Construction: pBEST-PX-UTRX-deGFP-T500, where X = A, B, D, F, G, L, and (P)L-L-PA-UTRA Backbone: pBEST-SphI // NcoI-deGFP-T500 Restriction enzymes: SphI GCATG^C (C^GTACG), NcoI C^CATGG (CCATG^G) Additional restriction sites for endpoint digestion: SphI -> SalI GTCGAC, NcoI -> HindIII AAGCTT PCR 5' hangover = 5'-SalI+SphI-3': GTCGACGCATGC PCR 3' hangover = 5'-HindIII+NcoI-3': AAGCTTCCATGG (RC = CCATGGAAGCTT) Sequences: PA-UTRA (61b) GTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTT PB-UTRB (212b) TTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCATCGCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTGGTTGGTTGTGGCCTGTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCTATGTGGCTAAATACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTAGGAGCTAAAGA PD-UTRD (71b) GTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGATGCTGTTCAACCACTAATAGGTAAGAAATC PF-UTRF (64b) TTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTGAGGATAAATT PG-UTRG (114b) GTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGCGGTAAAAATTTTAATTTTTGCCGCTGAGGGGTTGACCAAGCGAAGCGCGGTAGGTTTTCTGCTTAGGAGTTTAATC PL-UTRL (103b) ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTA PL-L-PA-UTRA (300b) ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTT ---------- Linkers = SphI-PX-UTRX-NcoI: PA-UTRA (73b) GCATGCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTCCATGG PB-UTRB (224b) GCATGCTTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCATCGCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTGGTTGGTTGTGGCCTGTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCTATGTGGCTAAATACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTAGGAGCTAAAGACCATGG PD-UTRD (82b) GCATGCGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGATGCTGTTCAACCACTAATAGGTAAGAAATCCATGG PF-UTRF (76b) GCATGCTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTGAGGATAAATTCCATGG PG-UTRG (125b) GCATGCGTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGCGGTAAAAATTTTAATTTTTGCCGCTGAGGGGTTGACCAAGCGAAGCGCGGTAGGTTTTCTGCTTAGGAGTTTAATCCATGG PL-UTRL (115b) GCATGCATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTACCATGG PL-L-PA-UTRA (312b) GCATGCATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTCCATGG ---------- Oligonucleotides to order: SphI-PA-UTRA-NcoI S LKR (61b): CGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTC SphI-PA-UTRA-NcoI AS LKR (71b): CATGGGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTGCATG SphI-PB-UTRB S PMR (30b): GTCGACGCATGCTTGCAAAATACGTGGCCT PB-UTRB-NcoI AS PMR (35b): CCATGGAAGCTTTCTTTAGCTCCTAGACCTTTAGC SphI-PD-UTRD-NcoI S LKR (72b): CGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGATGCTGTTCAACCACTAATAGGTAAGAAATC SphI-PD-UTRD-NcoI AS LKR (80b): CATGGATTTCTTACCTATTAGTGGTTGAACAGCATCGGACTCAGATAGTAATCCACGCTCTTTTAAAATGTCAACGCATG SphI-PF-UTRF-NcoI S LKR (66b): CTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTGAGGATAAATTC SphI-PF-UTRF-NcoI AS LKR (76b): CATGGAATTTATCCTCAAGTAAGGGGCCGAAGCCCCTGCAATTAAAATTGTTGACCACCTACATACCAAGCATG SphI-PG-UTRG S PMR (35b): GTCGACGCATGCGTGATAAAAGATTGAGTGTGAGG PG-UTRG-NcoI AS PMR (35b): CCATGGAAGCTTATTAAACTCCTAAGCAGAAAACC SphI-PL-UTRL S PMR (31b): GTCGACGCATGCATGACTCGCAAGGTTAGTG PL-UTRL-NcoI AS PMR (34b): CCATGGAAGCTTTAGAAATATCCTTTGCAGTAGC SphI-PL-L S (31b): same as SphI-PL-UTRL-NcoI S, (GTCGACGCATGCATGACTCGCAAGGTTAGTG) PA-UTRA-NcoI AS (31b): CCATGGAAGCTTAAAAAAGCCTCCAAGATTT ---------- Construction methods: 1. PA-UTRA (61b): SphI(sticky)-PA-UTRA-NcoI(sticky) sense linker (63b) *order CGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTC SphI(sticky)-PA-UTRA-NcoI(sticky) antisense (71b) *order CATGGGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTGCATG 2. PB-UTRB (212 bp) PRIMER PICKING RESULTS FOR PB-UTRB LKR No mispriming library specified Using 1-based sequence positions WARNING: Left primer is unacceptable: High 3' stability OLIGO start len tm gc% any 3' seq LEFT PRIMER 1 18 57.70 44.44 6.00 2.00 TTGCAAAATACGTGGCCT RIGHT PRIMER 212 23 55.69 43.48 4.00 2.00 TCTTTAGCTCCTAGACCTTTAGC SEQUENCE SIZE: 212 INCLUDED REGION SIZE: 212 PRODUCT SIZE: 212, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00 1 TTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCATCGCAGTTCGCTACACGCAGGAC >>>>>>>>>>>>>>>>>> 61 GCTTTTTCACGTTCTGGTTGGTTGTGGCCTGTTGATGCTAAAGGTGAGCCGCTTAAAGCT 121 ACCAGTTATATGGCTGTTGGTTTCTATGTGGCTAAATACGTTAACAAAAAGTCAGATATG 181 GACCTTGCTGCTAAAGGTCTAGGAGCTAAAGA <<<<<<<<<<<<<<<<<<<<<<< Final PCR product: 5'-SacI-SphI-PB-UTRB-NcoI-Hind3-3' (236b) GTCGACGCATGCTTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCATCGCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTGGTTGGTTGTGGCCTGTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCTATGTGGCTAAATACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTAGGAGCTAAAGACCATGGAAGCTT 3. PD-UTRD (71b): SphI(sticky)-PD-UTRD-NcoI(sticky) sense linker (72 b) *order CGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGATGCTGTTCAACCACTAATAGGTAAGAAATC SphI(sticky)-PD-UTRD-NcoI(sticky) antisense linker (80 b) *order CATGGATTTCTTACCTATTAGTGGTTGAACAGCATCGGACTCAGATAGTAATCCACGCTCTTTTAAAATGTCAACGCATG 4. PF-UTRF (64b): SphI(sticky)-PF-UTRF-NcoI(sticky) sense linker (66b) *order CTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTGAGGATAAATTC SphI(sticky)-PD-UTRD-NcoI(sticky) antisense linker (74b) *order CATGGAATTTATCCTCAAGTAAGGGGCCGAAGCCCCTGCAATTAAAATTGTTGACCACCTACATACCAAGCATG ---------- 5. PRIMER PICKING RESULTS FOR PG-UTRG LKR No mispriming library specified Using 1-based sequence positions OLIGO start len tm gc% any 3' seq LEFT PRIMER 1 23 55.57 39.13 2.00 0.00 GTGATAAAAGATTGAGTGTGAGG RIGHT PRIMER 113 23 55.27 34.78 5.00 0.00 ATTAAACTCCTAAGCAGAAAACC SEQUENCE SIZE: 113 INCLUDED REGION SIZE: 113 PRODUCT SIZE: 113, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00 1 GTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGCGGTAAAAATTTTAATTTTTGCC >>>>>>>>>>>>>>>>>>>>>>> 61 GCTGAGGGGTTGACCAAGCGAAGCGCGGTAGGTTTTCTGCTTAGGAGTTTAAT <<<<<<<<<<<<<<<<<<<<<<< Final PCR 5'-SacI-SphI-PG-UTRG-NcoI-HindIII-3' (137 bp) GTCGACGCATGCGTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGCGGTAAAAATTTTAATTTTTGCCGCTGAGGGGTTGACCAAGCGAAGCGCGGTAGGTTTTCTGCTTAGGAGTTTAATCCATGGAAGCTT 6. PRIMER PICKING RESULTS FOR PL-UTRL OLIGO start len tm gc% any 3' seq LEFT PRIMER 1 19 53.74 47.37 4.00 0.00 ATGACTCGCAAGGTTAGTG RIGHT PRIMER 103 22 53.43 36.36 4.00 2.00 TAGAAATATCCTTTGCAGTAGC SEQUENCE SIZE: 103 INCLUDED REGION SIZE: 103 PRODUCT SIZE: 103, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 3.00 1 ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGG >>>>>>>>>>>>>>>>>>> 61 TATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTA <<<<<<<<<<<<<<<<<<<<<< Final PCR product: 5'-SacI-SphI-PL-UTRL-NcoI-Hind3-3' (127) GTCGACGCATGCATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTACCATGGAAGCTT ---------- 7. PRIMER PICKING RESULTS FOR PL-L-PA-UTRA WARNING: Right primer is unacceptable: Long poly-X OLIGO start len tm gc% any 3' seq LEFT PRIMER 1 19 53.74 47.37 4.00 0.00 ATGACTCGCAAGGTTAGTG RIGHT PRIMER 300 19 53.30 31.58 4.00 4.00 AAAAAAGCCTCCAAGATTT SEQUENCE SIZE: 300 INCLUDED REGION SIZE: 300 PRODUCT SIZE: 300, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00 1 ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGG >>>>>>>>>>>>>>>>>>> 61 TATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCT 121 GCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC 181 AATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCG 241 TCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTT <<<<<<<<<<<<<<<<<<< Final PCR product: 5'-SacI-SphI-PL-L-PA-UTRA-NcoI-Hind3-3' (324) GTCGACGCATGCATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTCCATGGAAGCTT