pcons+RBS+SalI + pbs3C5 Pcons+RBS+NcoI + pbs3C5 Pcons+RBS+NdeI + pbs3C5 Pcons+RBS+XhoI+RBS+BFP+Ter + pbs1C3 Pcons+RBS+NdeI+RBS+BFP+Ter + pbs1C3 Pcons+RBS+SacI + pbs1C3 Pcons+RBS+NcoI + pbs1C3 DNA sequencing of: SalI-4 HindIII-4 Pcons+RBS+SalI-3+RBS+BFP+Ter Pcons+RBS+SalI-2 Pcons+RBS+NheI-4 NheI(9/26) Pcons+RBS+HindIII+RBS+BFP+Ter(9/30) Pcons+RBS+SalI+RBS+BFP+Ter(9/30)

2014/9/30 PCR and Electrophoresis: Pcons+RBS+NdeI+RBS+BFP+Ter NcoI Pcons+RBS+NheI+RBS+BFP+Ter Pcons+RBS+XhoI+RBS+BFP+Ter Pcons+RBS+SacI+RBS+BFP+Ter Cultivation of: Pcons+RBS+NheI-11+RBS+BFP+Ter Pcons+RBS+NheI-13+RBS+BFP+Ter Pcons+RBS+NdeI-3+RBS+BFP+Ter Pcons+RBS+NdeI-10+RBS+BFP+Ter Pcons+RBS+XhoI-3+RBS+BFP+Ter Pcons+RBS+XhoI-5+RBS+BFP+Ter Pcons+RBS+SacI-2+RBS+BFP+Ter Pcons+RBS+SacI-11+RBS+BFP+Ter NcoI-3 NcoI-12